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Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation
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Li Cui
Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.
Successful transplantation of in vitro expanded human corneal endothelial precursors to corneal endothelial surface using a nanocomposite sheet
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Parikumar P
Full Text Available Background: Though the transplantation of in vitro expanded human corneal endothelial precursors in animal models of endothelial damage by injecting into the anterior chamber has been reported, the practical difficulties of accomplishing such procedure in human patients have been a hurdle to clinical translation. Here we report the successful transplantation of in vitro expanded human corneal precursor cells to an animal eye using a transparent Nano-composite sheet and their engraftment.Materials and Methods: Human Corneal endothelial cells (HCEC were isolated from human cadaver eyes with informed consent and expanded in the lab using a sphere forming assay in a novel Thermoreversible Gelation Polymer (TGP for 26 days. HCEC obtained by sphere forming assay were seeded in a novel Nano-composite sheet, which was made of PNIPA-NC gels by in-situ, free-radical polymerization of NIPA monomer in the presence of exfoliated clay (synthetic hectorite "Laponite XLG" uniformly dispersed in aqueous media. After a further seven days in vitro culture of HCEC in the Nano-composite sheet, cells were harvested and transplanted on cadaver-bovine eyes (n=3. The cells were injected between the corneal endothelial layer and the Nano-composite sheet that had been placed prior to the injection in close proximity to the endothelial layer. After three hours, the transplanted Nano-composite sheets were removed from the bovine eyes and subjected to microscopic examination. The corneas were subjected to Histo-pathological studies along with controls. Results: HCEC formed sphere like colonies in TGP which expressed relevant markers as confirmed by RT-PCR. Microscopic studies of the Nanosheets and histopathological studies of the cornea of the Bull's eye revealed that the HCEC got engrafted to the corneal endothelial layer of the bovine eyes with no remnant cells in the Nanosheet. Conclusion: Transplantation of in vitro expanded donor human corneal endothelial cells
Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.
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Kathryn L McCabe
Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.
Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model.
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Natalia Vázquez
Full Text Available Corneal keratoplasty (penetrating or lamellar using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world's population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of
Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.
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Hannah J Levis
Full Text Available Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.
Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.
Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S
Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial
Trifluoperazine: corneal endothelial phototoxicity
International Nuclear Information System (INIS)
Hull, D.S.; Csukas, S.; Green, K.
Trifluoperazine is used for the treatment of psychiatric disorders. Perfusion of corneal endothelial cells with trifluoperazine-HC1 concurrent with exposure to long wavelength ultraviolet light resulted in a corneal swelling rate greater than that found in perfused corneas not exposed to ultraviolet light. Exposure of endothelial cells to 25 W incandescent light during perfusion with trifluoperazine-HC1 did not result in a higher corneal swelling rate compared to those perfused in the dark. The increased corneal swelling rate could be produced by pre-exposure of the trifluoperazine-HC1 perfusing solution to ultraviolet light suggesting the production of toxic photoproducts during exposure of trifluoperazine-HC1 to ultraviolet light. Perfusion of corneal endothelial cells with non-ultraviolet illuminated trifluoperazine-HC1 had no effect on endothelial cell membranes or ultrastructure. This is in contrast to cells perfused with trifluoperazine-HC1 that had been exposed to ultraviolet light in which there was an alteration of mitochondria and a loss of cytoplasmic homogeneity. The data imply that the trifluoperazine-HC1 photoproduct had an adverse effect on cellular transport mechanisms. The study also further demonstrates the value of the corneal endothelial cell model for identifying the physiological and anatomical changes occuring in photo-induced toxic reactions. (author)
Effects of phthalates on the human corneal endothelial cell line B4G12
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Krüger, Tanja; Cao, Yi; Kjærgaard, Søren K.
Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2...... toxicity was observed for DBP and BBP. Upon DBP exposure at nontoxic concentrations, a significant increased gene expression and cytokine cell secretion were observed for interleukin-1ß (IL-1ß) and IL-8, and also an increased IL-6 secretion was observed. In conclusion, the human corneal endothelial cell...
Cultivation of Human Microvascular Endothelial Cells on Topographical Substrates to Mimic the Human Corneal Endothelium
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Jie Shi Chua
Full Text Available Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet's membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+ adenine triphosphatase (ATPase expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.
Establishment of functioning human corneal endothelial cell line with high growth potential.
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Tadashi Yokoi
Full Text Available Hexagonal-shaped human corneal endothelial cells (HCEC form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+- and K(+-dependent ATPase (Na(+/K(+-ATPase. Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs in the Rb pathway (p16-CDK4/CyclinD1-pRb. In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7 and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin. Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7, THCEH (Cyclin and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7 and THCEH (Cyclin. THCEH (Cyclin expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na(+/K(+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7. This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.
Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium.
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Makiko Nakahara
Full Text Available Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs, even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM obtained from human bone marrow-derived mesenchymal stem cells (MSCs (MSC-CM for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na(+/K(+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27(Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary
Discovery of molecular markers to discriminate corneal endothelial cells in the human body
Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji; Clevers, J.C.; van de Wetering, M.L.
The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant
Discovery of Molecular Markers to Discriminate Corneal Endothelial Cells in the Human Body
Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji; Forrest, Alistair R. R.; Rehli, Michael; Baillie, J. Kenneth; de Hoon, Michiel J. L.; Haberle, Vanja; Lassmann, Timo; Kulakovskiy, Ivan V.; Lizio, Marina; Andersson, Robin; Mungall, Christopher J.; Meehan, Terrence F.; Schmeier, Sebastian; Bertin, Nicolas; Jørgensen, Mette; Dimont, Emmanuel; Arner, Erik; Schmidl, Christian; Schaefer, Ulf; Medvedeva, Yulia A.; Plessy, Charles; Vitezic, Morana; Severin, Jessica; Semple, Colin A.; Ishizu, Yuri; Francescatto, Margherita; Alam, Intikhab; Albanese, Davide; Altschuler, Gabriel M.; Archer, John A. C.; Arner, Peter; Babina, Magda; Baker, Sarah; Balwierz, Piotr J.; Beckhouse, Anthony G.; Pradhan-Bhatt, Swati; Blake, Judith A.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Geijtenbeek, Teunis B.
The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant
Revisited microanatomy of the corneal endothelial periphery: new evidence for continuous centripetal migration of endothelial cells in humans.
He, Zhiguo; Campolmi, Nelly; Gain, Philippe; Ha Thi, Binh Minh; Dumollard, Jean-Marc; Duband, Sébastien; Peoc'h, Michel; Piselli, Simone; Garraud, Olivier; Thuret, Gilles
The control of corneal transparency depends on the integrity of its endothelial monolayer, which is considered nonregenerative in adult humans. In pathological situations, endothelial cell (EC) loss, not offset by mitosis, can lead to irreversible corneal edema and blindness. However, the hypothesis of a slow, clinically insufficient regeneration starting from the corneal periphery remains debatable. The authors have re-evaluated the microanatomy of the endothelium in order to identify structures likely to support this homeostasis model. Whole endothelia of 88 human corneas (not stored, and stored in organ culture) with mean donor age of 80 ± 12 years were analyzed using an original flat-mounting technique. In 61% of corneas, cells located at the extreme periphery (last 200 μm of the endothelium) were organized in small clusters with two to three cell layers around Hassall-Henle bodies. In 68% of corneas, peripheral ECs formed centripetal rows 830 ± 295 μm long, with Descemet membrane furrows visible by scanning electron microscopy. EC density was significantly higher in zones with cell rows. When immunostained, ECs in the extreme periphery exhibited lesser differentiation (ZO-1, Actin, Na/K ATPase, CoxIV) than ECs in the center of the cornea but preferentially expressed stem cell markers (Nestin, Telomerase, and occasionally breast cancer resistance protein) and, in rare cases, the proliferation marker Ki67. Stored corneas had fewer cell clusters but more Ki67-positive ECs. We identified a novel anatomic organization in the periphery of the human corneal endothelium, suggesting a continuous slow centripetal migration, throughout life, of ECs from specific niches. Copyright © 2012 AlphaMed Press.
Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media
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Lucas Monferrari Monteiro Vianna
Full Text Available ABSTRACT Purpose: To compare cryopreserved human corneal endothelial cells (HCECs grown in human serum-supplemented media (HS-SM with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM. Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR for endothelial cell markers, and detachment time. Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages. Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.
A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function
Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.
Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors ("proliferative media") to media without those factors ("stabilizing media"). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488
Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.
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Ryohei Numata
Full Text Available The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.
Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.
Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru
To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.
In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins
International Nuclear Information System (INIS)
Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Soker, Shay; Khang, Gilson
The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation. (paper)
Human corneal endothelial cell transplantation using nanocomposite gel sheet in bullous keratopathy.
Parikumar, Periasamy; Haraguchi, Kazutoshi; Senthilkumar, Rajappa; Abraham, Samuel Jk
Transplantation of in vitro expanded human corneal endothelial precursors (HCEP) cells using a nanocomposite (D25-NC) gel sheet as supporting material in bovine's cornea has been earlier reported. Herein we report the transplantation of HCEP cells derived from a cadaver donor cornea to three patients using the NC gel sheet. In three patients with bullous keratopathy, one after cataract surgery, one after trauma and another in the corneal graft, earlier performed for congenital corneal dystrophy, not amenable to medical management HCEP cells isolated from a human cadaver donor cornea in vitro expanded using a thermoreversible gelation polymer (TGP) for 26 days were divided into three equal portions and 1.6 × 10 5 HCEP cells were injected on to the endothelium of the affected eye in each patient using the D25-NC gel sheet as a supporting material. The sheets were removed after three days. The bullae in the cornea disappeared by the 3 rd -11 th post-operative day in all the three patients. Visual acuity improved from Perception of light (PL)+/Projection of rays (PR)+ to Hand movements (HM)+ in one of the patients by post-operative day 3 which was maintained at 18 months follow-up. At 18 months follow-up, in another patient the visual acuity had improved from HM+ to 6/60 while in the third patient, visual acuity remained HM+ as it was prior to HCEP transplantation. There were no adverse effects during the follow-up in any of the patients.
Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.
Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla
To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.
Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.
Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz
The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. Copyright © 2014 Elsevier Ltd. All rights reserved.
Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro
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Jing Wang
Full Text Available AIM:To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2 on untransfected human corneal endothelial cells (HCECs in vitro.METHODS: After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L altered HCE cell morphology after treatment for 36h, increased the mean optical density (P<0.01 and the length of F-actin, reduced the mean optical density (P<0.01 of the collagen type IV in extracellular matrix (ECM and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. CONCLUTION:TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.
Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.
Ayaki, Masahiko; Yaguchi, Shigeo; Iwasawa, Atsuo; Koide, Ryohei
The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.
Assessment of the reliability of human corneal endothelial cell-density estimates using a noncontact specular microscope.
Doughty, M J; Müller, A; Zaman, M L
We sought to determine the variance in endothelial cell density (ECD) estimates for human corneal endothelia. Noncontact specular micrographs were obtained from white subjects without any history of contact lens wear, or major eye disease or surgery; subjects were within four age groups (children, young adults, older adults, senior citizens). The endothelial image was scanned, and the areas from > or =75 cells measured from an overlay by planimetry. The cell-area values were used to calculate the ECD repeatedly so that the intra- and intersubject variation in an average ECD estimate could be made by using different numbers of cells (5, 10, 15, etc.). An average ECD of 3,519 cells/mm2 (range, 2,598-5,312 cells/mm2) was obtained of counts of 75 cells/ endothelium from individuals aged 6-83 years. Average ECD estimates in each age group were 4,124, 3,457, 3,360, and 3,113 cells/mm2, respectively. Analysis of intersubject variance revealed that ECD estimates would be expected to be no better than +/-10% if only 25 cells were measured per endothelium, but approach +/-2% if 75 cells are measured. In assessing the corneal endothelium by noncontact specular microscopy, cell count should be given, and this should be > or =75/ endothelium for an expected variance to be at a level close to that recommended for monitoring age-, stress-, or surgery-related changes.
Chlorpromazine-induced corneal endothelial phototoxicity
International Nuclear Information System (INIS)
Hull, D.S.; Csukas, S.; Green, K.
Chlorpromazine, which has been used extensively for the treatment of psychiatric disorders, is known to accumulate in the posterior corneal stroma, lens, and uveal tract. Because it is a phototoxic compound, the potential exists for it to cause cellular damage after light exposure. Specular microscopic perfusion of corneal endothelial cells in darkness with 0.5 mM chlorpromazine HCl resulted in a swelling rate of 18 +/- 2 micrometer/hr, whereas corneas exposed to long-wavelength ultraviolet light for 3 min in the presence of 0.5 mM chlorpromazine swelled at 37 +/- 9 micrometer/hr (p less than 0.01). Preirradiation of 0.5 mM chlorpromazine solution with ultraviolet light for 30 min and subsequent corneal perfusion with the solution resulted in a corneal swelling rate of 45 +/- 19 micrometer/hr. Cornea endothelial cells perfused with 0.5 mM chlorpromazine that was preirradiated with ultraviolet light showed marked swelling on scanning electron microscopic examination, whereas those perfused with nonirradiated chlorpromazine were flat and showed a normal mosaic pattern. Combining either 500 U/ml catalase or 290 U/ml superoxide dismutase with chlorpromazine did not alter photoinduction of corneal swelling. The data suggest that corneal endothelial chlorpromazine phototoxicity is secondary to cytotoxic products resulting from the photodynamically induced decomposition of chlorpromazine and is not caused by hydrogen peroxide or superoxide anion generated during the phototoxic reaction
Corneal Endothelial Alterations in Chronic Renal Failure.
Sati, Alok; Jha, Ashok; Moulick, P S; Shankar, Sandeep; Gupta, Sandeep; Khan, M A; Dogra, Manu; Sangwan, Virender S
To evaluate the corneal endothelial changes in patients with chronic renal failure. A total of 128 corneas of 128 subjects were studied, and 3 groups were formed. The first, the dialyzed group, composed of 32 corneas of 32 patients; the second, the nondialyzed group, composed of 34 corneas of 34 patients; and the third, the age-matched control group, composed of 64 corneas of 64 healthy subjects were examined by a specular microscope and the endothelial parameters were compared. The dialyzed group (enhanced level of toxins in the blood) was further analyzed to assess the influence of blood urea, serum creatinine, serum calcium, and serum phosphorus including the duration of dialysis on corneal endothelium. On comparing the 3 groups using analysis of variance and posthoc tests, a significant difference was found in the central corneal thickness (CCT) and endothelial cell density (CD) between the control (CCT: 506 ± 29 μm, CD: 2760 ± 304 cells/mm) and dialyzed groups (CCT: 549 ± 30 μm, CD: 2337 ± 324 cells/mm) [P chronic renal failure, more marked in patients undergoing hemodialysis and with raised blood urea level.
Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.
Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas
The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.
Human corneal epithelial subpopulations
DEFF Research Database (Denmark)
Søndergaard, Chris Bath
Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity...... subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum...
The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.
Hongo, Akane; Okumura, Naoki; Nakahara, Makiko; Kay, EunDuck P; Koizumi, Noriko
We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.
Successful transplantation of in vitro expanded human cadaver corneal endothelial precursor cells on to a cadaver bovine's eye using a nanocomposite gel sheet.
Parikumar, Periyasamy; Haraguchi, Kazutoshi; Ohbayashi, Akira; Senthilkumar, Rajappa; Abraham, Samuel J K
In vitro expansion of human corneal endothelial precursor (HCEP) cells has been reported via production of cell aggregated spheres. However, to translate this procedure in human patients warrants maintaining the position of the eyeballs facing down for 36 h, which is not feasible. In this study, we report a method using a nanocomposite (NC) gel sheet to accomplish the integration of HCEP cells to the endothelium of cadaver bovine's eyes. HCEP cells were isolated from the corneal endothelium of a cadaver human eye and then expanded using a thermoreversible gelation polymer (TGP) as reported earlier. For the study, three cadaver bovine eyes were used. The NC gel sheets were inserted into the bovine eyes', aligned and suture-fixed in position under the host endothelium. HCEP cells previously expanded in the TGP were harvested and injected using a 26-gauge syringe between the endothelium and the NC gel sheet. The eyes were left undisturbed for three hours following which the NC gel sheets were gently removed. The corneas were harvested and subjected to histopathological studies. Histopathological studies showed that all the three corneas used for NC gel sheet implantation showed the presence of engrafted HCEP cells, seen as multi-layered cells over the native endothelium of the bovine cornea. Examination of the NC gel sheets used for implantation showed that only very few corneal endothelial cells remained on the sheets amounting to what could be considered negligible. The use of the NC gel sheet makes HCEP cell transplantation feasible for human patients. Further in vitro basic studies followed by translational studies are necessary to bring this method for clinical application in appropriate indications.
A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology.
Al-Fahdawi, Shumoos; Qahwaji, Rami; Al-Waisy, Alaa S; Ipson, Stanley; Ferdousi, Maryam; Malik, Rayaz A; Brahma, Arun
Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image. Copyright © 2018 Elsevier B.V. All rights reserved.
EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells.
Mergler, Stefan; Pleyer, Uwe; Reinach, Peter; Bednarz, Jürgen; Dannowski, Haike; Engelmann, Katrin; Hartmann, Christian; Yousif, Tarik
Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.
Validation of Na,K-ATPase pump function of corneal endothelial cells for corneal regenerative medicine.
Hatou, Shin; Higa, Kazunari; Inagaki, Emi; Yoshida, Satoru; Kimura, Erika; Hayashi, Ryuhei; Tsujikawa, Motokazu; Tsubota, Kazuo; Nishida, Kohji; Shimmura, Shigeto
Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.
Recovery of Corneal Endothelial Cells from Periphery after Injury.
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Sang Ouk Choi
Full Text Available Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.To investigate the recovery process of corneal endothelial cells (CECs from corneal endothelial injury.Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group. Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.
Polysaccharide coating of human corneal endothelium
DEFF Research Database (Denmark)
Schroder, H D; Sperling, S
Electron microscopy revealed the presence of a 600-1500 A thick layer of polysaccharide on the surface of human corneal endothelial cells. The surface layer was visualized by combined fixation and staining in a mixture of ruthenium red and osmium tetroxide. The coating material was stable for at ...... for at least 39 h post mortem and was retained on disintegrating cells....
The role of corneal endothelial morphology in graft assessment and prediction of endothelial cell loss during organ culture of human donor corneas.
Hermel, Martin; Salla, Sabine; Fuest, Matthias; Walter, Peter
Endothelial assessment is crucial in the release of corneas for grafting. We retrospectively analysed the role of endothelial morphology parameters in predicting endothelial cell loss during organ culture. Human donor corneas were cultured in minimal essential medium with 2% fetal calf serum and antibiotics. Initial endothelial morphology was assessed microscopically using score parameters polymegethism (POL), pleomorphism (PLE), granulation (GRA), vacuolization (VAC), segmentation of cell membranes (SEG), Descemet's folds (DF), trypan blue-positive cells (TBPC) and endothelial cell-free areas (ECFA). Some corneas were primarily rejected based on endothelial assessment. Endothelial cell density (ECD) was assessed at the beginning (I-ECD) and end of culture. Corneas were then placed in dehydration medium (as above + 5% dextran 500). In a subgroup, ECD was reassessed after dehydration. Endothelial cell loss during culture (ECL@Culture) and culture+dehydration (ECL-Culture&Dehydration) were calculated. Data were given as mean ± SD and analysed using multiple linear and logistic regression. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. I-ECD was 2812 ± 360/mm 2 (n = 2356). The decision to reject a cornea due to endothelial assessment was associated negatively with I-ECD (OR = 0.77/100 cells, CI 0.7-0.82) and positively with ECFA (OR = 2.7, CI 1.69-4.35), SEG (OR =1.3, CI 1.01-1.68) and donor age (OR = 1.26/decade, CI 1.33-1.41). ECL@Culture was 153 ± 201/mm 2 (n = 1277), ECL@Culture&Dehydration was 169 ± 183/mm 2 (n = 918). ECL@Culture was associated positively with donor age, I-ECD, GRA and TBPC, and negatively with PLE, and DF. ECL@Culture&Dehydration was associated positively with age, sex, initial ECD, POL, PLE, VAC and TBPC. Morphological parameters displayed associations with the exclusion of corneas from culture and with endothelial cell loss. Appropriate parameter selection for screening purposes may help improve
Corneal endothelial glutathione after photodynamic change
International Nuclear Information System (INIS)
Hull, D.S.; Riley, M.V.; Csukas, S.; Green, K.
Rabbit corneal endothelial cells perfused with 5 X 10(-6)M rose bengal and exposed to incandescent light demonstrated no alteration of either total of or percent oxidized glutathione after 1 hr. Addition of 5400 U/ml catalase to the perfusing solution had no effect on total glutathione levels but caused a marked reduction in percent oxidized glutathione in corneas exposed to light as well as in those not exposed to light. Substitution of sucrose for glucose in the perfusing solution had no effect on total or percent oxidized glutathione. Perfusion of rabbit corneal endothelium with 0.5 mM chlorpromazine and exposure to ultraviolet (UV) light resulted in no change in total glutathione content. A marked reduction in percent oxidized glutathione occurred, however, in corneas perfused with 0.5 mM chlorpromazine both in the presence and absence of UV light. It is concluded that photodynamically induced swelling of corneas is not the result of a failure of the glutathione redox system
Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.
Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro
To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.
Successful transportation of human corneal endothelial tissues without cool preservation in varying Indian tropical climatic conditions and in vitro cell expansion using a novel polymer.
Rao, Srinivas K; Sudhakar, John; Parikumar, Periyasamy; Natarajan, Sundaram; Insaan, Aditya; Yoshioka, Hiroshi; Mori, Yuichi; Tsukahara, Shigeo; Baskar, Subramani; Manjunath, Sadananda Rao; Senthilkumar, Rajappa; Thamaraikannan, Paramasivam; Srinivasan, Thangavelu; Preethy, Senthilkumar; Abraham, Samuel J K
Though the transplantation of human corneal endothelial tissue (CET) separated from cadaver cornea is in practice, its transportation has not been reported. We report the successful transportation of CET in varying Indian climatic conditions without cool preservation and the in vitro expansion of Human Corneal Endothelial Precursor Cells (HCEPCs) using a novel Thermo-reversible gelation polymer (TGP). CET from cadaver corneas (n = 67), unsuitable for transplantation, were used. In phase I, CET was transported in Basal Culture Medium (Group I) and TGP (Group II) and in Phase II, in TGP cocktail alone, from three hospitals 250-2500 km away, to a central laboratory. The transportation time ranged from 6 h to 72 h and the outdoor temperature between 20°C and 41°C. On arrival, CET were processed, cells were expanded upto 30 days in basal culture medium (Group A) and TGP scaffold (Group B). Cell viability and morphology were documented and Reverse transcription polymerase chain reaction (RT-PCR) characterization undertaken. In Phase I, TGP yielded more viable cells (0.11 × 10(6) cells) than Group I (0.04 × 10(6) cells). In Phase II, the average cell count was 5.44 × 10(4) cells. During expansion, viability of HCEPCs spheres in TGP was maintained for a longer duration. The cells from both the groups tested positive for B-3 tubulin and negative for cytokeratins K3 and K12, thereby proving them to be HCEPCs. TGP preserves the CET during transportation without cool preservation and supports in vitro expansion, with a higher yield of HCEPCs, similar to that reported in clinical studies.
Successful transportation of human corneal endothelial tissues without cool preservation in varying Indian tropical climatic conditions and in vitro cell expansion using a novel polymer
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Srinivas K Rao
Full Text Available Background: Though the transplantation of human corneal endothelial tissue (CET separated from cadaver cornea is in practice, its transportation has not been reported. We report the successful transportation of CET in varying Indian climatic conditions without cool preservation and the in vitro expansion of Human Corneal Endothelial Precursor Cells (HCEPCs using a novel Thermo-reversible gelation polymer (TGP. Materials and Methods: CET from cadaver corneas (n = 67, unsuitable for transplantation, were used. In phase I, CET was transported in Basal Culture Medium (Group I and TGP (Group II and in Phase II, in TGP cocktail alone, from three hospitals 250-2500 km away, to a central laboratory. The transportation time ranged from 6 h to 72 h and the outdoor temperature between 20°C and 41°C. On arrival, CET were processed, cells were expanded upto 30 days in basal culture medium (Group A and TGP scaffold (Group B. Cell viability and morphology were documented and Reverse transcription polymerase chain reaction (RT-PCR characterization undertaken. Results: In Phase I, TGP yielded more viable cells (0.11 × 10 6 cells than Group I (0.04 × 10 6 cells. In Phase II, the average cell count was 5.44 × 10 4 cells. During expansion, viability of HCEPCs spheres in TGP was maintained for a longer duration. The cells from both the groups tested positive for B-3 tubulin and negative for cytokeratins K3 and K12, thereby proving them to be HCEPCs. Conclusion: TGP preserves the CET during transportation without cool preservation and supports in vitro expansion, with a higher yield of HCEPCs, similar to that reported in clinical studies.
Corneal endothelial cell density and morphology in healthy Turkish eyes.
Arıcı, Ceyhun; Arslan, Osman Sevki; Dikkaya, Funda
Purpose. To describe the normative values of corneal endothelial cell density, morphology, and central corneal thickness in healthy Turkish eyes. Methods. Specular microscopy was performed in 252 eyes of 126 healthy volunteers (M : F, 42 : 84). Parameters studied included mean endothelial cell density (MCD), mean cell area (MCA), coefficient of variation (CV) in cell size, percentage of hexagonal cells, and central corneal thickness (CCT). Results. The mean age of volunteers was 44.3 ± 13.5 (range, 20 to 70) years. There was a statistically significant decrease in MCD (P Filipino eyes and higher than that described in Indian, Thai, and Iranian eyes.
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Nishat Sultana K
Full Text Available BACKGROUND Angle closure is characterised by apposition of the peripheral iris against the trabecular meshwork resulting in obstruction of aqueous outflow. Acute angle-closure glaucoma is characterised by pain, redness and blurred vision. The pain is typically a severe deep ache that follows the trigeminal distribution and maybe associated with nausea, vomiting, bradycardia and profuse sweating. The blurred vision, which is typically marked maybe caused by stretching of the corneal lamellae initially and later oedema of the cornea as well as a direct effect of the IOP on the optic nerve head. The modifications in corneal endothelial cell density after a crisis of angle-closure glaucoma is being evaluated. AIMS AND OBJECTIVES The objective of the study is to assess the corneal endothelial cell count (density by specular microscopy in patients presenting with acute angle-closure glaucoma. METHODS Corneal endothelial cell counts of 20 eyes of patients with PACG with an earlier documented symptomatic acute attack unilaterally were compared with 20 fellow eyes. Evaluation of patient included visual acuity, intraocular pressure, gonioscopy, disc findings and specular microscopy. RESULTS The mean endothelial cell density was 2104 cells/mm2 in the eye with acute attack and 2615 cells/mm2 in the fellow eye. The average endothelial cell count when the duration of attack lasted more than 72 hours was 1861 cells/mm2 . CONCLUSION Corneal endothelial cell density was found to be significantly reduced in eyes following an acute attack of primary angle closure glaucoma.
New therapeutic modality for corneal endothelial disease using Rho-associated kinase inhibitor eye drops.
Koizumi, Noriko; Okumura, Naoki; Ueno, Morio; Kinoshita, Shigeru
Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal endothelial transplantation. However, despite the value and potential of endothelial graft surgery, a strictly pharmacological approach for treating corneal endothelial dysfunction remains an attractive proposition. Previously, we reported that the selective Rho-associated kinase (ROCK) inhibitor Y-27632 promotes cell adhesion and proliferation, and inhibits the apoptosis of primate corneal endothelial cells in culture. These findings have led us to develop a novel medical treatment for the early phase of corneal endothelial disease using ROCK inhibitor eye drops. In rabbit and monkey models of partial endothelial dysfunction, we showed that corneal endothelial wound healing was accelerated via the topical application of ROCK inhibitor to the ocular surface, resulting in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. Based on these animal studies, we are now attempting to advance the clinical application of ROCK inhibitor eye drops for patients with corneal endothelial dysfunction. A pilot clinical study was performed at the Kyoto Prefectural University of Medicine, and the effects of Y-27632 eye drops after transcorneal freezing were evaluated in 8 patients with corneal endothelial dysfunction. We observed a positive effect of ROCK inhibitor eye drops in treating patients with central edema caused by Fuchs corneal endothelial dystrophy. We believe that our new findings will contribute to the establishment of a new approach for the treatment of corneal endothelial dysfunction.
Human tears reveal insights into corneal neovascularization.
Zakaria, Nadia; Van Grasdorff, Sigi; Wouters, Kristien; Rozema, Jos; Koppen, Carina; Lion, Eva; Cools, Nathalie; Berneman, Zwi; Tassignon, Marie-José
Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization.
Development of Cell Analysis Software for Cultivated Corneal Endothelial Cells.
Okumura, Naoki; Ishida, Naoya; Kakutani, Kazuya; Hongo, Akane; Hiwa, Satoru; Hiroyasu, Tomoyuki; Koizumi, Noriko
To develop analysis software for cultured human corneal endothelial cells (HCECs). Software was designed to recognize cell borders and to provide parameters such as cell density, coefficient of variation, and polygonality of cultured HCECs based on phase contrast images. Cultured HCECs with high or low cell density were incubated with Ca-free and Mg-free phosphate-buffered saline for 10 minutes to reveal the cell borders and were then analyzed with software (n = 50). Phase contrast images showed that cell borders were not distinctly outlined, but these borders became more distinctly outlined after phosphate-buffered saline treatment and were recognized by cell analysis software. The cell density value provided by software was similar to that obtained using manual cell counting by an experienced researcher. Morphometric parameters, such as the coefficient of variation and polygonality, were also produced by software, and these values were significantly correlated with cell density (Pearson correlation coefficients -0.62 and 0.63, respectively). The software described here provides morphometric information from phase contrast images, and it enables subjective and noninvasive quality assessment for tissue engineering therapy of the corneal endothelium.
TGF-β2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation
International Nuclear Information System (INIS)
Lu Jiawei; Lu Zhenyu; Reinach, Peter
The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-β2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-β2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-β2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-β2 and FGF-2 oppositely affect BCE cell proliferation and TGF-β2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-β2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-β2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-β2-induced suppression of the PI3-kinase/AKT signaling pathway
Applications of Biomaterials in Corneal Endothelial Tissue Engineering.
Wang, Tsung-Jen; Wang, I-Jong; Hu, Fung-Rong; Young, Tai-Horng
When corneal endothelial cells (CECs) are diseased or injured, corneal endothelium can be surgically removed and tissue from a deceased donor can replace the original endothelium. Recent major innovations in corneal endothelial transplantation include replacement of diseased corneal endothelium with a thin lamellar posterior donor comprising a tissue-engineered endothelium carried or cultured on a thin substratum with an organized monolayer of cells. Repairing CECs is challenging because they have restricted proliferative ability in vivo. CECs can be cultivated in vitro and seeded successfully onto natural tissue materials or synthetic polymeric materials as grafts for transplantation. The optimal biomaterials for substrata of CEC growth are being investigated. Establishing a CEC culture system by tissue engineering might require multiple biomaterials to create a new scaffold that overcomes the disadvantages of single biomaterials. Chitosan and polycaprolactone are biodegradable biomaterials approved by the Food and Drug Administration that have superior biological, degradable, and mechanical properties for culturing substratum. We successfully hybridized chitosan and polycaprolactone into blended membranes, and demonstrated that CECs proliferated, developed normal morphology, and maintained their physiological phenotypes. The interaction between cells and biomaterials is important in tissue engineering of CECs. We are still optimizing culture methods for the maintenance and differentiation of CECs on biomaterials.
Corneal Endothelial Cell Density and Morphology in Healthy Turkish Eyes
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Ceyhun Arıcı
Full Text Available Purpose. To describe the normative values of corneal endothelial cell density, morphology, and central corneal thickness in healthy Turkish eyes. Methods. Specular microscopy was performed in 252 eyes of 126 healthy volunteers (M : F, 42 : 84. Parameters studied included mean endothelial cell density (MCD, mean cell area (MCA, coefficient of variation (CV in cell size, percentage of hexagonal cells, and central corneal thickness (CCT. Results. The mean age of volunteers was 44.3±13.5 (range, 20 to 70 years. There was a statistically significant decrease in MCD (P<0.001; correlation, −0.388 and percentage of hexagonal cells, (P<0.001; correlation, −0.199 with age. There was also a statistically significant increase in MCA (P<0.001; correlation, 0.363 with increasing age. There was no statistically significant difference in MCD, MCA, CV in cell size, percentage of hexagonal cells, and CCT between genders and there was also no significant difference in these parameters between fellow eyes of subjects. Conclusions. Normotive data for the endothelium in the Turkish population are reported. Endothelial cell density in the Turkish eyes is less than that described in the Japanese, American, Chinese, and Filipino eyes and higher than that described in Indian, Thai, and Iranian eyes.
CD147 required for corneal endothelial lactate transport.
Li, Shimin; Nguyen, Tracy T; Bonanno, Joseph A
CD147/basigin is a chaperone for lactate:H(+) cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema. CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity. In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls. CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
Corneal endothelial cell density and morphology in Phramongkutklao Hospital
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Narumon Sopapornamorn
Full Text Available Narumon Sopapornamorn1, Manapon Lekskul1, Suthee Panichkul21Department of Ophthalmology, Phramongkutklao Hospital, Bangkok, Thailand; 2Department of Obstetrics and Gynecology, Phramongkutklao College of Medicine, Bangkok, ThailandObjective: To describe the corneal endothelial density and morphology in patients of Phramongkutklao Hospital and the relationship between endothelial cell parameters and other factors.Methods: Four hundred and four eyes of 202 volunteers were included. Noncontact specular microscopy was performed after taking a history and testing the visual acuity, intraocular pressure measurement, Schirmer's test and routine eye examination by slit lamp microscope. The studied parameters included mean endothelial cell density (MCD, coefficient of variation (CV, and percentage of hexagonality.Results: The mean age of volunteers was 45.73 years; the range being 20 to 80 years old. Their MCD (SD, mean percentage of CV (SD and mean (SD percentage of hexagonality were 2623.49(325 cell/mm2, 39.43(8.23% and 51.50(10.99%, respectively. Statistically, MCD decreased significantly with age (p < 0.01. There was a significant difference in the percentage of CV between genders. There was no statistical significance between parameters and other factors.Conclusion: The normative data of the corneal endothelium of Thai eyes indicated that, statistically, MCD decreased significantly with age. Previous studies have reported no difference in MCD, percentage of CV, and percentage of hexagonality between gender. Nevertheless, significantly different percentages of CV between genders were presented in this study.Keywords: Corneal endothelial cell, parameters, age, gender, smoking, Thailand
[Influence AquaLase at corneal endothelial cells].
Jirásková, N; Rozsíval, P; Ludvíková, M; Burova, M; Nekolová, J
To assess the effect of the cleaning of the posterior capsule using pulses of balanced salt solution (BSS) on the corneal endothelial cells. This pilot study involves 43 patients with bilateral cataracts having lens removal using torsional phacoemulsification (Ozil, Infiniti, Alcon) and bimanul irrigation/aspiration (I/A). Posterior capsule of the right eye of each patient was cleaned using pulses of BSS (AquaLase, Infiniti, Alcon). Surgery was performed by one of 2 surgeons (NJ, PR), both eyes of each patient was operated on by the same surgeon. Best corrected visual acuity (BCVA), endotelial cell count and pachymetry were evaluated pre- and postoperatively as well as occurence af peri- and postoperative complications. Preoperative mean pachymetry (P) was 566 +/- 45 microm in the right eye (RE) and 562 +/- 42 microm in the left eye (LE), mean endotelial cell count (ECC) 2541 +/- 317 cells/mm2 (RE) and 2567 +/- 311 cells/mm2 (LE). Three months after surgery P was 557 +/- 43 microm (RE) and 558 +/- 45 microm (LE) and ECC 2368 +/- 416 cells/mm2 (RE) and 2396 +/- 417 cells/mm2 (LE). There was no statistical difference in postoperative changes of both corneal parameters between right and left eyes. Best corrected visual acuity improved in all eyes and no peri-or postoperative complications occured. Cleaning of the posterior capsule using AquaLase is safe for corneal endothelial cells.
Corneal Endothelial Safety of Intracameral Preservative-free 1% Xylocaine
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Shah Alpesh
Full Text Available Purpose : To evaluate the effect of intracameral preservative-free 1% xylocaine on the corneal endothelium as an adjuvant to topical anaesthesia during phacoemulsification and Acrysof foldable IOL implantation. Material & Methods: This is a prospective, controlled, randomised, double-masked study. 106 patients with soft to moderately dense (Grade 1-3 senile cataract and corneal endothelial cell density of >1500/mm2 were randomised to the xylocaine group (n=53 and control group(n=53. Central endothelial specular microscopy and ultrasound corneal pachymetry were performed preoperatively. On the first postoperative day the eyes were evaluated for corneal oedema and Descemet′s folds. Ultrasound corneal pachymetry was performed at 1, 3 and 12 months. Specular microscopy was performed at 3 and 12 months. Cell loss was expressed as a percentage of preoperative cell density. Six patients could not complete one year follow-up. Chi-square and paired t test (2 tail statistical tests were applied for analysis. Results: Four (7.54% patients in the xylocaine group and 5 (9.43% in the control group had a few Descemet′s folds associated with mild central stromal oedema. Corneal thickness increased from 549.3µ ± 37.2µ to 555.5µ ± 36.5µ in the xylocaine group and from 553.1µ ± 36.2µ to 559.3µ ± 40.5µ in the control group at the one-month postoperative visit. Thickness returned to the preoperative level in xylocaine group 549.6µ ± 34.5µ and control group 554.7µ ± 41.1µ at three months. (P=0.484 The percentage of cell loss was 4.47 ± 2.53% in the xylocaine group and 4.49 ± 3.09 % in the control group at one year. (P=0.97 Conclusion: Intracameral preservative-free 1% xylocaine does not appear to affect corneal endothelium adversely during phacoemulsification.
Corneal endothelial cell density and morphology in patients with acromegaly.
Hatipoglu, Esra; Arici, Ceyhun; Arslan, Osman Sevki; Dikkaya, Funda; Sultan, Pinar; Kadioglu, Pinar; Gundogdu, Sadi
Acromegaly has various impacts on many organs. The ophthalmologic effects of acromegaly have not yet been investigated in detail. The aim of the current study was to evaluate qualitative and quantitative changes in corneal endothelial cells and central corneal thickness (CCT) of the patients with acromegaly. In this prospective, cross-sectional study, 128 eyes of 64 patients with acromegaly (female/male=40/24) and 208 eyes of 104 age and gender-matched healthy volunteers (female/male=69/35) were included. Endothelial cell density (ECD), cellular area (CA), coefficient of variation (CV) in cell size, percentage of hexagonal cells, and CCT were measured in patients with acromegaly and in healthy volunteers using the noncontact specular microscopy (SP-3000P: Topcon Corporation, Tokyo, Japan). ECD and CA were lower in cases with acromegaly than in controls (ECD in acromegaly: 2615.65 cell/mm(2) and in controls: 2700.35 cell/mm(2); p=0.002. CA in acromegaly: 382.30μm(2) and in controls: 400.30μm(2); p=0.02). In the entire group with acromegaly, the time elapsed since diagnosis was positively correlated with CA and was negatively correlated with ECD (r=+0.39, p=0.001 and r=-0.42, p=0.001). The endothelial layer of the cornea may be under risk of impairment with prolonged disease duration in acromegaly. Consistency of the corneal endothelium should be also sought during long-term follow-up of the cases with acromegaly. Copyright © 2014 Elsevier Ltd. All rights reserved.
Corneal endothelial cell changes associated with cataract surgery in patients with type 2 diabetes mellitus
DEFF Research Database (Denmark)
Hugod, Mikkel; Storr-Paulsen, Allan; Norregaard, Jens Christian
To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation.......To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation....
Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells
Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.
Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B
Effects of vitrectomy combined with cataract surgery on the corneal endothelial cells in diabetic retinopathy
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Lei Zhan
Full Text Available AIM: To investigate the effects of vitrectomy combined with cataract surgery on the corneal endothelial cells in diabetic retinopathy. METHODS: A retrospective study was designed. 160 patients(160 eyeswith diabetic retinopathy from Jan 2015 to Feb 2017 were divided into two groups according to cataract. 74 patients(74 eyeswere operated on vitrectomy, and 86 patients(86 eyeson vitrectomy combined with phacoemulsification cataract surgery and capsular bag implantation of foldable intraocular lens. To record the change of corneal endothelial cells density, average cellular area, coefficient of variation and percentage of hexagonal endothelial cell before and after treatment with Topcon corneal specular microscope. RESULTS: Before and after surgery, the results of corneal endothelial cells density, average cellular area, coefficient of variation and percentage of hexagonal endothelial cell in simple vitrectomy group were no significant difference(P>0.05; After treatment corneal endothelial cells density and percentage of hexagonal endothelial cell were changed with statistical difference as the same as average cellular area and coefficient of variation(PPCONCLUSION: It has certain influence on the corned endothelial cells when using vitrectomy combined with cataract surgery in diabetic retinopathy. For patients with indications, it should be paid attention to protecting the corneal endothelial cells.
Cornea stress test--evaluation of corneal endothelial function in vivo by contact lens induced stress
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Saini Jagjit
Full Text Available Reliable and valid assessment of corneal endothelial function is a critical input for diagnosing, prognosticating and monitoring progression of disorders affecting corneal endothelium. In 123 eyes, corneal endothelial function was assessed employing data from the corneal hydration recovery dynamics. Serial pachometric readings were recorded on Haag-Striet pachometer with Mishima-Hedbys modification before and after two hours of thick soft contact lens wear. Percentage Recovery Per Hour (PRPH was derived from raw data as an index of endothelial function. Assessed PRPH in pseudophakic corneal oedema and Fuchs′ endothelial dystrophy eyes (35.9 +/- 9.8% was significantly lower than normal controls (61.9 +/- 10.5%. On employing receiver operation characteristics curve analysis the tested results demonstrated high sensitivity (87% and specificity (92% for detection of low endothelial function at PRPH cut off of 47.5%. Using this PRPH cut off, 80% of Fuchs′ endothelial dystrophy and 93.3% of pseudophakic corneal oedema eyes could be demonstrated to have low endothelial function. A total of 66.7% of diabetic eyes also demonstrated PRPH of lower than 47.5%. Clear corneal grafts demonstrated PRPH values of 24.6% to 73.0%. Of 6 corneal grafts that demonstrated initial PRPH of lower than 47.5%, 4 failed within 4 to 6 months. Our data demonstrated high sensitivity and specificity of this corneal stress test. PRPH index was useful in quantifying endothelial function in clinical disorders including diabetes mellitus. The index PRPH was demonstrated to be useful in monitoring and prognosticating outcome of corneal grafts.
Corneal endothelial cell density and morphology in normal Iranian eyes
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Fallah Mohammad
Full Text Available Abstract Background We describe corneal endothelial cell density and morphology in normal Iranian eyes and compare endothelial cell characteristics in the Iranian population with data available in the literature for American and Indian populations. Methods Specular microscopy was performed in 525 eyes of normal Iranian people aged 20 to 85 years old. The studied parameters including mean endothelial cell density (MCD, mean cell area (MCA and coefficient of variation (CV in cell area were analyzed in all of the 525 eyes. Results MCD was 1961 ± 457 cell/mm2 and MCA was 537.0 ± 137.4 μm2. There was no statistically significant difference in MCD, MCA and CV between genders (Student t-test, P = 0.85, P = 0.97 and P = 0.15 respectively. There was a statistically significant decrease in MCD with age (P r = -0.64. The rate of cell loss was 0.6% per year. There was also a statistically significant increase in MCA (P r = 0.56 and CV (P r = 0.30 from 20 to 85 years of age. Conclusion The first normative data for the endothelium of Iranian eyes seems to confirm that there are no differences in MCD, MCA and CV between genders. Nevertheless, the values obtained in Iranian eyes seem to be different to those reported by the literature in Indian and American populations.
Femtosecond laser cutting of multiple thin corneal stromal lamellae for endothelial bioengineering.
Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Gauthier, Anne-Sophie; Peoc'h, Michel; Dumollard, Jean-Marc; Acquart, Sophie; Montard, Romain; Delbosc, Bernard; Gain, Philippe; Thuret, Gilles
To assess the feasibility of cutting multiple thin stromal lamellae in human donor corneas using a commercial femtosecond laser (FSL) to provide cell carriers for future endothelial graft bioengineering. Eight edematous organ-cultured corneas not suitable for grafting for endothelial reasons were mounted on a Ziemer anterior chamber and cut with a Z6 FSL with 6 successive parallel cuts, from depth to surface. Target thickness of each lamella ranged from 100 to 150 μm depending on initial corneal thickness. Thickness was measured using anterior segment optical coherence tomography before and after cutting on mounted corneas, and on each stromal lamella after detachment. Scanning electron microscopy observation was performed on 4 lamellae and histological cross sections on 1 cornea before detachment. A median of 5 (minimum 3, maximum 7) lamellae was obtained per cornea. All lamellae still attached were the most posterior ones, suggesting that FSL was less efficient because of light scattering by edematous stroma. Cut precision and postdetachment swelling were correlated with anterior-posterior position within the cornea. Median lamella thickness was 127 μm (56-222 μm) before detachment and 196 μm (80-304 μm) after detachment. Surface state was consistent with previously reported FSL lamellar cuts during Descemet stripping automated endothelial keratoplasty. Up to 7 thin lamellae can be cut in stored corneas with an FSL. This method, once optimized primarily by using deswelled, more transparent corneas, could prove effective for recycling unsuitable donor corneas in corneal bioengineering processes.
Derivation of corneal endothelial cell-like cells from rat neural crest cells in vitro.
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Chengqun Ju
Full Text Available The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC to differentiate to functional corneal endothelial cell (CEC-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na(+/K(+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet's membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.
Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo
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Ying Miao
Full Text Available AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells in vitro and cat corneal endothelial cells(CCE cells in vivo, providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology.METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology, growth status, plasma membrane permeability, DNA fragmentation, and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope, 3-(4,5-dimethylthiahiazo (-z-y1-3,5-di-phenytetrazoliumromide (MTT assay, acridine orange (AO/ethidium bromide (EB double-fluorescent staining, DNA agarose gel electrophoresis, and transmission electron microscope (TEM. The in vivo density, morphology, and ultrastructure of CCE cells, corneal thickness, and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy, applanation tonometer, alizarin red staining, scanning electron microscope (SEM, and TEM.RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation, cellular shrinkage, structural disorganization, chromatin condensation, and apoptotic body appearance. Simultaneously, betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore, betaxolol at a dose of 2.8g/L also induced decrease of density of CCE cells in vivo, and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia.CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells, and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis
[Analysis of the Effect of Non-phacoemulsification Cataract Operation on Corneal Endothelial Cell Nucleus Division].
Huang, Zufeng; Miao, Xiaoqing
To investigate the effect of non-phacoemulsification cataract operation in two different patterns of nucleus delivery on the quantity and morphology of corneal endothelial cells and postoperative visual acuity. Forty patients diagnosed with cataract underwent cataract surgery and were assigned into the direct nuclear delivery and semi-nuclear delivery groups. Lens density was measured and divided into the hard and soft lenses according to Emery-little lens nucleus grading system. Non-phacoemulsification cataract operation was performed. At 3 d after surgery, the quantity and morphology of corneal endothelium were counted and observed under corneal endothelial microscope. During 3-month postoperative follow-up, the endothelial cell loss rate, morphological changes and visual acuity were compared among four groups. Corneal endothelial cell loss rate in the direct delivery of hard nucleus group significantly differed from those in the other three groups before and 3 months after operation (P nucleus, semi-delivery of hard nucleus and semi-delivery soft nucleus groups (all P > 0.05). Preoperative and postoperative 2-d visual acuity did not differ between the semi-delivery of hard nucleus and direct delivery of soft nucleus groups (P = 0.49), significantly differed from those in the semi-delivery of soft nucleus (P = 0.03) and direct delivery of hard nucleus groups (P = 0.14). Visual acuity at postoperative four months did not differ among four groups (P = 0.067). During non-phacoemulsification cataract surgery, direct delivery of hard nucleus caused severe injury to corneal endothelium and semi-delivery of soft nucleus yielded mild corneal endothelial injury. Slight corneal endothelial injury exerted no apparent effect upon visual acuity and corneal endothelial morphology at three months after surgery.
N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering
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Bernadette K. Madathil
Full Text Available Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.
Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering.
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Jui-Yang Lai
Full Text Available Hyaluronic acid (HA is a linear polysaccharide naturally found in the eye and therefore is one of the most promising biomaterials for corneal endothelial regenerative medicine. This study reports, for the first time, the development of overrun-processed porous HA hydrogels for corneal endothelial cell (CEC sheet transplantation and tissue engineering applications. The hydrogel carriers were characterized to examine their structures and functions. Evaluations of carbodiimide cross-linked air-dried and freeze-dried HA samples were conducted simultaneously for comparison. The results indicated that during the fabrication of freeze-dried HA discs, a technique of introducing gas bubbles in the aqueous biopolymer solutions can be used to enlarge pore structure and prevent dense surface skin formation. Among all the groups studied, the overrun-processed porous HA carriers show the greatest biological stability, the highest freezable water content and glucose permeability, and the minimized adverse effects on ionic pump function of rabbit CECs. After transfer and attachment of bioengineered CEC sheets to the overrun-processed HA hydrogel carriers, the therapeutic efficacy of cell/biopolymer constructs was tested using a rabbit model with corneal endothelial dysfunction. Clinical observations including slit-lamp biomicroscopy, specular microscopy, and corneal thickness measurements showed that the construct implants can regenerate corneal endothelium and restore corneal transparency at 4 weeks postoperatively. Our findings suggest that cell sheet transplantation using overrun-processed porous HA hydrogels offers a new way to reconstruct the posterior corneal surface and improve endothelial tissue function.
Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.
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Ming-Li Chou
Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices
[Contribution of confocal microscopy and anterior chamber OCT to the study of corneal endothelial pathologies].
Fayol, N; Labbé, A; Dupont-Monod, S; Dupas, B; Baudouin, C
To describe the appearance of various endothelial diseases with in vivo confocal microscopy and anterior chamber optical coherence tomography (AC OCT). In this study, ten patients with five different corneal endothelial pathologies were evaluated. Three patients had cornea guttata, three had corneal endothelial precipitates, two had irido-corneo-endothelial (ICE) syndrome, one had endothelial folds, and one had breaks in the Descemet membrane. All patients had bilateral ophthalmologic examinations, in vivo confocal microscopy, and AC OCT analysis. In cases of cornea guttata, AC OCT showed a finely embossed line corresponding to the empty intercellular cavities found with in vivo confocal microscopy. Corneal endothelium precipitates had the aspect of round formations suspended with the endothelium. Iris atrophy and irido-corneal synechiae resulting from ICE syndrome were precisely visualized with the AC OCT. High-resolution images of the anterior segment could be obtained using the AC OCT. Associated with in vivo confocal microscopy, these two new imaging techniques provide a precise evaluation of endothelial pathologies.
Atypical Presentation of Iridocorneal Endothelial Syndrome With Band Keratopathy but No Corneal Edema Managed With Descemet Membrane Endothelial Keratoplasty.
Zygoura, Vasiliki; Lavy, Itay; Verdijk, Robert M; Santander-García, Diana; Baydoun, Lamis; Dapena, Isabel; Melles, Gerrit R J
To report an unusual presentation of iridocorneal endothelial (ICE) syndrome associated with band keratopathy and its management with ethylenediamine-tetraacetic acid (EDTA) chelation and Descemet membrane endothelial keratoplasty (DMEK). A 57-year-old female patient presented with unilateral progressive painless visual impairment, corneal band keratopathy, and morphological corneal endothelial changes without corneal edema or any previous ophthalmic, medical, or family history. Routine specular and confocal microscopy imaging, as well as biomicroscopy, best-corrected visual acuity, and pachymetry measurements were performed before and after the surgical procedures. Histopathologic and immunohistochemical evaluations of the surgically excised diseased DM-endothelium were performed. Superficial epithelial keratectomy with EDTA chelation was performed. After an initial period of a few months of corneal clearance, the patient presented with recurrence of visually significant band keratopathy. After 1 year, she underwent retreatment with superficial epithelial keratectomy and EDTA chelation, followed by DMEK. Histopathologic and immunohistochemical analysis showed ICE syndrome. Two years after DMEK surgery, the cornea was still clear and band keratopathy had not recurred. To the best of our knowledge, this is the first case in the literature that reports the association of ICE syndrome with band keratopathy. As band keratopathy recurred shortly after EDTA chelation, endothelial keratoplasty (DMEK) may be indicated to successfully treat such cases.
Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells
a cell population particularly suitable for low serum propagation, provided that appropriate growth factors are available. A low serum medium...of MGK. 15. SUBJECT TERMS Cornea, chemical warfare agent, corneal endothelial cell, endothelium, growth , isolation, mouse, rabbit, porcine, in...with corneal SM exposure.2 A primary requirement in achieving this goal is to develop methods that enable the isolation of a pure CEC population and
Corneal endothelial morphology and function after torsional and longitudinal ultrasound mode phacoemulsification.
Módis, László Jr; Szalai, Eszter; Flaskó, Zsuzsa; Németh, Gábor
To study the endothelial cell morphology and corneal thickness changes after phacoemulsification by using the OZil torsional and longitudinal ultrasound techniques (Infiniti Vision System, Alcon Laboratories). Department of Ophthalmology, Clinical Center, University of Debrecen, Debrecen, Hungary. 52 patients with cataract were randomly assigned to longitudinal ultrasound and torsional mode group. All surgeries were performed through a 2.2 mm clear corneal incision, the method employed being divide and conquer. The endothelial morphometry such as cell density (ECD), mean cell area, coefficient of variation of cell area, and central corneal thickness were examined with specular microscopy (EM-1000, Tomey) preoperatively and 4, 8 weeks postoperatively. ECD values decreased significantly in both surgical groups (P .05). No significant correlation was found between the endothelial cell loss and the nucleus density. Both phacoemulsification techniques were safe and effective. The torsional handpiece performs oscillatory movements and delivers less energy into the eye than the longitudinal ultrasound technique, therefore providing more favorable energy and thermal safety profile.
Real-time assessment of corneal endothelial cell damage following graft preparation and donor insertion for DMEK.
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Maninder Bhogal
Full Text Available To establish a method for assessing graft viability, in-vivo, following corneal transplantation.Optimization of calcein AM fluorescence and toxicity assessment was performed in cultured human corneal endothelial cells and ex-vivo corneal tissue. Descemet membrane endothelial keratoplasty grafts were incubated with calcein AM and imaged pre and post preparation, and in-situ after insertion and unfolding in a pig eye model. Global, macroscopic images of the entire graft and individual cell resolution could be attained by altering the magnification of a clinical confocal scanning laser microscope. Patterns of cell loss observed in situ were compared to those seen using standard ex-vivo techniques.Calcein AM showed a positive dose-fluorescence relationship. A dose of 2.67μmol was sufficient to allow clear discrimination between viable and non-viable areas (sensitivity of 96.6% with a specificity of 96.1% and was not toxic to cultured endothelial cells or ex-vivo corneal tissue. Patterns of cell loss seen in-situ closely matched those seen on ex-vivo assessment with fluorescence viability imaging, trypan blue/alizarin red staining or scanning electron microscopy. Iatrogenic graft damage from preparation and insertion varied between 7-35% and incarceration of the graft tissue within surgical wounds was identified as a significant cause of endothelial damage.In-situ graft viability assessment using clinical imaging devices provides comparable information to ex-vivo methods. This method shows high sensitivity and specificity, is non-toxic and can be used to evaluate immediate cell viability in new grafting techniques in-vivo.
Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.
Diao, Jin-Mei; Pang, Xin; Qiu, Yue; Miao, Ying; Yu, Miao-Miao; Fan, Ting-Jun
A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.
Corneal endothelial morphology and central thickness in patients with type II diabetes mellitus
DEFF Research Database (Denmark)
Storr-Paulsen, Allan; Singh, Amardeep; Jeppesen, Helene
size was based on a power calculation (power = 0.90; p = 0.05). The diabetic patients had on average more than four HbA1c tests performed (mean 4.1; range 2-14) with intervals of at least 3 months as a reflection of the long-term glycaemic status. The controls had no diabetes confirmed by two causal......PURPOSE: To investigate corneal endothelial cell density and morphology in type II diabetic and non-diabetic patients and to relate potential differences to the glycaemic status. METHODS: A prospective clinical study including 107 patients with type II diabetes and 128 non-diabetic patients. Sample...... blood tests. The endothelial cell density, the variation in endothelial cell size (CV), the percentage of hexagonal cells, and the central corneal thickness (CCT) were recorded. RESULTS: Type II diabetic subjects did not differ from the non-diabetic control subjects with regards to endothelial cell...
SLC4A11 Prevents Osmotic Imbalance Leading to Corneal Endothelial Dystrophy, Deafness, and Polyuria*
Gröger, Nicole; Fröhlich, Henning; Maier, Hannes; Olbrich, Andrea; Kostin, Sawa; Braun, Thomas; Boettger, Thomas
Maintenance of ion concentration gradients is essential for the function of many organs, including the kidney, the cornea, and the inner ear. Ion concentrations and fluid content in the cornea are regulated by endothelial cells that separate the collagenous avascular corneal stroma from the anterior eye chamber. Failure to maintain correct ion concentrations leads to swelling and destruction of the cornea. In the inner ear, the stria vascularis is responsible for generating proper ion concentrations in the endolymph, which is essential for hearing. Mutations of SLC4A11 in humans lead to syndromes associated with corneal dystrophy and perceptive deafness. The molecular mechanisms underlying these symptoms are poorly understood, impeding therapeutic interventions. The ion transporter SLC4A11 mediates sodium-dependent transport of borate as well as flux of sodium and hydroxyl ions in vitro. Here, we show that SLC4A11 is expressed in the endothelial cells of the cornea where it prevents severe morphological changes of the cornea caused by increased sodium chloride concentrations in the stroma. In the inner ear, SLC4A11 is located in fibrocytes underlying the stria vascularis. Loss of SLC4A11 leads to morphological changes in the fibrocytes and deafness. We demonstrate that SLC4A11 is essential for the generation of the endocochlear potential but not for regulation of potassium concentrations in the endolymph. In the kidney, SLC4A11 is expressed in the thin descending limb of Henle loop. SLC4A11 is essential for urinary concentration, suggesting that SLC4A11 participates in the countercurrent multiplication that concentrates urine in the kidney medulla. PMID:20185830
Hormonal regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells.
Hatou, Shin
Na- and K-dependent ATPase (Na,K-ATPase) in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in these cells. Mouse corneal endothelial cells were exposed to dexamethasone or insulin. ATPase activity was evaluated by spectrophotometric measurement, and pump function was measured using an Ussing chamber. Western blotting and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. Dexamethasone increased Na,K-ATPase activity and the pump function of endothelial cells. Western blot analysis indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit but decreased the ratio of active to inactive Na,K-ATPase α1-subunit. Insulin increased Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were transient and blocked by protein kinase C inhibitors and inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A). Western blot analysis indicated that insulin decreased the amount of inactive Na,K-ATPase α1-subunit, but the expression of total Na,K-ATPase α1-subunit was unchanged. Immunocytochemistry showed that insulin increased cell surface expression of the Na,K-ATPase α1-subunit. Our results suggest that dexamethasone and insulin stimulate Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells was mediated by Na,K-ATPase synthesis and an increased enzymatic activity because of dephosphorylation of Na,K-ATPase α1-subunits. The effect of insulin is mediated by the protein kinase C, PP1, and/or PP2A pathways.
Proteomics of Fuchs' Endothelial Corneal Dystrophy support that the extracellular matrix of Descemet's membrane is disordered
DEFF Research Database (Denmark)
Poulsen, Ebbe Toftgaard; Dyrlund, Thomas F; Runager, Kasper
Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer we determined...... that the morphological changes observed in FECD is caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer......., respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been implicated in familial early onset forms of the disease. Using the second relative quantitation method iTRAQ we identified 22...
Changes in corneal endothelial cell density and the cumulative risk of corneal decompensation after Ahmed glaucoma valve implantation.
Kim, Kyoung Nam; Lee, Sung Bok; Lee, Yeon Hee; Lee, Jong Joo; Lim, Hyung Bin; Kim, Chang-Sik
To evaluate changes in the corneal endothelial cell density (ECD) and corneal decompensation following Ahmed glaucoma valve (AGV) implantation. This study was retrospective and observational case series. Patients with refractory glaucoma who underwent AGV implantation and were followed >5 years were consecutively enrolled. We reviewed the medical records, including the results of central corneal specular microscopy. Of the 127 enrolled patients, the annual change in ECD (%) was determined using linear regression for 72 eyes evaluated at least four times using serial specular microscopic examination and compared with 31 control eyes (fellow glaucomatous eyes under medical treatment). The main outcome measures were cumulative risk of corneal decompensation and differences in the ECD loss rates between subjects and controls. The mean follow-up after AGV implantation was 43.1 months. There were no cases of postoperative tube-corneal touch. The cumulative risk of corneal decompensation was 3.3%, 5 years after AGV implantation. There was a more rapid loss of ECD in the 72 subject eyes compared with the 31 controls (-7.0% and -0.1%/year, respectively; p<0.001). However, the rate of loss decreased over time and statistical significance compared with control eyes disappeared after 2 years postoperatively: -10.7% from baseline to 1 year (p<0.01), -7.0% from 1 year to 2 years (p=0.037), -4.2% from 2 years to 3 years (p=0.230) and -2.7% from 3 years to the final follow-up (p=0.111). In case of uncomplicated AGV implantation, the cumulative risk of corneal decompensation was 3.3%, 5 years after the operation. The ECD loss was statistically greater in eyes with AGV than in control eyes without AGV, but the difference was significant only up to 2 years post surgery. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
Efficient and safe gene delivery to human corneal endothelium using magnetic nanoparticles.
Czugala, Marta; Mykhaylyk, Olga; Böhler, Philip; Onderka, Jasmine; Stork, Björn; Wesselborg, Sebastian; Kruse, Friedrich E; Plank, Christian; Singer, Bernhard B; Fuchsluger, Thomas A
To develop a safe and efficient method for targeted, anti-apoptotic gene therapy of corneal endothelial cells (CECs). Magnetofection (MF), a combination of lipofection with magnetic nanoparticles (MNPs; PEI-Mag2, SO-Mag5, PalD1-Mag1), was tested in human CECs and in explanted human corneas. Effects on cell viability and function were investigated. Immunocompatibility was assessed in human peripheral blood mononuclear cells. Silica iron-oxide MNPs (SO-Mag5) combined with X-tremeGENE-HP achieved high transfection efficiency in human CECs and explanted human corneas, without altering cell viability or function. Magnetofection caused no immunomodulatory effects in human peripheral blood mononuclear cells. Magnetofection with anti-apoptotic P35 gene effectively blocked apoptosis in CECs. Magnetofection is a promising tool for gene therapy of corneal endothelial cells with potential for targeted on-site delivery.
Effects of vitrectomy combined with cataract surgery on the corneal endothelial cells in diabetic retinopathy
Lei Zhan; Si-Ying Xiong; Meng-Xin Gan; Li-Hui Wen
AIM: To investigate the effects of vitrectomy combined with cataract surgery on the corneal endothelial cells in diabetic retinopathy. METHODS: A retrospective study was designed. 160 patients(160 eyes)with diabetic retinopathy from Jan 2015 to Feb 2017 were divided into two groups according to cataract. 74 patients(74 eyes)were operated on vitrectomy, and 86 patients(86 eyes)on vitrectomy combined with phacoemulsification cataract surgery and capsular bag implantation of foldable intraocular...
Comparison of the corneal endothelial cell count in type II diabetic patients with healthy adults
International Nuclear Information System (INIS)
Rizvi, B.Z.; Zafar, O.
To compare the mean corneal endothelial cell count in type II diabetic patients with healthy adults. Study Design: Case control. Place and Duration of Study: Out-patient Department of Armed Forces Institute of Ophthalmology, Rawalpindi from September 10, 2013 to March 25, 2014. Material and Methods: A hospital-based case-control study was carried out at out-patient department of Armed Forces Institute of Ophthalmology in which 130 eyes (65 diabetic eyes and 65 controls) were included. Non-probability consecutive sampling was adopted. Relevant detailed history including information about age, gender, duration of diabetes, any other medical illness and current medical treatment being taken by patient was recorded. Results: Data entry and analysis was done in SPSS version 10. Total 130 eyes (65 diabetic and 65 non-diabetic eyes) were included in our study according to the inclusion criteria. Mean age (years) of patient in both the groups was 59.55 +- 8.01 and 53.85 +- 10.07. Mean corneal endothelial cell count in both the groups was 2368.35 +- 389.58 and 2588.64 +- 269.84 respectively which was statistically significant (p-value=0.001) in both the groups. Conclusion: The conclusion of the study was that the mean corneal endothelial cell count in type II diabetic patients was significantly less as compared to healthy adults. (author)
Changes in corneal sensation, epithelial damage, and tear function after descemet stripping automated endothelial keratoplasty.
Hirayama, Yumiko; Satake, Yoshiyuki; Hirayama, Masatoshi; Shimazaki-Den, Seika; Konomi, Kenji; Shimazaki, Jun
To study the ocular surface changes in eyes after Descemet stripping automated endothelial keratoplasty (DSAEK) compared with those after penetrating keratoplasty (PKP). This prospective study compared the changes in 31 eyes of 28 patients who underwent DSAEK (DSAEK group) with those in 15 disease-matched eyes of 15 patients who underwent PKP (PKP group). Corneal epithelial integrity was evaluated using a fluorescein staining score. Corneal sensation was measured with a Cochet-Bonnet esthesiometer. Tear function was evaluated using the Schirmer test, tear clearance test, tear function index, and tear break-up time. The postoperative fluorescein staining score was significantly higher in the PKP group than in the DSAEK group (P = 0.02). Postoperative corneal sensation was significantly better in the DSAEK group than in the PKP group (P sensation after DSAEK was significantly better than the preoperative value (P = 0.02). There were no statistically significant changes in the Schirmer test, tear clearance test, tear function index, or break-up time before and after the surgery in both the DSAEK and PKP groups. No significant differences were observed between the DSAEK and PKP groups after the surgery. Corneal sensation was preserved, and epithelial damage was less severe after DSAEK compared with PKP. Preservation of corneal sensation may contribute to the early recovery of visual function and long-term maintenance of ocular surface health after DSAEK.
Safety of intracameral injection of gatifloxacin, levofloxacin on corneal endothelial structure and viability.
Choi, Jin A; Chung, Sung Kun
To investigate the safety of intracameral injection of gatifloxacin, levofloxacin in a rabbit model as prophylaxis against endophthalmitis. Twenty-four eyes of New Zealand white rabbits were randomly divided into 3 treatment groups: levofloxacin, gatifloxacin, and balanced salt solution (BSS) control groups. After 100 microL of each was injected into the anterior chamber, endothelial toxicity was evaluated by measuring the central corneal thicknesses and the clinical toxicity scores using a slit-lamp at post-procedure days 3 and 7. The percent of dead cells was determined by vital staining with alizarin red and trypan blue at 7 days after injection. Finally, in each group, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed for the evaluation of structural integrity. The toxicity scores were increased at post-procedure days 3 and 7, but the difference among the groups was not statistically significant (P = 0.661, 0.216, respectively). With regard to baseline corneal thickness, only the levofloxacin group exhibited a significant increase from baseline (P = 0.028), whereas the other treatment groups showed no difference from baseline (P = 0.128 in gatifloxacin, 0.161 in BSS group). The mean corneal endothelial damage was 0.81 +/- 0.31% in the levofloxacin group, 0.56 +/- 0.47% in the gatifloxacin group, and 0.53 +/- 0.52% in the BSS group, with no statistically significant difference noted among the groups (P = 0.582). SEM revealed a well-preserved hexagonal endothelial cell mosaic and normal microvilli on the endothelial cell surface in the gatifloxacin and control groups. However, the levofloxacin group showed slightly disintegrated cellular borders. TEM revealed that each group maintained normal intracellular organization, whereas the levofloxacin group exhibited slightly flat cell configuration with irregular folds on the apical cell surface. Intracameral injection of gatifloxacin and levofloxacin was nontoxic in terms of
Coefficient of Friction of Human Corneal Tissue.
Wilson, Tawnya; Aeschlimann, Rudolf; Tosatti, Samuele; Toubouti, Youssef; Kakkassery, Joseph; Osborn Lorenz, Katherine
A novel property evaluation methodology was used to determine the elusive value for the human corneal coefficient of friction (CoF). Using a microtribometer on 28 fresh human donor corneas with intact epithelia, the CoF was determined in 4 test solutions (≥5 corneas/solution): tear-mimicking solution (TMS) in borate-buffered saline (TMS-PS), TMS in phosphate-buffered saline (TMS-PBS), TMS with HEPES-buffered saline (TMS-HEPES), and tear-like fluid in PBS (TLF-PBS). Mean (SD) CoF values ranged from 0.006 to 0.015 and were 0.013 (0.010) in TMS-PS, 0.006 (0.003) in TMS-PBS, 0.014 (0.005) in TMS-HEPES, and 0.015 (0.009) in TLF-PBS. Statistically significant differences were shown for TMS-PBS versus TLF (P = 0.0424) and TMS-PBS versus TMS-HEPES (P = 0.0179), but not for TMS-PBS versus TMS-PS (P = 0.2389). Successful measurement of the fresh human corneal tissue CoF was demonstrated, with values differing in the evaluated buffer solutions, within this limited sample size.
Corneal endothelial rejection after penetrating keratoplasty treated with intravenous and topic corticosteroid: one year follow up
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Ricardo Yuji Abe
Full Text Available OBJECTIVE: To analyze the recovery of visual acuity (VA and graft survival after first episode of endothelial rejection in penetrating keratoplasty (PKP treated with intravenous (IV and topic corticosteroid. METHODS: Interventional, prospective, non-comparative case series study evolving 32 PKP patients in one year follow up, who presented first episode of corneal endothelial rejection. The patients were submitted to 500 mg IV injection of methylprednisolone in association with topical prednisolone. Main outcome measures included VA recovery and corneal edema regression. Second outcome included new rejections and graft failure. Multivariate analysis techniques were used to estimate rates of graft outcome events and the impact of risk factors. RESULTS: A total of 32 eyes from 32 patients (13 male and 19 female were included in the study. The mean VA (in number of letters before rejection was 48 (22 to 88 letters. Patients treated within 7 days or less of initial symptoms had better VA recovery, corneal edema regression and less graft failure (p<0.001. Patients with previous ocular surgery had worse VA recovery and more graft failure (p<0.047. CONCLUSION: The association between the other risk factors and the outcomes did not reach statistical significance in the multivariate model because of the small numbers of patients. Methylprednisolone in association with topical prednisolone is an alternative treatment for graft rejection. Our study showed that patients treated within 7 days of symptoms and no previous anterior segment surgery had better visual outcome and graft survival after treatment.
Corneal endothelial cell density and morphology in low and moderate myopic Chinese eyes
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Jane Mei Chun
Full Text Available AIM: To describe and compare the corneal endothelial cell density and morphology in young, low and moderate myopic Chinese adults in Malaysian Chinese population.METHODS: Non-contact specular microscopy (Topcon SP3000P, Tokyo, Japan was performed in low (n=78; 21.22±1.51 years and moderate (n=78; 21.82±1.40 years myopic subjects. The mean of three consecutive measurements of endothelial cell density (MCD, coefficient of variation (CV in the cell size, and hexagonal appearance of the cell were obtained.RESULTS: In low myopic eyes the MCD was 3 063.0±176.2/mm2, the mean CV was 33.4±4.0% and the mean hexagonal appearance of the cell was 57.9±2.7%. In moderate myopic eyes the MCD was 2961.6±159.0/mm2, the mean CV was 33.9±3.6% and mean hexagonal appearance of the cell was 56.2±4.7%. There were statistically significant differences in MCD (PPCONCLUSION:The corneal endothelial cell layer in more myopic eyes tends to have less MCD and cell hexagonality compared to lower myopic eyes. Nevertheless, there is no significant difference in CV between low and moderate myopic eyes.
In vivo human corneal hydration control dynamics: A new model
Odenthal, M.T.P.; Nieuwendaal, C.P.; Venema, H.W.; Oosting, J.; Kok, J.H.C.; Kijlstra, A.
PURPOSE. To introduce a new model describing human in vivo corneal deswelling after hypoxic contact lens wear, based on a damped harmonic oscillator, which can describe an overshoot in corneal deswelling, to compare this new model with the currently used exponential model, and also to test whether a
In vivo human corneal hydration control dynamics: a new model
Odenthal, M. T.; Nieuwendaal, C. P.; Venema, H. W.; Oosting, J.; Kok, J. H. C.; Kijlstra, A.
PURPOSE: To introduce a new model describing human in vivo corneal deswelling after hypoxic contact lens wear, based on a damped harmonic oscillator, which can describe an overshoot in corneal deswelling, to compare this new model with the currently used exponential model, and also to test whether a
Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells.
Hatou, Shin; Yamada, Masakazu; Akune, Yoko; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo; Tsubota, Kazuo
The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.
Analysis of corneal endothelial cell density and morphology after laser in situ keratomileusis using two types of femtosecond lasers
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Tomita M
Full Text Available Minoru Tomita,1,2,* George O Waring IV,3,4 Miyuki Watabe,1,* 1Shinagawa LASIK Center, Chiyoda-ku, Tokyo, Japan; 2Department of Ophthalmology, Wenzhou Medical College, Wenzhou, China; 3Medical University of South Carolina, Storm Eye Institute, Charleston, SC, USA; 4Magill Laser Center, Charleston, SC, USA*These authors contributed equally to this studyPurpose: To compare two different femtosecond lasers used for flap creation during laser-assisted in situ keratomileusis (LASIK surgery in terms of their effects on the corneal endothelium.Methods: We performed LASIK surgery on 254 eyes of 131 patients using IntraLase FS60 (Abbott Medical Optics, Inc, Irvine, CA; IntraLase group and 254 eyes of 136 patients using Femto LDV (Ziemer Group AG, Port, Switzerland; LDV group for corneal flap creation. The mean cell density, coefficient of variation, and hexagonality of the corneal endothelial cells were determined and the results were statistically compared.Results: There were no statistically significant differences in the corneal morphology between pre and post LASIK results in each group, nor were there significant differences between the results of both groups at 3 months post LASIK.Conclusions: Both IntraLase FS60 and Ziemer Femto LDV are able to create flaps without significant adverse effects on the corneal endothelial morphology through 3 months after LASIK surgery.Keywords: LASIK, corneal endothelium, femtosecond laser, IntraLase FS60, Ziemer LDV
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Yann Gavet
Full Text Available The human corneal endothelium can be observed with two types of microscopes: classical optical microscope for ex-vivo imaging, and specular optical microscope for in-vivo imaging. The quality of the cornea is correlated to the endothelial cell density and morphometry. Automatic methods to analyze the human corneal endothelium images are still not totally efficient. Image analysis methods that focus only on cell contours do not give good results in presence of noise and of bad conditions of acquisition. More elaborated methods introduce regional informations in order to performthe cell contours completion, thus implementing the duality contour-region. Their good performance can be explained by their connections with several basic principles of human visual perception (Gestalt Theory and Marr's computational theory.
Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.
Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C
Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in
Cell pattern in adult human corneal endothelium.
Directory of Open Access Journals (Sweden)
Carlos H Wörner
Full Text Available A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.
A study of corneal endothelial changes in soft contact lens wearers using non-contact specular microscopy
Directory of Open Access Journals (Sweden)
Renu M Magdum
Full Text Available Aim: To study the corneal endothelial changes after soft contact lens wear, to correlate these changes with the duration of soft contact lens wear, and to study the pattern of use and preferences of contact lens among young adults. Materials and Methods: This observational study was carried out in 100 eyes of 50 soft contact lens users aged between 19 and 27 years. Both eyes of 50 medical students who had never worn contact lenses served as controls. Data from each subject were collected using a structured questionnaire of 24 items that included demographic profile, pattern of contact lens use, symptoms, brand name, number of years worn, and hours of daily wear. These data were analyzed using Chi square for association. Specular microscopy was done using TOPCON SP-3000P. Computerized morphometry was used to evaluate central corneal thickness, size, shape, mean cellular density, hexagonality, coefficient of variation, and polymegathism of the corneal cells . Results: It was found that central corneal thickness was 0.532 ± 0.0309 mm in lens users and 0.514 ± 0.03 mm in controls, cell density was 2570.91 ± 432.06 cells/mm 2 in lens users and 2723.17 ± 327.64 cells/mm 2 in controls, while hexagonality was 54.81 ± 39.72% in lens users and 67.65 ± 36.49% in controls. Conclusion: Despite the known effects of long duration of soft contact lens use on corneal endothelial cell morphology, this study could not draw a significant correlation between them. However, a significant difference was found in the corneal endothelial thickness, cell density, and hexagonality. Among the soft contact lens users, 62% used soft disposable type while 38% used soft extended wear contact lens. Contact lenses were preferred over spectacles for better cosmetic appearance, comfort, and wider visual field.
Signaling hierarchy regulating human endothelial cell development.
Kelly, Melissa A; Hirschi, Karen K
Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.
The effects of dexamethasone on the Na,K-ATPase activity and pump function of corneal endothelial cells.
Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo
The Na(+)- and K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase alpha(1)-subunit. Dexamethasone (1 or 10 microM) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase alpha(1)-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase alpha(1)-subunit. Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase alpha(1)-subunits.
Corneal endothelial cell changes after cataract surgery in patients on systemic sympathetic α-1a antagonist medication (tamsulosin).
Storr-Paulsen, Allan; Jørgensen, Jesper Skovlund; Norregaard, Jens Christian; Thulesen, Jesper
The purpose of this study was to assess the incidence of intraoperative floppy iris syndrome (IFIS) and the morphology of the corneal endothelium after cataract extraction in Caucasian male patients exposed to the α-1a adrenergic receptor antagonist tamsulosin. In a clinical prospective study, 23 male patients (23 eyes) treated with tamsulosin due to benign prostatic hyperplasia and 25 male patients (25 eyes) with no tamsulosin treatment had cataract surgery. The divide-and-conquer technique was used with the Infinity OZil(®) machine. A combination of Healon and Healon5 was used in all patients, but the use of additional Vision Blue, iris retractors or intracameral phenylephrine in the tamsulosin group was at the discretion of the surgeon. The endothelial cell density, variation in endothelial cell size (CV), percentage of hexagonal cells and central corneal thickness (CCT) were recorded at baseline and at 3 months postoperatively. In the tamsulosin-treated group, 19 of 23 eyes (83%) developed IFIS, compared with no IFIS in the control group. Compared with the control group, the tamsulosin group showed significantly less dilatation at the start of the operation, significant miosis during surgery and significantly greater corneal endothelial cell loss 3 months postoperatively (12% versus 3%; ptamsulosin-treated male patients. Patients on tamsulosin showed less preoperative dilatation, significant miosis during surgery, and had significantly greater postoperative endothelial cell loss compared with nontreated patients despite recommended precautions. © 2013 Acta Ophthalmologica Scandinavica Foundation. Published by Blackwell Publishing Ltd.
Changes in central corneal thickness and endothelial cell count pediatric cataract surgery
International Nuclear Information System (INIS)
Memon, M.N.
To evaluate the mean changes in Central Corneal Thickness (CCT) and Endothelial Cell Count (ECC) in eyes after pediatric cataract surgery with foldable intraocular lens using scleral tunnel incision micro-surgical technique. Study Design: Qausi experimental study. Place and Duration of Study: Department of Pediatric Ophthalmology and Strabismus, Al-Shifa Trust Eye Hospital, Rawalpindi, from May 2011 to March 2012. Methodology: Fifty-two eyes of 37 children with pediatric cataract were included in the study. Extracapsular Cataract Extraction (ECE) with foldable Intra Ocular Lens (IOL) implantation using sclera tunnel incision was performed in all children. Endothelial Cell Count (ECC) and Central Corneal Thickness (CCT) were recorded before surgery and 1 month, 3 months and 6 months after surgery and the effect of currently practiced surgical technique on ECC and CCTwas evaluated. Results: The mean age at the time of surgery was 8.8 ± 2.7 years (range: 4 to 15 years). The postoperative ECC and CCT were significantly different from the pre-operative values. Mean pre-operative ECC was 3175.3 ± 218.4 cell/mm2 and in first postoperative month the mean ECC was 3113.4 ± 210.8 cell/mm2 (p<0.0001). In the 3rd and 6th month postoperative means ECC were 3052 ± 202.5 cell/mm2 (p<0.0001) and 3015 ±190.6 cell/mm2 (p<0.0001), respectively. The mean cell loss at first postoperative month was 1.95% and at 3rd and 6th postoperative month were 3.9% and 5.05%, respectively. Mean pre-operative CCT was 514 ± 49.9 micro m and first postoperative mean CCT after 1 month was 524.1 ± 25 micro m (p = 0.084). After the 3rd and 6th months postoperative, mean CCT were 527.3 ± 24.6 micro m, and 530 ± 24.5 micro m, respectively. Third and 6th months postoperative means were significantly higher than baseline CCT, p = 0.024 and 0.007, respectively. Conclusion: Endothelial cell loss with closed chamber micro-surgical technique using scleral tunnel incision is within acceptable limits and
Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization
Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li
Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD) peptide–hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV
Signaling hierarchy regulating human endothelial cell development
Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...
Comparison of ultrasonic energy expenditures and corneal endothelial cell density reductions during modulated and non-modulated phacoemulsification.
Davison, James A
To compare the Legacy 20000 Advantec continuous and Infiniti hyperpulse modes (Alcon Laboratories, Fort Worth, TX) with respect to average power, machine-measured phacoemulsification time, total stopwatch real time spent within the phacoemulsification process, balanced salt solution (BSS) volume, and corneal endothelial cell density losses. A background study was done of consecutive patients operated on with the Legacy (n = 60) and Infiniti (n = 40) machines programmed with identical parameters and using the continuous mode only. A primary study of another set of consecutive cases was operated on using the Legacy (n = 87) and Infiniti (n = 94) with the same parameters, but using the hyperpulse mode during quadrant removal with the Infiniti. Measurements for each set included average power and phacoemulsification time with corneal endothelial cell densities, BSS volume, and time spent in the phacoemulsification process. Similarities were found in the background study for average power percent and average minutes of phacoemulsification time. In the primary study, similarities were found for total minutes in the phacoemulsification process, BSS usage, and ECD losses, and differences were found for average power percent (PInfiniti performed similarly in continuous mode. With the Infiniti hyperpulse mode, a total ultrasonic energy reduction of 66% was noted. The machines required the same amount of total stopwatch measured time to accomplish phacoemulsification and produced the same 5% corneal endothelial cell loss. Therefore, clinically, these two machines behave in a comparable manner relative to safety and effectiveness.
Response of human corneal fibroblasts on silk film surface patterns.
Gil, Eun Seok; Park, Sang-Hyug; Marchant, Jeff; Omenetto, Fiorenzo; Kaplan, David L
Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37-342 nm depths and 445-3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions.
Serial corneal endothelial cell loss with lathe-cut and injection-molded posterior chamber intraocular lenses.
Kraff, M C; Sanders, D R; Lieberman, H L
We compared endothelial cell loss of patients implanted with lathe-cut posterior chamber lenses and those implanted with injection-molded lenses over a three-year postoperative period. Results were based on more than 2,500 measurements of corneal endothelial density. Although the technique of cataract extraction (anterior chamber phacoemulsification, posterior chamber phacoemulsification, or planned extracapsular extraction) significantly affected cell loss (P less than .01), the type of implant (lathe-cut or injection-molded) did not. Significant continuing endothelial cell loss did not occur during the first three postoperative years with injection-molded lenses. There was, however, a statistically significant 7% to 15% additional cell loss after surgery over the first two to three postoperative years with lathe-cut implants. There have been no cases of corneal endothelial decompensation developing after implantation of injection-molded or lathe-cut lenses. Because a standard field clinical specular microscope was used in this study, cell counting errors cannot be ruled out as a cause of these findings.
Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization
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Chang CY
Full Text Available Che-Yi Chang,1,2,* Ming-Chen Wang,2,* Takuya Miyagawa,1 Zhi-Yu Chen,1 Feng-Huei Lin,3,4 Ko-Hua Chen,5,6 Guei-Sheung Liu,7 Ching-Li Tseng1 1Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, 2Department of Biomedical Engineering, Chung Yuan Christian University, Taoyuan, 3Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Zhunan, 4Institute of Biomedical Engineering, National Taiwan University, 5Department of Ophthalmology, Taipei Veterans General Hospital, 6Department of Ophthalmology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 7Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia *These authors contributed equally to this work Abstract: Neovascularization (NV of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG, presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD peptide–hyaluronic acid (HA-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV, with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a
In vitro effects of three blood derivatives on human corneal epithelial cells.
Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia
We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.
Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.
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Ryuhei Hayashi
Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.
Characterization of bicarbonate-dependent potassium uptake in cultured corneal endothelial cells
International Nuclear Information System (INIS)
Savion, N.; Farzame, N.; Berlin, H.B.
Bovine corneal endothelial (BCE) cells in culture demonstrated 86Rb+ uptake which was mostly ouabain-sensitive with some (15 to 50%) ouabain-insensitive uptake that was dependent on the presence of bicarbonate in the incubation medium. Bovine smooth muscle (SM) cells demonstrated ouabain-sensitive 86Rb+ uptake but the ouabain-insensitive 86Rb+ uptake was not bicarbonate-dependent. Although omission of bicarbonate from the incubation buffer resulted in some reduction in the pH, this change was not responsible for the reduction in the ouabain-insensitive 86Rb+ uptake. Furthermore, the removal of bicarbonate decreased the 86Rb+ influx but not its efflux. This ouabain-insensitive and bicarbonate-dependent 86Rb+ influx in BCE cells proceeded at a linear rate for at least 60 min and increased as a function of bicarbonate concentration such that almost maximal uptake was observed at a concentration of about 10 to 15 mM. Saturation of the bicarbonate-dependent 86Rb+ pump in BCE cells occurred at a concentration of 2 mM Rb+ in the incubation buffer, similar to the previously observed value for the Na+, K+-ATPase. Competition experiments with both unlabeled Rb+ and K+ demonstrated that likewise in the Na+, K+-ATPase the 86Rb+ influx represented physiological influx of K+. Furthermore, the energy requirements of the bicarbonate-dependent 86Rb+ uptake were similar to those of the 86Rb+ uptake via the Na+, K+-ATPase. The results described in this work demonstrated a novel bicarbonate-dependent K+ pump in addition to the Na+, K+-ATPase pump.(ABSTRACT TRUNCATED AT 250 WORDS)
Comparison of manual & automated analysis methods for corneal endothelial cell density measurements by specular microscopy.
Huang, Jianyan; Maram, Jyotsna; Tepelus, Tudor C; Modak, Cristina; Marion, Ken; Sadda, SriniVas R; Chopra, Vikas; Lee, Olivia L
To determine the reliability of corneal endothelial cell density (ECD) obtained by automated specular microscopy versus that of validated manual methods and factors that predict such reliability. Sharp central images from 94 control and 106 glaucomatous eyes were captured with Konan specular microscope NSP-9900. All images were analyzed by trained graders using Konan CellChek Software, employing the fully- and semi-automated methods as well as Center Method. Images with low cell count (input cells number <100) and/or guttata were compared with the Center and Flex-Center Methods. ECDs were compared and absolute error was used to assess variation. The effect on ECD of age, cell count, cell size, and cell size variation was evaluated. No significant difference was observed between the Center and Flex-Center Methods in corneas with guttata (p=0.48) or low ECD (p=0.11). No difference (p=0.32) was observed in ECD of normal controls <40 yrs old between the fully-automated method and manual Center Method. However, in older controls and glaucomatous eyes, ECD was overestimated by the fully-automated method (p=0.034) and semi-automated method (p=0.025) as compared to manual method. Our findings show that automated analysis significantly overestimates ECD in the eyes with high polymegathism and/or large cell size, compared to the manual method. Therefore, we discourage reliance upon the fully-automated method alone to perform specular microscopy analysis, particularly if an accurate ECD value is imperative. Copyright © 2017. Published by Elsevier España, S.L.U.
Accuracy of Corneal Thickness by Swept-Source Optical Coherence Tomography and Scheimpflug Camera in Virgin and Treated Fuchs Endothelial Dystrophy.
Arnalich-Montiel, Francisco; Ortiz-Toquero, Sara; Auladell, Clara; Couceiro, Ana
To assess intraobserver repeatability, intersession reproducibility, and agreement of swept-source Fourier-domain optical coherence tomography (SS-OCT) and the Scheimpflug camera in measuring corneal thickness in virgin and grafted eyes with Fuchs endothelial corneal dystrophy (FECD). Thirty-six control eyes, 35 FECD eyes, 30 FECD with corneal edema eyes, 25 Descemet stripping automated endothelial keratoplasty (DSAEK) eyes, and 29 Descemet membrane endothelial keratoplasty (DMEK) eyes were included. The apical center, pupillary center, and thinnest corneal thickness were determined in 3 consecutive images and repeated 2 weeks later. Repeatability and reproducibility coefficients, intraclass correlation coefficients, and 95% limits of agreement (LOA) between measurements were calculated. Agreement between devices was assessed using Bland-Altman analysis. Corneal thickness measurements were highly reproducible and repeatable with both systems. SS-OCT showed better repeatability in all corneal locations in the normal, FECD, FECD with edema, DSAEK, and DMEK groups (coefficient of variation ≤0.60%, ≤0.36%, ≤0.43%, ≤1.09%, and ≤0.48%, respectively) than the Scheimpflug (coefficient of variation ≤1.15%, ≤0.92%, ≤1.10%, ≤1.25%, and ≤1.14%, respectively). Between-session 95% LOA for SS-OCT was less than 3% for all groups except for the FECD with edema group, being almost double using the Scheimpflug camera. Differences between instruments were statistically significant in all groups and locations (P group (P ≤ 0.51); however, SS-OCT underestimated all measurements. SS-OCT provides better reproducible and repeatable measurements of corneal thickness than those obtained with the Scheimpflug camera in patients with FECD or an endothelial transplant. Variations between examinations higher than the 95% LOA observed in our study should raise awareness of changes in the endothelial function.
Microspectroscopy of spectral biomarkers associated with human corneal stem cells
Nakamura, Takahiro; Kelly, Jemma G.; Trevisan, J?lio; Cooper, Leanne J.; Bentley, Adam J.; Carmichael, Paul L.; Scott, Andrew D.; Cotte, Marine; Susini, Jean; Martin-Hirsch, Pierre L.; Kinoshita, Shigeru; Fullwood, Nigel J.; Martin, Francis L.
Purpose Synchrotron-based radiation (SRS) Fourier-transform infrared (FTIR) microspectroscopy potentially provides novel biomarkers of the cell differentiation process. Because such imaging gives a ?biochemical-cell fingerprint? through a cell-sized aperture, we set out to determine whether distinguishing chemical entities associated with putative stem cells (SCs), transit-amplifying (TA) cells, or terminally-differentiated (TD) cells could be identified in human corneal epithelium. Methods D...
Corneal endothelial cell density after femtosecond thin-flap LASIK and PRK for myopia: a contralateral eye study.
Smith, Ryan T; Waring, George O; Durrie, Daniel S; Stahl, Jason E; Thomas, Priscilla
To compare the effect of femtosecond thinflap LASIK and photorefractive keratectomy (PRK) on postoperative endothelial cell density. In a prospective, randomized, contralateral, single-center clinical trial, 25 patients (mean age: 30+/-5 years [range: 21 to 38 years]) underwent PRK in one eye and thin-flap LASIK in the fellow eye for the correction of myopia using a wavefront-guided platform. The central corneal endothelial cell density was measured using the NIDEK Confoscan 4 preoperatively, and at 1 and 3 months postoperatively. Changes in endothelial cell density were analyzed over time between the two refractive techniques. In PRK, the average preoperative endothelial cell density was 3011+/-329 cells/mm(2), which decreased to 2951+/-327 cells/mm(2) at 1 month (P=.5736) and 2982+/-365 cells/mm(2) at 3 months (P=.6513). In thinflap LASIK, the average preoperative endothelial cell density was 2995+/-325 cells/mm(2), which decreased to 2977+/-358 cells/mm(2) at 1 month (P=.5756) and 2931+/-369 cells/mm(2) at 3 months (P=.4106). No statistically significant difference was found between the two groups at 1 (P=.7404) or 3 (P=.3208) months postoperatively. No statistically significant change was noted in endothelial cell density following either PRK or thin-flap LASIK for the treatment of myopia. Furthermore, no statistically significant difference was found between the two groups out to 3 months postoperatively, indicating that thin-flap LASIK is as safe as PRK with regards to endothelial health.
Construction of Anterior Hemi-Corneal Equivalents Using Nontransfected Human Corneal Cells and Transplantation in Dog Models.
Xu, Bin; Song, Zhan; Fan, Tingjun
Tissue-engineered human anterior hemi-cornea (TE-aHC) is a promising equivalent for treating anterior lamellar keratopathy to surmount the severe shortage of donated corneas. This study was intended to construct a functional TE-aHC with nontransfected human corneal stromal (ntHCS) and epithelial (ntHCEP) cells using acellular porcine corneal stromata (aPCS) as a carrier scaffold, and evaluate its biological functions in a dog model. To construct a TE-aHC, ntHCS cells were injected into an aPCS scaffold and cultured for 3 days; then, ntHCEP cells were inoculated onto the Bowman's membrane of the scaffold and cultured for 5 days under air-liquid interface condition. After its morphology and histological structure were characterized, the constructed TE-aHC was transplanted into dog eyes via lamellar keratoplasty. The corneal transparency, thickness, intraocular pressure, epithelial integrity, and corneal regeneration were monitored in vivo, and the histological structure and histochemical property were examined ex vivo 360 days after surgery, respectively. The results showed that the constructed TE-aHC was highly transparent and composed of a corneal epithelium of 7-8 layer ntHCEP cells and a corneal stroma of regularly aligned collagen fibers and well-preserved glycosaminoglycans with sparsely distributed ntHCS cells, mimicking a normal anterior hemi-cornea (aHC). Moreover, both ntHCEP and ntHCS cells maintained positive expression of their marker and functional proteins. After transplantation into dog eyes, the constructed TE-aHC acted naturally in terms of morphology, structure and inherent property, and functioned well in maintaining corneal clarity, thickness, normal histological structure, and composition in dog models by reconstructing a normal aHC, which could be used as a promising aHC equivalent in corneal regenerative medicine and aHC disorder therapy. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
The effect of an Ahmed glaucoma valve implant on corneal endothelial cell density in children with glaucoma secondary to uveitis.
Kalinina Ayuso, Viera; Scheerlinck, Laura M; de Boer, Joke H
To assess the effect of Ahmed glaucoma valve implants on corneal endothelial cell density (ECD) in children with uveitic glaucoma. Cross-sectional study. setting: Institutional. patientpopulation: Eighty eyes from 42 patients diagnosed with uveitis before the age of 16. Twenty-eight eyes had an Ahmed glaucoma valve implant because of secondary glaucoma. Fifty-two eyes without an implant served as controls. intervention orobservationprocedure(s): Corneal ECD was examined cross-sectionally using a noncontact specular microscope. Univariate and multivariate generalized estimating equations analyses with correction for paired eyes were performed. mainoutcomemeasure(s): Correlation of ECD with the presence of an Ahmed glaucoma valve implant and with the time following implantation. ECD was significantly lower in the Ahmed glaucoma valve group than in controls (2359 and 3088 cells/mm(2), respectively; P Ahmed glaucoma valve implantation. Presence of an Ahmed glaucoma valve implant, previous intraocular surgery, age, duration of uveitis, and history of corneal touch by the implant tube were all significantly associated with decreased ECD. Following a multivariate analysis, presence of an Ahmed glaucoma valve implant (B = -340; adjusted P Ahmed glaucoma valve implantation was highly correlated with decreased ECD (B = -558, P Ahmed glaucoma valve implants in children with uveitic glaucoma are independently associated with decreased ECD, and this effect is associated with the time interval following Ahmed glaucoma valve implantation. Copyright © 2013 Elsevier Inc. All rights reserved.
Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells
Harris, Deshea L.
Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that
Changes in Corneal Endothelial Cell after Ahmed Glaucoma Valve Implantation and Trabeculectomy: 1-Year Follow-up.
Kim, Min Su; Kim, Kyoung Nam; Kim, Chang-Sik
To compare changes in corneal endothelial cell density (CECD) after Ahmed glaucoma valve (AGV) implantation and trabeculectomy. Changes in corneal endothelium in patients that underwent AGV implantation or trabeculectomy were prospectively evaluated. Corneal specular microscopy was performed at the central cornea using a non-contact specular microscope before surgery and 6 months and 12 months after surgery. The CECD, hexagonality of the endothelial cells, and the coefficient of variation of the cell areas were compared between the two groups. Forty eyes of 40 patients with AGV implantation and 28 eyes of 28 patients with trabeculectomy were studied. Intraocular pressure in the AGV implantation group was significantly higher than that in the trabeculectomy group ( p < 0.001), but there was no significant difference in other clinical variables between the two groups. In the AGV implantation group, the mean CECD significantly decreased by 9.4% at 6 months and 12.3% at 12 months compared with baseline values (both, p < 0.001), while it decreased by 1.9% at 6 months and 3.2% at 12 months in the trabeculectomy group ( p = 0.027 and p = 0.015, respectively). The changes at 6 months and 12 months in the AGV implantation group were significantly higher than those in the trabeculectomy group ( p = 0.030 and p = 0.027, respectively). In the AGV implantation group, there was a significant decrease in the CECD between baseline and 6 months and between 6 months and 12 months ( p < 0.001 and p = 0.005, respectively). However, in the trabeculectomy group, a significant decrease was observed only between baseline and 6 months ( p = 0.027). Both the AGV implantation group and the trabeculectomy group showed statistically significant decreases in the CECD 1 year after surgery. The decrease in CECD in the AVG implantation group was greater and persisted longer than that in the trabeculectomy group.
Pseudophakodonesis and corneal endothelial contact: direct observations by high-speed cinematography.
Jacobs, P M; Cheng, H; Price, N C
High-speed cinematography was used to observe the movement of Federov type I lens implants within the anterior chamber. Our measurements suggest that in most patients contact between the lens implant and corneal endothelium does not occur.
Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.
Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog; Jurkunas, Ula V
Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.
Corneal regeneration by induced human buccal mucosa cultivated on an amniotic membrane following alkaline injury.
Man, Rohaina Che; Yong, Then Kong; Hwei, Ng Min; Halim, Wan Haslina Wan Abdul; Zahidin, Aida Zairani Mohd; Ramli, Roszalina; Saim, Aminuddin Bin; Idrus, Ruszymah Binti Hj
Various clinical disorders and injuries, such as chemical, thermal, or mechanical injuries, may lead to corneal loss that results in blindness. PURPOSE : The aims of this study were to differentiate human buccal mucosa (BMuc) into corneal epithelial-like cells, to fabricate engineered corneal tissue using buccal mucosal epithelial cells, and to reconstruct a damaged corneal epithelium in a nude rat model. BMuc were subjected to 10 d of induction factors to investigate the potential of cells to differentiate into corneal lineages. Corneal stem cell markers β1-integrin, C/EBPδ, ABCG2, p63, and CK3 were upregulated in the gene expression analysis in induced BMuc, whereas CK3 and p63 showed significant protein expression in induced BMuc compared to the uninduced cells. BMuc were then left to reach 80% confluency after differential trypsinization. The cells were harvested and cultivated on a commercially available untreated air-dried amniotic membrane (AM) in a Transwell system in induction medium. The corneal constructs were fabricated and then implanted into damaged rat corneas for up to 8 weeks. A significant improvement was detected in the treatment group at 8 weeks post-implantation, as revealed by slit lamp biomicroscopy analysis. The structure and thickness of the corneal layer were also analyzed using histological staining and time-domain optical coherence tomography scans and were found to resemble a native corneal layer. The protein expression for CK3 and p63 were continuously detected throughout the corneal epithelial layer in the corneal construct. In conclusion, human BMuc can be induced to express a corneal epithelial-like phenotype. The addition of BMuc improves corneal clarity, prevents vascularization, increases corneal thickness and stromal alignment, and appears to have no adverse effect on the host after implantation.
Aloe vera extract activity on human corneal cells.
Woźniak, Anna; Paduch, Roman
Ocular diseases are currently an important problem in modern societies. Patients suffer from various ophthalmologic ailments namely, conjunctivitis, dry eye, dacryocystitis or degenerative diseases. Therefore, there is a need to introduce new treatment methods, including medicinal plants usage. Aloe vera [Aloe barbadensis Miller (Liliaceae)] possesses wound-healing properties and shows immunomodulatory, anti-inflammatory or antioxidant activities. NR uptake, MTT, DPPH• reduction, Griess reaction, ELISA and rhodamine-phalloidin staining were used to test toxicity, antiproliferative activity, reactive oxygen species (ROS) reduction, nitric oxide (NO) and cytokine level, and distribution of F-actin in cells, respectively. The present study analyzes the effect of Aloe vera extracts obtained with different solvents on in vitro culture of human 10.014 pRSV-T corneal cells. We found no toxicity of ethanol, ethyl acetate and heptane extracts of Aloe vera on human corneal cells. No ROS reducing activity by heptane extract and trace action by ethanol (only at high concentration 125 µg/ml) extract of Aloe vera was observed. Only ethyl acetate extract expressed distinct free radical scavenging effect. Plant extracts decreased NO production by human corneal cells as compared to untreated controls. The cytokine (IL-1β, IL-6, TNF-α and IL-10) production decreased after the addition of Aloe vera extracts to the culture media. Aloe vera contains multiple pharmacologically active substances which are capable of modulating cellular phenotypes and functions. Aloe vera ethanol and ethyl acetate extracts may be used in eye drops to treat inflammations and other ailments of external parts of the eye such as the cornea.
Viability of human corneal keratocytes during organ culture
DEFF Research Database (Denmark)
Møller-Pedersen, T; Møller, H J
The viability of human corneal keratocytes was assessed during four weeks of 'closed system' organ culture at 31 degrees C. After 28 days of culturing, the entire keratocyte population was still alive and viable because all cells incorporated uridine; a parameter for RNA-synthesis. During the first...... of keratan sulphate proteoglycan suggested that approximately 1% of the total content was lost during the period. In conclusion, our current organ culture technique can maintain a viable keratocyte population for four weeks; a viable stroma can be grafted within this period....
[Management of corneal endothelial decompensation with Descemet's membrane endothelial keratoplasty in a patient with Ahmed glaucoma valve implant].
Röck, T; Bartz-Schmidt, K-U; Röck, D; Yoeruek, E
Currently, the main causes for developing bullous keratopathy are from problems related to intraocular surgery, trauma, infection, Fuchs' endothelial dystrophy and chronically elevated intraocular pressure. In the 1990s penetrating keratoplasty was once considered the therapy of choice for treatment of bullous keratopathy but in recent years it has been replaced by posterior lamellar keratoplasty. The Descemet membrane endothelial keratoplasty (DMEK) procedure represents the final development of posterior lamellar keratoplasty. The question now arises whether DMEK can be used in patients with bullous keratopathy and Ahmed glaucoma valve implant. A 72-year-old man was referred to our hospital for further evaluation with the diagnosis of bullous keratopathy and pseudoexfoliative glaucoma. The bullous keratopathy was caused by a variety of previous operations as well as decompensation of intraocular pressure. This article describes the therapy of bullous keratopathy by DMEK with existing Ahmed glaucoma valve implant. After surgery the cornea became clear and the best-corrected visual acuity improved from hand movement to 0.2. The intraocular pressure remained normal (10-14 mmHg) without antiglaucoma medication and the endothelial cell count decreased only slightly over a follow-up of 13 months. No complications were encountered. The DMEK surgical procedure seems to be possible in patients with Ahmed glaucoma valve implant and endothelial decompensation. However, further studies with a larger number of patients should follow to validate the replacement of penetrating keratoplasty and other posterior lamellar procedures by DMEK.
Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells
purposes of advertisement . REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public reporting burden for this collection of...widespread stockpiling and the lack of definitive medical treatment. The vast majority of SM exposure cases involve corneal injury. At low-exposure doses
Pseudophakodonesis and corneal endothelial contact: direct observations by high-speed cinematography.
Jacobs, P M; Cheng, H; Price, N C
High-speed cinematography was used to observe the movement of Federov type I lens implants within the anterior chamber. Our measurements suggest that in most patients contact between the lens implant and corneal endothelium does not occur. Images PMID:6615750
Role of protein kinase C in regulation of Na+- and K +-dependent ATPase activity and pump function in corneal endothelial cells.
Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Nishida, Teruo
Na+- and K+-dependent ATPase (Na,K-ATPase) plays an important role in the pump function of the corneal endothelium. We investigated the possible role of protein kinase C (PKC) in regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to phorbol 12,13-dibutyrate (PDBu) to induce activation of PKC. ATPase activity of the cells was evaluated by using ammonium molybdate in spectrophotometric measurement of phosphate released from ATP, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with a Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. PDBu (10(-7) M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of PDBu were potentiated by the cyclooxygenase inhibitor indomethacin and the cytochrome P(450) inhibitor resorufin and were blocked by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results suggest that PKC bidirectionally regulates Na,K-ATPase activity in mouse corneal endothelial cells: it inhibits Na,K-ATPase activity in a cyclooxygenase- and cytochrome P(450)-dependent manner, whereas it stimulates such activity by activating protein phosphatases 1 or 2A.
Generation of Corneal Keratocytes from Human Embryonic Stem Cells.
Hertsenberg, Andrew J; Funderburgh, James L
Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.
Comparison of corneal endothelial changes following phacoemulsification with transversal and torsional phacoemulsification machines.
Ataş, Mustafa; Demircan, Süleyman; Karatepe Haşhaş, Arzu Seyhan; Gülhan, Ahmet; Zararsız, Gökmen
To compare and evaluate the phacoemulsification parameters and postoperative endothelial cell changes of two different phacoemulsification machines, each with different modes, but also to assess the relationship between postoperative endothelial cell loss and the phacoemulsification parameters, as well as the other factors in both groups. This prospective observational study was comprised of consecutive eligible cataract patients operated with phacoemulsification technique performed by the same surgeon using either a WHITESTAR Signature Ellips FX (transversal, group 1) or Infiniti OZil IP (torsional, group 2) machine. The study included 86 patients. Baseline characteristics in the groups were similar. The median nuclear sclerosis grade was 3 (2-4) in the first group and 2 (2-4) in the second group (P=0.265). Both groups had similar phacoemulsification needle times (group 1: 60.63±36 s; group 2: 55.98±30 s; P=0.789). The percentage of endothelial cell loss 30d after surgery ranged from 3% to 15% with a median of 7% in group 1, and from 2% to 13% with a median of 6% in group 2; however, there was no statistically significant difference between the groups (P=0.407). Hexagonality (P=0.794) and the coefficient of variation (CV; P=0.142) did not differ significantly between the groups before and 30d after surgery. A significant positive correlation was found between the endothelial cell loss and nuclear sclerosis grade (group 1: P<0.001; group 2: P<0.001) and between the endothelial cell loss and average phacoemulsification power (group 1: P=0.007; group 2: P=0.008). Both of these machines were efficient, with similar endothelial cell loss. This endothelial cell loss was related to the increased nuclear sclerosis grade and increased phacoemulsification power.
Organ culture storage of pre-prepared corneal donor material for Descemet's membrane endothelial keratoplasty.
Bhogal, Maninder; Matter, Karl; Balda, Maria S; Allan, Bruce D
To evaluate the effect of media composition and storage method on pre-prepared Descemet's membrane endothelial keratoplasty (DMEK) grafts. 50 corneas were used. Endothelial wound healing and proliferation in different media were assessed using a standard injury model. DMEK grafts were stored using three methods: peeling with free scroll storage; partial peeling with storage on the stroma and fluid bubble separation with storage on the stroma. Endothelial cell (EC) phenotype and the extent of endothelial overgrowth were examined. Global cell viability was assessed for storage methods that maintained a normal cell phenotype. 1 mm wounds healed within 4 days. Enhanced media did not increase EC proliferation but may have increased EC migration into the wounded area. Grafts that had been trephined showed evidence of EC overgrowth, whereas preservation of a physical barrier in the bubble group prevented this. In grafts stored in enhanced media or reapposed to the stroma after trephination, endothelial migration occurred sooner and cells underwent endothelial-mesenchymal transformation. Ongoing cell loss, with new patterns of cell death, was observed after returning grafts to storage. Grafts stored as free scrolls retained more viable ECs than grafts prepared with the fluid bubble method (74.2± 3% vs 60.3±6%, p=0.04 (n=8). Free scroll storage is superior to liquid bubble and partial peeling techniques. Free scrolls only showed overgrowth of ECs after 4 days in organ culture, indicating a viable time window for the clinical use of pre-prepared DMEK donor material using this method. Methods for tissue preparation and storage media developed for whole corneas should not be used in pre-prepared DMEK grafts without prior evaluation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
Effect of laser peripheral iridotomy using argon and neodymium-YAG lasers on corneal endothelial cell density: 7-year longitudinal evaluation.
Ono, Takashi; Iida, Masaharu; Sakisaka, Toshihiro; Minami, Keiichiro; Miyata, Kazunori
To evaluate the changes in corneal endothelial cell density (ECD) over a 7-year period after laser peripheral iridotomy (LPI) using argon and neodymium-doped yttrium aluminum garnet (Nd:YAG) lasers. Retrospective case series. Eyes that underwent prophylactic LPI using argon and Nd:YAG lasers were followed up for 7 years. Central corneal endothelial cells were observed by use of noncontact specular microscopy preoperatively and at 1 and 7 years postoperatively. Changes in ECD and the associations between preoperative ECD and the total energy of the Nd:YAG laser were evaluated. Fifty-one eyes of 51 patients were followed up for 7 years. The ECD significantly decreased after LPI (P laser energy. Long-term evaluation indicated that the reduction in ECD after argon-Nd:YAG laser LPI was present but small during the initial year and was negligible after 1 year.
Replication of TCF4 through association and linkage studies in late-onset Fuchs endothelial corneal dystrophy.
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Yi-Ju Li
Full Text Available Fuchs endothelial corneal dystrophy (FECD is a common, late-onset disorder of the corneal endothelium. Although progress has been made in understanding the genetic basis of FECD by studying large families in which the phenotype is transmitted in an autosomal dominant fashion, a recently reported genome-wide association study identified common alleles at a locus on chromosome 18 near TCF4 which confer susceptibility to FECD. Here, we report the findings of our independent validation study for TCF4 using the largest FECD dataset to date (450 FECD cases and 340 normal controls. Logistic regression with sex as a covariate was performed for three genetic models: dominant (DOM, additive (ADD, and recessive (REC. We found significant association with rs613872, the target marker reported by Baratz et al.(2010, for all three genetic models (DOM: P = 9.33×10(-35; ADD: P = 7.48×10(-30; REC: P = 5.27×10(-6. To strengthen the association study, we also conducted a genome-wide linkage scan on 64 multiplex families, composed primarily of affected sibling pairs (ASPs, using both parametric and non-parametric two-point and multipoint analyses. The most significant linkage region localizes to chromosome 18 from 69.94cM to 85.29cM, with a peak multipoint HLOD = 2.5 at rs1145315 (75.58cM under the DOM model, mapping 1.5 Mb proximal to rs613872. In summary, our study presents evidence to support the role of the intronic TCF4 single nucleotide polymorphism rs613872 in late-onset FECD through both association and linkage studies.
Comparison of mean corneal endothelial cell loss after trabeculectomy with and without mitomycin C
International Nuclear Information System (INIS)
Shaheer, M.; Amjad, A.; Ahmed, N.
Objective:To compare mean endothelial cell loss in patients of primary open angle glaucoma undergoing trabeculectomy with and without mitomycin C. Study Design:Randomised control study. Place and Duration of Study:Institute of Ophthalmology, Mayo Hospital, Lahore, from May 2016 to April 2017. Methodology:Patients with primary open angle glaucoma, not controlled with medication, were selected from the outpatient department. Patients with secondary glaucomas and concomitant ocular disease were excluded. Selected patients were divided into two groups of 30 each. Group A patients underwent trabeculectomy with adjunctive mitomycin C while group B patients underwent trabeculectomy alone. Pre- and post-trabeculectomy endothelial cell counts were recorded with the help of specular microsope and entered on proforma. Results:Median cell loss was 283 (66.50) when trabeculectomy was done with the use of adjunctive mitomycin C in group A while median endothelial cell loss was 72.50 (19.25) when the trabeculectomy was done without the use of adjunctive mitomycin C in group B. Conclusion:Use of mitomycin C causes more endothelial cell loss during trabeculectomy as compared to when done without it. (author)
Comparison of central corneal thickness and endothelial cell measurements by Scheimpflug camera system and two noncontact specular microscopes.
Karaca, Irmak; Yilmaz, Suzan Guven; Palamar, Melis; Ates, Halil
To investigate the correlation of Scheimpflug camera system and two noncontact specular microscopes in terms of central corneal thickness (CCT) and corneal endothelial cell morphology measurements. One hundred eyes of 50 healthy subjects were examined by Pentacam Scheimpflug Analyzer, CEM-530 (Nidek Co, Ltd, Gamagori, Japan) and CellChek XL (Konan Medical, California, USA) via fully automated image analysis with no corrections made. Measurement differences and agreement between instruments were determined by intraclass correlation analysis. The mean age of the subjects was 36.74 ± 8.59 (range 22-57). CCTs were well correlated among all devices, with having CEM-530 the thinnest and CellChek XL the thickest measurements (intraclass correlation coefficient (ICC) = 0.83; p < 0.001 and ICC = 0.78; p < 0.001, respectively). Mean endothelial cell density (ECD) given by CEM-530 was lower than CellChek XL (2613.17 ± 228.62 and 2862.72 ± 170.42 cells/mm 2 , respectively; ICC = 0.43; p < 0.001). Mean value for coefficient of variation (CV) was 28.57 ± 3.61 in CEM-530 and 30.30 ± 3.53 in CellChek XL. Cell hexagonality (HEX) with CEM-530 was higher than with CellChek XL (68.70 ± 4.16% and 45.19 ± 6.58%, respectively). ECDs with CellChek XL and CEM-530 have good correlation, but the values obtained by CellChek XL are higher than CEM-530. Measurements for HEX and CV differ significantly and show weak correlation. Thus, we do not recommend interchangeable use of CellChek XL and CEM-530. In terms of CCTs, Pentacam, CEM-530 and CellChek XL specular microscopy instruments are reliable devices.
Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells
Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.
ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal 'crosstalk' between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571
Evaluation of the Endothelial Cell Density and the Central Corneal Thickness in Pseudoexfoliation Syndrome and Pseudoexfoliation Glaucoma
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Bożydar T. Tomaszewski
Full Text Available Purpose. Evaluation of central corneal thickness (CCT and endothelial cell density (ECD in patients with senile cataract and coexisting pseudoexfoliation (PEX syndrome with glaucoma (PEXG and without glaucoma using specular microscopy. Participants and Methods. The study included 122 patients (217 eyes. In this group of patients we identified 133 eyes with PEX syndrome (65 with glaucoma, 68 without glaucoma and 84 eyes without PEX syndrome. ECD and CCT were measured in each eye by specular microscopy. Results. ECD in eyes with PEX syndrome without glaucoma (2297 ± 359 cell/mm2 and in eyes with PEXG (2241 ± 363 cell/mm2 was lower than in the control group (2503 ± 262 cell/mm2 (P<0.001. CCT in eyes with PEXG (508.2 ± 32.6 μm was thinner than in eyes with PEX syndrome without glaucoma (529.7 ± 30.3 μm and control group (527.7 ± 29.4 μm (P<0.001. Conclusions. This research shows that in eyes with PEX syndrome, both with and without glaucoma, ECD was statistically significantly lower than in the control group. In patients with PEXG, CCT was statistically significantly thinner than in the PEX syndrome and control group.
IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells
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Roni M. Shtein
Full Text Available Purpose. To determine time course of effect of lipopolysaccharide (LPS on production of interleukin-8 (IL-8 and monocyte chemotactic protein (MCP by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05 after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.
Sickle erythrocytes inhibit human endothelial cell DNA synthesis
International Nuclear Information System (INIS)
Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.
Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling
Corneal endothelial cell loss and corneal biomechanical characteristics after two-step sequential or combined phaco-vitrectomy surgery for idiopathic epiretinal membrane
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Hamoudi, Hassan; Christensen, Ulrik Correll; La Cour, Morten
with non-contact specular microscopy. Pachymetry [central cornea thickness (CCT)], keratometry and cornea volume (CV) were measured with Pentacam Scheimpflug camera. Primary outcome was change in CED after 12 months; secondary outcomes were changes in CCT and CV after 12 months. RESULTS: Sixty-two eyes......PURPOSE: To assess the impact of sequential and combined surgery [cataract surgery and 23-gauge pars plana vitrectomy (PPV) with peeling] on corneal endothelium cell density (CED) and corneal biomechanical characteristics. METHODS: Phakic eyes with epiretinal membrane (ERM) were prospectively...... allocated to (i) cataract surgery and subsequent PPV (CAT group), (ii) PPV and subsequent cataract surgery (VIT group) or (iii) phacovitrectomy (COMBI group). Eyes were examined at baseline, 1 month after each surgery, and at 3 and 12 months follow-up. Corneal endothelium cell density (CED) was assessed...
TGC repeat expansion in the TCF4 gene increases the risk of Fuchs' endothelial corneal dystrophy in Australian cases.
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Abraham Kuot
Full Text Available Fuchs' endothelial corneal dystrophy (FECD is a progressive, vision impairing disease. Common single nucleotide polymorphisms (SNPs and a trinucleotide repeat polymorphism, thymine-guanine-cytosine (TGC, in the TCF4 gene have been associated with the risk of FECD in some populations. We previously reported association of SNPs in TCF4 with FECD risk in the Australian population. The aim of this study was to determine whether TGC repeat polymorphism in TCF4 is associated with FECD in the Australian population. In 189 unrelated Australian cases with advanced late-onset FECD and 183 matched controls, the TGC repeat polymorphism located in intron 3 of TCF4 was genotyped using a short tandem repeat (STR assay. The repeat length was verified by direct sequencing in selected homozygous carriers. We found significant association between the expanded TGC repeat (≥ 40 repeats in TCF4 and advanced FECD (P = 2.58 × 10-22; OR = 15.66 (95% CI: 7.79-31.49. Genotypic analysis showed that 51% of cases (97 compared to 5% of controls (9 were heterozygous or homozygous for the expanded repeat allele. Furthermore, the repeat expansion showed stronger association than the most significantly associated SNP, rs613872, in TCF4, with the disease in the Australian cohort. This and haplotype analysis of both the polymorphisms suggest that considering both the polymorphisms together rather than either of the two alone would better predict susceptibility to FECD in the Australian population. This is the first study to report association of the TGC trinucleotide repeat expansion in TCF4 with advanced FECD in the Australian population.
Topical versus subconjunctival anti-vascular endothelial growth factor therapy (Bevacizumab, Ranibizumab and Aflibercept for treatment of corneal neovascularization
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Tariq Al-Debasi
Anti-VEGF agents (Bevacizumab, Ranibizumab and Aflibercept seem to have a higher efficiency and efficacy for corneal NV treatment. Both subconjunctival therapy and topical therapy of bevacizumab prohibit corneal NV, while early treatment with subconjunctival administration of ranibizumab may successfully reduce corneal NV. Therefore, establishment of safe doses is highly important before these drugs can be involved in the clinical setting. Further investigations and studies are highly warranted to adjust the dose and route of administration for the antibodies directed against VEGF to be the key therapeutic agents in the corneal NV treatment.
Visual outcome and changes in corneal endothelial cell density following aphakic iris-fixated intraocular lens implantation in pediatric eyes with subluxated lenses.
Siddiqui, Sorath Noorani; Khan, Ayesha
To evaluate the visual outcome and corneal endothelial cell density after Artisan aphakic intraocular lens (IOL) implantation (Ophtec, Groningen, the Netherlands) in pediatric eyes with subluxated lenses. Artisan aphakic IOLs were implanted in 18 eyes of 11 children with subluxated lenses. Idiopathic subluxations and ectopia lentis due to Marfan syndrome were included, whereas subluxations due to trauma or buphthalmos were excluded. Best-corrected visual acuity (BCVA) and endothelial cell density were monitored. Mean postoperative BCVA and endothelial cell density at last follow-up visit were calculated. The age of children at the time of Artisan aphakic IOL implantation ranged from 8 to 16 years (mean: 11.58 ± 2.9 years). Mean follow-up was 9.12 ± 4.30 months. Mean postoperative logarithm of the minimum angle of resolution BCVA was 0.26 ± 0.13 (P = .001) and mean postoperative endothelial cell density was 2,860 ± 435 cells/mm(2) (P = .000). Mean endothelial cell loss was 17.1%. Artisan aphakic IOL implantation is a safe surgical choice in the management of ectopia lentis in the pediatric age group. It has minimal complications and is less traumatic to pediatric eyes. However, long-term follow-up of these children is required.[J Pediatr Ophthalmol Strabismus 2013;50(3):178-182.]. Copyright 2013, SLACK Incorporated.
Rapid, automated mosaicking of the human corneal subbasal nerve plexus.
Vaishnav, Yash J; Rucker, Stuart A; Saharia, Keshav; McNamara, Nancy A
Corneal confocal microscopy (CCM) is an in vivo technique used to study corneal nerve morphology. The largest proportion of nerves innervating the cornea lie within the subbasal nerve plexus, where their morphology is altered by refractive surgery, diabetes and dry eye. The main limitations to clinical use of CCM as a diagnostic tool are the small field of view of CCM images and the lengthy time needed to quantify nerves in collected images. Here, we present a novel, rapid, fully automated technique to mosaic individual CCM images into wide-field maps of corneal nerves. We implemented an OpenCV image stitcher that accounts for corneal deformation and uses feature detection to stitch CCM images into a montage. The method takes 3-5 min to process and stitch 40-100 frames on an Amazon EC2 Micro instance. The speed, automation and ease of use conferred by this technique is the first step toward point of care evaluation of wide-field subbasal plexus (SBP) maps in a clinical setting.
Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.
Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A
It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.
Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.
Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A
Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.
Verocytotoxin-induced apoptosis of human microvascular endothelial cells.
Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W
The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.
Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: Implications for human health.
Xiang, Ping; Liu, Rong-Yan; Li, Chao; Gao, Peng; Cui, Xin-Yi; Ma, Lena Q
Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most detected organophosphorus flame retardants (OPFRs) in the environment, especially in indoor dust. Continuous daily exposure to TDCPP-containing dust may adversely impact human cornea. However, its detrimental effects on human corneal epithelium are largely unknown. In this study, we investigated the cell apoptosis in normal human corneal epithelial cells (HCECs) after TDCPP exposure and elucidated the underlying molecular mechanisms. Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC 50 at 202 μg/mL. A concentration-dependent apoptotic sign was observed in HCECs after exposing to ≥2 μg/mL TDCPP. Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes (ATF-4, CHOP, BiP, and XBP1). Furthermore, alternation of Bcl-2/Bax expression, mitochondrial membrane potential loss, cellular ATP content decrease, and caspase-3 and -9 activity increase were observed after exposing to 2 or 20 μg/mL TDCPP. Taken together, the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis, probably mediated by mitochondrial apoptotic pathway. Our findings showed TDCPP exposure induced toxicity to human cornea. Due to TDCPP's presence at high levels in indoor dust, further study is warranted to evaluate its health risk on human corneas. Published by Elsevier Ltd.
Isolated corneal papilloma-like lesion associated with human papilloma virus type 6.
Park, Choul Yong; Kim, Eo-Jin; Choi, Jong Sun; Chuck, Roy S
To report a case of a corneal papilloma-like lesion associated with human papilloma virus type 6. A 48-year-old woman presented with a 2-year history of ocular discomfort and gradual visual deterioration in her right eye. Ophthalmic examination revealed an elevated, semitranslucent, well-defined vascularized mass approximately 4 × 2.5 mm in size localized to the right cornea. The surface of the mass appeared smooth and many small, shallow, and irregular elevations were noted. An excisional biopsy was performed. The underlying cornea was markedly thinned, and fine ramifying vasculature was also noted on the exposed corneal stroma. Typical koilocytic change was observed on the histopathologic examination. Polymerase chain reaction revealed the existence of human papilloma virus type 6 DNA. Here we describe a case of an isolated corneal papilloma-like lesion. Although the corneal extension of the limbal or the conjunctival papillomas has been commonly observed, an isolated corneal papilloma-like lesion with underlying stromal destruction has only rarely been reported.
Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells
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Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)
Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.
Infection of endothelial cells by common human viruses.
Friedman, H M
Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.
Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium
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Wenlin Zhang
Full Text Available Corneal endothelium (CE is among the most metabolically active tissues in the body. This elevated metabolic rate helps the CE maintain corneal transparency by its ion and fluid transport properties, which when disrupted, leads to visual impairment. Here we demonstrate that glutamine catabolism (glutaminolysis through TCA cycle generates a large fraction of the ATP needed to maintain CE function, and this glutaminolysis is severely disrupted in cells deficient in NH3:H+ cotransporter Solute Carrier Family 4 Member 11 (SLC4A11. Considering SLC4A11 mutations leads to corneal endothelial dystrophy and sensorineural deafness, our results indicate that SLC4A11-associated developmental and degenerative disorders result from altered glutamine catabolism. Overall, our results describe an important metabolic mechanism that provides CE cells with the energy required to maintain high level transport activity, reveal a direct link between glutamine metabolism and developmental and degenerative neuronal diseases, and suggest an approach for protecting the CE during ophthalmic surgeries.
Interference figures of polarimetric interferometry analysis of the human corneal stroma.
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Rodolfo Mastropasqua
Full Text Available A rotating polarimetric 90°-cross linear-filter interferometry system was used to detect the morphological characteristics and features of interference patterns produced in in-vivo corneal stroma in healthy human corneas of 23 subjects. The characteristic corneal isogyres presenting with an evident cross-shaped pattern, grossly aligned with the fixation axis, were observed in all patients with centers within the pupillary dark area, impeding the exact determination of the center point. During the rotational scan in 78.3% of the eyes the cross-shaped pattern of the isogyre gradually separated to form two distinct hyperbolic arcs in opposite quadrants, reaching their maximal separation at 45 degrees with respect to angle of cross-shaped pattern formation. The corneal cross and hyperbolic-pattern repeated every 90° throughout the 360° rotational scan. While the interpretation of the isogyres presents particular difficulties, two summary parameters can be extracted for each cornea: the presence/orientation of a single or two dark areas in post-processed images and isochromes. However, the development of dedicated software for semi-quantitative analysis of these parameters and enantiomorphism may become available in the near future. The possible application of polarimetric interferometry in the field of both corneal pathologies and corneal surgery may be of great interest for clinical purposes.
Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices
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Stafiej, Piotr; Küng, Florian [Department of Ophthalmology, Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen (Germany); Institute of Polymer Materials, Universität Erlangen-Nürnberg, Martensstraße 7, 91054 Erlangen (Germany); Thieme, Daniel; Czugala, Marta; Kruse, Friedrich E. [Department of Ophthalmology, Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen (Germany); Schubert, Dirk W. [Institute of Polymer Materials, Universität Erlangen-Nürnberg, Martensstraße 7, 91054 Erlangen (Germany); Fuchsluger, Thomas A., E-mail: firstname.lastname@example.com [Department of Ophthalmology, Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen (Germany)
In this work, polycaprolactone (PCL) was used as a basic polymer for electrospinning of random and aligned nanofiber matrices. Our aim was to develop a biocompatible substrate for ophthalmological application to improve wound closure in defects of the cornea as replacement for human amniotic membrane. We investigated whether blending the hydrophobic PCL with poly (glycerol sebacate) (PGS) or chitosan (CHI) improves the biocompatibility of the matrices for cell expansion. Human corneal epithelial cells (HCEp) and human corneal keratocytes (HCK) were used for in vitro biocompatibility studies. After optimization of the electrospinning parameters for all blends, scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and water contact angle were used to characterize the different matrices. Fluorescence staining of the F-actin cytoskeleton of the cells was performed to analyze the adherence of the cells to the different matrices. Metabolic activity of the cells was measured by cell counting kit-8 (CCK-8) for 20 days to compare the biocompatibility of the materials. Our results show the feasibility of producing uniform nanofiber matrices with and without orientation for the used blends. All materials support adherence and proliferation of human corneal cell lines with oriented growth on aligned matrices. Although hydrophobicity of the materials was lowered by blending PCL, no increase in biocompatibility or proliferation, as was expected, could be measured. All tested matrices supported the expansion of human corneal cells, confirming their potential as substrates for biomedical applications. - Highlights: • PCL was blended with chitosan and poly(glycerol sebacate) for electrospinning. • Biocompatibility was proven with two human corneal cell lines. • Both cell lines adhered and proliferated on random and aligned nanofiber matrices. • Cytoskeletal orientation is shown on aligned nanofiber matrices. �
Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes.
Ponec, Maria; El Ghalbzouri, Abdoelwaheb; Dijkman, Remco; Kempenaar, Johanna; van der Pluijm, Gabri; Koolwijk, Pieter
A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.
Acrylamide induces accelerated endothelial aging in a human cell model.
Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas
Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging. Copyright © 2015 Elsevier Ltd. All rights reserved.
New Details of the Human Corneal Limbus Revealed With Second Harmonic Generation Imaging.
Park, Choul Yong; Lee, Jimmy K; Zhang, Cheng; Chuck, Roy S
To report novel findings of the human corneal limbus by using second harmonic generation (SHG) imaging. Corneal limbus was imaged by using an inverted two-photon excitation fluorescence microscope. Laser (Ti:Sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of SHG and autofluorescence (AF) were collected through a 425/30-nm emission filter and a 525/45-emission filter, respectively. Multiple, consecutive, and overlapping image stacks (z-stack) were acquired for the corneal limbal area. Two novel collagen structures were revealed by SHG imaging at the limbus: an anterior limbal cribriform layer and presumed anchoring fibers. Anterior limbal cribriform layer is an intertwined reticular collagen architecture just beneath the limbal epithelial niche and is located between the peripheral cornea and Tenon's/scleral tissue. Autofluorescence imaging revealed high vascularity in this structure. Central to the anterior limbal cribriform layer, radial strands of collagen were found to connect the peripheral cornea to the limbus. These presumed anchoring fibers have both collagen and elastin and were found more extensively in the superficial layers than deep layer and were absent in very deep limbus near Schlemm's canal. By using SHG imaging, new details of the collagen architecture of human corneal limbal area were elucidated. High resolution images with volumetric analysis revealed two novel collagen structures.
Axenfeld-Rieger anomaly and corneal endothelial dystrophy: a case series Anomalia de Axenfeld-Rieger e distrofia corneana endotelial: uma série de casos
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Mariana Borges Oliveira
Full Text Available Report of a study of a family with a remarkable combination of endothelial corneal dystrophy, and anterior chamber dysgenesis classified as Axenfeld-Rieger anomaly. A 56 year-old female had blurred vision complaints. A slit lamp evaluation showed a bilateral posterior embryotoxon and guttata with corneal edema, Descemet membrane's folds, characterizing Fuchs endothelial dystrophy. A 27 year-old daughter was asymptomatic. The biomicroscopic exam disclosed a bilateral nasal and temporal posterior embryotoxon and a central guttata. Gonioscopy showed iridocorneal strands with a prominent Schwalbe´s line. The intraocular pressure was normal. Her sister, a 25 year-old female presented very similar abnormalities along with her brother (22 years old. A 19 year-old female sister presented the iridocorneal angle alterations, without Fuchs endothelial dystrophy, until this moment. This paper presents a family with a central cornea guttata or Fuchs dystrophy associated with Axenfeld-Rieger anomaly. Two different inherited diseases appearing together in an entire family may suggest a single molecular and genetic etiology, with high penetrance.Relato dos resultados do estudo de uma família com distrofia corneana endotelial associada a uma disgenesia do segmento anterior, a anomalia de Axenfeld-Rieger. Paciente de 56 anos, feminina, queixava-se de visão borrada. O exame na lâmpada de fenda evidenciou embriotoxo posterior bilateral e guttata no centro da córnea, edema estromal e dobras na membrana de Descemet, caracterizando distrofia endothelial de Fuchs. A filha de 27 anos estava assintomática. A biomicroscopia revelou embriotoxo posterior nasal e temporal bilateral e guttata central. A gonioscopia evidenciou processos iridocorneanos até a linha de Schwalbe, que se encontrava anteriorizada e proeminente. A pressão intra-ocular estava normal. Não foram observadas alterações sistêmicas. Sua irmã, 25 anos, sexo feminino, apresentava altera
Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells
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Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik
PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....
Full Text Available ... Ophthalmology/Strabismus Ocular Pathology/Oncology Oculoplastics/Orbit Refractive ... Corneal Laceration Sections What Is Corneal Laceration? Corneal Laceration Symptoms What Causes ...
Small GTP-binding proteins in human endothelial cells
de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.
Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of
Effect of propionyl-L-carnitine on human endothelial cells
Hinsbergh, V.W.M. van; Scheffer, M.A.
A possible protective effect of propionyl-L-carnitine on human endothelial cells was studied both under basal culture conditions and in the presence of agents capable of influencing oxidative damage, such as glucose/glucose oxidase and oxidized low-density lipoproteins. Propionyl-L-carnitine had no
Endothelial and lipoprotein lipases in human and mouse placenta
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Lindegaard, Marie L S; Olivecrona, Gunilla; Christoffersen, Christina
Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharos...
Small-Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells
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Alexandra Mikhailova
Full Text Available Human induced pluripotent stem cells (hiPSCs offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation.
Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits
Kim, Tae-Hyun; Park, Young-Woo; Ahn, Jae-Sang; Ahn, Jeong-Taek; Kim, Se-Eun; Jeong, Man-Bok; Seo, Min-Su; Kang, Kyung-Sun
This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits. PMID:23388445
Corneal epithelial wound healing and bactericidal effect of conditioned medium from human uterine cervical stem cells.
Bermudez, Maria A; Sendon-Lago, Juan; Eiro, Noemi; Treviño, Mercedes; Gonzalez, Francisco; Yebra-Pimentel, Eva; Giraldez, Maria Jesus; Macia, Manuel; Lamelas, Maria Luz; Saa, Jorge; Vizoso, Francisco; Perez-Fernandez, Roman
To evaluate the effect of conditioned medium from human uterine cervical stem cells (CM-hUCESCs) on corneal epithelial healing in a rat model of dry eye after alkaline corneal epithelial ulcer. We also tested the bactericidal effect of CM-hUCESCs. Dry eye was induced in rats by extraocular lacrimal gland excision, and corneal ulcers were produced using NaOH. Corneal histologic evaluation was made with hematoxylin-eosin (H&E) staining. Real-time PCR was used to evaluate mRNA expression levels of proinflammatory cytokines. We also studied the bactericidal effect of CM-hUCESCs in vitro and on infected corneal contact lenses (CLs) using Escherichia coli and Staphylococcus epidermidis bacteria. In addition, in order to investigate proteins from CM-hUCESCs that could mediate these effects, we carried out a human cytokine antibody array. After injury, dry eyes treated with CM-hUCESCs significantly improved epithelial regeneration and showed reduced corneal macrophage inflammatory protein-1 alpha (MIP-1α) and TNF-α mRNA expression as compared to untreated eyes and eyes treated with culture medium or sodium hyaluronate ophthalmic drops. In addition, we found in CM-hUCESCs high levels of proteins, such as tissue inhibitors of metalloproteinases 1 and 2, fibroblast growth factor 6 and 7, urokinase receptor, and hepatocyte growth factor, that could mediate these effects. In vitro, CM-hUCESCs showed a clear bactericidal effect on both E. coli and S. epidermidis and CLs infected with S. epidermidis. Analyses of CM-hUCESCs showed elevated levels of proteins that could be involved in the bactericidal effect, such as the chemokine (C-X-C motif) ligands 1, 6, 8, 10, and the chemokine (C-C motif) ligands 5 and 20. Treatment with CM-hUCESCs improved wound healing of alkali-injured corneas and showed a strong bactericidal effect on CLs. Patients using CLs and suffering from dry eye, allergies induced by commercial solutions, or small corneal injuries could benefit from this treatment
Uptake of gold nanoparticles in primary human endothelial cells
DEFF Research Database (Denmark)
Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin
Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy......–3 or more particles. Pre-treatment with chlorpromazine inhibited the AuNP-uptake in HUVECs, indicating that internalisation occurred mainly by clathrin-mediated endocytosis. Cell activation by exposure to tumour necrosis factor or lipopolysaccharide had a slight or no effect on the uptake of Au...
Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation
Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.
Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.
Radiosensitization of human endothelial cells by IL-24
International Nuclear Information System (INIS)
Meyn, R.E.
Radiation therapy remains an important cancer treatment modality but despite improvements in dose delivery many patients still fail at their primary tumor site. Therefore, new strategies designed to improve local control are needed. Protocols combining radiation with anti-angiogenic agents might be of particular advantage based on their documented low toxicity. In this regard, we have been conducting preclinical investigations of a novel cytokine, mda7/IL-24. Our collaborators have shown that mda7/IL-24 protein targets the endothelial cells of the tumor microvascular system and has potent anti-angiogenic properties in both in vitro and in vivo assays. Recently, we have demonstrated that recombinant mda7/IL-24 protein radiosensitizes human endothelial cells in vitro. Specifically, 10 ng/ml of recombinant human IL-24 protein for 12 hrs reduced the survival at 2 Gy for human umbilical vein endothelial cells (HUVECs) from 0.33 to 0.12. We are also working on understanding the molecular basis for this radiosensitizing effect. Preliminary data suggest a model whereby mda7/IL-24 engages a specific receptor on the surface of endothelial cells and initiates a signal transduction pathway that modulates the cell's propensity for radiation-induced apoptosis and capacity for repairing radiation-induced DNA double strand breaks. Mechanistic insight gained from these studies may have implications for the actions of other anti-angiogenic agents and may generally explain the regulation of radiosensitivity imparted by growth factors and cytokines
Detection and Quantification of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases in Primary Human Endothelial Cells.
Fearnley, Gareth W; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan
Proteins differ widely in their pattern of expression depending on organism, tissue, and regulation in response to changing conditions. In the mammalian vasculature, the endothelium responds to vascular endothelial growth factors (VEGFs) via membrane-bound receptor tyrosine kinases (VEGFRs) to modulate many aspects of vascular physiology including vasculogenesis, angiogenesis, and blood pressure. Studies on VEGFR biology are thus dependent on detecting expression levels in different cell types and evaluating how changes in protein levels correlate with changing conditions including circulating VEGF levels. Here, we present a robust immunoblot-based protocol for detecting and quantifying VEGFRs in human endothelial cells. Using internal and external standards, we can rapidly evaluate receptor copy number and assess how this is altered in response to the cellular environment.
Corneal thickness: measurement and implications.
Ehlers, Niels; Hjortdal, Jesper
The thickness of the cornea was reported in more than 100-year-old textbooks on physiological optics (Helmholtz, Gullstrand). Physiological interest was revived in the 1950s by David Maurice, and over the next 50 years, this 'simple' biological parameter has been studied extensively. Several techniques for its measurement have been described and physiological and clinical significance have been studied. In this review, the different methods and techniques of measurement are briefly presented (optical, ultrasound). While the corneal thickness of many animals are the same over a considerable part of the surface, in the human cornea anterior and posterior curvature are not concentric giving rise to a problem of definition. Based on this the precision and accuracy of determining the central corneal thickness are discussed. Changes in corneal thickness reflects changes in function of the boundary layers, in particular the endothelial barrier. The absolute value of thickness is of importance for the estimation of IOP but also in diagnosis of corneal and systemic disorders. Finally it is discussed to what extent the thickness is a biometric parameter of significance, e.g. in the progression of myopia or in the development of retinal detachment.
Human cadaver retina model for retinal heating during corneal surgery with a femtosecond laser
Sun, Hui; Fan, Zhongwei; Yun, Jin; Zhao, Tianzhuo; Yan, Ying; Kurtz, Ron M.; Juhasz, Tibor
Femtosecond lasers are widely used in everyday clinical procedures to perform minimally invasive corneal refractive surgery. The intralase femtosecond laser (AMO Corp. Santa Ana, CA) is a common example of such a laser. In the present study a numerical simulation was developed to quantify the temperature rise in the retina during femtosecond intracorneal surgery. Also, ex-vivo retinal heating due to laser irradiation was measured with an infrared thermal camera (Fluke Corp. Everett, WA) as a validation of the simulation. A computer simulation was developed using Comsol Multiphysics to calculate the temperature rise in the cadaver retina during femtosecond laser corneal surgery. The simulation showed a temperature rise of less than 0.3 degrees for realistic pulse energies for the various repetition rates. Human cadaver retinas were irradiated with a 150 kHz Intralase femtosecond laser and the temperature rise was measured withan infrared thermal camera. Thermal camera measurements are in agreement with the simulation. During routine femtosecond laser corneal surgery with normal clinical parameters, the temperature rise is well beneath the threshold for retina damage. The simulation predictions are in agreement with thermal measurements providing a level of experimental validation.
Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.
Turner, Helen C; Budak, Murat T; Akinci, M A Murat; Wolosin, J Mario
To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that
ITE inhibits growth of human pulmonary artery endothelial cells.
Pang, Ling-Pin; Li, Yan; Zou, Qing-Yun; Zhou, Chi; Lei, Wei; Zheng, Jing; Huang, Shi-An
Pulmonary arterial hypertension (PAH), a deadly disorder is associated with excessive growth of human pulmonary artery endothelial (HPAECs) and smooth muscle (HPASMCs) cells. Current therapies primarily aim at promoting vasodilation, which only ameliorates clinical symptoms without a cure. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous aryl hydrocarbon receptor (AhR) ligand, and mediates many cellular function including cell growth. However, the roles of ITE in human lung endothelial cells remain elusive. Herein, we tested a hypothesis that ITE inhibits growth of human pulmonary artery endothelial cells via AhR. Immunohistochemistry was performed to localize AhR expression in human lung tissues. The crystal violet method and MTT assay were used to determine ITE's effects on growth of HPAECs. The AhR activation in HPAECs was confirmed using Western blotting and RT-qPCR. The role of AhR in ITE-affected proliferation of HPAECs was assessed using siRNA knockdown method followed by the crystal violet method. Immunohistochemistry revealed that AhR was present in human lung tissues, primarily in endothelial and smooth muscle cells of pulmonary veins and arteries, as well as in bronchial and alveolar sac epithelia. We also found that ITE dose- and time-dependently inhibited proliferation of HPAECs with a maximum inhibition of 83% at 20 µM after 6 days of treatment. ITE rapidly decreased AhR protein levels, while it increased mRNA levels of cytochrome P450 (CYP), family 1, member A1 (CYP1A1) and B1 (CYP1B1), indicating activation of the AhR/CYP1A1 and AhR/CYP1B1 pathways in HPAECs. The AhR siRNA significantly suppressed AhR protein expression, whereas it did not significantly alter ITE-inhibited growth of HPAECs. ITE suppresses growth of HPAECs independent of AhR, suggesting that ITE may play an important role in preventing excessive growth of lung endothelial cells.
Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks.
Sorkio, Anni; Koch, Lothar; Koivusalo, Laura; Deiwick, Andrea; Miettinen, Susanna; Chichkov, Boris; Skottman, Heli
There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted
Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.
Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P
The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the
Epigalloccatechin-3-gallate Inhibits Ocular Neovascularization and Vascular Permeability in Human Retinal Pigment Epithelial and Human Retinal Microvascular Endothelial Cells via Suppression of MMP-9 and VEGF Activation
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Hak Sung Lee
Full Text Available Epigalloccatechin-3-gallate (EGCG is the main polyphenol component of green tea (leaves of Camellia sinensis. EGCG is known for its antioxidant, anti-inflammatory, antiviral, and anti-carcinogenic properties. Here, we identify EGCG as a new inhibitor of ocular angiogenesis and its vascular permeability. Matrix metalloproteinases (MMPs and vascular endothelial growth factor (VEGF play a key role in the processes of extracellular matrix (ECM remodeling and microvascular permeability during angiogenesis. We investigated the inhibitory effects of EGCG on ocular neovascularization and vascular permeability using the retina oriented cells and animal models induced by VEGF and alkaline burn. EGCG treatment significantly decreased mRNA and protein expression levels of MMP-9 in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA and tumor necrosis factor alpha (TNF-α in human retinal pigment epithelial cells (HRPECs. EGCG also effectively protected ARPE-19 cells from cell death and attenuated mRNA expressions of key angiogenic factors (MMP-9, VEGF, VEGF Receptor-2 by inhibiting generation of reactive oxygen species (ROS. EGCG significantly inhibited proliferation, vascular permeability, and tube formation in VEGF-induced human retinal microvascular endothelial cells (HRMECs. Furthermore, EGCG significantly reduced vascular leakage and permeability by blood-retinal barrier breakdown in VEGF-induced animal models. In addition, EGCG effectively limited upregulation of MMP-9 and platelet endothelial cell adhesion molecule (PECAM/CD31 on corneal neovascularization (CNV induced by alkaline burn. Our data suggest that MMP-9 and VEGF are key therapeutic targets of EGCG for treatment and prevention of ocular angiogenic diseases such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization.
Isolation and Characterization of Human Lung Lymphatic Endothelial Cells
Lorusso, Bruno; Falco, Angela; Madeddu, Denise; Frati, Caterina; Cavalli, Stefano; Graiani, Gallia; Gervasi, Andrea; Rinaldi, Laura; Lagrasta, Costanza; Maselli, Davide; Gnetti, Letizia; Silini, Enrico M.; Quaini, Eugenio; Ampollini, Luca; Carbognani, Paolo; Quaini, Federico
Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. PMID:26137493
Organizational behavior of human umbilical vein endothelial cells
Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization. PMID:6813338
Reproducibility of Corneal Graft Thickness measurements with COLGATE in patients who have undergone DSAEK (Descemet Stripping Automated Endothelial Keratoplasty
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Wong Melissa HY
Full Text Available Abstract Background The CorneaL GrAft Thickness Evaluation (COLGATE system was recently developed to facilitate the evaluation of corneal graft thickness from OCT images. Graft thickness measurement can be a surrogate indicator for detecting graft failure or success. The purpose of this study was to determine the reproducibility of the COLGATE system in measuring DSAEK graft area between two observers. Methods This was a prospective case series in which 50 anterior segment OCT images of patients who had undergone DSAEK in either eye were analysed. Two observers (MW, AC independently obtained the image analysis for the graft area using both semi automated and automated method. One week later, each observer repeated the analysis for the same set of images. Bland-Altman analysis was performed to analyze inter and intra observer agreement. Results There was strong intraobserver correlation between the 2 semi automated readings obtained by both observers. (r = 0.936 and r = 0.962. Intraobserver ICC for observer 1 was 0.936 (95% CI 0.890 to 0.963 and 0.967 (95% CI 0.942 to 0.981 for observer 2. Likewise, there was also strong interobserver correlation (r = 0.913 and r = 0.969. The interobserver ICC for the first measurements was 0.911 (95% CI 0.849 to 0.949 and 0.968 (95% CI 0.945 to 0.982 for the second. There was statistical difference between the automatic and the semi automated readings for both observers (p = 0.006, p = 0.003. The automatic readings gave consistently higher values than the semi automated readings especially in thin grafts. Conclusion The analysis from the COLGATE programme can be reproducible between different observers. Care must be taken when interpreting the automated analysis as they tend to over estimate measurements.
In vitro analysis of human periodontal microvascular endothelial cells.
Tsubokawa, Mizuki; Sato, Soh
Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites
Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2
Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.
Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipa...
The tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing.
Couture, Camille; Zaniolo, Karine; Carrier, Patrick; Lake, Jennifer; Patenaude, Julien; Germain, Lucie; Guérin, Sylvain L
Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Copyright © 2015 Elsevier Ltd. All rights reserved.
Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells
Czech Academy of Sciences Publication Activity Database
Rárová, L.; Zahler, S.; Liebl, J.; Kryštof, Vladimír; Sedlák, David; Bartůněk, Petr; Kohout, Ladislav; Strnad, Miroslav
Roč. 77, č. 13 (2012), s. 1502-1509 ISSN 0039-128X R&D Projects: GA MŠk(CZ) LC06077 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50520514; CEZ:AV0Z40550506 Keywords : Angiogenesis * Human umbilical vein endothelial cells * Migration Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.803, year: 2012
Transplantation with cultured stem cells derived from the human amniotic membrane for corneal alkali burns: an experimental study.
Zeng, Wei; Li, Yanwei; Zeng, Guangwei; Yang, Bo; Zhu, Yu
Amniotic membranes (AM) have been used in a wide range of clinical applications. We successfully extracted mesenchymal stem cells (MSCs) from human AM, but little is known about the use and efficacy of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) for the treatment of alkali burns. We utilized hAM-dMSCs transplantation, AM grafting, and their combined use in the treatment of alkali burns. An experimental model in rabbits was devised to analyze the use of these techniques with immunocytochemistry and ELISA. The survival and migration of hAM-dMSCs labeled by SPION in the host were assessed with Prussian blue staining. Compared with the control group, the treated groups demonstrated faster reconstruction of the corneal epithelium, and lower levels of corneal opacification and neovascularization within corneal alkali burns. Furthermore, dark blue-stained particles were detected in the limbus corneae at day 28. These results demonstrated the ability of hAM-dMSCs to enhance epithelial healing and reduce corneal opacification and neovascularization in corneal alkali wounds.
Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin.
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Young Yu
Full Text Available The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin αVβ3 blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. αVβ3 blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving αVβ3. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.
Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.
Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan
Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by
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Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression
International Nuclear Information System (INIS)
Yu Hui; Wu Jihong; Li Huiming; Wang Zhanli; Chen Xiafang; Tian Yuhua; Yi Miaoying; Ji Xunda; Ma Jialie; Huang Qian
The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (10 8 PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization
Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.
Rossman, Matthew J; Kaplon, Rachelle E; Hill, Sierra D; McNamara, Molly N; Santos-Parker, Jessica R; Pierce, Gary L; Seals, Douglas R; Donato, Anthony J
Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P age-related differences were not present (all P > 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 ( r = -0.49, P = 0.003), p21 ( r = -0.38, P = 0.03), and p16 ( r = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P healthy older sedentary (63 ± 1 yr, n = 18) versus young sedentary (25 ± 1 yr, n = 9) adults; age-related changes in arterial EC p53 and p21 expression were not observed ( P > 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age. NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function
Diclofenac protects cultured human corneal epithelial cells against hyperosmolarity and ameliorates corneal surface damage in a rat model of dry eye.
Sawazaki, Ryoichi; Ishihara, Tomoaki; Usui, Shinya; Hayashi, Erika; Tahara, Kayoko; Hoshino, Tatsuya; Higuchi, Akihiro; Nakamura, Shigeru; Tsubota, Kazuo; Mizushima, Tohru
Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.
Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures
Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto
The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.
VIP and VIP gene silencing modulation of differentiation marker N-cadherin and cell shape of corneal endothelium in human corneas ex vivo.
Koh, Shay-Whey M; Chandrasekara, Krish; Abbondandolo, Cara J; Coll, Timothy J; Rutzen, Allan R
Vasoactive intestinal peptide (VIP) is expressed by corneal endothelial (CE) cells and is present in the aqueous humor, which bathes CE cells in vivo. This study demonstrated the role of CE cell VIP in maintaining the expression level of a CE differentiation marker, N-cadherin, and the hexagonal cell shape. To determine the most effective VIP concentration, bovine corneoscleral explants were treated with 0 (control) and 10(-12) to 10(-6) M VIP. Paired human corneas (nine donors) from an eye bank were used as control; the other corneas were treated with VIP. To silence endogenous VIP, paired fresh human donor corneas (from seven cadavers) were transduced with VIP shRNA or the control lentiviral particles and then bisected/quartered for quantitative analysis by semiquantitative RT-PCR (for mRNA) and Western blot analysis/immunocytochemistry (for protein), whereas alizarin red S staining revealed CE cell shape. VIP concentration dependently increased bovine CE cell N-cadherin mRNA levels, with the maximal effect observed between 10(-10) (1.47 +/- 0.06-fold; P = 0.002) and 10(-8) M VIP (1.48 +/- 0.18-fold; P = 0.012). VIP (10(-8) M) treatment increased N-cadherin protein levels in bovine and human CE cells to 1.98 +/- 0.28-fold (P = 0.005) and 1.17 +/- 0.10 (range, 0.91-1.87)-fold (P = 0.050) of their respective controls. VIP antagonist (SN)VIPhyb diminished the VIP effect. VIP silencing resulted in deterioration of the hexagonal cell shape and decreased levels of VIP protein and mRNA, N-cadherin (but not connexin-43) mRNA and protein, and the antiapoptotic Bcl-2 protein. Through its autocrine VIP, CE cells play an active role in maintaining the differentiated state and suppressing apoptosis in the corneal endothelium in situ.
Perfluorooctane sulfonate (PFOS) induces reactive oxygen species (ROS) production in human microvascular endothelial cells: role in endothelial permeability.
Qian, Yong; Ducatman, Alan; Ward, Rebecca; Leonard, Steve; Bukowski, Valerie; Lan Guo, Nancy; Shi, Xianglin; Vallyathan, Val; Castranova, Vincent
Perfluorooctane sulfonate (PFOS) is a member of the perfluoroalkyl acids (PFAA) containing an eight-carbon backbone. PFOS is a man-made chemical with carbon-fluorine bonds that are among the strongest in organic chemistry, and PFOS is widely used in industry. Human occupational and environmental exposure to PFOS occurs globally. PFOS is non-biodegradable and is persistent in the human body and environment. In this study, data demonstrated that exposure of human microvascular endothelial cells (HMVEC) to PFOS induced the production of reactive oxygen species (ROS) at both high and low concentrations. Morphologically, it was found that exposure to PFOS induced actin filament remodeling and endothelial permeability changes in HMVEC. Furthermore, data demonstrated that the production of ROS plays a regulatory role in PFOS-induced actin filament remodeling and the increase in endothelial permeability. Our results indicate that the generation of ROS may play a role in PFOS-induced aberrations of the endothelial permeability barrier. The results generated from this study may provide a new insight into the potential adverse effects of PFOS exposure on humans at the cellular level.
Effects of Nebivolol on Endothelial Gene Expression during Oxidative Stress in Human Umbilical Vein Endothelial Cells
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Ulisse Garbin
Full Text Available The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L for 24 hours and oxidative stress was induced by the addition of oxidized (ox-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin; proteins linked to inflammation (IL-6 and TNFalpha, thrombotic state (tissue factor, PAI-1 and uPA, hypertension such as endothelin-1 (ET-1, and vascular remodeling such as metalloproteinases (MMP-2, MMP-9 and protease inhibitor (TIMP-1. The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.
A novel minimally-invasive method to sample human endothelial cells for molecular profiling.
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Stephen W Waldo
Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.
Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.
Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S
In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.
Low-Intensity Pulsed Ultrasound Prevents the Oxidative Stress Induced Endothelial-Mesenchymal Transition in Human Aortic Endothelial Cells
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Jiamin Li
Full Text Available Background/Aims: Endothelial-mesenchymal transition (EndMT has been shown to take part in the generation and progression of diverse diseases, involving a series of changes leading to a loss of their endothelial characteristics and an acquirement of properties typical of mesenchymal cells. Low-intensity pulsed ultrasound (LIPUS is a new therapeutic option that has been successfully used in fracture healing. However, whether LIPUS can inhibit oxidative stress-induced endothelial cell damages through inhibiting EndMT remained unknown. This study aimed to investigate the protective effects of LIPUS against oxidative stress-induced endothelial cell damages and the underlying mechanisms. Methods: EndMT was induced by H2O2 (100 µm for seven days. Human aortic endothelial cells (HAECs were exposed to H2O2 with or without LIPUS treatment for seven days. The expression of EndMT markers (CD31, VE-cadherin, FSP1 and α-SMA were analyzed. The levels of total and phosphorylated PI3K and AKT proteins were detected by Western Blot analysis. Cell chemotaxis was determined by wound healing and transwell assay. Results: LIPUS relieved EndMT by decreasing ROS accumulation and increasing activation of the PI3K signaling cascade. LIPUS alleviated the migration of EndMT-derived mesenchymal-like cells through reducing extracellular matrix (ECM deposition that is associated with matrix metallopeptidase (MMP proteolytic activity and collagen production. Conclusion: LIPUS produces cytoprotective effects against oxidative injuries to endothelial cells through suppressing the oxidative stress-induced EndMT, activating the PI3K/AKT pathway under oxidative stress, and limiting cell migration and excessive ECM deposition.
Radiation-induced inhibition of human endothelial cells replicating in culture
International Nuclear Information System (INIS)
DeGowin, R.L.; Lewis, L.J.; Mason, R.E.; Borke, M.K.; Hoak, J.C.
The radiosensitivity of some tumors may depend upon the sensitivity of their microvasculature to radiation. Heretofore, the dose-response of human endothelial cells replicating in tissue culture has not been published. In studies reported here, we exposed flasks containing 4 to 7 x 10 4 genetically identical human endothelial cells to doses of x irradiation from 125 to 1000 rad. During the phase of logarithmic growth, cell counts were compared to those of an unirradiated control to construct a dose--response curve. Similar studies were performed with normal fibroblasts. We found that 160 rad suppressed endothelial cell replication by 37 percent. Although recovery was evident with doses of 500 rad, no net increase in cell number occurred in 3 weeks in flasks of endothelial cells that received 750 or 1000 rad. Fibroblasts were slightly less sensitive under these conditions. To our knowledge, this is the first report of a radiation dose--response curve for human endothelial cells replicating in culture
Double suicide genes selectively kill human umbilical vein endothelial cells
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Liu Lunxu
Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful
Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions
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Bartels, Emil D; Nielsen, John E; Lindegaard, Marie Louise Skakkebæk
Endothelial lipase (EL) is expressed in endothelial cells, and affects plasma lipoprotein metabolism by hydrolyzing phospholipids in HDL. To determine the cellular expression of EL mRNA and protein in human atherosclerotic lesions, we performed in situ hybridization and immunohistochemical studies...
Development of a human corneal epithelium model utilizing a collagen vitrigel membrane and the changes of its barrier function induced by exposing eye irritant chemicals.
Takezawa, Toshiaki; Nishikawa, Kazunori; Wang, Pi-Chao
The brief TEER (trans-epithelial electrical resistance) assay after exposing chemicals to corneal epithelium in vivo is known as a suitable method for evaluating corneal irritancy and permeability quantitatively and continuously. A collagen vitrigel membrane we previously developed is a thin (about 20 μm thick) and transparent membrane composed of high density collagen fibrils equivalent to connective tissues in vivo, e.g. corneal Bowman's membrane. To develop such a TEER assay system in vitro utilizing a human corneal epithelial model, HCE-T cells (a human corneal epithelial cell line) were cultured on the collagen vitrigel membrane substratum prepared in a Millicell chamber suitable for TEER measurement. Human corneal epithelium model possessing 5-6 cell layers sufficient for TEER assay was successfully reconstructed on the substratum in the Millicell chamber by culturing the cells in monolayer for 2 days and subsequently in air-liquid interface for 7 days. The exposure of chemicals to the model induced the time-dependent relative changes of TEER in response to the characteristic of each chemical within a few minutes. These results suggest that the TEER assay using the human corneal epithelial model is very useful for an ocular irritancy evaluation as an alternative to the Draize eye irritation test. Copyright © 2011 Elsevier Ltd. All rights reserved.
The structural and optical properties of type III human collagen biosynthetic corneal substitutes
Hayes, Sally; Lewis, Phillip; Islam, M. Mirazul; Doutch, James; Sorensen, Thomas; White, Tomas; Griffith, May; Meek, Keith M.
The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2–9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. PMID:26159106
Human umbilical vein endothelium-derived exosomes play a role in foetoplacental endothelial dysfunction in gestational diabetes mellitus
Sáez, Tamara; Salsoso, Rocío; Leiva, Andrea; Toledo, Fernando; de Vos, Paul; Faas, Marijke; Sobrevia, Luis
Gestational diabetes mellitus (GDM) characterizes by foetoplacental endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) from women with GDM show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1). Moreover, expression of endothelial nitric
Human umbilical vein endothelium-derived exosomes play a role in foetoplacental endothelial dysfunction in gestational diabetes mellitus
Sáez, Tamara; Salsoso, Rocío; Leiva, Andrea; Toledo, Fernando; de Vos, Paul; Faas, Marijke; Sobrevia, Luis
Gestational diabetes mellitus (GDM) characterizes by foetoplacental endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) from women with GDM show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1). Moreover, expression of endothelial nitric
Vps35-deficiency impairs SLC4A11 trafficking and promotes corneal dystrophy.
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Wei Liu
Full Text Available Vps35 (vacuolar protein sorting 35 is a major component of retromer that selectively promotes endosome-to-Golgi retrieval of transmembrane proteins. Dysfunction of retromer is a risk factor for the pathogenesis of Parkinson's disease (PD and Alzheimer's disease (AD. However, Vps35/retromer's function in the eye or the contribution of Vps35-deficiency to eye degenerative disorders remains to be explored. Here we provide evidence for a critical role of Vps35 in mouse corneal dystrophy. Vps35 is expressed in mouse and human cornea. Mouse cornea from Vps35 heterozygotes (Vps35+/- show features of dystrophy, such as loss of both endothelial and epithelial cell densities, disorganizations of endothelial, stroma, and epithelial cells, excrescences in the Descemet membrane, and corneal edema. Additionally, corneal epithelial cell proliferation was reduced in Vps35-deficient mice. Intriguingly, cell surface targeting of SLC4A11, a membrane transport protein (OH- /H+ /NH3 /H2O of corneal endothelium, whose mutations have been identified in patients with corneal dystrophy, was impaired in Vps35-deficient cells and cornea. Taken together, these results suggest that SLC4A11 appears to be a Vps35/retromer cargo, and Vps35-regulation of SLC4A11 trafficking may underlie Vps35/retromer regulation of corneal dystrophy.
A SAGE based approach to human glomerular endothelium : defining the transcriptome, finding a novel molecule and highlighting endothelial diversity
Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere
BACKGROUND: Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis
Microtubules Growth Rate Alteration in Human Endothelial Cells
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Irina B. Alieva
Full Text Available To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with "normal" (similar to those in monolayer EC and "fast" (three times as much growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.
SNEV overexpression extends the life span of human endothelial cells
International Nuclear Information System (INIS)
Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes
In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells
Endothelial Dysfunction in Human Diabetes Is Mediated by Wnt5a-JNK Signaling.
Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G; Fetterman, Jessica L; Linder, Erika A; Berk, Brittany D; Masaki, Nobuyuki; Weisbrod, Robert M; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J; Walsh, Kenneth; Hamburg, Naomi M
Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus. © 2016 American Heart
Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components
Qu, Mingli; Wang, Yao; Yang, Lingling; Zhou, Qingjun
Purpose To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. Methods The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were...
In vivo high resolution human corneal imaging using full-field optical coherence tomography.
Mazlin, Viacheslav; Xiao, Peng; Dalimier, Eugénie; Grieve, Kate; Irsch, Kristina; Sahel, José-Alain; Fink, Mathias; Boccara, A Claude
We present the first full-field optical coherence tomography (FFOCT) device capable of in vivo imaging of the human cornea. We obtained images of the epithelial structures, Bowman's layer, sub-basal nerve plexus (SNP), anterior and posterior stromal keratocytes, stromal nerves, Descemet's membrane and endothelial cells with visible nuclei. Images were acquired with a high lateral resolution of 1.7 µm and relatively large field-of-view of 1.26 mm x 1.26 mm - a combination, which, to the best of our knowledge, has not been possible with other in vivo human eye imaging methods. The latter together with a contactless operation, make FFOCT a promising candidate for becoming a new tool in ophthalmic diagnostics.
Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells
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Thilo, Florian; Vorderwülbecke, Bernd J; Marki, Alex
in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis......The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined...
Caveolin-1 as a Novel Indicator of Wound-Healing Capacity in Aged Human Corneal Epithelium
Rhim, Ji Heon; Kim, Jae Hoon; Yeo, Eui-Ju; Kim, Jae Chan; Park, Sang Chul
Excess caveolin-1 has been reported to play a role in age-dependent hyporesponsiveness to growth factors in vitro. Therefore, we hypothesized that caveolin-1–dependent hyporesponsiveness to growth factors in aged corneal epithelial cells might be responsible for delayed wound healing in vivo. To test this hypothesis, we evaluated corneal wound-healing time by vital staining using fluorescein after laser epithelial keratomileusis (LASEK). We compared wound-healing times in young, middle-aged a...
Comparison of the effects of ophthalmic solutions on human corneal epithelial cells using fluorescent dyes.
Xu, Manlong; Sivak, Jacob G; McCanna, David J
To investigate the effect of differently preserved ophthalmic solutions on the viability and barrier function of human corneal epithelial cells (HCEC) using fluorescent dyes. HCEC monolayers were exposed to the ophthalmic solutions containing benzalkonium chloride (BAK), edetate disodium, polyquad, stabilized oxychloro complex (Purite), sodium perborate, or sorbic acid for 5 min, 15 min, and 1 h. At 24 h after exposure, the cultures were assessed for metabolic activity using alamarBlue. The enzyme activity, membrane integrity, and apoptosis were evaluated using confocal microscopy. Barrier function was assessed using sodium fluorescein. The metabolic assay showed that the BAK-preserved ophthalmic solutions significantly reduced cell viability after a 5-min exposure compared to the phosphate buffered saline treated control (POphthalmic solutions with new preservatives had varying time-dependent adverse effects on cell viability, and the preservative-free solution had the least effect on HCEC. Sodium fluorescein permeability showed that HCEC monolayers treated with BAK-preserved solutions were more permeable to sodium fluorescein than those treated by the other ophthalmic solutions (Psolutions had greater adverse effects on metabolic activity, enzyme activity, membrane integrity, cell viability, and barrier function than the solutions that were not preserved with BAK. Our study suggests that BAK-free especially, preservative-free ophthalmic solutions are safer alternatives to BAK-preserved ones.
Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells
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Yélian Marc Bossou
Full Text Available Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B1 (AFB1 and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB1 but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity.
Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2.
Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X
Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappaB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappaB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1), antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules.
Sensing inhomogeneous mechanical properties of human corneal Descemet's membrane with AFM nano-indentation.
Di Mundo, Rosa; Recchia, Giuseppina; Parekh, Mohit; Ruzza, Alessandro; Ferrari, Stefano; Carbone, Giuseppe
The paper describes a highly space-resolved characterization of the surface mechanical properties of the posterior human corneal layer (Descemet's membrane). This has been accomplished with Atomic Force Microscopy (AFM) nano-indentation by using a probe with a sharp tip geometry. Results indicate that the contact with this biological tissue in liquid occurs with no (or very low) adhesion. More importantly, under the same operating conditions, a broad distribution of penetration depth can be measured on different x-y positions of the tissue surface, indicating a high inhomogeneity of surface stiffness, not yet clearly reported in the literature. An important contribution to such inhomogeneity should be ascribed to the discontinuous nature of the collagen/proteoglycans fibers matrix tissue, as can be imaged by AFM when the tissue is semi-dry. Using classical contact mechanics calculations adapted to the specific geometry of the tetrahedral tip it has been found that the elastic modulus E of the material in the very proximity of the surface ranges from 0.23 to 2.6 kPa. Copyright © 2017 Elsevier Ltd. All rights reserved.
Corneal biomechanical properties from air-puff corneal deformation imaging
Marcos, Susana; Kling, Sabine; Bekesi, Nandor; Dorronsoro, Carlos
The combination of air-puff systems with real-time corneal imaging (i.e. Optical Coherence Tomography (OCT), or Scheimpflug) is a promising approach to assess the dynamic biomechanical properties of the corneal tissue in vivo. In this study we present an experimental system which, together with finite element modeling, allows measurements of corneal biomechanical properties from corneal deformation imaging, both ex vivo and in vivo. A spectral OCT instrument combined with an air puff from a non-contact tonometer in a non-collinear configuration was used to image the corneal deformation over full corneal cross-sections, as well as to obtain high speed measurements of the temporal deformation of the corneal apex. Quantitative analysis allows direct extraction of several deformation parameters, such as apex indentation across time, maximal indentation depth, temporal symmetry and peak distance at maximal deformation. The potential of the technique is demonstrated and compared to air-puff imaging with Scheimpflug. Measurements ex vivo were performed on 14 freshly enucleated porcine eyes and five human donor eyes. Measurements in vivo were performed on nine human eyes. Corneal deformation was studied as a function of Intraocular Pressure (IOP, 15-45 mmHg), dehydration, changes in corneal rigidity (produced by UV corneal cross-linking, CXL), and different boundary conditions (sclera, ocular muscles). Geometrical deformation parameters were used as input for inverse finite element simulation to retrieve the corneal dynamic elastic and viscoelastic parameters. Temporal and spatial deformation profiles were very sensitive to the IOP. CXL produced a significant reduction of the cornea indentation (1.41x), and a change in the temporal symmetry of the corneal deformation profile (1.65x), indicating a change in the viscoelastic properties with treatment. Combining air-puff with dynamic imaging and finite element modeling allows characterizing the corneal biomechanics in-vivo.
Human endothelial progenitor cells internalize high-density lipoprotein.
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Kaemisa Srisen
Full Text Available Endothelial progenitor cells (EPCs originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL, and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate, cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal
Caveolin-1 as a novel indicator of wound-healing capacity in aged human corneal epithelium.
Rhim, Ji Heon; Kim, Jae Hoon; Yeo, Eui-Ju; Kim, Jae Chan; Park, Sang Chul
Excess caveolin-1 has been reported to play a role in age-dependent hyporesponsiveness to growth factors in vitro. Therefore, we hypothesized that caveolin-1-dependent hyporesponsiveness to growth factors in aged corneal epithelial cells might be responsible for delayed wound healing in vivo. To test this hypothesis, we evaluated corneal wound-healing time by vital staining using fluorescein after laser epithelial keratomileusis (LASEK). We compared wound-healing times in young, middle-aged and elderly patients. We also examined caveolin-1 levels and other aging markers, such as p53 and p21, in the corneal epithelium. Elderly patients generally had higher caveolin-1 levels in the corneal epithelia than young patients. There were, however, variations among individuals with increased caveolin-1 in some young patients and decreased levels in some elderly patients. Wound-healing time after LASEK correlated well with the corneal caveolin-1 status. Therefore, we suggest that caveolin-1 status might be responsible for delayed wound healing in elderly patients after LASEK. Caveolin-1 status might be a regulator for wound-healing capacity and a novel target for in vivo adjustment.
Collagen VII deficient mice show morphologic and histologic corneal changes that phenotypically mimic human dystrophic epidermolysis bullosa of the eye.
Chen, Vicki M; Shelke, Rajani; Nyström, Alexander; Laver, Nora; Sampson, James F; Zhiyi, Cao; Bhat, Najma; Panjwani, Noorjahan
Absence of collagen VII causes blistering of the skin, eyes and many other tissues. This disease is termed dystrophic epidermolysis bullosa (DEB). Corneal fibrosis occurs in up to 41% and vision loss in up to 64% of patients. Standard treatments are supportive and there is no cure. The immune-histologic and morphologic changes in the corneas of the mouse model for this disease have not been described in the literature. Our purpose is to characterize the eyes of these mice to determine if this is an appropriate model for study of human therapeutics. Western blot analysis (WB) and immunohistochemistry (IHC) were performed to assess the relative collagen VII protein levels and its location within the cornea. Additional IHC for inflammatory and fibrotic biomarkers alpha-smooth muscle actin (α-SMA), transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), proteinase 3, tenascin C and collagen III were performed. Clinical photographs documenting opacification of the corneas of animals of differing ages were assessed and scored independently by 2 examiners. Histology was then used to investigate morphologic changes. IHC and WB confirmed that these mice are deficient in collagen VII production at the level of the basement membrane when compared with wild-types. IHC showed anomalous deposition of collagen III throughout the stroma. Of the 5 biomarkers tested, TGF-β showed the strongest and most consistently staining. Photographs documented corneal opacities only in mice older than 10 weeks, opacities were not seen in younger animals. Histology showed multiple abnormalities, including epithelial hyperplasia, ulceration, fibrosis, edema, dysplasia, neovascularization and bullae formation. The collagen VII hypomorphic mouse shows reduced collagen VII production at the level of the corneal basement membrane. Corneal changes are similar to pathology seen in humans with this disease. The presence of anomalous stromal collagen III and TGF-β appear to be
A nanoengineered peptidic delivery system with specificity for human brain capillary endothelial cells
DEFF Research Database (Denmark)
Wu, Linping; Moghimi, Seyed Moein
, without manipulating the integrity of the BBB. This may be achieved by simultaneous and appropriate nanoparticle surface decoration with polymers that protect nanoparticles against rapid interception by body's defenses and ligands specific for cerebral capillary endothelial cells. To date, the binding...... avidity of the majority of the so-called 'brain-specific' nanoparticles to the brain capillary endothelial cells has been poor, even during in vitro conditions. We have addressed this issue and designed a versatile peptidic nanoplatform with high binding avidity to the human cerebral capillary endothelial...... cells. This was achieved by selecting an appropriate phage-derived peptide with high specificity for human brain capillary endothelial cells, which following careful structural modifications spontaneously formed a nanoparticle-fiber network. The peptidic network was characterized fully and its uptake...
An ibuprofen-antagonized plasmin inhibitor released by human endothelial cells.
Rockwell, W B; Ehrlich, H P
Serum-free culture medium harvested from endothelial cell monolayer cultures derived from human scars and dermis was examined for inhibition of fibrinolysis using a fibrin plate assay. Human cultured fibroblasts and smooth muscle cells did not produce any detectable inhibitory activity. The inhibitor is spontaneously released from the cultured endothelial cells over time. In the fibrin plate assay of plasmin-induced fibrinolysis, one nonsteroidal antiinflammatory (NSAI) drug, ibuprofen, was demonstrated to antagonize the inhibition of fibrinolysis. The antagonistic activity of ibuprofen appears unrelated to its NSAI drug activity because other NSAI drugs such as indomethacin and tolmetin have minimal antagonistic activity. Heating the cultured endothelial cells to 42 degrees C stimulates greater release of the inhibitor in a shorter period of time. This plasmin inhibitor, which is produced by endothelial cells, may contribute to postburn vascular occlusion, leading to secondary progressive necrosis in burn-traumatized patients.
A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.
Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R
Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.
BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma.
Williams, Christopher S; Zhang, Baolin; Smith, J Joshua; Jayagopal, Ashwath; Barrett, Caitlyn W; Pino, Christopher; Russ, Patricia; Presley, Sai H; Peng, DunFa; Rosenblatt, Daniel O; Haselton, Frederick R; Yang, Jin-Long; Washington, M Kay; Chen, Xi; Eschrich, Steven; Yeatman, Timothy J; El-Rifai, Wael; Beauchamp, R Daniel; Chang, Min S
The acquisition of a mesenchymal phenotype is a critical step in the metastatic progression of epithelial carcinomas. Adherens junctions (AJs) are required for suppressing this epithelial-mesenchymal transition (EMT) but less is known about the role of tight junctions (TJs) in this process. Here, we investigated the functions of blood vessel epicardial substance (BVES, also known as POPDC1 and POP1), an integral membrane protein that regulates TJ formation. BVES was found to be underexpressed in all stages of human colorectal carcinoma (CRC) and in adenomatous polyps, indicating its suppression occurs early in transformation. Similarly, the majority of CRC cell lines tested exhibited decreased BVES expression and promoter DNA hypermethylation, a modification associated with transcriptional silencing. Treatment with a DNA-demethylating agent restored BVES expression in CRC cell lines, indicating that methylation represses BVES expression. Reexpression of BVES in CRC cell lines promoted an epithelial phenotype, featuring decreased proliferation, migration, invasion, and anchorage-independent growth; impaired growth of an orthotopic xenograft; and blocked metastasis. Conversely, interfering with BVES function by expressing a dominant-negative mutant in human corneal epithelial cells induced mesenchymal features. These biological outcomes were associated with changes in AJ and TJ composition and related signaling. Therefore, BVES prevents EMT, and its epigenetic silencing may be an important step in promoting EMT programs during colon carcinogenesis.
Full Text Available ... Tips & Prevention News Ask an Ophthalmologist Patient Stories Español Eye Health / Eye Health A-Z Corneal Laceration ... Laceration Treatment What Is Corneal Laceration? Leer en Español: ¿Qué es una laceración de la córnea? Written ...
DEFF Research Database (Denmark)
Hjortdal, Jesper Østergaard
with less risk of rejection episodes. Besides covering updated chapters on penetrating keratoplasty, and anterior and posterior lamellar procedures, this textbook also gives a thorough overview of the history of corneal transplantation and a detailed presentation of the microstructural components...... and to assist fellows and corneal surgeons in their advice and selection of patients for the best surgical procedure considering benefi ts and risks....
Progress in corneal wound healing
Ljubimov, Alexander V.; Saghizadeh, Mehrnoosh
Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and
Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae
International Nuclear Information System (INIS)
Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.
Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51 Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51 Cr release from radiolabeled monolayers
The high-risk corneal regraft model: a justification for tissue matching in humans
Czech Academy of Sciences Publication Activity Database
Vitova, A.; Kuffova, L.; Klaska, I.; Holáň, Vladimír; Cornall, R.J.; Forrester, J.V.
Roč. 26, č. 4 (2013), s. 453-461 ISSN 0934-0874 Institutional support: RVO:68378050 Keywords : accelerated rejection * corneal transplantation * dendritic cell s * regraft * T- cell memory * transgenic mouse Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.120, year: 2013
Breakdown evaluation of corneal epithelial barrier caused by antiallergic eyedrops using an electrophysiologic method.
Nakashima, Mikiro; Nakamura, Tadahiro; Teshima, Mugen; To, Hideto; Uematsu, Masafumi; Kitaoka, Takashi; Taniyama, Kotaro; Nishida, Koyo; Nakamura, Junzo; Sasaki, Hitoshi
The aim of this study was to examine the usefulness of an electrophysiologic method for predicting corneal epithelial breakdown by antiallergic eyedrops and comparing the results with those in other appraisal methods. Six kinds of antiallergic eyedrops, including benzalkonium chloride (BK) as an ophthalmic preservative and two kinds of BK-free antiallergic eyedrops, were used in this study. Eyedrops were applied to excise rabbit corneas and monitoring was performed according to an electrophysiologic method, using a commercially available chamber system to mimic human tear turnover. Changes in transepithelial electrical resistance (TEER) in the corneal surface were recorded. The cytotoxicity of each kind of eyedrops in a normal rabbit corneal epithelial (NRCE) cell line and a human endothelial cell line EA.hy926 was also examined. The extent of decrease in the corneal TEER after applying antiallergic eyedrops was dependent on the concentration of the BK included as a preservative, but it was also affected by the different kinds of drugs when the BK concentration was low. Higher cytotoxicity of the eyedrops against the NRCE and EA.hy926 cell lines was observed with a reduction of TEER. Monitoring changes in the corneal TEER, according to the electrophysiologic method with the application of antiallergic eyedrops, is useful for predicting corneal epithelial breakdown caused by their instillation.
Cellular toxicity of calf blood extract on human corneal epithelial cells in vitro.
Park, Young Min; Kim, Su Jin; Han, Young Sang; Lee, Jong Soo
To investigate the biologic effects of the calf blood extract on corneal epithelial cells in vitro. The effects on corneal epithelial cells were evaluated after 1, 4, 12, and 24 h of exposure to various concentrations of calf blood extract (3, 5, 8 and 16%). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was performed to measure levels of cellular metabolic activity. The lactate dehydrogenase (LDH) assay was performed to determine the extent of cellular damage. Cellular morphology was examined using phase-contrast microscopy. The scratch wound assay was performed to quantify the migration of corneal epithelial cells. At the 3 and 5% concentrations of calf blood extract, MTT values were similar to those observed in the control group. However, at a concentration of 8 and 16%, cellular metabolic activity was significantly decreased after 4 h of exposure to calf blood extract. After 12 h of exposure to 8 and 16% concentrations of calf blood extract, LDH activity and cellular morphological damage to the corneal epithelial cells were significantly increased. There was no evidence of cellular migration after 12 h exposure to 5% or higher concentration of calf blood extract because of cellular toxicity. Compared with normal corneal epithelial cells, the cellular activity was decreased, and toxicity was increased after over 12 h of exposure to more than 5% concentration of calf blood extract. Further clinical studies will be necessary to determine the optimal concentration and exposure time for the topical application of eye drops containing calf blood extract.
Clinical applications of corneal confocal microscopy
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Mitra Tavakoli
Full Text Available Mitra Tavakoli1, Parwez Hossain2, Rayaz A Malik11Division of Cardiovascular Medicine, University of Manchester and Manchester Royal Infirmary, Manchester, UK; 2University of Southampton, Southampton Eye Unit, Southampton General Hospital, Southampton, UKAbstract: Corneal confocal microscopy is a novel clinical technique for the study of corneal cellular structure. It provides images which are comparable to in-vitro histochemical techniques delineating corneal epithelium, Bowman's layer, stroma, Descemet's membrane and the corneal endothelium. Because, corneal confocal microscopy is a non invasive technique for in vivo imaging of the living cornea it has huge clinical potential to investigate numerous corneal diseases. Thus far it has been used in the detection and management of pathologic and infectious conditions, corneal dystrophies and ecstasies, monitoring contact lens induced corneal changes and for pre and post surgical evaluation (PRK, LASIK and LASEK, flap evaluations and Radial Keratotomy, and penetrating keratoplasty. Most recently it has been used as a surrogate for peripheral nerve damage in a variety of peripheral neuropathies and may have potential in acting as a surrogate marker for endothelial abnormalities.Keywords: corneal confocal microscopy, cornea, infective keratitis, corneal dystrophy, neuropathy
A new 3D reconstituted human corneal epithelium model as an alternative method for the eye irritation test.
Jung, Kyoung-Mi; Lee, Su-Hyon; Ryu, Yang-Hwan; Jang, Won-Hee; Jung, Haeng-Sun; Han, Ju-Hee; Seok, Seung-Hyeok; Park, Jae-Hak; Son, Youngsook; Park, Young-Ho; Lim, Kyung-Min
Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. Here we report a new reconstructed human corneal epithelial model (MCTT HCE model) prepared from primary-cultured human limbal epithelial cells as a new alternative in vitro eye irritation test method. In histological and immunohistochemical observation, MCTT HCE model displayed a morphology and biomarker expressions similar to intact human cornea. Moreover, the barrier function was well preserved as measured by high transepithelial electrical resistance, effective time-50 for Triton X-100, and corneal thickness. To employ the model as a new alternative method for eye irritation test, protocol refinement was performed and optimum assay condition was determined including treatment time, treatment volume, post-incubation time and rinsing method. Using the refined protocol, 25 reference chemicals with known eye irritation potentials were tested. With the viability cut-off value at 50%, chemicals were classified to irritant or non-irritant. When compared with GHS classification, the MCTT HCE model showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results suggest that the MCTT HCE model might be useful as a new alternative eye irritation test method. Copyright © 2010 Elsevier Ltd. All rights reserved.
Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.
Yamamoto, Naoki; Kato, Yoshinao; Sato, Atsushi; Hiramatsu, Noriko; Yamashita, Hiromi; Ohkuma, Mahito; Miyachi, Ei-Ichi; Horiguchi, Masayuki; Hirano, Koji; Kojima, Hajime
In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.
Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA
DEFF Research Database (Denmark)
Barnathan, E S; Kuo, A; Karikó, K
Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence...
Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells.
Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X
To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces
Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa–Infected Human Corneal Epithelial Cells
Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X.
PURPOSE To determine the role of epidermal growth factor (EGF) receptor (EGFR)–mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa–infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase–mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis
Interleukin 1 is an autocrine regulator of human endothelial cell growth
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Cozzolino, F.; Torcia, M.; Aldinucci, D.; Ziche, M.; Bani, D.; Almerigogna, F.; Stern, D.M.
Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, the authors demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G 1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth the suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells
Protein Kinase-C Beta Contributes to Impaired Endothelial Insulin Signaling in Humans with Diabetes Mellitus
Tabit, Corey E; Shenouda, Sherene M; Holbrook, Monica; Fetterman, Jessica L; Kiani, Soroosh; Frame, Alissa A; Kluge, Matthew A; Held, Aaron; Dohadwala, Mustali; Gokce, Noyan; Farb, Melissa; Rosenzweig, James; Ruderman, Neil; Vita, Joseph A; Hamburg, Naomi M
Background Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition. Conclusions We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance. PMID:23204109
Full Text Available ... by something sharp flying into the eye. It can also be caused by something striking the eye ... If the corneal laceration is deep enough it can cause a full thickness laceration. This is when ...
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Corneal densitometry and its correlation with age, pachymetry, corneal curvature, and refraction.
Garzón, Nuria; Poyales, Francisco; Illarramendi, Igor; Mendicute, Javier; Jáñez, Óscar; Caro, Pedro; López, Alfredo; Argüeso, Francisco
To determine normative corneal densitometry values in relation to age, sex, refractive error, corneal thickness, and keratometry, measured using the Oculus Pentacam system. Three hundred and thirty-eight healthy subjects (185 men; 153 women) with no corneal disease underwent an exhaustive ocular examination. Corneal densitometry was expressed in standardized grayscale units (GSU). The mean corneal densitometry over the total area was 16.46 ± 1.85 GSU. The Pearson correlation coefficient for total densitometry was r = 0.542 (p 0.05). This is the first report of normative corneal densitometry values in relation to keratometry, corneal thickness, and spherical equivalent measured with the latest Oculus Pentacam software. Corneal densitometry increases with age, but corneal keratometry and refractive parameters do not affect light scattering in the human cornea.
Deposition of cellular fibronectin and desorption of human serum albumin during adhesion and spreading of human endothelial cells on polymers
Dekker, A.; Dekker, A.; Beugeling, T.; Beugeling, T.; Wind, H.; Poot, Andreas A.; Bantjes, A.; Bantjes, A.; Feijen, Jan; van Aken, W.G.
More insight into the mechanism of adhesion of human endothelial cells (HEC) on to polymeric surfaces may lead to the development of improved small-diameter vascular grafts. HEC suspended in 20% human serum-containing culture medium adhere and spread well on moderately water-wettable polymers such
Cyclooxygenase inhibition improves endothelial vasomotor dysfunction of visceral adipose arterioles in human obesity
Farb, Melissa G.; Tiwari, Stephanie; Karki, Shakun; Ngo, Doan TM; Carmine, Brian; Hess, Donald T.; Zuriaga, Maria A.; Walsh, Kenneth; Fetterman, Jessica L.; Hamburg, Naomi M.; Vita, Joseph A.; Apovian, Caroline M.; Gokce, Noyan
Objective The purpose of this study was to determine whether cyclooxygenase inhibition improves vascular dysfunction of adipose microvessels from obese humans. Design and Methods In 20 obese subjects (age 37±12 yrs, BMI 47±8 kg/m2) we collected subcutaneous and visceral fat during bariatric surgery and characterized adipose depot-specific gene expression, endothelial cell phenotype, and microvascular function. Vasomotor function was assessed in response to endothelium-dependent agonists using videomicroscopy of small arterioles from fat. Results Arterioles from visceral fat exhibited impaired endothelium-dependent, acetylcholine-mediated vasodilation, compared to the subcutaneous depot (p<0.001). Expression of mRNA transcripts relevant to the cyclooxygenase pathway were upregulated in visceral compared to subcutaneous fat. Pharmacological inhibition of cyclooxygenase with indomethacin improved endothelium-dependent vasodilator function of arterioles from visceral fat by 2-fold (p=0.01), whereas indomethacin had no effect in the subcutaneous depot. Indomethacin increased activation via serine-1177 phosphorylation of endothelial nitric oxide synthase in response to acetylcholine in endothelial cells from visceral fat. Inhibition of endothelial nitric oxide synthase with Nω-nitro-L-arginine methyl ester abrogated the effects of cyclooxygenase-inhibition suggesting that vascular actions of indomethacin were related to increased nitric oxide bioavailability. Conclusions Our findings suggest that cyclooxygenase-mediated vasoconstrictor prostanoids partly contribute to endothelial dysfunction of visceral adipose arterioles in human obesity. PMID:23640904
Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration
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Shumei Man
Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.
Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells
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Pan, Hong; Wu, Xinyi
Highlights: ► Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-β. ► Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. ► Hypoxia inhibits Acanthamoeba-induced the activation of NF-κB and ERK1/2 in HCECs. ► Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. ► LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion
Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles
van Leeuwen, E B; van der Veen, A Y; Hoekstra, D; Engberts, J B; Halie, M R; van der Meer, J; Ruiters, M H
OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a
Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP
Effect of apple extracts on NF-kappaB activation in human umbilical vein endothelial cells
Czech Academy of Sciences Publication Activity Database
Davis, P.A.; Polagruto, J.A.; Valacchi, G.; Phung, A.; Souček, Karel; Keen, C.L.; Gershwin, M.E.
Roč. 231, č. 5 (2006), s. 594-598 ISSN 1535-3702 Institutional research plan: CEZ:AV0Z50040507 Keywords : human umbilical vascular endothelial cells * NF-kappaB * antioxidants Subject RIV: BO - Biophysics Impact factor: 2.845, year: 2006
Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge
van Wachem, P.B.; Hogt, A.H.; Beugeling, T.; Feijen, Jan; Bantjes, A.; Detmers, J.P.; van Aken, W.G.
The adhesion of human endothelial cells (HEC) onto a series of well-characterized methacrylate polymer surfaces with varying wettabilities and surface charges was studied either in serum-containing (CMS) or in serum-free (CM) culture medium. HEC adhesion in CMS onto (co)polymers * of hydroxyethyl
Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿
Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed
Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment.
Alteration of human umbilical vein endothelial cell gene expression in different biomechanical environments.
Shoajei, Shahrokh; Tafazzoli-Shahdpour, Mohammad; Shokrgozar, Mohammad Ali; Haghighipour, Nooshin
Biomechanical environments affect the function of cells. In this study we analysed the effects of five mechanical stimuli on the gene expression of human umbilical vein endothelial cells (HUVECs) in mRNA level using real-time PCR. The following loading regimes were applied on HUVECs for 48 h: intermittent (0-5 dyn/cm(2) , 1 Hz) and uniform (5 dyn/cm(2) ) shear stresses concomitant by 10% intermittent equiaxial stretch (1 Hz), uniform shear stress alone (5 dyn/cm(2) ), and intermittent uniaxial and equiaxial stretches (10%, 1 Hz). A new bioreactor was made to apply uniform/cyclic shear and tensile loadings. Three endothelial suggestive specific genes (vascular endothelial growth factor receptor-2 (VEGFR-2, also known as FLK-1), von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin)), and two smooth muscle genes (α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC)) were chosen for assessment of alteration in gene expression of endothelial cells and transdifferentiation toward smooth cells following load applications. Shear stress alone enhanced the endothelial gene expression significantly, while stretching alone was identified as a transdifferentiating factor. Cyclic equiaxial stretch contributed less to elevation of smooth muscle genes compared to uniaxial stretch. Cyclic shear stress in comparison to uniform shear stress concurrent with cyclic stretch was more influential on promotion of endothelial genes expression. Influence of different mechanical stimuli on gene expression may open a wider horizon to regulate functions of cell for tissue engineering purposes. © 2013 International Federation for Cell Biology.
Molecular mechanism of ocular surface damage: application to an in vitro dry eye model on human corneal epithelium.
Meloni, Marisa; De Servi, Barbara; Marasco, Daniela; Del Prete, Salvatore
The present study was concerned with the development of a new experimental model of dry eye using human reconstructed in vitro corneal epithelium (HCE). The model is based on the use of adapted culture conditions that induce relevant modifications at the cellular and molecular level thus mimicking dry eye. The HCE model was maintained in a controlled environmental setting (relative humidity eye. The evolution of the dry eye condition was assessed by histology, immunohistochemistry staining, scanning electron microscopy, and gene expression by using TaqMan gene assay technology (mucin-4 [MUC4], matrix metallopeptidase-9 [MMP9], tumor necrosis factor-α [TNF-α], and defensin β-2 [DEFB2). The effects of different commercially available tear substitutes on the induced dry eye condition were tested. This in vitro dry eye HCE model, that was well established within 24 h, has the characteristic features of a dry eye epithelium and could be satisfactorily used for preliminary assessment of the protective activity of some artificial tears. The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression. By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears.
The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.
Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J
Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.
Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells
Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu
Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104
Vasodilatory effect and endothelial integrity in papaverine- and milrinone-treated human radial arteries.
Rudzinski, P; Wegrzyn, P; Lis, G J; Piatek, J; Konstanty-Kalandyk, J; Nosalski, R; Mikolajczyk, T; Jasinska, M; Pyka-Fosciak, G; Guzik, T; Litwin, J A; Korbut, R; Sadowski, J
Prevention of the vasospasm is an important aspect of coronary artery bypass grafting (CABG) with the use of radial artery (RA) as the conduit. We compared the effect of two phosphodiesterase inhibitors papaverine and milrinone on vasodilation and endothelial integrity of human RA segments harvested from 20 CABG patients. Vasodilatory effect of the drugs were assessed by organ bath technique in RA rings precontracted with KCl and phenylephrine. Endothelial integrity was evaluated by CD34 immunofluorescence in frozen sections. Vasorelaxation induced by papaverine was significantly greater as compared to that induced by milrinone (90.47% ± 10.16% vs. 78.98% ± 19.56%, pmilrinone in the preservation of endothelial integrity (75.3% ± 12.9% vs. 51.8% ± 18.0%, pmilrinone for prevention of vasospasm in radial artery conduits used for CABG.
Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis
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Zhou Yijun; Wang Jiahe; Zhang Jin
Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-κB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-κB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease
Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture
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Ruth Olmer
Full Text Available Summary: Endothelial cells (ECs are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability. : In this article, U. Martin and colleagues show the generation of hiPSC endothelial cells in scalable cultures in up to 100 mL culture volume. The generated ECs show in vitro proliferation capacity and a high degree of chromosomal stability after in vitro expansion. The established protocol allows to generate hiPSC-derived ECs in relevant numbers for regenerative approaches. Keywords: hiPSC differentiation, endothelial cells, scalable culture
Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin
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Bar, R.S.; Dake, B.L.; Spanheimer, R.G.
The ( 35 S)glycosaminoglycans (( 35 S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with 35 SO 4 for 72 h, the ( 35 S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of ( 35 S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of ( 35 S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author)
Sulfated glycosaminoglycans in cultured endothelial cells from capillaries and large vessels of human and bovine origin
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Bar, R.S.; Dake, B.L.; Spanheimer, R.G.
The (/sup 35/S)glycosaminoglycans ((/sup 35/S)GAG) synthesized by capillary endothelial cells were analyzed and compared to GAG synthesized by endothelial cells cultured from 4 larger vessels. Two separate cultures of endothelial cells were established from bovine fat capillaries and from 4 larger vessels of human origin (umbilical vein) and bovine origin (pulmonary artery, pulmonary vein and aorta). After incubation with /sup 35/SO/sub 4/ for 72 h, the (/sup 35/S)glycosaminoglycans (GAG) composition of the media, pericellular and cellular fractions of each culture were determined by selective degradation with nitrous acid, chondroitinase ABC and chondroitinase AC. All endothelial cells produced large amounts of (/sup 35/S)GAG with increased proportions of heparinoids (heparan sulfate and heparin) in the cellular and pericellular fractions. Each culture showed a distinct distribution of (/sup 35/S)GAG in the media, pericellular and cellular fractions with several specific differences found among the 5 cultures. The differences in GAG content were confirmed in a second group of separate cultures from each of the 5 vessels indicating that, although having several features of GAG metabolism in common, each endothelial cell culture demonstrated a characteristic complement of synthesized, secreted and cell surface-sulfated glycosaminoglycans. (author). 16 refs.
Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells
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Wu Hualin; Lin ChiIou; Huang Yuanli; Chen, Pin-Shern; Kuo, Cheng-Hsiang; Chen, Mei-Shing; Wu, G.C.-C.; Shi, G.-Y.; Yang, H.-Y.; Lee Hsinyu
Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation
Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane.
Rohaina, Che Man; Then, Kong Yong; Ng, Angela Min Hwei; Wan Abdul Halim, Wan Haslina; Zahidin, Aida Zairani Mohd; Saim, Aminuddin; Idrus, Ruszymah B H
The cornea can be damaged by a variety of clinical disorders or chemical, mechanical, and thermal injuries. The objectives of this study were to induce bone marrow mesenchymal stem cells (BMSCs) to corneal lineage, to form a tissue engineered corneal substitute (TEC) using BMSCs, and to treat corneal surface defects in a limbal stem cell deficiency model. BMSCs were induced to corneal lineage using limbal medium for 10 days. Induced BMSCs demonstrated upregulation of corneal stem cell markers; β1-integrin, C/EBPδ, ABCG2, and p63, increased protein expression of CK3 and p63 significantly compared with the uninduced ones. For TEC formation, passage 1 BMSCs were trypsinized and seeded on amniotic membrane in a transwell co-culture system and were grown in limbal medium. Limbal stem cell deficiency models were induced by alkaline injury, and the TEC was implanted for 8 weeks. Serial slit lamp evaluation revealed remarkable improvement in corneal regeneration in terms of corneal clarity and reduced vascularization. Histologic and optical coherence tomography analyses demonstrated comparable corneal thickness and achieved stratified epithelium with a compact stromal layer resembling that of normal cornea. CK3 and p63 were expressed in the newly regenerated cornea. In conclusion, BMSCs can be induced into corneal epithelial lineage, and these cells are viable for the formation of TEC, to be used for the reconstruction of the corneal surface in the limbal stem cell deficient model. Copyright © 2014 Mosby, Inc. All rights reserved.
Correlations between corneal and total wavefront aberrations
Mrochen, Michael; Jankov, Mirko; Bueeler, Michael; Seiler, Theo
Purpose: Corneal topography data expressed as corneal aberrations are frequently used to report corneal laser surgery results. However, the optical image quality at the retina depends on all optical elements of the eye such as the human lens. Thus, the aim of this study was to investigate the correlations between the corneal and total wavefront aberrations and to discuss the importance of corneal aberrations for representing corneal laser surgery results. Methods: Thirty three eyes of 22 myopic subjects were measured with a corneal topography system and a Tschernig-type wavefront analyzer after the pupils were dilated to at least 6 mm in diameter. All measurements were centered with respect to the line of sight. Corneal and total wavefront aberrations were calculated up to the 6th Zernike order in the same reference plane. Results: Statistically significant correlations (p the corneal and total wavefront aberrations were found for the astigmatism (C3,C5) and all 3rd Zernike order coefficients such as coma (C7,C8). No statistically significant correlations were found for all 4th to 6th order Zernike coefficients except for the 5th order horizontal coma C18 (p equals 0.003). On average, all Zernike coefficients for the corneal aberrations were found to be larger compared to Zernike coefficients for the total wavefront aberrations. Conclusions: Corneal aberrations are only of limited use for representing the optical quality of the human eye after corneal laser surgery. This is due to the lack of correlation between corneal and total wavefront aberrations in most of the higher order aberrations. Besides this, the data present in this study yield towards an aberration balancing between corneal aberrations and the optical elements within the eye that reduces the aberration from the cornea by a certain degree. Consequently, ideal customized ablations have to take both, corneal and total wavefront aberrations, into consideration.
Changes in the corneal Na-K ATPase levels in eyes stored in moist chamber at 4°C
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Devi B
Full Text Available This report deals with a chronological measurement of Na-K ATPase enzyme activity in human and bovine corneas stored in a moist chamber at 4°C. Paired human and bovine eyes were sterilized by the standard eye bank procedure and stored up to 6 days. At the desired time, the corneal endothelium was assayed for Na-K ATPase activity. The protein content of each tissue sample was also determined. In a parallel set of experiments, the viability of identical stored corneas was determined by trypan blue and alizarin red staining technique, and morphometric analysis was done to quantify the extent of the corneal endothelial damage. The human corneas showed that there was a significant progressive decrease in the Na-K ATPase activity as the storage time increased. The decrease was related to morphological endothelial damage.
Generation of iPSC line epiHUVEC from human umbilical vein endothelial cells
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Peggy Matz
Full Text Available Human umbilical vein endothelial cells (HUVECs were used to generate the iPSC line epiHUVEC employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHUVEC and the human embryonic stem cell line — H1 have a Pearson correlation of 0.899.
Reconstituted human corneal epithelium: a new alternative to the Draize eye test for the assessment of the eye irritation potential of chemicals and cosmetic products.
Doucet, O; Lanvin, M; Thillou, C; Linossier, C; Pupat, C; Merlin, B; Zastrow, L
The aim of this study was to evaluate the interest of a new three-dimensional epithelial model cultivated from human corneal cells to replace animal testing in the assessment of eye tolerance. To this end, 65 formulated cosmetic products and 36 chemicals were tested by means of this in vitro model using a simplified toxicokinetic approach. The chemicals were selected from the ECETOC data bank and the EC/HO International validation study list. Very satisfactory results were obtained in terms of concordance with the Draize test data for the formulated cosmetic products. Moreover, the response of the corneal model appeared predictive of human ocular response clinically observed by ophthalmologists. The in vitro scores for the chemicals tested strongly correlated with their respective scores in vivo. For all the compounds tested, the response of the corneal model to irritants was similar regardless of their chemical structure, suggesting a good robustness of the prediction model proposed. We concluded that this new three-dimensional epithelial model, developed from human corneal cells, could be promising for the prediction of eye irritation induced by chemicals and complex formulated products, and that these two types of materials should be tested using a similar protocol. A simple shortening of the exposure period was required for the chemicals assumed to be more aggressively irritant to the epithelial tissues than the cosmetic formulae.
Regulation of Ecto-5´-Nucleotidase by Docosahexaenoic Acid in Human Endothelial Cells
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Vu Thi Thom
Full Text Available Background/Aims: Modulation of extracellular adenine nucleotide and adenosine concentrations is one potential mechanism by which docosahexaenoic acid (DHA may exert beneficial effects in critically ill patients. This study assessed DHA effects on extracellular adenine purines. Methods: Experiments used human pulmonary endothelial cells (HPMEC and umbilical vein endothelial cells (HUVEC treated with DHA (48 h. mRNA level (real-time PCR, expression (western blot, flow cytometry and activities (hydrolysis of etheno(ε-purines and fluorescence HPLC of CD73 (ecto-5´-nucleotidase and CD39 (ecto-NTPDase-1 were quantified. Results: DHA elevated total CD73 membrane protein expression concentration-dependently but CD73 mRNA level did not change. Increased expression was paralleled by increased enzyme activity. Effects observed on membrane level were reversed in intact cells, in which ε-AMP hydrolysis decreased after DHA. In intact endothelial cells ATP release was enhanced and CD39 activity blunted following DHA treatment. Hence, extracellular ATP and ADP concentrations increased and this inhibited ε-AMP hydrolysis. Conclusion: In human endothelial cells DHA caused 1 up-regulation of CD73 protein content and increased AMP hydrolysis at the cell membrane level, 2 increased cellular ATP release, and 3 decreased extracellular ATP/ADP hydrolysis. Thus, reorganization of the extracellular adenine-nucleotide-adenosine axis in response to DHA resulted in an increased extracellular ATP/adenosine ratio.
Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release
International Nuclear Information System (INIS)
Li Aihua; Cheng Guangli; Zhu Genghui; Tarnawski, Andrzej S.
Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increased in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling
PX-18 Protects Human Saphenous Vein Endothelial Cells under Arterial Blood Pressure.
Kupreishvili, Koba; Stooker, Wim; Emmens, Reindert W; Vonk, Alexander B A; Sipkens, Jessica A; van Dijk, Annemieke; Eijsman, Leon; Quax, Paul H; van Hinsbergh, Victor W M; Krijnen, Paul A J; Niessen, Hans W M
Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A 2 (sPLA 2 -IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA 2 -IIA herein. The effects of PX-18, an inhibitor of both sPLA 2 -IIA and apoptosis, on residual endothelium and the presence of sPLA 2 -IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs. The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA 2 -IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA 2 -IIA. In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA 2 -IIA inhibition. Copyright © 2017 Elsevier Inc. All rights reserved.
Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction
Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha
Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was
DEFF Research Database (Denmark)
Andersen, J.; Koch-Jensen, P.; Østerby, Ole
The central corneal zone is depicted on keratoscope photographs using a small target aperture and a large object distance. Information on the peripheral area is included by employing a hemispherical target with a dense circular and radial pattern. On a 16 mm (R = 8 mm) reference steel sphere the ...
Biomaterials trigger endothelial cell activation when co-incubated with human whole blood.
Herklotz, Manuela; Hanke, Jasmin; Hänsel, Stefanie; Drichel, Juliane; Marx, Monique; Maitz, Manfred F; Werner, Carsten
Endothelial cell activation resulting from biomaterial contact or biomaterial-induced blood activation may in turn also affect hemostasis and inflammatory processes in the blood. Current in vitro hemocompatibility assays typically ignore these modulating effects of the endothelium. This study describes a co-incubation system of human whole blood, biomaterial and endothelial cells (ECs) that was developed to overcome this limitation. First, human endothelial cells were characterized in terms of their expression of coagulation- and inflammation-relevant markers in response to various activators. Subsequently, their capacity to regulate hemostasis as well as complement and granulocyte activation was monitored in a hemocompatibility assay. After blood contact, quiescent ECs exhibited anticoagulant and anti-inflammatory properties. When they were co-incubated with surfaces exhibiting pro-coagulant or pro-inflammatory characteristics, the ECs down-regulated coagulation but not complement or leukocyte activation. Analysis of intracellular levels of the endothelial activation markers E-selectin and tissue factor showed that co-incubation with model surfaces and blood significantly increased the activation state of ECs. Finally, the coagulation- and inflammation-modulating properties of the ECs were tested after blood/biomaterial exposure. Pre-activation of ECs by biomaterials in the blood induced a pro-coagulant and pro-inflammatory state of the ECs, wherein the pro-coagulant response was higher for biomaterial/blood pre-activated ECs than for TNF-α-pre-activated cells. This work provides evidence that biomaterials, even without directly contacting the endothelium, affect the endothelial activation state with and have consequences for plasmatic and cellular reactions in the blood. Copyright © 2016 Elsevier Ltd. All rights reserved.
Urea immunoliposome inhibits human vascular endothelial cell proliferation for hemangioma treatment
Background Urea injection has been used in hemangioma treatment as sclerotherapy. It shrinks vascular endothelial cells and induces degeneration, necrosis, and fibrosis. However, this treatment still has disadvantages, such as lacking targeting and difficulty in controlling the urea dosage. Thus, we designed a urea immunoliposome to improve the efficiency of treatment. Methods The urea liposome was prepared by reverse phase evaporation. Furthermore, the urea immunoliposome was generated by coupling the urea liposome with a vascular endothelial growth factor receptor (VEGFR) monoclonal antibody using the glutaraldehyde cross-linking method. The influence of the urea immunoliposome on cultured human hemangioma vascular endothelial cells was observed preliminarily. Results Urea immunoliposomes showed typical liposome morphology under a transmission electron microscope, with an encapsulation percentage of 54.4% and a coupling rate of 36.84% for anti-VEGFR. Treatment with the urea immunoliposome significantly inhibited the proliferation of hemangioma vascular endothelial cells (HVECs) in a time- and dose-dependent manner. Conclusions The urea immunoliposome that we developed distinctly and persistently inhibited the proliferation of HVECs and is expected to be used in clinical hemangioma treatment. PMID:24266957
Effect of transfer of donor corneal tissue from McCarey–Kaufmann medium to Optisol-GS on corneal endothelium
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Neha Kapur
Full Text Available Purpose: The purpose of this study is to evaluate the effect of transfer of donor corneal tissue from McCarey–Kaufmann (MK medium to Optisol-GS on corneal endothelium. Methods: This was a prospective, randomized comparative study. Twenty paired human donor corneal tissues of optical quality were retrieved. One tissue of the pair was preserved in Optisol-GS preservative medium (Group A and other tissue of the pair in MK medium (Group B at the time of corneoscleral disc excision. Within 12 h of retrieval, each cornea was evaluated using slit-lamp biomicroscopic examination and specular microscopic analysis. Group B corneas were transferred to Optisol-GS medium within 48–53 h of retrieval. Specular analysis of the paired corneas was repeated 3 h after transferring to Optisol-GS. On day 7 of storage, specular analysis of both the tissues was repeated. Results: The average age of the donor at the time of death was 29 years (16–68 years. The reduction in endothelial cell count, from baseline, in Groups A and B was 5.5% and 5.8% (P = 0.938 on the 3rd day and 8.2% and 12.6% (P = 0.025 on the 7th day, respectively, postretrieval. The coefficient of variation (CV increased by 36% (P = 0.021 and hexagonality reduced by 19% (P = 0.007 on day 7. All tissues retained an endothelial cell density higher than the accepted critical level for penetrating keratoplasty. Conclusion: Significant endothelial cell loss was noted while transferring tissues from one medium to another, necessitating the need for reevaluation of transferred tissues before utilization.
Effect of polysaccharide of dendrobium candidum on proliferation and apoptosis of human corneal epithelial cells in high glucose
Li, Qiangxiang; Chen, Jing; Li, Yajia; Chen, Ting; Zou, Jing; Wang, Hua
Abstract Background: The aim of the study was to observe the effect of polysaccharide of dendrobium candidum (PDC) and high glucose on proliferation, apoptosis of human corneal epithelial cells (HCEC). Methods: The MTT method was used to screen and take the optimal high-glucose concentration, treatment time, and PDC concentration using HCEC and divide it into 4 groups: control group (C), high glucose group (HG), PDC group, and HG + PDC group. We observed and compared the effect of the 4 groups on HCEC proliferation by MTT, apoptosis by Annexin V-FITC/PI double fluorescent staining and flow cytometry (FCM), and expression of bax mRNA and bcl-2 mRNA by RT-qPCR. Results: Compared with the control group, proliferative activity of HCEC cells was reduced; the cells apoptosis ratio was increased; the expression of bax mRNA was increased, and the expression of bcl-2 mRNA was reduced in the HG group. Proliferative activity of HCEC cells in the PDC group was increased, and the expression of bcl-2 mRNA was increased but that of bax mRNA was decreased. Proliferative activity of HCEC cells in the HG + PDC group was increased, but it could not restore to the normal level; the expression of bax mRNA was significantly decreased but the expression of bcl-2 mRNA was significantly increased. Conclusions: Our results demonstrate that high glucose can inhibit proliferative activity and induce apoptosis of HCEC. PDC can improve the proliferative activity of HCEC cells under the high glucose environment and reduce the apoptosis of cells by regulating the expression of bax and bcl-2. PDC play a very important role on protecting and repairing of corneal epithelial cells damage in high glucose. PMID:28796073
Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components.
Qu, Mingli; Wang, Yao; Yang, Lingling; Zhou, Qingjun
To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were measured using 3-(4,5)-dimethylthiahiazo (-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and transwell migration assay. Cell damage was determined with the lactate dehydrogenase (LDH) assay. Cell viability and median lethal time (LT₅₀) were measured by 7-amino-actinomycin D (7-AAD) staining and flow cytometry analysis. Cellular effects after exposure of HCECs to the four anti-inflammatory eye drops were concentration dependent. The differences of cellular toxicity on cell proliferation became significant at lower concentrations (Eye Drops showed significant increasing effects on cell damage and viability when compared with the other three solutions. Tobramycin & Dex Eye Drops inhibited the migration of HCECs significantly. Tobramycin & Dex Eye Drops showed the quickest effect on cell viability: the LT₅₀ was 3.28, 9.23, 10.38, and 23.80 min for Tobramycin & Dex Eye Drops, Diclofenac Sodium Eye Drops, Pranoprofen Eye Drops, and Bromfenac Sodium Hydrate Eye Drops, respectively. However, the comparisons of cellular toxicity revealed significant differences between the eye drops and their active components under the same concentration. The corneal epithelial toxicity differences among the active components of the four eye drops became significant as higher concentration (>0.020%). The four anti-inflammatory eye drops showed different cellular effects on HCECs, and the toxicity was not related with their active components, which provides new reference for the clinical application and drug
Biophysical Assessment of Single Cell Cytotoxicity: Diesel Exhaust Particle-Treated Human Aortic Endothelial Cells
Wu, Yangzhe; Yu, Tian; Gilbertson, Timothy A.; Zhou, Anhong; Xu, Hao; Nguyen, Kytai Truong
Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model ...
2-Chlorohexadecanal and 2-chlorohexadecanoic acid induce COX-2 expression in human coronary artery endothelial cells
Messner, Maria C.; Albert, Carolyn J.; Ford, David A.
2-Chlorohexadecanal (2-ClHDA), a 16-carbon chain chlorinated fatty aldehyde that is produced by reactive chlorinating species attack of plasmalogens, is elevated in atherosclerotic plaques, infarcted myocardium, and activated leukocytes. We tested the hypothesis that 2-ClHDA and its metabolites, 2-chlorohexadecanoic acid (2-ClHA) and 2-chlorohexadecanol (2-ClHOH), induce COX-2 expression in human coronary artery endothelial cells (HCAEC). COX-2 protein expression increased in response to 2-Cl...
Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿
Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed
Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991
An easy and inexpensive method for quantitative analysis of endothelial damage by using vital dye staining and Adobe Photoshop software.
Saad, Hisham A; Terry, Mark A; Shamie, Neda; Chen, Edwin S; Friend, Daniel F; Holiman, Jeffrey D; Stoeger, Christopher
We developed a simple, practical, and inexpensive technique to analyze areas of endothelial cell loss and/or damage over the entire corneal area after vital dye staining by using a readily available, off-the-shelf, consumer software program, Adobe Photoshop. The purpose of this article is to convey a method of quantifying areas of cell loss and/or damage. Descemet-stripping automated endothelial keratoplasty corneal transplant surgery was performed by using 5 precut corneas on a human cadaver eye. Corneas were removed and stained with trypan blue and alizarin red S and subsequently photographed. Quantitative assessment of endothelial damage was performed by using Adobe Photoshop 7.0 software. The average difference for cell area damage for analyses performed by 1 observer twice was 1.41%. For analyses performed by 2 observers, the average difference was 1.71%. Three masked observers were 100% successful in matching the randomized stained corneas to their randomized processed Adobe images. Vital dye staining of corneal endothelial cells can be combined with Adobe Photoshop software to yield a quantitative assessment of areas of acute endothelial cell loss and/or damage. This described technique holds promise for a more consistent and accurate method to evaluate the surgical trauma to the endothelial cell layer in laboratory models. This method of quantitative analysis can probably be generalized to any area of research that involves areas that are differentiated by color or contrast.
Human haemato-endothelial precursors: cord blood CD34+ cells produce haemogenic endothelium.
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Elvira Pelosi
Full Text Available Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144-, triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45- capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.
Treating fat grafts with human endothelial progenitor cells promotes their vascularization and improves their survival in diabetes mellitus.
Hamed, Saher; Ben-Nun, Ohad; Egozi, Dana; Keren, Aviad; Malyarova, Nastya; Kruchevsky, Danny; Gilhar, Amos; Ullmann, Yehuda
Bone marrow-derived endothelial progenitor cells are required for vascularization of a fat graft to form a functional microvasculature within the graft and to facilitate its integration into the surrounding tissues. Organ transplantation carries a high risk of graft loss and rejection in patients with diabetes mellitus because endothelial progenitor cell function is impaired. The authors investigated the influence of endothelial progenitor cell treatment on the phenotype and survival of human fat grafts in immunocompromised mice with experimentally induced diabetes mellitus. The authors injected 1 ml of human fat tissue into the scalps of 14 nondiabetic and 28 diabetic immunocompromised mice, and then treated some of the grafts with endothelial progenitor cells that was isolated from the blood of a human donor. The phenotype of the endothelial progenitor cell-treated fat grafts from the 14 diabetic mice was compared with that of the untreated fat grafts from 14 nondiabetic and 14 diabetic mice, 18 days and 15 weeks after fat transplantation. Determination of graft phenotype included measurements of weight and volume, vascular endothelial growth factor levels, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase, and caspase 3 expression levels, and histologic analysis of the extent of vascularization. The untreated grafts from the diabetic mice were fully resorbed 15 weeks after fat transplantation. The phenotype of endothelial progenitor cell-treated fat grafts from the diabetic mice was similar to that of the untreated fat grafts from the nondiabetic mice. Endothelial progenitor cell treatment of transplanted fat can increase the survival of a fat graft by inducing its vascularization and decreasing the extent of apoptosis.
Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier
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van Doorn Ruben
Full Text Available Abstract Background The sphingosine 1-phosphate (S1P receptor modulator FTY720P (Gilenya® potently reduces relapse rate and lesion activity in the neuroinflammatory disorder multiple sclerosis. Although most of its efficacy has been shown to be related to immunosuppression through the induction of lymphopenia, it has been suggested that a number of its beneficial effects are related to altered endothelial and blood–brain barrier (BBB functionality. However, to date it remains unknown whether brain endothelial S1P receptors are involved in the maintenance of the function of the BBB thereby mediating immune quiescence of the brain. Here we demonstrate that the brain endothelial receptor S1P5 largely contributes to the maintenance of brain endothelial barrier function. Methods We analyzed the expression of S1P5 in human post-mortem tissues using immunohistochemistry. The function of S1P5 at the BBB was assessed in cultured human brain endothelial cells (ECs using agonists and lentivirus-mediated knockdown of S1P5. Subsequent analyses of different aspects of the brain EC barrier included the formation of a tight barrier, the expression of BBB proteins and markers of inflammation and monocyte transmigration. Results We show that activation of S1P5 on cultured human brain ECs by a selective agonist elicits enhanced barrier integrity and reduced transendothelial migration of monocytes in vitro. These results were corroborated by genetically silencing S1P5 in brain ECs. Interestingly, functional studies with these cells revealed that S1P5 strongly contributes to brain EC barrier function and underlies the expression of specific BBB endothelial characteristics such as tight junctions and permeability. In addition, S1P5 maintains the immunoquiescent state of brain ECs with low expression levels of leukocyte adhesion molecules and inflammatory chemokines and cytokines through lowering the activation of the transcription factor NFκB. Conclusion Our
Birth weight and characteristics of endothelial and smooth muscle cell cultures from human umbilical cord vessels
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Lurbe Empar
Full Text Available Abstract Background Low birth weight has been related to an increased risk for developing high blood pressure in adult life. The molecular and cellular analysis of umbilical cord artery and vein may provide information about the early vascular characteristics of an individual. We have assessed several phenotype characteristics of the four vascular cell types derived from human umbilical cords of newborns with different birth weight. Further follow-up studies could show the association of those vascular properties with infancy and adulthood blood pressure. Methods Endothelial and smooth muscle cell cultures were obtained from umbilical cords from two groups of newborns of birth weight less than 2.8 kg or higher than 3.5 kg. The expression of specific endothelial cell markers (von Willebrand factor, CD31, and the binding and internalization of acetylated low-density lipoprotein and the smooth muscle cell specific α-actin have been evaluated. Cell culture viability, proliferation kinetic, growth fraction (expression of Ki67 and percentage of senescent cells (detection of β-galactosidase activity at pH 6.0 have been determined. Endothelial cell projection area was determined by morphometric analysis of cell cultures after CD31 immunodetection. Results The highest variation was found in cell density at the confluence of endothelial cell cultures derived from umbilical cord arteries (66,789 ± 5,093 cells/cm2 vs. 45,630 ± 11,927 cells/cm2, p 2, p Conclusion The analysis of umbilical cord artery endothelial cells, which demonstrated differences in cell size related to birth weight, can provide hints about the cellular and molecular links between lower birth weight and increased adult high blood pressure risk.
Microvascular Endothelial Dysfunction in Sedentary, Obese Humans is mediated by NADPH Oxidase; Influence of Exercise Training
La Favor, Justin D.; Dubis, Gabriel S.; Yan, Huimin; White, Joseph D.; Nelson, Margaret A.M.; Anderson, Ethan J.; Hickner, Robert C.
Objective The objectives of this study were to determine the impact of in vivo reactive oxygen species (ROS) on microvascular endothelial function in obese human subjects and to determine the efficacy of an aerobic exercise intervention on alleviating obesity-associated dysfunctionality. Approach and Results Young, sedentary men and women were divided into lean (BMI 18–25; n=14), intermediate (BMI 28–32.5; n=13), and obese (BMI 33–40; n=15) groups. A novel microdialysis technique was utilized to detect elevated interstitial hydrogen peroxide (H2O2) and superoxide levels in the vastus lateralis of obese compared to both lean and intermediate subjects. Nutritive blood flow was monitored in the vastus lateralis via the microdialysis-ethanol technique. A decrement in acetylcholine-stimulated blood flow revealed impaired microvascular endothelial function in the obese subjects. Perfusion of apocynin, an NADPH oxidase (Nox) inhibitor, lowered (normalized) H2O2 and superoxide levels and reversed microvascular endothelial dysfunction in obese subjects. Following 8-weeks of exercise, H2O2 levels were decreased in the obese subjects and microvascular endothelial function in these subjects was restored to levels similar to lean subjects. Skeletal muscle protein expression of the Nox subunits p22phox, p47phox, and p67phox were increased in obese relative to lean subjects, where p22phox and p67phox expression was attenuated by exercise training in obese subjects. Conclusions This study implicates Nox as a source of excessive ROS production in skeletal muscle of obese individuals, and links excessive Nox derived ROS to microvascular endothelial dysfunction in obesity. Furthermore, aerobic exercise training proved to be an effective strategy for alleviating these maladies. PMID:27765769
A novel adipocytokine, chemerin exerts anti-inflammatory roles in human vascular endothelial cells
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Yamawaki, Hideyuki, E-mail: firstname.lastname@example.com [Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori 034-8628 (Japan); Kameshima, Satoshi; Usui, Tatsuya; Okada, Muneyoshi; Hara, Yukio [Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori 034-8628 (Japan)
Highlights: Black-Right-Pointing-Pointer Chemerin is a novel adipocytokine with almost unknown function in vasculature. Black-Right-Pointing-Pointer Chemerin activates Akt/eNOS/NO pathways in endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-{alpha}-induced monocyte adhesion to endothelial cells. Black-Right-Pointing-Pointer Chemerin inhibits TNF-induced VCAM-1 via suppressing NF-{kappa}B and p38 signal. Black-Right-Pointing-Pointer Chemerin is anti-inflammatory through producing NO in vascular endothelium. -- Abstract: Chemerin is a recently identified adipocytokine which plays a role on inflammation and adipocytes metabolism. However, its function in vasculature is largely unknown. We examined the effects of chemerin on vascular endothelial inflammatory states. Treatment of human umbilical vein endothelial cells with chemerin (300 ng/ml, 20 min) induced phosphorylation of Akt (Ser473) and endothelial nitric oxide (NO) synthase (eNOS) (Ser1177). Consistently, chemerin increased intracellular cyclic GMP content. Pretreatment with chemerin (1-300 ng/ml, 24 h) significantly inhibited phosphorylation of nuclear factor (NF)-{kappa}B p65 (Ser536) and p38 as well as vascular cell adhesion molecule (VCAM)-1 expression induced by tumor necrosis factor (TNF)-{alpha} (5 ng/ml, 20 min-6 h). Inhibitor of NF-{kappa}B or p38 significantly inhibited the TNF-{alpha}-induced VCAM-1 expression. Chemerin also inhibited TNF-{alpha}-induced VCAM-1 expression in rat isolated aorta. Moreover, chemerin significantly inhibited monocytes adhesion to TNF-{alpha}-stimulated endothelial cells. The inhibitory effect of chemerin on TNF-{alpha}-induced VCAM-1 was reversed by a NOS inhibitor. Conversely, an NO donor, sodium nitroprusside significantly inhibited TNF-{alpha}-induced VCAM-1. The present results for the first time demonstrate that chemerin plays anti-inflammatory roles by preventing TNF-{alpha}-induced VCAM-1 expression and monocytes adhesion in vascular
Influence of the corneal optical zone on the point-spread function of the human eye
Rol, Pascal O.; Parel, Jean-Marie A.
In refractive surgery, a number of surgical techniques have been developed to correct ametropia (refractive defaults) of the eye by changing the exterior shape of the cornea. Because the air-cornea interface makes up for about two thirds of the refractive power of the eye, a refractive correction can be obtained by a suitable reshaping of the cornea. Postoperatively, it is usually observed that the corneal region consists of two or more zones which are characterized by different optical parameters exhibiting in particular different focal distances. Under normal circumstances, only the central area of the cornea is involved in the formation of the retinal image. However, if part of the light entering the eye through peripheral portions of the cornea with refractive properties different from the central area can pass the pupil, an out-of-focus `ghost' image may be overlaid on the retina causing a blur. In such a case the resolution, and the contrast performance of the eye which is expected from a successful operation, may be reduced. This study is an attempt to quantify the vision blur as a function of the diameter of the central zone, i.e., the optical zone which is of importance for vision.
Therapeutic effects of zerumbone in an alkali-burned corneal wound healing model.
Kim, Jong Won; Jeong, Hyuneui; Yang, Myeon-Sik; Lim, Chae Woong; Kim, Bumseok
Cornea is an avascular transparent tissue. Ocular trauma caused by a corneal alkali burn induces corneal neovascularization (CNV), inflammation, and fibrosis, leading to vision loss. The purpose of this study was to examine the effects of Zerumbone (ZER) on corneal wound healing caused by alkali burns in mice. CNV was induced by alkali-burn injury in BALB/C female mice. Topical ZER (three times per day, 3μl each time, at concentrations of 5, 15, and 30μM) was applied to treat alkali-burned mouse corneas for 14 consecutive days. Histopathologically, ZER treatment suppressed alkali burn-induced CNV and decreased corneal epithelial defects induced by alkali burns. Corneal tissue treated with ZER showed reduced mRNA levels of pro-angiogenic genes, including vascular endothelial growth factor, matrix metalloproteinase-2 and 9, and pro-fibrotic factors such as alpha smooth muscle actin and transforming growth factor-1 and 2. Immunohistochemical analysis demonstrated that the infiltration of F4/80 and/or CCR2 positive cells was significantly decreased in ZER-treated corneas. ZER markedly inhibited the mRNA and protein levels of monocyte chemoattractant protein-1 (MCP-1) in human corneal fibroblasts and murine peritoneal macrophages. Immunoblot analysis revealed that ZER decreased the activation of signal transducer and activator of transcription 3 (STAT3), with consequent reduction of MCP-1 production by these cells. In conclusion, topical administration of ZER accelerated corneal wound healing by inhibition of STAT3 and MCP-1 production. Copyright © 2017. Published by Elsevier B.V.
Mapping the distinctive populations of lymphatic endothelial cells in different zones of human lymph nodes.
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Saem Mul Park
Full Text Available The lymphatic sinuses in human lymph nodes (LNs are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs. A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs; in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.
Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells
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Ray, Ratna B.; Basu, Arnab; Steele, Robert; Beyene, Aster; McHowat, Jane; Meyer, Keith; Ghosh, Asish K.; Ray, Ranjit
Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences
Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.
Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R
The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.
Transcriptional responses of human aortic endothelial cells to nanoconstructs used in biomedical applications.
Moos, Philip J; Honeggar, Matthew; Malugin, Alexander; Herd, Heather; Thiagarajan, Giridhar; Ghandehari, Hamidreza
Understanding the potential toxicities of manufactured nanoconstructs used for drug delivery and biomedical applications may help improve their safety. We sought to determine if surface-modified silica nanoparticles and poly(amido amine) dendrimers elicit genotoxic responses on vascular endothelial cells. The nanoconstructs utilized in this study had a distinct geometry (spheres vs worms) and surface charge, which were used to evaluate the contributions of these parameters to any potential adverse effects of these materials. Time-dependent cytotoxicity was found for surfaced-functionalized but geometrically distinct silica materials, while amine-terminated dendrimers displayed time-independent cytotoxicity and carboxylated dendrimers were nontoxic in our assays. Transcriptomic evaluation of human aortic endothelial cell (HAEC) responses indicated time-dependent gene induction following silica exposure, consisting of cell cycle gene repression and pro-inflammatory gene induction. However, the dendrimers did not induce genomic toxicity, despite displaying general cytotoxicity.
Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells
Tóth, Andrea E.; Walter, Fruzsina R.; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G.; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A.
Background Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. PMID:25033388
Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells.
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Andrea E Tóth
Full Text Available Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line treated with methylglyoxal.Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging.Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound.These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases.
Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells
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Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: email@example.com [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)
Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.
Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells
Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.
Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.
Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells
International Nuclear Information System (INIS)
Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K
Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (∼97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.
Glucosamine exposure reduces proteoglycan synthesis in primary human endothelial cells in vitro
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Trine M. Reine
Full Text Available Purpose: Glucosamine (GlcN supplements are promoted for medical reasons, for example, for patients with arthritis and other joint-related diseases. Oral intake of GlcN is followed by uptake in the intestine, transport in the circulation and thereafter delivery to chondrocytes. Here, it is postulated to have an effect on synthesis and turnover of extracellular matrix constituents expressed by these cells. Following uptake in the intestine, serum levels are transiently increased, and the endothelium is exposed to increased levels of GlcN. We investigated the possible effects of GlcN on synthesis of proteoglycans (PGs, an important matrix component, in primary human endothelial cells. Methods: Primary human endothelial cells were cultured in vitro in medium with 5 mM glucose and 0–10 mM GlcN. PGs were recovered and analysed by western blotting, or by SDS-PAGE, gel chromatography or ion-exchange chromatography of 35S-PGs after 35S-sulphate labelling of the cells. Results: The synthesis and secretion of 35S-PGs from cultured endothelial cells were reduced in a dose- and time-dependent manner after exposure to GlcN. PGs are substituted with sulphated glycosaminoglycan (GAG chains, vital for PG function. The reduction in 35S-PGs was not related to an effect on GAG chain length, number or sulphation, but rather to the total expression of PGs. Conclusion: Exposure of endothelial cells to GlcN leads to a general decrease in 35S-PG synthesis. These results suggest that exposure to high levels of GlcN can lead to decreased matrix synthesis, contrary to what has been claimed by supporters of such supplements.
Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.
Quan, S; Yang, L; Abraham, N G; Kappas, A
Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.
Evaluation of Human Corneal Lenticule Quality After SMILE With Different Cap Thicknesses Using Scanning Electron Microscopy.
Weng, Shengbei; Liu, Manli; Yang, Xiaonan; Liu, Fang; Zhou, Yugui; Lin, Haiqin; Liu, Quan
To evaluate the surface characteristics of lenticules created by small-incision lenticule extraction (SMILE) with different cap thicknesses. This prospective study included 20 consecutive patients who underwent bilateral SMILE. Surface regularity of the extracted corneal lenticule was analyzed using scanning electron microscopy (SEM) combined with 2 methods: qualitative and quantitative regularity. Qualitative regularity of SEM images was graded by masked observers using an established scoring system. Quantitative regularity of SEM images was assessed by counting the total number and areas of tissue bridges using Image-Pro Plus software. Four different cap thickness of 120, 130, 140, and 150 μm were compared. Refractive outcomes of patients were measured at baseline and 1 month after surgery. As 10 specimens were not analyzable, only 30 eyes were included. Postoperatively, all eyes had postoperative uncorrected distance visual acuity of 20/20 or better; 43% had an unchanged corrected distance visual acuity; 43% gained 1 line; 10% lost 1 line. Ultrastructurally, surface irregularity was primarily caused by tissue bridges. The average surface regularity score obtained was 10.87 ± 2.40 for 120 μm, 10.78 ± 2.60 for 130 μm, 8.76 ± 2.16 for 140 μm, and 8.70 ± 2.66 for 150 μm (P < 0.001). The total number and areas of tissue bridges of 120 to 130 μm were significantly less than 140 to 150 μm (P < 0.05). Surface regularity decreased as cap thickness increased (P < 0.05). There is smoother appearance of the lenticular surface as seen through SEM when a thin cap is created compared with a thick cap qualitatively and quantitatively.
GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells
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Dawid Krokowski
Full Text Available Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR. In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR
Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model.
Tanaka, Yohei; Nakayama, Jun
Humans are increasingly exposed to near-infrared (NIR) radiation from both natural (eg, solar) and artificial (eg, electrical appliances) sources. Although the biological effects of sun and ultraviolet (UV) exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues. DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C). The water-filter allowed 1,000-1,800 nm wavelengths and excluded 1,400-1,500 nm wavelengths. A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR) was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm(2) irradiation (Psolar energy reaching the Earth is in the NIR region, which cannot be adequately blocked by eyewear and thus can induce eye damage with intensive or long-term exposure, protection from both UV and NIR radiation may prevent changes in gene expression and in turn eye damage.
Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.
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Jorge S Burns
Full Text Available BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC strain (hMSC-TERT20 immortalized by retroviral vector mediated human telomerase (hTERT gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+ and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1
The chemotactic activity of beta-carotene in endothelial cell progenitors and human umbilical vein endothelial cells: A microarray analysis
Polus, A.; Kiec-wilk, B.; Hartwich, J.; Balwierz, A.; Stachura, J.; Dyduch, G.; Laidler, P.; Zagajewski, J.; Langman, T.; Schmitz, G.; Goralcsky, R.; Wertz, K.; Riss, G.; Keijer, J.; Dembinska-Kiec, A.
Objectives: Endothelial cells and their progenitors play an important role in angiogenesis that is essential for organogenesis and tissue remodelling, as well as for inflammatory responses and carcinogenesis in all periods of life. In the present study, the authors concentrated on the direct effect
Diffusion and Monod kinetics model to determine in vivo human corneal oxygen-consumption rate during soft contact lens wear
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Luis F. Del Castillo
Conclusion: Present results are relevant for the calculation on the partial pressure of oxygen, available at different depths into the corneal tissue behind contact lenses of different oxygen transmissibility.
Anterior corneal profile with variable asphericity.
Rosales, Marco A; Juárez-Aubry, Montserrat; López-Olazagasti, Estela; Ibarra, Jorge; Tepichín, Eduardo
We present a corneal profile in which the eccentricity, e(Q=-e(2)), has a nonlinear continuous variation from the center outwards. This nonlinear variation is intended to fit and reproduce our current experimental data in which the anterior corneal surface of the human eye exhibits different values of e at different diameters. According to our clinical data, the variation is similar to an exponential decay. We propose a linear combination of two exponential functions to describe the variation of e. We then calculate the corneal sagittal height by substituting e in the first-order aspherical surface equation to obtain the corneal profile. This corneal profile will be used as a reference to analyze the resultant profiles of the customized corneal ablation in refractive surgery.
Loss of CD34 expression in aging human choriocapillaris endothelial cells.
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Elliott H Sohn
Full Text Available Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD. In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40, age-matched controls (> age 60 without AMD, or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization. Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10(-6. Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse or a trend toward decreased CD34 (human in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.
Vascular Endothelial Growth Factor Receptor 1 Contributes to Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway▿ †
Zhao, Wei-Dong; Liu, Wei; Fang, Wen-Gang; Kim, Kwang Sik; Chen, Yu-Hua
Escherichia coli is the most common Gram-negative organism causing neonatal meningitis. Previous studies demonstrated that E. coli K1 invasion of brain microvascular endothelial cells (BMEC) is required for penetration into the central nervous system, but the microbe-host interactions that are involved in this process remain incompletely understood. Here we report the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) expressed on human brain microvascular endothelial cells...
A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells
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Laura Giusti
Full Text Available Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs, bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG, on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation.
Brugia malayi microfilariae adhere to human vascular endothelial cells in a C3-dependent manner.
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Jan-Hendrik Schroeder
Full Text Available Brugia malayi causes the human tropical disease, lymphatic filariasis. Microfilariae (Mf of this nematode live in the bloodstream and are ingested by a feeding mosquito vector. Interestingly, in a remarkable co-evolutionary adaptation, Mf appearance in the peripheral blood follows a circadian periodicity and reaches a peak when the mosquito is most likely to feed. For the remaining hours, the majority of Mf sequester in the lung capillaries. This circadian phenomenon has been widely reported and is likely to maximise parasite fitness and optimise transmission potential. However, the mechanism of Mf sequestration in the lungs remains largely unresolved. In this study, we demonstrate that B. malayi Mf can, directly adhere to vascular endothelial cells under static conditions and under flow conditions, they can bind at high (but not low flow rates. High flow rates are more likely to be experienced diurnally. Furthermore, a non-periodic nematode adheres less efficiently to endothelial cells. Strikingly C3, the central component of complement, plays a crucial role in the adherence interaction. These novel results show that microfilariae have the ability to bind to endothelial cells, which may explain their sequestration in the lungs, and this binding is increased in the presence of inflammatory mediators.
Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells
International Nuclear Information System (INIS)
Schwalm, Stephanie; Doell, Frauke; Roemer, Isolde; Bubnova, Svetlana; Pfeilschifter, Josef; Huwiler, Andrea
Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1α and HIF-2α, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1α or HIF-2α by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression
The influence of statins on the free intracellular calcium concentration in human umbilical vein endothelial cells
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Figulla Hans R
Full Text Available Abstract Background Statins are cholesterol-lowering drugs that are widely used to reduce the risk of cardiac infarction. Their beneficial clinical effects, however, are not restricted to their influence on cholesterol production. As several studies have shown that they have a potency of relaxing blood vessels. Methods We measured the effects of statins on the intracellular free calcium concentration ([Ca2+]i in human umbilical vein endothelial cells (HUVEC after acute application and 24-h-preincubation of statins. Results Incubation of the cells for 24 h with cerivastatin or fluvastatin significantly increased the resting [Ca2+]i. For cerivastatin this effect manifested at a concentration of 1 μM. Increase of resting [Ca2+]i in the presence of cerivastatin also occurred when the nitric oxide synthase was inhibited. Transient Ca2+ release induced by histamine was not affected. Conclusions The increase of resting [Ca2+]i after incubation with cerivastatin or fluvastatin may provide an explanation for the direct effects of statins on the endothelial-dependent vasodilatation and restoration of endothelial activity in vivo.
Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells
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Ana-María Zaske
Full Text Available Although atomic force microscopy (AFM has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.
[The status quo and expectation of corneal research in China].
Shi, Weiyun; Xie, Lixin
In China, corneal disease is currently the second leading cause of blindness. Severe donor shortage, insufficient technique supports and promotion, and the lack of corneal disease specialists due to poor systematic training are all urgent problems to be resolved. The last 5 years have witnessed a considerable progress in basic and clinical researches of corneal disease. Investigations on the pathogenesis and treatment of fungal keratitis have won an international reputation. Results from the study of corneal reconstruction with tissue-engineered and acellular matrix corneas have been tested in clinical trials with good preliminary performance. Moreover, the clinical researches of corneal refractive surgery have kept pace with the latest international progresses. However, Descemet's membrane endothelial keratoplasty needs further promotion, and the development and application of keratoprosthesis remains a blank. Although keratoprosthesis and corneal collagen cross-linking have been widely applied in Europe with satisfactory clinical efficacy, they are still under assessment by China Food and Drug Administration for approval of use.
Leonurine protects against tumor necrosis factor-α-mediated inflammation in human umbilical vein endothelial cells.
Liu, Xinhua; Pan, Lilong; Wang, Xianli; Gong, Qihai; Zhu, Yi Zhun
Leonurine, a bioactive alkaloid compound in Herba leonuri, has various pharmacological activities, including antioxidant and anti-apoptotic capacities. This study was conducted to test the hypothesis that leonurine was able to attenuate tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) activation and the underlying molecular mechanisms. Mitogen-activated protein kinases (MAPK) activation, nuclear factor-κB (NF-κB) activation, and inflammatory mediators expression were detected by Western blot or enzyme-liked immunosorbent assay, intracellular reactive oxygen species (ROS) and NF-κB p65 translocation were measured by immunofluorescence, endothelial cell-monocyte interaction was detected by microscope. Leonurine inhibited U937 cells adhesion to TNF-α-activated HUVEC in a concentration dependent manner. Treatment with leonurine blocked TNF-α-induced mRNA and protein expression of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), cyclooxygenase-2, and monocyte chemoattractant protein-1 in endothelial cells. In addition, leonurine attenuated TNF-α-induced intracellular ROS production in HUVEC. Furthermore, leonurine also suppressed the TNF-α-activated p38 phosphorylation and IκBα degradation. Subsequently, reduced NF-κB p65 phosphorylation, nuclear translocation, and DNA-binding activity were also observed. Our results demonstrated for the first time that the anti-inflammatory properties of leonurine in endothelial cells, at least in part, through suppression of NF-κB activation, which may have a potential therapeutic use for inflammatory vascular diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.
Pérez-Cremades, Daniel; Bueno-Betí, Carlos; García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Hermenegildo, Carlos; Pallardó, Federico V; Novella, Susana
Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
Mutations in LOXHD1, a Recessive-Deafness Locus, Cause Dominant Late-Onset Fuchs Corneal Dystrophy
Riazuddin, S. Amer; Parker, David S.; McGlumphy, Elyse J.; Oh, Edwin C.; Iliff, Benjamin W.; Schmedt, Thore; Jurkunas, Ula; Schleif, Robert; Katsanis, Nicholas; Gottsch, John D.
Fuchs corneal dystrophy (FCD) is a genetic disorder of the corneal endothelium and is the most common cause of corneal transplantation in the United States. Previously, we mapped a late-onset FCD locus, FCD2, on chromosome 18q. Here, we present next-generation sequencing of all coding exons in the FCD2 critical interval in a multigenerational pedigree in which FCD segregates as an autosomal-dominant trait. We identified a missense change in LOXHD1, a gene causing progressive hearing loss in humans, as the sole variant capable of explaining the phenotype in this pedigree. We observed LOXHD1 mRNA in cultured human corneal endothelial cells, whereas antibody staining of both human and mouse corneas showed staining in the corneal epithelium and endothelium. Corneal sections of the original proband were stained for LOXHD1 and demonstrated a distinct increase in antibody punctate staining in the endothelium and Descemet membrane; punctate staining was absent from both normal corneas and FCD corneas negative for causal LOXHD1 mutations. Subsequent interrogation of a cohort of >200 sporadic affected individuals identified another 15 heterozygous missense mutations that were absent from >800 control chromosomes. Furthermore, in silico analyses predicted that these mutations reside on the surface of the protein and are likely to affect the protein's interface and protein-protein interactions. Finally, expression of the familial LOXHD1 mutant allele as well as two sporadic mutations in cells revealed prominent cytoplasmic aggregates reminiscent of the corneal phenotype. All together, our data implicate rare alleles in LOXHD1 in the pathogenesis of FCD and highlight how different mutations in the same locus can potentially produce diverse phenotypes. PMID:22341973
Development of Acyclovir-Loaded Albumin Nanoparticles and Improvement of Acyclovir Permeation Across Human Corneal Epithelial T Cells.
Suwannoi, Panita; Chomnawang, Mullika; Sarisuta, Narong; Reichl, Stephan; Müller-Goymann, Christel C
The aim of the present study was to develop acyclovir (ACV) ocular drug delivery systems of bovine serum albumin (BSA) nanoparticles as well as to assess their in vitro transcorneal permeation across human corneal epithelial (HCE-T) cell multilayers. The ACV-loaded BSA nanoparticles were prepared by desolvation method along with physicochemical characterization, cytotoxicity, as well as in vitro transcorneal permeation studies across HCE-T cell multilayers. The nanoparticles appeared to be spherical in shape and nearly uniform in size of about 200 nm. The size of nanoparticles became smaller with decreasing BSA concentration, while the ratios of water to ethanol seemed not to affect the size. Increasing the amount of ethanol in desolvation process led to significant reduction of drug entrapment of nanoparticles with smaller size and more uniformity. The ACV-loaded BSA nanoparticles prepared were shown to have no cytotoxic effect on HCE-T cells used in permeation studies. The in vitro transcorneal permeation results revealed that ACV could permeate through the HCE-T cell multilayers significantly higher from BSA nanoparticles than from aqueous ACV solutions. The ACV-loaded BSA nanoparticles could be prepared by desolvation method without glutaraldehyde in the formulation. ACV could increasingly permeate through the multilayers of HCE-T cells from the ACV-loaded BSA nanoparticles. Therefore, the ACV-loaded BSA nanoparticles could be a highly potential ocular drug delivery system.
Sulfation of chlorotyrosine and nitrotyrosine by human lung endothelial and epithelial cells: Role of the human SULT1A3
International Nuclear Information System (INIS)
Yasuda, Shin; Yasuda, Tomoko; Liu, Ming-Yih; Shetty, Sreerama; Idell, Steven; Boggaram, Vijayakumar; Suiko, Masahito; Sakakibara, Yoichi; Fu Jian; Liu, Ming-Cheh
During inflammation, potent reactive oxidants formed may cause chlorination and nitration of both free and protein-bound tyrosine. In addition to serving as biomarkers of inflammation-mediated oxidative stress, elevated levels of chlorotyrosine and nitrotyrosine have been linked to the pathogenesis of lung and vascular disorders. The current study was designed to investigate whether the lung cells are equipped with mechanisms for counteracting these tyrosine derivatives. By metabolic labeling, chlorotyrosine O-[ 35 S]sulfate and nitrotyrosine O-[ 35 S]sulfate were found to be generated and released into the labeling media of human lung endothelial and epithelial cells labeled with [ 35 S]sulfate in the presence of added chlorotyrosine and nitrotyrosine. Enzymatic assays using the eleven known human cytosolic sulfotransferases (SULTs) revealed SULT1A3 as the enzyme responsible for catalyzing the sulfation of chlorotyrosine and nitrotyrosine. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the expression of SULT1A3 in the lung endothelial and epithelial cells used in this study. Kinetic constants of the sulfation of chlorotyrosine and nitrotyrosine by SULT1A3 were determined. Collectively, these results suggest that sulfation by SULT1A3 in lung endothelial and epithelial cells may play a role in the inactivation and/or disposal of excess chlorotyrosine and nitrotyrosine generated during inflammation.
Melatonin prevents human pancreatic carcinoma cell PANC-1-induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression.
Cui, Peilin; Yu, Minghua; Peng, Xingchun; Dong, Lv; Yang, Zhaoxu
Melatonin is an important natural oncostatic agent, and our previous studies have found its inhibitory action on tumor angiogenesis, but the mechanism remains unclear. It is well known that vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis and has become an important target for antitumor therapy. Pancreatic cancer is a representative of the most highly vascularized and angiogenic solid tumors, which responds poorly to chemotherapy and radiation. Thus, seeking new treatment strategies targeting which have anti-angiogenic capability is urgent in clinical practice. In this study, a co-culture system between human umbilical vein endothelial cells (HUVECs) and pancreatic carcinoma cells (PANC-1) was used to investigate the direct effect of melatonin on the tumor angiogenesis and its possible action on VEGF expression. We found HUVECs exhibited an increased cell proliferation and cell migration when co-cultured with PANC-1 cells, but the process was prevented when melatonin added to the incubation medium. Melatonin at concentrations of 1 μm and 1 mm inhibited the cell proliferation and migration of HUVECs and also decreased both the VEGF protein secreted to the cultured medium and the protein produced by the PANC-1 cells. In addition, the VEGF mRNA expression was also down-regulated by melatonin. Taken together, our present study shows that melatonin at pharmacological concentrations inhibited the elevated cell proliferation and cell migration of HUVECs stimulated by co-culturing them with PANC-1 cells; this was associated with a suppression of VEGF expression in PANC-1 cells. © 2011 John Wiley & Sons A/S.
The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.
Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick
Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.
The New Zealand National Eye Bank study: trends in the acquisition and storage of corneal tissue over the decade 2000 to 2009.
Cunningham, William J; Moffatt, S Louise; Brookes, Nigel H; Twohill, Helen C; Pendergrast, David G C; Stewart, Joanna M; McGhee, Charles N J
To evaluate trends in the acquisition, storage, and utilization of donated corneal tissue in New Zealand, 2000 to 2009. The New Zealand National Eye Bank records were analyzed for the decade January 2000 to December 2009. Variables analyzed included donor demographics (age, sex, and ethnicity), donor source, donor cause of death, death-to-preservation interval (DPI), corneal storage time, tissue contamination, endothelial assessment, cornea suitability for transplantation, and corneal tissue utilization. A total of 1268 eye donors were identified during the 10-year period. Overall, 36% (n = 457) were female and 64% male (n = 813). Median donor age was 67 years, and 23% of donors were younger than 50 years (range, 5-90 years). There was a decrease in donor age over the decade (P = 0.006). The median DPI was 18.5 hours. No relationship was identified between cornea suitability for transplantation and DPI (P = 0.28) or donor gender (P = 0.54). There was a low microbial contamination rate (1%). Human immunodeficiency virus, hepatitis B, or hepatitis C serology was positive in 48 donors (4%). Overall, 90% of corneas were suitable for transplantation with a high utilization rate (88%). A novel association was identified between male sex and lower corneal endothelial cell density (P = 0.03). This New Zealand National Eye Bank analysis identified trends in the acquisition, storage, and utilization of donated corneal tissue throughout New Zealand over the past decade and provides valuable additional information to the international eye bank data.
Upregulated epidermal growth factor receptor expression following near-infrared irradiation simulating solar radiation in a three-dimensional reconstructed human corneal epithelial tissue culture model
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Tanaka Y
Full Text Available Yohei Tanaka,1,2 Jun Nakayama2 1Department of Plastic Surgery, Clinica Tanaka Plastic, Reconstructive Surgery and Anti-aging Center, 2Department of Molecular Pathology, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan Background and objective: Humans are increasingly exposed to near-infrared (NIR radiation from both natural (eg, solar and artificial (eg, electrical appliances sources. Although the biological effects of sun and ultraviolet (UV exposure have been extensively investigated, the biological effect of NIR radiation is still unclear. We previously reported that NIR as well as UV induces photoaging and standard UV-blocking materials, such as sunglasses, do not sufficiently block NIR. The objective of this study was to investigate changes in gene expression in three-dimensional reconstructed corneal epithelial tissue culture exposed to broad-spectrum NIR irradiation to simulate solar NIR radiation that reaches human tissues.Materials and methods: DNA microarray and quantitative real-time polymerase chain reaction analysis were used to assess gene expression levels in a three-dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water-filtered broad-spectrum NIR irradiation with a contact cooling (20°C. The water-filter allowed 1,000–1,800 nm wavelengths and excluded 1,400–1,500 nm wavelengths.Results: A DNA microarray with >62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold, respectively, after NIR irradiation. In particular, epidermal growth factor receptor (EGFR was upregulated by 19.4-fold relative to control cells. Quantitative real-time polymerase chain reaction analysis revealed that two variants of EGFR in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 J/cm2 irradiation (P<0.05.Conclusion: We found that NIR irradiation induced the
Human β-Defensin 3 Reduces TNF-α-Induced Inflammation and Monocyte Adhesion in Human Umbilical Vein Endothelial Cells
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Tianying Bian
Full Text Available The aim of this study was to investigate the role of human β-defensin 3 (hBD3 in the initiation stage of atherosclerosis with human umbilical vein endothelial cells (HUVECs triggered by tumor necrosis factor- (TNF- α. The effects of hBD3 on TNF-α-induced endothelial injury and inflammatory response were evaluated. Our data revealed that first, hBD3 reduced the production of interleukin-6 (IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage migration inhibitory factor (MIF in HUVECs in a dose-dependent manner. In addition, hBD3 significantly prevented intracellular reactive oxygen species (ROS production by HUVECs. Second, western blot analysis demonstrated that hBD3 dose-dependently suppressed the protein levels of intracellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1 in TNF-α-induced HUVECs. As a result, hBD3 inhibited monocyte adhesion to TNF-α-treated endothelial cells. Additionally, hBD3 suppressed TNF-α-induced F-actin reorganization in HUVECs. Third, hBD3 markedly inhibited NF-κB activation by decreasing the phosphorylation of IKK-α/β, IκB, and p65 subunit within 30 min. Moreover, the phosphorylation of p38 and c-Jun N-terminal protein kinase (JNK in the mitogen-activated protein kinase (MAPK pathway were also inhibited by hBD3 in HUVECs. In conclusion, hBD3 exerts anti-inflammatory and antioxidative effects in endothelial cells in response to TNF-α by inhibiting NF-κB and MAPK signaling.
α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.
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Chih-Ping Chung
Full Text Available Endothelial cells (ECs express fibroblast growth factor (FGF receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs and human brain microvascular endothelial cells (HBMECs were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS expression, nitric oxide (NO production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.
RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.
Alghanem, Ahmad F; Wilkinson, Emma L; Emmett, Maxine S; Aljasir, Mohammad A; Holmes, Katherine; Rothermel, Beverley A; Simms, Victoria A; Heath, Victoria L; Cross, Michael J
Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.
Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent
James, Daylon; Nam, Hyung-song; Seandel, Marco; Nolan, Daniel; Janovitz, Tyler; Tomishima, Mark; Studer, Lorenz; Lee, Gabsang; Lyden, David; Benezra, Robert; Zaninovic, Nikica; Rosenwaks, Zev; Rabbany, Sina Y; Rafii, Shahin
Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application. PMID:20081865
Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle
DEFF Research Database (Denmark)
Høier, Birgitte; Prats Gavalda, Clara; Qvortrup, Klaus
The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations...... regions and between the contractile elements within the muscle fibers; and in pericytes situated on the skeletal muscle capillaries. Quantitation of the subsarcolemmal density of VEGF vesicles, calculated on top of myonuclei, in the muscle fibers revealed a ∼50% increase (P...
Transcriptome of E. coli K1 bound to human brain microvascular endothelial cells
Xie, Yi; Parthasarathy, Geetha; Di Cello, Francescopaolo; Teng, Ching-Hao; Paul-Satyaseela, Maneesh; Kim, Kwang Sik
Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis. Binding to human brain microvascdular endothelial cells (HBMEC) is an essential step for E. coli K1 traversal of the blood-brain barrier. In this study, we examined expression profiles of E. coli K1 strain RS218 during its binding to HBMEC. Comparison of HBMEC-bound E. coli K1 with collagen-bound E. coli revealed more than one hundred genes whose expression patterns were significantly changed in HBMEC-b...
Sprouting angiogenesis in human midterm uterus and fallopian tube is guided by endothelial tip cells.
Rusu, M C; Motoc, A G M; Pop, F; Folescu, R
Five samples of human midterm fetal uterus and fallopian tube (four donor bodies) were used to assess whether or not processes of angiogenesis are guided by endothelial tip cells (ETCs), and if cytokine-receptors, such as CD117/c-kit and PDGFR-α, are expressed in the microenvironment of the endothelial tubes. CD34 labeled microvessels in the uterine wall (myometrium and endometrium) and in the wall of the uterine (fallopian) tube, and accurately identified ETCs in both organs. We conclude that sprouting angiogenesis in the developing human female tract is guided by ETCs. Moreover, CD117/c-kit antibodies labeled mural networks of pericytes, α-SMA-positive and desmin-negative, related to the endometrial (but not myometrial) microvessels, and similar labeling was identified in the wall of the uterine tube. PDGFR-α positive labeling, stromal and pericytary, was also found. Thus, sprouting angiogenesis in human fetal genital organs appears to be guided by tip cells and is influenced by tyrosine kinase receptor signaling.
Human Skin Constructs with Spatially Controlled Vasculature Using Primary and iPSC-Derived Endothelial Cells.
Abaci, Hasan E; Guo, Zongyou; Coffman, Abigail; Gillette, Brian; Lee, Wen-Han; Sia, Samuel K; Christiano, Angela M
Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival, functionality, and viable engraftment. In this study, the latest microfabrication techniques are used and a novel bioengineering approach is established to micropattern spatially controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells. Using 3D printing technology makes it possible to control the geometry of the micropatterned vascular networks. It is verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function, which allows for the recapitulation of both topical and systemic delivery of drugs. In addition, the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice is examined and it is demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall, this innovative bioengineering approach can enable in vitro evaluation of topical and systemic drug delivery as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Binding of tissue plasminogen activator to human umbilical vein endothelial cells
International Nuclear Information System (INIS)
Beebe, D.P.
The binding of purified, recombinant tissue plasminogen activator (tPA) to human umbilical vein endothelial cells (HUVEC) was studied in vitro using immunofluorescence as well as radiolabeled tPA. Immunofluorescence was performed on HUVEC grown on round glass coverslips using rabbit anti-human tPA and fluorescein-conjugated anti-rabbit immunoglobulin. Positive fluorescence was observed only after incubation of HUVEC with tPA. HUVEC were grown to confluence in 24-well tissue culture plates, washed, and incubated with a constant amount of 125 I-tPA and various concentrations of unlabeled tPA. The binding of tPA to HUVEC was found to be specific, saturable, and reversible. Scatchard analysis yielded as equilibrium constant (K/sub eq/) of 4.2 x 10 6 M -1 and 1.2 x 10 7 binding sites per cell. Binding was inhibited by positively charged amino acids and by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone but not by carbohydrates including mannose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. Neat human plasma abrogates but does not totally inhibit binding of tPA to HUVEC. Binding was neither enhanced nor inhibited by fibronectin. Although the affinity of binding of tPA to HUVEC is low, the endothelial cell may be involved in regulating plasma levels of tPA in vivo which may have therapeutic significance
Human haemodynamic frequency harmonics regulate the inflammatory phenotype of vascular endothelial cells.
Feaver, Ryan E; Gelfand, Bradley D; Blackman, Brett R
Haemodynamic variations are inherent to blood vessel geometries (such as bifurcations) and correlate with regional development of inflammation and atherosclerosis. However, the complex frequency spectrum characteristics from these haemodynamics have never been exploited to test whether frequency variations are critical determinants of endothelial inflammatory phenotype. Here we utilize an experimental Fourier transform analysis to systematically manipulate individual frequency harmonics from human carotid shear stress waveforms applied in vitro to human endothelial cells. The frequency spectrum, specifically the 0 th and 1st harmonics, is a significant regulator of inflammation, including NF-κB activity and downstream inflammatory phenotype. Further, a harmonic-based regression-model predicts eccentric NF-κB activity observed in the human internal carotid artery. Finally, short interfering RNA-knockdown of the mechanosensor PECAM-1 reverses frequency-dependent regulation of NF-κB activity. Thus, PECAM-1 may have a critical role in the endothelium's exquisite sensitivity to complex shear stress frequency harmonics and provide a mechanism for the focal development of vascular inflammation.
Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells.
Yamanegi, Koji; Kawabe, Mutsuki; Futani, Hiroyuki; Nishiura, Hiroshi; Yamada, Naoko; Kato-Kogoe, Nahoko; Kishimoto, Hiromitsu; Yoshiya, Shinichi; Nakasho, Keiji
The level of vascular endothelial growth inhibitor (VEGI) has been reported to be negatively associated with neovascularization in malignant tumors. The soluble form of VEGI is a potent anti-angiogenic factor due to its effects in inhibiting endothelial cell proliferation. This inhibition is mediated by death receptor 3 (DR3), which contains a death domain in its cytoplasmic tail capable of inducing apoptosis that can be subsequently blocked by decoy receptor 3 (DcR3). We investigated the effects of sodium valproate (VPA) and trichostatin A (TSA), histone deacetylase inhibitors, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Consequently, treatment with VPA and TSA increased the VEGI and DR3 expression levels without inducing DcR3 production in the OS cell lines. In contrast, the effect on the HMVE cells was limited, with no evidence of growth inhibition or an increase in the DR3 and DcR3 expression. However, VPA-induced soluble VEGI in the OS cell culture medium markedly inhibited the vascular tube formation of HMVE cells, while VEGI overexpression resulted in enhanced OS cell death. Taken together, the HDAC inhibitor has anti-angiogenesis and antitumor activities that mediate soluble VEGI/DR3-induced apoptosis via both autocrine and paracrine pathways. This study indicates that the HDAC inhibitor may be exploited as a therapeutic strategy modulating the soluble VEGI/DR3 pathway in osteosarcoma patients.
A biomimetic physiological model for human adipose tissue by adipocytes and endothelial cell cocultures with spatially controlled distribution
International Nuclear Information System (INIS)
Yao, Rui; Zhang, Renji; Lin, Feng; Du, Yanan; Luan, Jie
An in vitro model that recapitulates the characteristics of native human adipose tissue would largely benefit pathology studies and therapy development. In this paper, we fabricated a physiological model composed of both human adipocytes and endothelial cells with spatially controlled distribution that biomimics the structure and composition of human adipose tissue. Detailed studies into the cell–cell interactions between the adipocytes and endothelial cells revealed a mutual-enhanced effect which resembles the in vivo routine. Furthermore, comparisons between planar coculture and model coculture demonstrated improved adipocyte function as well as endothelial cell proliferation under the same conditions. This research provided a reliable model for human adipose tissue development studies and potential obesity-related therapy development. (paper)
Saccharomyces cerevisiae show low levels of traversal across human endothelial barrier in vitro [version 2; referees: 2 approved
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Roberto Pérez-Torrado
Full Text Available Background: Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans. Methods: In this work, we studied S. cerevisiae isolates in an in vitro human endothelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. Results: The results showed that this food related yeast is able to cross the endothelial barrier in vitro. However, in contrast to C. glabrata and C. albicans, S. cerevisiae showed very low levels of traversal. Conclusions: We conclude that using an in vitro human endothelial barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods.
Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.
Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C
The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.
Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells
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Gao, Zhong-Xiu-Zi [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Huang, Da-Yong [Department of Oncology, The Second Clinical Hospital, Harbin Medical University, Harbin (China); Li, Hai-Xia; Zhang, Li-Na; Lv, Yan-Hong; Cui, Hai-Dong [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Zheng, Jin-Hua, E-mail: firstname.lastname@example.com [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China)
Research highlights: {yields} It has been shown that scutellarin exhibits a variety of pharmacological actions, including anti-oxidative, anti-inflammatory, vasodilator as well as cardiovascular and cerebrovascular ischemia protective effects, indicating beneficial vascular effects of scutellarin. Therefore, it is speculated that scutellarin may be able to stimulate angiogenesis, which could be beneficial in the treatment of ischemic disease, wound healing and tissue regeneration. {yields} The purpose of the present study was to elucidate the direct angiogenic actions of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. {yields} Our results showed that scutellarin to directly induce in vitro angiogenesis, which is closely correlated with upregulated MMP-2 expression, suggesting a potential for increasing angiogenesis. -- Abstract: Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin's effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin's angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly
The cytotoxicity evaluation of magnetic iron oxide nanoparticles on human aortic endothelial cells
Ge, Gaoyuan; Wu, Hengfang; Xiong, Fei; Zhang, Yu; Guo, Zhirui; Bian, Zhiping; Xu, Jindan; Gu, Chunrong; Gu, Ning; Chen, Xiangjian; Yang, Di
One major obstacle for successful application of nanoparticles in medicine is its potential nanotoxicity on the environment and human health. In this study, we evaluated the cytotoxicity effect of dimercaptosuccinic acid-coated iron oxide (DMSA-Fe2O3) using cultured human aortic endothelial cells (HAECs). Our results showed that DMSA-Fe2O3 in the culture medium could be absorbed into HAECs, and dispersed in the cytoplasm. The cytotoxicity effect of DMSA-Fe2O3 on HAECs was dose-dependent, and the concentrations no more than 0.02 mg/ml had little toxic effect which were revealed by tetrazolium dye assay. Meanwhile, the cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without DMSA-Fe2O3). However, the endocrine function for endothelin-1 and prostacyclin I-2, as well as the urea transporter function, was altered even without obvious evidence of cell injury in this context. We also showed by real-time PCR analysis that DMSA-Fe2O3 exposure resulted in differential effects on the expressions of pro- and anti-apoptosis genes of HAECs. Meanwhile, it was noted that DMSA-Fe2O3 exposure could activate the expression of genes related to oxidative stress and adhesion molecules, which suggested that inflammatory response might be evoked. Moreover, we demonstrated by in vitro endothelial tube formation that even a small amount of DMSA-Fe2O3 (0.01 and 0.02 mg/ml) could inhibit angiogenesis by the HAECs. Altogether, these results indicate that DMSA-Fe2O3 have some cytotoxicity that may cause side effects on normal endothelial cells.
Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.
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Philip E Morgan
Full Text Available Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH. We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC were incubated with high glucose (25 mM, 24 h, 37°C, or methylglyoxal (MGO, glyoxal, or glycolaldehyde (100-500 µM, 1 h, 37°C, before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05 decreased total thiols (∼35%, further experiments with MGO showed significant losses of GSH (∼40% and NADPH (∼10%; these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10% NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE formed; lower levels of N(ε-(carboxyethyllysine (CEL and N(ε-(carboxymethyllysine (CML were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.
Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells
International Nuclear Information System (INIS)
Gao, Zhong-Xiu-Zi; Huang, Da-Yong; Li, Hai-Xia; Zhang, Li-Na; Lv, Yan-Hong; Cui, Hai-Dong; Zheng, Jin-Hua
Research highlights: → It has been shown that scutellarin exhibits a variety of pharmacological actions, including anti-oxidative, anti-inflammatory, vasodilator as well as cardiovascular and cerebrovascular ischemia protective effects, indicating beneficial vascular effects of scutellarin. Therefore, it is speculated that scutellarin may be able to stimulate angiogenesis, which could be beneficial in the treatment of ischemic disease, wound healing and tissue regeneration. → The purpose of the present study was to elucidate the direct angiogenic actions of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. → Our results showed that scutellarin to directly induce in vitro angiogenesis, which is closely correlated with upregulated MMP-2 expression, suggesting a potential for increasing angiogenesis. -- Abstract: Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin's effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin's angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and m
Identification and characterization of angiogenesis targets through proteomic profiling of endothelial cells in human cancer tissues.
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Mehdi Mesri
Full Text Available Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.
The coffee diterpene kahweol inhibits tumor necrosis factor-α-induced expression of cell adhesion molecules in human endothelial cells
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Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil; Lee, Kyung Jin; Lee, Kwang Youl; Kim, Dong Hee; Kim, Dong Hyun; Jeong, Hye Gwang
Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNFα-induced monocytes to endothelial cells and suppressed the TNFα-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNFα-induced JAK2-PI3K/Akt-NF-κB activation pathway in these cells. Overall, kahweol has anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells
Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C
Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.
An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of
Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells
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Preis, M.; Cohen, T.; Sarnatzki, Y.; Ben Yosef, Y.; Schneiderman, J.; Gluzman, Z.; Koren, B.; Lewis, B.S.; Shaul, Y.; Flugelman, M.Y.
Background: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF 165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. Methods and results: Fibulin-5 and VEGF 165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24 h after EC seeding. The effects of fibulin-5 and VEGF 165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF 165 co-expression ameliorated this effect. Conclusion: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF 165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses
Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells
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Mishima Satoshi
Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ, bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs. Methods In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. Results RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Conclusion Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.
Human aortic endothelial cell morphology influenced by topography of porous silicon substrates.
Formentín, Pilar; Catalán, Úrsula; Fernández-Castillejo, Sara; Alba, Maria; Baranowska, Malgorzata; Solà, Rosa; Pallarès, Josep; Marsal, Lluís F
Porous silicon has received much attention because of its optical properties and for its usefulness in cell-based biosensing, drug delivery, and tissue engineering applications. Surface properties of the biomaterial are associated with cell adhesion and with proliferation, migration, and differentiation. The present article analyzes the behavior of human aortic endothelial cells in macro- and nanoporous collagen-modified porous silicon samples. On both substrates, cells are well adhered and numerous. Confocal microscopy and scanning electron microscopy were employed to study the effects of porosity on the morphology of the cells. On macroporous silicon, filopodia is not observed but the cell spreads on the surface, increasing the lamellipodia surface which penetrates the macropore. On nanoporous silicon, multiple filopodia were found to branch out from the cell body. These results demonstrate that the pore size plays a key role in controlling the morphology and growth rate of human aortic endothelial cells, and that these forms of silicon can be used to control cell development in tissue engineering as well as in basic cell biology research. © The Author(s) 2015.
Pharmacological activities of an eye drop containing Matricaria chamomilla and Euphrasia officinalis extracts in UVB-induced oxidative stress and inflammation of human corneal cells.
Bigagli, Elisabetta; Cinci, Lorenzo; D'Ambrosio, Mario; Luceri, Cristina
Ultraviolet B (UVB) exposure is a risk factor for corneal damage resulting in oxidative stress, inflammation and cell death. The aim of this study was to investigate the potential protective effects of a commercial eye drop (Dacriovis™) containing Matricaria chamomilla and Euphrasia officinalis extracts on human corneal epithelial cells (HCEC-12) against UVB radiation-induced oxidative stress and inflammation as well as the underlying mechanisms. The antioxidant potential of the eye drops was evaluated by measuring the ferric reducing antioxidant power and the total phenolic content by Folin-Ciocalteu reagent. HCEC-12 cells were exposed to UVB radiation and treated with the eye drops at various concentrations. Cell viability, wound healing assay, reactive oxygen species (ROS) levels, protein and lipid oxidative damage and COX-2, IL-1β, iNOS, SOD-2, HO-1 and GSS gene expression, were assessed. Eye drops were able to protect corneal epithelial cells from UVB-induced cell death and ameliorated the wound healing; the eye drops exhibited a strong antioxidant activity, decreasing ROS levels and protein and lipid oxidative damage. Eye drops also exerted anti-inflammatory activities by decreasing COX-2, IL-1β, iNOS expression, counteracted UVB-induced GSS and SOD-2 expression and restored HO-1 expression to control levels. These findings suggest that an eye drop containing Matricaria chamomilla and Euphrasia officinalis extracts exerts positive effects against UVB induced oxidative stress and inflammation and may be useful in protecting corneal epithelial cells from UVB exposure. Copyright © 2017 Elsevier B.V. All rights reserved.
Therapeutic Effects of Topical Netrin-4 Inhibits Corneal Neovascularization in Alkali-Burn Rats
Han, Yun; Shao, Yi; Liu, Tingting; Qu, Yang-Luowa; Li, Wei; Liu, Zuguo
Netrins are secreted molecules involved in axon guidance and angiogenesis. However, the role of netrins in the vasculature remains unclear. Netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors. Previously, we found that netrin-1 acts as an anti-angiogenic factor in rats by inhibiting alkali burn-induced corneal neovascularization. Here, we further investigate the effects of netrin-4, another member of the same netrin family, on neovascularization in vitro and in vivo. We found that netrin-4 functions similarly as netrin-1 in angiogenesis. In vitro angiogenesis assay shows that netrin-4 affected human umbilical vein endothelial cell (HUVEC) tube formation, viability and proliferation, apoptosis, migration, and invasion in a dose-dependent manner. Netrin-4 was topically applied in vivo to alkali-burned rat corneas on day 0 (immediately after injury) and/or day 10 post-injury. Netrin-4 subsequently suppressed and reversed corneal neovascularization. Netrin-4 inhibited corneal epithelial and stromal cell apoptosis, inhibited vascular endothelial growth factor (VEGF), but promoted pigment epithelium-derived factor (PEDF) expression, decreased NK-KB p65 expression, and inhibits neutrophil and macrophage infiltration. These results indicate that netrin-4 shed new light on its potential roles in treatmenting for angiogenic diseases that affect the ocular surface, as well as other tissues. PMID:25853509
The Role of Titanium Surface Microtopography on Adhesion, Proliferation, Transformation, and Matrix Deposition of Corneal Cells.
Zhou, Chengxin; Lei, Fengyang; Chodosh, James; Paschalis, Eleftherios I
Titanium (Ti) is an excellent implantable biomaterial that can be further enhanced by surface topography optimization. Despite numerous data from orthopedics and dentistry, the effect of Ti surface topography on ocular cells is still poorly understood. In light of the recent adaptation of Ti in the Boston Keratoprosthesis artificial cornea, we attempted to perform an extended evaluation of the effect of Ti surface topography on corneal cell adhesion, proliferation, cytotoxicity, transformation, and matrix deposition. Different surface topographies were generated on medical grade Ti-6Al-4V-ELI (extra-low interstitial), with linearly increased roughness (polished to grit blasted). Biological response was evaluated in vitro using human corneal limbal epithelial (HCLE) cells, stromal fibroblasts (HCF), and endothelial cells (HCEnC). None of the Ti surface topographies caused cytotoxicity to any of the three corneal cell types. However, rough Ti surface inhibited HCLE and HCF cell adhesion and proliferation, while HCEnC proliferation was unaffected. Long-term experiments with HCF revealed that rough Ti surface with R(a) (the arithmetic average of the profile height from the mean line) ≥ 1.15 μm suppressed HCF focal adhesion kinase phosphorylation, changed fibroblast morphology, and caused less aligned and reduced deposition of collagen matrix as compared to smooth Ti (R(a) ≤ 0.08 μm). In the presence of transforming growth factor β1 (TGFβ1) stimulation, rough Ti inhibited alpha-smooth muscle actin (α-SMA) expression and collagen deposition, leading to decreased myofibroblast transformation and disorganization of the collagen fibrils as compared to smooth Ti. This study suggests that Ti surface topography regulates corneal cell behavior in a tissue-dependent manner that varies across the corneal strata. Contrary to the accepted paradigm, smooth surface topography can enhance cell adhesion and proliferation and increase matrix deposition by corneal cells.
Immunohistochemical markers for corneal stem cells in the early developing human eye
DEFF Research Database (Denmark)
Lyngholm, Mikkel; Høyer, Poul E; Vorum, Henrik
niche (week 14) in human embryos and fetuses. The expression of SOD2 and CK15 was investigated together with other recently identified limbal proteins. Previously suggested LSC and differentiation markers (PAX6, aquaporin-1 and nestin) were also investigated. Both SOD2 and CK15 were present...... markers and potential markers for LSCs and early transient amplifying cells in human adults. In this study, we describe the development of the ectodermally derived LSCs and the mesodermally derived niche cells from the time at which the cornea is defined (week 6) until the formation of the early limbal...
Effect of peritoneal dialysis fluid containing osmo-metabolic agents on human endothelial cells
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Bonomini M
Full Text Available Mario Bonomini,1,2 Sara Di Silvestre,3,4 Pamela Di Tomo,3,4 Natalia Di Pietro,2,4 Domitilla Mandatori,3,4 Lorenzo Di Liberato,1 Vittorio Sirolli,1,2 Francesco Chiarelli,2,4 Cesare Indiveri,5 Assunta Pandolfi,3,4 Arduino Arduini6 1Unit of Nephrology and Dialysis, 2Department of Medicine and Aging Sciences, 3Department of Medical, Oral and Biotechnological Sciences, 4Aging Research Center and Translational Medicine, CeSI-MeT, University "G. d'Annunzio", Chieti-Pescara, 5Department DiBEST (Biologia, Ecologia, Scienze della Terra, Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende CS, Italy; 6CoreQuest, Manno, Switzerland Background: The use of glucose as the only osmotic agent in peritoneal dialysis (PD solutions (PDSs is believed to exert local (peritoneal and systemic detrimental actions, particularly in diabetic PD patients. To improve peritoneal biocompatibility, we have developed more biocompatible PDSs containing xylitol and carnitine along with significantly less amounts of glucose and have tested them in cultured Human Vein Endothelial Cells (HUVECs obtained from the umbilical cords of healthy (C and gestational diabetic (GD mothers. Methods: Primary C- and GD-HUVECs were treated for 72 hours with our PDSs (xylitol 0.7% and 1.5%, whereas carnitine and glucose were fixed at 0.02% and 0.5%, respectively and two glucose-based PDSs (glucose 1.36% or 2.27%. We examined their effects on endothelial cell proliferation (cell count, viability (3-(4,5-dimethylthiazolyl-2-2,5-diphenyltetrazolium bromide assay, intracellular nitro-oxidative stress (peroxynitrite levels, Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 membrane exposure (flow cytometry, and HUVEC-monocyte interactions (U937 adhesion assay. Results: Compared to glucose-based PDSs, our in vitro studies demonstrated that the tested PDSs did not change the proliferative potential both in C- and GD-HUVECs. Moreover, our
In Vitro Model for Predicting the Protective Effect of Ultraviolet-Blocking Contact Lens in Human Corneal Epithelial Cells.
Abengózar-Vela, Antonio; Arroyo, Cristina; Reinoso, Roberto; Enríquez-de-Salamanca, Amalia; Corell, Alfredo; González-García, María Jesús
To develop an in vitro method to determine the protective effect of UV-blocking contact lenses (CLs) in human corneal epithelial (HCE) cells exposed to UV-B radiation. SV-40-transformed HCE cells were covered with non-UV-blocking CL, UV-blocking CL or not covered, and exposed to UV-B radiation. As control, HCE cells were covered with both types of CLs or not covered, but not exposed to UV-B radiation. Cell viability at 24, 48 and 72 h, after UV-B exposure and removing CLs, was determined by alamarBlue(®) assay. Percentage of live, dead and apoptotic cells was also assessed by flow cytometry after 24 h of UV-B exposure. Intracellular reactive oxygen species (ROS) production after 1 h of exposure was assessed using the dye H(2)DCF-DA. Cell viability significantly decreased, apoptotic cells and intracellular ROS production significantly increased when UVB-exposed cells were covered with non-UV-blocking CL or not covered compared to non-irradiated cells. When cells were covered with UV-blocking CL, cell viability significantly increased and apoptotic cells and intracellular ROS production did not increase compared to exposed cells. UV-B radiation induces cell death by apoptosis, increases ROS production and decreases viable cells. UV-blocking CL is able to avoid these effects increasing cell viability and protecting HCE cells from apoptosis and ROS production induced by UV-B radiation. This in vitro model is an alternative to in vivo methods to determine the protective effect of UV-blocking ophthalmic biomaterials because it is a quicker, cheaper and reliable model that avoids the use of animals.
The preservative polyquaternium-1 increases cytoxicity and NF-kappaB linked inflammation in human corneal epithelial cells
Paimela, Tuomas; Ryhänen, Tuomas; Kauppinen, Anu; Marttila, Liisa; Salminen, Antero
Purpose In numerous clinical and experimental studies, preservatives present in eye drops have had detrimental effects on ocular epithelial cells. The aim of this study was to compare the cytotoxic and inflammatory effects of the preservative polyquaternium-1 (PQ-1) containing Travatan (travoprost 0.004%) and Systane Ultra eye drops with benzalkonium chloride (BAK) alone or BAK-preserved Xalatan (0.005% latanoprost) eye drops in HCE-2 human corneal epithelial cell culture. Methods HCE-2 cells were exposed to the commercial eye drops Travatan, Systane Ultra, Xalatan, and the preservative BAK. Cell viability was determined using colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by release of lactate dehydrogenase (LDH). Induction of apoptosis was measured with a using a colorimetric caspase-3 assay kit. DNA binding of the nuclear factor kappa B (NF-κB) transcription factor, and productions of the proinflammatory cytokines, interleukins IL-6 and IL-8, were determined using an enzyme-linked immunosorbent assay (ELISA) method. Results Cell viability, as measured by the MTT assay, declined by up to 50% after exposure to Travatan or Systane Ultra solutions which contain 0.001% PQ-1. BAK at 0.02% rather than at 0.001% concentration evoked total cell death signs on HCE-2 cells. In addition, cell membrane permeability, as measured by LDH release, was elevated by sixfold with Travatan and by a maximum threefold with Systane Ultra. Interestingly, Travatan and Systane Ultra activated NF-κB and elevated the secretion of inflammation markers IL-6 by 3 to eightfold and IL-8 by 1.5 to 3.5 fold, respectively, as analyzed with ELISA. Conclusions Eye drops containing PQ-1 evoke cytotoxicity and enhance the NF-κB driven inflammation reaction in cultured HCE-2 cells. Our results indicate that these harmful effects of ocular solutions preserved with PQ-1 should be further evaluated in vitro and in vivo. PMID:22605930
Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.
Mukhey, Dev; Phillips, James B; Daniels, Julie T; Kureshi, Alvena K
The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils
HJURP regulates cellular senescence in human fibroblasts and endothelial cells via a p53-dependent pathway.
Heo, Jong-Ik; Cho, Jung Hee; Kim, Jae-Ryong
Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.
Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells.
Wieghaus, Kristen A; Gianchandani, Erwin P; Neal, Rebekah A; Paige, Mikell A; Brown, Milton L; Papin, Jason A; Botchwey, Edward A
We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode of action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray data set of PNF1-treated (vs. control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-alpha) and, subsequently, transforming growth factor-beta (TGF-beta) signaling networks by PNF1. Here we validate this microarray data set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this data set and identify three specific TGF-beta-induced genes with regulation by PNF1 conserved over multiple timepoints-amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)-that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1-48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode of action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.
Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels
Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.
We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319
Effect of a rosmarinic acid supplemented hemodialysis fluid on inflammation of human vascular endothelial cells
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W-J. Wang
Full Text Available Chronic systemic inflammation and repetitive damage of vascular endothelia by incompatible dialysis system are probable causes of cardiovascular disease in patients on dialysis. The present study aimed to assess in vitro biocompatibility and anti-inflammatory effect of hemodialysis fluid supplemented with rosmarinic acid (RA using human umbilical vein endothelial cells (HUVEC. HUVECs (5×106 cells/mL were pre-exposed to 1 μg/mL of lipopolysaccharides (LPS and incubated with RA-supplemented hemodialysis fluid (HDF. Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by MTT assay. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR and production of NO was quantified. Phosphorylation of AKT and nuclear localization of nuclear factor kappa B (NF-κB were examined using western blotting. Exposure of HUVECs to RA-supplemented HDF had no influence on morphology and viability. Inhibition of proinflammatory mediator production in HUVECs by RA supplementation to HDF was significant in a dose-dependent manner. Exposure to RA-supplemented HDF resulted in a decrease in nitric oxide synthase expression and reduction of NO production in LPS-stimulated HUVECs. RA supplementation of HDF suppressed Akt activation in LPS-stimulated HUVECs. In addition, the level of cellular IκB was increased in parallel to a reduced nuclear translocation of NF-κB in LPS-induced endothelial cells. Our results suggest that RA-supplemented HDF is biocompatible and significantly suppressed inflammation induced in endothelial cells. In this respect, the use of HDF supplemented with RA could alleviate inflammation and improve long-term treatment of patients with renal failure on dialysis. Further clinical studies are required to confirm the effects.
Differential sex-specific effects of oxygen toxicity in human umbilical vein endothelial cells
International Nuclear Information System (INIS)
Zhang, Yuhao; Lingappan, Krithika
Despite the well-established sex-specific differences in the incidence of bronchopulmonary dysplasia (BPD), the molecular mechanism(s) behind these are not completely understood. Pulmonary angiogenesis is critical for alveolarization and arrest in vascular development adversely affects lung development. Human neonatal umbilical vein endothelial cells (HUVECs) provide a robust in vitro model for the study of endothelial cell physiology and function. Male and Female HUVECs were exposed to room air (21% O 2 , 5% CO 2 ) or hyperoxia (95% O 2 , 5% CO 2 ) for up to 72 h. Cell viability, proliferation, H 2 O 2 production and angiogenesis were analyzed. Sex-specific differences in the expression of VEGFR2 and modulation of NF-kappa B pathway were measured. Male HUVECs have decreased survival, greater oxidative stress and impairment in angiogenesis compared to similarly exposed female cells. There is differential expression of VEGFR2 between male and female HUVECs and greater activation of the NF-kappa B pathway in female HUVECs under hyperoxic conditions. The results indicate that sex differences exist between male and female HUVECs in vitro after hyperoxia exposure. Since endothelial dysfunction has a major role in the pathogenesis of BPD, these differences could explain in part the mechanisms behind sex-specific differences in the incidence of this disease. - Highlights: • Cellular sex effects viability and oxidative stress in HUVECs exposed to hyperoxia. • Male HUVECs show greater impairment in angiogenesis compared to female cells. • Sex-specific modulation of VEGFR2 and the NF-kappaB pathway was noted.
Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.
Sato, Hiromi; Coburn, Jenifer
Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways
Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.
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Hiromi Sato
Full Text Available Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1 extracellular matrix, 2 intercellular adhesion molecules and cell surface receptors, 3 intracellular proteins, 4 cell-cell junction proteins, and 5 a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or
Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells
Sato, Hiromi
Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways
Mitotic and antiapoptotic effects of nanoparticles coencapsulating human VEGF and human angiopoietin-1 on vascular endothelial cells
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Khan AA
Full Text Available Afshan Afsar Khan, Arghya Paul, Sana Abbasi, Satya PrakashBiomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering Faculty of Medicine, McGill University Montreal, Québec, CanadaBackground: Research towards the application of nanoparticles as carrier vehicles for the delivery of therapeutic agents is increasingly gaining importance. The angiogenic growth factors, human vascular endothelial growth factor (VEGF and human angiopoietin-1 are known to prevent vascular endothelial cell apoptosis and in fact to stimulate human vascular endothelial cell (HUVEC proliferation. This paper aims to study the combined effect of these bioactive proteins coencapsulated in human serum albumin nanoparticles on HUVECs and to evaluate the potential application of this delivery system towards therapeutic angiogenesis.Methods and results: The angiogenic proteins, human VEGF and human angiopoietin-1, were coencapsulated in albumin nanoparticles for better controlled delivery of the proteins. The application of a nanoparticle system enabled efficient and extended-release kinetics of the proteins. The size of the nanoparticles crosslinked with glutaraldehyde was 101.0 ± 0.9 nm and the zeta potential was found to be -18 ± 2.9 mV. An optimal concentration of glutaraldehyde for the nanoparticle coating process was determined, and this provided stable and less toxic nanoparticles as protein carriers. The results of the study indicate that nanoparticles crosslinked with glutaraldehyde produced nanoparticles with tolerable toxicity which provided efficient and controlled release of the coencapsulated proteins. The nanoparticles were incubated for two weeks to determine the release profiles of the proteins. At the end of the two-week incubation period, it was observed that 49% ± 1.3% of human angiopoietin-1 and 59% ± 2.1% of human VEGF had been released from the nanoparticles. The proliferation and percent apoptosis of the HUVECs in
Human iPSC-Derived Endothelial Cells and Microengineered Organ-Chip Enhance Neuronal Development
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Samuel Sances
Full Text Available Summary: Human stem cell-derived models of development and neurodegenerative diseases are challenged by cellular immaturity in vitro. Microengineered organ-on-chip (or Organ-Chip systems are designed to emulate microvolume cytoarchitecture and enable co-culture of distinct cell types. Brain microvascular endothelial cells (BMECs share common signaling pathways with neurons early in development, but their contribution to human neuronal maturation is largely unknown. To study this interaction and influence of microculture, we derived both spinal motor neurons and BMECs from human induced pluripotent stem cells and observed increased calcium transient function and Chip-specific gene expression in Organ-Chips compared with 96-well plates. Seeding BMECs in the Organ-Chip led to vascular-neural interaction and specific gene activation that further enhanced neuronal function and in vivo-like signatures. The results show that the vascular system has specific maturation effects on spinal cord neural tissue, and the use of Organ-Chips can move stem cell models closer to an in vivo condition. : Sances et al. combine Organ-Chip technology with human induced pluripotent stem cell-derived spinal motor neurons to study the maturation effects of Organ-Chip culture. By including microvascular cells also derived from the same patient line, the authors show enhancement of neuronal function, reproduction of vascular-neuron pathways, and specific gene activation that resembles in vivo spinal cord development. Keywords: organ-on-chip, spinal cord, iPSC, disease modeling, amyotrophic lateral sclerosis, microphysiological system, brain microvascular endothelial cells, spinal motor neurons, vasculature, microfluidic device
Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis
International Nuclear Information System (INIS)
Sun, Hui-Yan; Wei, Shu-Ping; Xu, Rui-Cheng; Xu, Peng-Xiao; Zhang, Wen-Cheng
Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.
Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis
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Sun, Hui-Yan, E-mail: firstname.lastname@example.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Wei, Shu-Ping, E-mail: email@example.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Xu, Rui-Cheng, E-mail: firstname.lastname@example.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Xu, Peng-Xiao, E-mail: email@example.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Zhang, Wen-Cheng, E-mail: firstname.lastname@example.com [Department of Physiology and Pathophysiology, Medical College of Chinese People' s Armed Police Forces, Tianjin 300162 (China)
Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.
Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.
Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S
Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.
Endothelial cell chimerism associated with graft rejection after human lung transplantation.
Ratajczak , Philippe; Murata , Hideyuki; Meignin , Véronique; Groussard , Odile; Fournier , Michel; Socié , Gérard; Mal , Hervé; Janin , Anne
International audience; Endotheliitis is a major sign of graft rejection. Recipient-derived endothelial cells found in two series of liver and kidney transplants were related to graft rejection. Here, we assessed the presence and the number of chimeric endothelial cells in lung transplants, and their relation with graft rejection. In six males grafted with female lungs out of 193 lung transplantations, endothelial chimerism was studied by combined XY-fluorescent in situ hybridization with CD3...
[The effects of intraocular irrigating solutions on the human corneal endothelium (author's transl)].
Weekers, J F; Dethinne, M
Human corneas from enucleated eyes get thicker during perfusion with B.S.S. On the contrary, their thickness decreases when perfused with T.C. Earle solution. Addition of reduced glutathion and adenosine does not change the results obtained with T.C. Earle. Histological study of the endothelium after a 24 hours perfusion demonstrates a better conservation of the cells with T.C. Earle and T.C. Earle glutathion--adenosine than with B.S.S.
Endothelial differentiation of human stem cells seeded onto electrospun polyhydroxybutyrate/polyhydroxybutyrate-co-hydroxyvalerate fiber mesh.
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Alessandra Zonari
Full Text Available Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM. Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF, the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in
Endothelial differentiation of human stem cells seeded onto electrospun polyhydroxybutyrate/polyhydroxybutyrate-co-hydroxyvalerate fiber mesh.
Zonari, Alessandra; Novikoff, Silviene; Electo, Naira R P; Breyner, Natália M; Gomes, Dawidson A; Martins, Albino; Neves, Nuno M; Reis, Rui L; Goes, Alfredo M
Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with
Regulation of thrombomodulin expression and release in human aortic endothelial cells by cyclic strain.
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Fiona A Martin
Full Text Available Thrombomodulin (TM, an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain intrinsic to endothelial-mediated vascular homeostasis.This study employed human aortic endothelial cells (HAECs to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs, and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs, on TM expression and release. Time-, dose- and frequency-dependency studies were performed.Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0-7.5% and frequency (0.5-2.0 Hz dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2, receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml and ox-LDL (10-50 µg/ml appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001 nor rhomboids (3,4-dichloroisocoumarin had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs.A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM
The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles
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Guillermo Bodega
Full Text Available We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs and their microvesicles (MVs. Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.
Medroxyprogesterone acetate attenuates estrogen-induced nitric oxide production in human umbilical vein endothelial cells
International Nuclear Information System (INIS)
Oishi, Akira; Ohmichi, Masahide; Takahashi, Kazuhiro; Takahashi, Toshifumi; Mori-Abe, Akiko; Kawagoe, Jun; Otsu, Reiko; Mochizuki, Yoshiko; Inaba, Noriyuki; Kurachi, Hirohisa
We report the novel observation that medroxyprogesterone acetate (MPA) attenuates the induction by 17β estradiol (E2) of both nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells. Although MPA had no effect on basal NO production or basal eNOS phosphorylation or activity, it attenuated the E2-induced NO production and eNOS phosphorylation and activity. Moreover, we examined the mechanism by which MPA attenuated the E2-induced NO production and eNOS phosphorylation. MPA attenuated the E2-induced phosphorylation of Akt, a kinase that phosphorylates eNOS. Treatment with pure progesterone receptor (PR) antagonist RU486 completely abolished the inhibitory effect of MPA on E2-induced Akt phosphorylation and eNOS phosphorylation. In addition, the effects of actinomycin D were tested to rule out the influence of genomic events mediated by nuclear PRs. Actinomycin D did not affect the inhibitory effect of MPA on E2-induced Akt phosphorylation. Furthermore, the potential roles of PRA and PRB were evaluated. In COS cells transfected with either PRA or PRB, MPA attenuated E2-induced Akt phosphorylation. These results indicate that MPA attenuated E2-induced NO production via an Akt cascade through PRA or PRB in a non-genomic manner
Thioredoxin reductase 1 upregulates MCP-1 release in human endothelial cells
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Liu, Zhen-Bo [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China); Shen, Xun, E-mail: firstname.lastname@example.com [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China)
To know if thioredoxin reductase 1 (TrxR1) plays a role in antioxidant defense mechanisms against atherosclerosis, effect of TrxR1 on expression/release of monocyte chemoattractant protein (MCP-1) was investigated in activated human endothelial-like EAhy926 cells. The MCP-1 release and expression, cellular generation of reactive oxygen species (ROS), nuclear translocation and DNA-binding activity of NF-{kappa}B subunit p65 were assayed in cells either overexpressing recombinant TrxR1 or having their endogenous TrxR1 knocked down. It was found that overexpression of TrxR1 enhanced, while knockdown of TrxR1 reduced MCP-1 release and expression. Upregulation of MCP-1 by TrxR1 was associated with increasing generation of intracellular ROS generation, enhanced nuclear translocation and DNA-binding activity of NF-{kappa}B. Assay using NF-{kappa}B reporter revealed that TrxR1 upregulated transcriptional activity of NF-{kappa}B. This study suggests that TrxR1 enhances ROS generation, NF-{kappa}B activity and subsequent MCP-1 expression in endothelial cells, and may promote rather than prevent vascular endothelium from forming atherosclerotic plaque.
LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells
Figarola, James L.; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay
Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis. PMID:24615331
Novel effects of edaravone on human brain microvascular endothelial cells revealed by a proteomic approach.
Onodera, Hidetaka; Arito, Mitsumi; Sato, Toshiyuki; Ito, Hidemichi; Hashimoto, Takuo; Tanaka, Yuichiro; Kurokawa, Manae S; Okamoto, Kazuki; Suematsu, Naoya; Kato, Tomohiro
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a free radical scavenger used for acute ischemic stroke. However, it is not known whether edaravone works only as a free radical scavenger or possess other pharmacological actions. Therefore, we elucidated the effects of edaravone on human brain microvascular endothelial cells (HBMECs) by 2 dimensional fluorescence difference gel electrophoresis (2D-DIGE). We found 38 protein spots the intensity of which was significantly altered 1.3 fold on average (pedaravone treatment and successfully identified 17 proteins of those. Four of those 17 proteins were cytoskeleton proteins or cytoskeleton-regulating proteins. Therefore, we subsequently investigated the change of size and shape of the cells, the actin network, and the tight junction of HBMEC by immunocytochemistry. As a result, most edaravone-treated HBMECs became larger and rounder compared with those that were not treated. Furthermore, edaravone-treated HBMECs formed gathering zona occludens (ZO)-1, a tight junction protein, along the junction of the cells. In addition, we found that edaravone suppressed interleukin (IL)-1β-induced secretion of monocyte chemoattractant protein-1 (MCP-1), which was reported to increase cell permeability. We found a novel function of edaravone is the promotion of tight junction formations of vascular endothelial cells partly via the down-regulation of MCP-1 secretion. These data provide fundamental and useful information in the clinical use of edaravone in patients with cerebral vascular diseases. © 2013 Elsevier B.V. All rights reserved.
Buddleja officinalis inhibits high glucose-induced matrix metalloproteinase activity in human umbilical vein endothelial cells.
Lee, Yun Jung; Kang, Dae Gill; Kim, Jin Sook; Lee, Ho Sub
The aim of the present investigation was to investigate whether an aqueous extract of Buddleja officinalis (ABO), a traditional Korean herbal medicine, suppresses the endothelial extracellular matrix degradation under high glucose condition. The incubation with high concentration of glucose (25 mM) increased significantly matrix metalloproteinase (MMP)-2/-9 expressions and activities in primary cultured human umbilical vein endothelial cells (HUVEC). Pretreatment with ABO decreased high glucose-induced increase of MMP-2/-9 activities in a dose-dependent manner. Real time qRT-PCR revealed that high glucose-induced MMP-2/-9 mRNA expression levels were attenuated by pretreatment with ABO. High glucose-induced MCP-1 and IL-8 mRNA expression levels also decreased by ABO. ABO decreased high glucose-induced hydrogen peroxide production, oxidative stress marker. These results provide new insights into the pathophysiological mechanisms for anti-inflammatory properties of ABO in vascular diseases associated with diabetes mellitus. (c) 2008 John Wiley & Sons, Ltd.
Anti-Inflammatory effect of Buddleja officinalis on vascular inflammation in human umbilical vein endothelial cells.
Lee, Yun Jung; Moon, Mi Kyoung; Hwang, Sun Mi; Yoon, Jung Joo; Lee, So Min; Seo, Kwan Soo; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub
Vascular inflammation process has been suggested to be an important risk factor in the initiation and development of atherosclerosis. In this study, we investigated whether and by what mechanisms an aqueous extract of Buddleja officinalis (ABO) inhibited the expressions of cellular adhesion molecules, which are relevant to inflammation and atherosclerosis. Pretreatment of human umbilical vein endothelial cells (HUVEC) with ABO (1-10 microg/ml) for 18 hours dose-dependently inhibited TNF-alpha-induced adhesion U937 monocytic cells, as well as mRNA and protein expressions of vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule-1 (ICAM-1). Pretreatment with ABO also blocked TNF-alpha-induced ROS formation. Nuclear factor-kappa B (NF-kappaB) is required in the transcription of these adhesion molecule genes. Western blot analysis revealed that ABO inhibits the translocation of the p65 subunit of NF-kappaB to the nucleus. ABO inhibited the TNF-alpha-induced degradation of IkappaB-alpha, an inhibitor of NF-kappaB, by inhibiting the phosphorylation of IkappaB-alpha in HUVEC. Taken together, ABO could reduce cytokine-induced endothelial adhesiveness throughout down-regulating intracellular ROS production, NF-kappaB, and adhesion molecule expression in HUVEC, suggesting that the natural herb Buddleja officinalis may have potential implications in atherosclerosis.
Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC Damages.
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Yumei Zhang
Full Text Available Here, we studied the underlying mechanism of aldosterone (Aldo-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L inhibited human umbilical vein endothelial cells (HUVEC survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18 production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P, an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS inhibitor PDMP or the ceramide (C6 potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1 is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.
Butein Inhibits Angiogenesis of Human Endothelial Progenitor Cells via the Translation Dependent Signaling Pathway
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Ching-Hu Chung
Full Text Available Compelling evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs can contribute to postnatal neovascularization and tumor angiogenesis. EPCs have been shown to play a "catalytic" role in metastatic progression by mediating the angiogenic switch. Understanding the pharmacological functions and molecular targets of natural products is critical for drug development. Butein, a natural chalcone derivative, has been reported to exert potent anticancer activity. However, the antiangiogenic activity of butein has not been addressed. In this study, we found that butein inhibited serum- and vascular endothelial growth factor- (VEGF- induced cell proliferation, migration, and tube formation of human EPCs in a concentration dependent manner without cytotoxic effect. Furthermore, butein markedly abrogated VEGF-induced vessels sprouting from aortic rings and suppressed microvessel formation in the Matrigel implant assay in vivo. In addition, butein concentration-dependently repressed the phosphorylation of Akt, mTOR, and the major downstream effectors, p70S6K, 4E-BP1, and eIF4E in EPCs. Taken together, our results demonstrate for the first time that butein exhibits the antiangiogenic effect both in vitro and in vivo by targeting the translational machinery. Butein is a promising angiogenesis inhibitor with the potential for treatment of cancer and other angiogenesis-related diseases.
A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.
Sengoelge, Guerkan; Winnicki, Wolfgang; Kupczok, Anne; von Haeseler, Arndt; Schuster, Michael; Pfaller, Walter; Jennings, Paul; Weltermann, Ansgar; Blake, Sophia; Sunder-Plassmann, Gere
Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.
HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells.
Nauta, Tessa D; Duyndam, Monique C A; Weijers, Ester M; van Hinsbergh, Victor M W; Koolwijk, Pieter
During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis. We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling. Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction. Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.
Efficient generation of endothelial cells from human pluripotent stem cells and characterization of their functional properties.
Song, Wei; Kaufman, Dan S; Shen, Wei
Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs), large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34, respectively, from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGFβ-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542 + hESC-ECs, SB431542 - hESC-ECs, and HUVECs showed similar permeability to 10,000 Da dextran, but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542 + hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542 - hESC-ECs and HUVECs responded differently to VEGF and bFGF, which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542 - hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 678-687, 2016. © 2015 Wiley Periodicals, Inc.
Evaluation of intracameral injection of ranibizumab and bevacizumab on the corneal endothelium by scanning electron microscopy.
Ari, Seyhmus; Nergiz, Yusuf; Aksit, Ihsan; Sahin, Alparslan; Cingu, Kursat; Caca, Ihsan
To evaluate the effects of intracameral injection of ranibizumab and bevacizumab on the corneal endothelium by scanning electron microscopy (SEM). Twenty-eight female rabbits were randomly divided into four equal groups. Rabbits in groups 1 and 2 underwent intracameral injection of 1 mg/0.1 mL and 0.5 mg/0.05 mL ranibizumab, respectively; group 3 was injected with 1.25 mg/0.05 mL bevacizumab. All three groups were injected with a balanced salt solution (BSS) into the anterior chamber of the left (fellow) eye. None of the rabbits in group 4 underwent an injection. Corneal thickness and intraocular pressure were measured before the injections, on the first day, and in the first month after injection. The rabbits were sacrificed and corneal tissues were excised in the first month after injection. Specular microscopy was used for the corneal endothelial cell count. Endothelial cell density was assessed and comparisons drawn between the groups and the control. Micrographs were recorded for SEM examination. The structure of the corneal endothelial cells, the junctional area of the cell membrane, the distribution of microvillus, and the cell morphology of the eyes that underwent intracameral injection of vascular endothelial growth factor (VEGF), BSS, and the control group were compared. Corneal thickness and intraocular pressure were not significantly different between the groups that underwent anti-VEGF or BSS injection and the control group on the first day and in the first month of injection. The corneal endothelial cell count was significantly diminished in all three groups; predominantly in group 1 and 2 (P<0.05). The SEM examination revealed normal corneal endothelial histology in group 3 and the control group. Eyes in group 1 exhibited indistinctness of corneal endothelial cell borders, microvillus loss in the luminal surface, excessive blebbing, and disintegration of intercellular junctions. In group 2, the cell structure of the corneal endothelium
Safety of Cultivated Limbal Epithelial Stem Cell Transplantation for Human Corneal Regeneration
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J. Behaegel
Full Text Available Ex vivo cultivated limbal stem cell transplantation is a promising technique for the treatment of limbal stem cell deficiency. While the results of the clinical trials have been extensively reported since the introduction of the technique in 1997, little has been reported regarding the potential health risks associated with production processes and transplantation techniques. Culture procedures require the use of animal and/or human-derived products, which carry the potential of introducing toxic or infectious agents through contamination with known or unknown additives. Protocols vary widely, and the risks depend on the local institutional methods. Good manufacturing practice and xeno-free culture protocols could reduce potential health risks but are not yet a common practice worldwide. In this review, we focus on the safety of both autologous- and allogeneic-cultivated limbal stem cell transplantation, with respect to culture processes, surgical approaches, and postoperative strategies.
Mechanisms of allograft rejection of corneal endothelium
International Nuclear Information System (INIS)
Tagawa, Y.; Silverstein, A.M.; Prendergast, R.A.
The local intraocular graft-vs.-host (GVH) reaction, involving the destruction of the corneal endothelial cells of the rabbit host by sensitized donor lymphoid cells, has been used to study the mechanism of corneal allograft rejection. Pretreatment of donor cells with a specific mouse monoclonal hybridoma anti-T cell antibody and complement suppresses the destructive reaction, suggesting that a cellular-immune mechanism is primarily involved. Pretreatment of donor cells with mitomycin-C completely abolishes the local GVH reaction, indicating that the effector lymphocytes must undergo mitosis within the eye before they can engage in target cell destruction. Finally, studies of the local GVH reaction in irradiated leukopenic recipients or in preinflamed rabbit eyes suggest that host leukocytes may contribute nonspecifically to enhance the destructive process. These studies show that the local ocular GVH reaction may provide a useful model for the study of the mechanisms involved in the rejection of corneal allografts
Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering.
Denecke, Bernd; Horsch, Liska D; Radtke, Stefan; Fischer, Johannes C; Horn, Peter A; Giebel, Bernd
One of the major challenges in tissue engineering is to supply larger three-dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)-derived endothelial colony-forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue-culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo-expanded ECFCs are a novel, very potent cell source for scaffold-based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.
Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice
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Takamitsu Sasaki
Full Text Available The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR and vascular endothelial growth factor receptor (VEGFR signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α and vascular endothelial growth factor (VEGF but were negative for EGFR, human epidermal growth factor receptor 2 (HER2, VEGFR. Double immunofluorescence staining revealed that tumorassociated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR, phosphorylated VEGFR (pVEGFR. Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01; this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001. AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, increased the level of apoptosis in both tumorassociated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.
Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.
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Yanwei Li
Full Text Available An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.
Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.
Li, Yanwei; Liu, Haifeng; Zeng, Wei; Wei, Jing
An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.
Antiangiogenic properties of cafestol, a coffee diterpene, in human umbilical vein endothelial cells
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Wang, Shuaiyu; Yoon, Yeo Cho; Sung, Mi-Jeong; Hur, Haeng-Jeon; Park, Jae-Ho
Highlights: ► Cafestol inhibits tube formation and migration of VEGF-stimulated HUVEC. ► Cafestol inhibits phosphorylation of FAK and Akt. ► Cafestol decreases NO production. -- Abstract: As angiogenesis plays important roles in tumor growth and metastasis, searching for antiangiogenic compounds is a promising tactic for treating cancers. Cafestol, a diterpene found mainly in unfiltered coffee, provides benefit through varied biological activity, including antitumorigenic, antioxidative, and anti-inflammatory effects. This study aimed to investigate the effects of cafestol on angiogenesis and to uncover the associated mechanism. We show that cafestol inhibits angiogenesis of human umbilical vascular endothelial cells. This inhibition affects the following specific steps of the angiogenic process: proliferation, migration, and tube formation. The inhibitory effects of cafestol are accompanied by decreasing phosphorylation of FAK and Akt and by a decrease in nitric oxide production. Overall, cafestol inhibits angiogenesis by affecting the angiogenic signaling pathway.
Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans
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Jinah Han
Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.
Development and Characterization of a Brain Endothelial Cell Phenotype using Human Induced Pluripotent Stem Cells
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Goldeman, Charlotte; Saaby, Lasse; Holst, Bjørn
for experiments the following day. The model was monitored by measuring the trans-endothelial electrical resistance (TEER). RA had an inductive effect on the model, shown by an elevation in barrier tightness which correlated with the presence of tight junction proteins, shown by confocal microscopy images which...... be used to investigate drug transport in vitro, and screen candidates for permeation properties. One recent approach is to develop in vitro models of the BBB using human induced pluripotent stem cells (hIPSCs) as described by Stebbins et al. (2015).The aim of the present study was to investigate whether...... the published protocols were generically applicable and thus to develop and characterize in vitro models of the BBB using hIPSCs from different sources. Two stem cell lines, Bioni010-C and WTSli024-A, were seeded and maintained on Matrigel in mTesR1 media. Cells were then seeded as single cells at different...
Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation
Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.
Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771
Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.
Hollmann, Emma K; Bailey, Amanda K; Potharazu, Archit V; Neely, M Diana; Bowman, Aaron B; Lippmann, Ethan S
Due to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods. Induced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines. The use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm 2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm 2 and fluorescein permeability below 1.95 × 10 -7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm 2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated
Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.
Chen, Dong-Bao; Feng, Lin; Hodges, Jennifer K; Lechuga, Thomas J; Zhang, Honghai
Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study
Behaviour of human endothelial cells on surface modified NiTi alloy.
Plant, Stuart D; Grant, David M; Leach, Lopa
Intravascular stents are being designed which utilise the shape memory properties of NiTi alloy. Despite the clinical advantages afforded by these stents their application has been limited by concerns about the large nickel ion content of the alloy. In this study, the surface chemistry of NiTi alloy was modified by mechanical polishing and oxidising heat treatments and subsequently characterised using X-ray photon spectroscopy (XPS). The effect of these surfaces on monolayer formation and barrier integrity of human umbilical vein endothelial cells (HUVEC) was then assessed by confocal imaging of the adherens junctional molecule VE-cadherin, perijunctional actin and permeability to 42kDa dextrans. Dichlorofluoroscein assays were used to measure oxidative stress in the cells. XPS analysis of NiTi revealed its surface to be dominated by TiO(2). However, where oxidation had occurred after mechanical polishing or post polishing heat treatments at 300 and 400 degrees C in air, a significant amount of metallic nickel or nickel oxide species (10.5 and 18.5 at%) remained on the surface. Exposure of HUVECs to these surfaces resulted in increased oxidative stress within the cells, loss of VE-cadherin and F-actin and significantly increased paracellular permeability. These pathological phenomena were not found in cells grown on NiTi which had undergone heat treatment at 600 degrees C. At this temperature thickening of the TiO(2) layer had occurred due to diffusion of titanium ions from the bulk of the alloy, displacing nickel ions to sub-surface areas. This resulted in a significant reduction in nickel ions detectable on the sample surface (4.8 at%). This study proposes that the integrity of human endothelial monolayers on NiTi is dependent upon the surface chemistry of the alloy and that this can be manipulated, using simple oxidising heat treatments.
Increased endothelial apoptotic cell density in human diabetic erectile tissue--comparison with clinical data.
Costa, Carla; Soares, Raquel; Castela, Angela; Adães, Sara; Hastert, Véronique; Vendeira, Pedro; Virag, Ronald
Erectile dysfunction (ED) is a common complication of diabetes. Endothelial cell (EC) dysfunction is one of the main mechanisms of diabetic ED. However, loss of EC integrity has never been assessed in human diabetic corpus cavernosum. To identify and quantify apoptotic cells in human diabetic and normal erectile tissue and to compare these results with each patient's clinical data and erection status. Eighteen cavernosal samples were collected, 13 from diabetics with ED and 5 from nondiabetic individuals. Cavernosal structure and cell proliferation status were evaluated by immunohistochemistry. Tissue integrity was assessed by terminal transferase dUTP nick end labeling assay, an index of apoptotic cell density (ACD) established and compared with each patient age, type of diabetes, arterial risk factors number, arterial/veno-occlusive disease, response to intracavernous vasoactive injections (ICI), and penile nitric oxide release test (PNORT). Establish an index of ACD and correlate those results with patient clinical data. Nondiabetic samples presented few scattered cells in apoptosis and an ACD of 7.15 +/- 0.44 (mean apoptotic cells/tissue area mm(2) +/- standard error). The diabetic group showed an increased ACD of 23.82 +/- 1.53, and apoptotic cells were located specifically at vascular sites. Rehabilitation of these endothelial lesions seemed impaired, as no evidence of EC proliferation was observed. Furthermore, higher ACD in diabetic individuals correlated to poor response to PNORT and to ICI. We provided evidence for the first time that loss of cavernosal EC integrity is a crucial event involved in diabetic ED. Furthermore, we were able to establish a threshold between ACD values and cavernosal tissue functionality, as assessed by PNORT and vasoactive ICI.
Human endothelial progenitor cells rescue cortical neurons from oxygen-glucose deprivation induced death.
Bacigaluppi, Susanna; Donzelli, Elisabetta; De Cristofaro, Valentina; Bragazzi, Nicola Luigi; D'Amico, Giovanna; Scuteri, Arianna; Tredici, Giovanni
Cerebral ischemia is characterized by both acute and delayed neuronal injuries. Neuro-protection is a major issue that should be properly addressed from a pharmacological point of view, and cell-based treatment approaches are of interest due to their potential pleiotropic effects. Endothelial progenitor cells have the advantage of being mobilized from the bone marrow into the circulation, but have been less studied than other stem cells, such as mesenchymal stem cells. Therefore, the comparison between human endothelial progenitor cells (hEPC) and human mesenchymal progenitor cells (hMSC) in terms of efficacy in rescuing neurons from cell death after transitory ischemia is the aim of the current study, in the effort to address further directions. In vitro model of oxygen-glucose deprivation (OGD) on a primary culture of rodent cortical neurons was set up with different durations of exposure: 1, 2 and 3hrs with assessment of neuron survival. The 2hrs OGD was chosen for the subsequent experiments. After 2hrs OGD neurons were either placed in indirect co-culture with hMSC or hEPC or cultured in hMSC or hEPC conditioned medium and cell viability was evaluated by MTT assay. At day 2 after 2hrs OGD exposure, mean neuronal survival was 47.9±24.2%. In contrast, after treatment with hEPC and hMSC indirect co-culture was 74.1±27.3%; and 69.4±18.8%, respectively. In contrast, treatment with conditioned medium did not provide any advantage in terms of survival to OGD neurons The study shows the efficacy of hEPC in indirect co-culture to rescue neurons from cell death after OGD, comparable to that of hMSC. hEPC deserve further studies given their potential interest for ischemia. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.
Kusters, B.; Waal, R.M.W. de; Wesseling, P.; Verrijp, K.; Maass, C.N.; Heerschap, A.; Barentsz, J.O.; Sweep, C.G.J.; Ruiter, D.J.; Leenders, W.P.J.
We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood
Effects of the PPARγ agonist troglitazone on endothelial cells in vivo and in vitro: Differences between human and mouse
International Nuclear Information System (INIS)
Kakiuchi-Kiyota, Satoko; Vetro, Joseph A.; Suzuki, Shugo; Varney, Michelle L.; Han, Huai-Yun; Nascimento, Merielen; Pennington, Karen L.; Arnold, Lora L.; Singh, Rakesh K.; Cohen, Samuel M.
Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists have been or are being developed for clinical use in the treatment of type 2 diabetes mellitus and hyperlipidemias. A common tumor finding in rodent carcinogenicity studies for these agonists is hemangioma/hemangiosarcoma in mice but not in rats. We hypothesized that increased endothelial cell proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice, and we investigated the effects on endothelial cells utilizing troglitazone, the first clinically used PPARγ agonist, in vivo and in vitro. Troglitazone (400 and 800 mg/kg/day) induced hemangiosarcomas in mice in a 2-year bioassay. We showed that troglitazone increased endothelial cell proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses after 4 weeks of treatment. Troglitazone was cytotoxic both to human dermal microvascular endothelial cells (HMEC1) and mouse mammary fat pad microvascular endothelial cells (MFP MVEC) at high concentrations. However, MFP MVEC were more resistant to the cytotoxic effects of troglitazone based on the much lower LC 50 in HMEC1 (17.4 μM) compared to MFP MVEC (92.2 μM). Troglitazone increased the proliferation and survival of MFP MVEC but not HMEC1 in growth factor reduced conditions. Our data demonstrate that troglitazone may induce hemangiosarcomas in mice, at least in part, through enhancement of survival and proliferation of microvascular endothelial cells. Such an effect does not occur with human cells, suggesting that human may react differently to exposure to PPAR agonists compared with mice.
Seasonal superoxide overproduction and endothelial activation in guinea-pig heart; seasonal oxidative stress in rats and humans.
Konior, Anna; Klemenska, Emilia; Brudek, Magdalena; Podolecka, Ewa; Czarnowska, Elżbieta; Beręsewicz, Andrzej
Seasonality in endothelial dysfunction and oxidative stress was noted in humans and rats, suggesting it is a common phenomenon of a potential clinical relevance. We aimed at studying (i) seasonal variations in cardiac superoxide (O(2)(-)) production in rodents and in 8-isoprostane urinary excretion in humans, (ii) the mechanism of cardiac O(2)(-) overproduction occurring in late spring/summer months in rodents, (iii) whether this seasonal O(2)(-)-overproduction is associated with a pro-inflammatory endothelial activation, and (iv) how the summer-associated changes compare to those caused by diabetes, a classical cardiovascular risk factor. Langendorff-perfused guinea-pig and rat hearts generated ~100% more O(2)(-), and human subjects excreted 65% more 8-isoprostane in the summer vs. other seasons. Inhibitors of NADPH oxidase, xanthine oxidase, and NO synthase inhibited the seasonal O(2)(-)-overproduction. In the summer vs. other seasons, cardiac NADPH oxidase and xanthine oxidase activity, and protein expression were increased, the endothelial NO synthase and superoxide dismutases were downregulated, and, in guinea-pig hearts, adhesion molecules upregulation and the endothelial glycocalyx destruction associated these changes. In guinea-pig hearts, the summer and a streptozotocin-induced diabetes mediated similar changes, yet, more severe endothelial activation associated the diabetes. These findings suggest that the seasonal oxidative stress is a common phenomenon, associated, at least in guinea-pigs, with the endothelial activation. Nonetheless, its biological meaning (regulatory vs. deleterious) remains unclear. Upregulated NADPH oxidase and xanthine oxidase and uncoupled NO synthase are the sources of the seasonal O(2)(-)-overproduction. Copyright © 2010 Elsevier Ltd. All rights reserved.
Degradation of endothelial basement membrane by human breast cancer cell lines
International Nuclear Information System (INIS)
Yee, C.; Shiu, R.P.
During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of [35S]methionine-labeled and [3H]proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer
Comparison of Endothelial Cell Loss by Specular Microscopy ...
African Journals Online (AJOL)
Endothelial cell loss was also compared in phacoemulsification group by temporal clear corneal incision (CCI) and by superior scleral incision (SI) technique. ..... vs manual sutureless small-incision extracapsular cataract surgery in Nepal.
The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells.
García-Morales, Verónica; Luaces-Regueira, María; Campos-Toimil, Manuel
3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca 2+ concentration ([Ca 2+ ] c ) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca 2+ ] c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca 2+ , was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca 2+ ] c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.
Effect of Interleukin-10 and Laminar Shear Stress on Endothelial Nitric Oxide Synthase and Nitric Oxide in African American Human Umbilical Vein Endothelial Cells.
Babbitt, Dianne M; Kim, Ji-Seok; Forrester, Steven J; Brown, Michael D; Park, Joon-Young
African Americans have a predisposition to heightened systemic inflammation and a high prevalence of hypertension. The purpose of this study was to evaluate the influence of interleukin-10 (IL-10) and laminar shear stress (LSS) on African American endothelial cells by measuring total endothelial nitric oxide synthase (eNOS) protein expression and its phosphorylated form (p-eNOS) at Serine 1177, and nitric oxide (NO) levels, in response to IL-10 incubation and high physiological levels of LSS, used as an in vitro mimetic for aerobic exercise training (AEXT). Human umbilical vein endothelial cells (HUVEC) from an African American donor were cultured. The experimental conditions included Static, Static with IL-10 Incubation, LSS at 20 dynes/cm², and LSS at 20 dynes/cm² with IL-10 Incubation. Western blotting was used to measure eNOS and p-eNOS protein expression in the cells. A modified Griess assay was used to measure NO metabolites in the cell culture media. There were significant increases in p-eNOS, eNOS, and NO in the LSS at 20 dynes/cm² and LSS at 20 dynes/cm² with IL-10 Incubation experimental conditions when compared to the Static experimental condition. There were no other statistically significant differences demonstrating that IL-10 did not have an additive effect on eNOS activity in our study. The significant increases in p-eNOS, eNOS, and NO as a result of LSS in African American HUVECs suggest that AEXT may be a viable, nonpharmacologic method to improve vascular inflammation status and vasodilation, and thereby contribute to hypertension reduction in the African American population.
Angiogenesis in mucous retention cyst: a human in vivo-like model of endothelial cell differentiation in mucous substrate.
Swelam, Wael; Ida-Yonemochi, Hiroko; Saku, Takashi
Mucous retention cysts contain a mucous pool in the lumina, in which pure angiogenic processes are occasionally observed. By using this unique human material, our aim was to understand the in vivo angiogenic process. Fifteen surgical tissue samples of mucous retention cysts of the lip were examined for expression of vascular endothelial markers and extracellular matrix molecules by immunohistochemistry and in situ hybridization (ISH). Endothelial cells forming new vascular channels showed immunopositivities for CD31, CD34, vascular endothelial growth factor (VEGF), and von Willebrand factor (vWF). These newly formed capillaries were surrounded by tenascin-positive matrices and further by a dense infiltration of CD68-positive cells with foamy to epitheloid appearances. Some of these cells were simultaneously positive for CD34, VEGF, and one of its receptors, Flk-1, and they showed definite mRNA as well as protein signals for tenascin. In addition, these cells often tended to be aligned, which suggested tubule formation. The results suggest that monocyte/macrophage lineage cells are a major source for endothelial cells at least in mucous retention cysts and that tenascin produced by those cells plays an important role in differentiation of endothelial cells.
Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).
Meinert, Christian; Gembardt, Florian; Böhme, Ilka; Tetzner, Anja; Wieland, Thomas; Greenberg, Barry; Walther, Thomas
The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like "Molecular Mechanism of Cancer" and "p53 signaling" in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.
Equine corneal stromal abscesses
DEFF Research Database (Denmark)
Henriksen, M. D. L.; Andersen, P. H.; Plummer, C. E.
The last 30 years have seen many changes in the understanding of the pathogenesis and treatment of equine corneal stromal abscesses (SAs). Stromal abscesses were previously considered an eye problem related to corneal bacterial infection, equine recurrent uveitis, corneal microtrauma and corneal....... Medical and surgical treatments are now directed towards elimination of fungal and bacterial infections, reduction and replacement of diseased corneal stroma, and suppression of iridocyclitis. If the abscess and anterior uveitis do not respond satisfactorily to medical therapy, full thickness or split...
Carvacrol promotes angiogenic paracrine potential and endothelial differentiation of human mesenchymal stem cells at low concentrations.
Matluobi, Danial; Araghi, Atefeh; Maragheh, Behnaz Faramarzian Azimi; Rezabakhsh, Aysa; Soltani, Sina; Khaksar, Majid; Siavashi, Vahid; Feyzi, Adel; Bagheri, Hesam Saghaei; Rahbarghazi, Reza; Montazersaheb, Soheila
Phenolic monoterpene compound, named Carvacrol, has been found to exert different biological outcomes. It has been accepted that the angiogenic activity of human mesenchymal stem cells was crucial in the pursuit of appropriate regeneration. In the current experiment, we investigated the contribution of Carvacrol on the angiogenic behavior of primary human mesenchymal stem cells. Mesenchymal stem cells were exposed to Carvacrol in a dose ranging from 25 to 200μM for 48h. We measured cell survival rate by MTT assay and migration rate by a scratch test. The oxidative status was monitored by measuring SOD, GPx activity. The endothelial differentiation was studied by evaluating the level of VE-cadherin and vWF by real-time PCR and ELISA analyses. The content of VEGF and tubulogenesis behavior was monitored in vitro. We also conducted Matrigel plug in vivo CAM assay to assess the angiogenic potential of conditioned media from human mesenchymal stem cells after exposure to Carvacrol. Carvacrol was able to increase mesenchymal stem cell survival and migration rate (pcells by detecting vWF and VE-cadherin expression (pmesenchymal stem cells conditioned media improved angiogenesis tube formation in vitro (pmesenchymal stem cells by modulating cell differentiation and paracrine angiogenic response. Copyright © 2017 Elsevier Inc. All rights reserved.
In vitro model for human endothelial cell seeding of a small diameter vascular graft
International Nuclear Information System (INIS)
Kent, K.C.; Oshima, A.; Ikemoto, T.; Whittemore, A.D.
A precise system was devised to measure the kinetics of attachment of human venous endothelium to a variety of materials and substrates. Cells were labelled in a postconfluent state with tritiated thymidine, harvested, and a cell suspension seeded into a 4 mm PTFE graft. After a 90 minute incubation period, one half of the graft segment was sacrificed and the remaining portion placed in a perfusion system (225 cc/min) for 1 hour. Graft segments, effluents, and seeding suspension were assayed in a beta scintillation counter. The percentage of cells that attached pre- and postperfusion were determined, as well as the retrieval of tritium from the system. Initially, 71% of seeded cells attached to grafts coated with fibronectin, with significantly less (60%) remaining attached after perfusion. Only 10% of cells initially attached to uncoated grafts, with 4% retained postperfusion. Retrieval of tritium averaged 102 +/- 10% for all experiments. This system determines both pre- and postperfusion attachment of human endothelial cells to vascular grafts following manipulation of numerous variables, including graft material, substrate, incubation time, and seeding density. An optimal seeding protocol for human trials can thus be determined
Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.
Lee, Kwang Hoon; Chung, Hae-Shin; Kim, Hyoung Sup; Oh, Sang-Ho; Ha, Moon-Kyung; Baik, Ja-Hyun; Lee, Sungnack; Bang, Dongsik
To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.
cGMP and nitric oxide modulate thrombin-induced endothelial permeability : Regulation via different pathways in human aortic and umbilical vein endothelial cells
Draijer, R.; Atsma, D.E.; Laarse, A. van der; Hinsbergh, V.W.M. van
Previous studies have demonstrated that cGMP and cAMP reduce the endothelial permeability for fluids and macromolecules when the endothelial permeability is increased by thrombin. In this study, we have investigated the mechanism by which cGMP improves the endothelial barrier function and examined
Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells.
Dong, Lixue; Krewson, Elizabeth A; Yang, Li V
Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the "Warburg effect"), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4- induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.
Alcohol enhances oxysterol-induced apoptosis in human endothelial cells by a calcium-dependent mechanism.
Spyridopoulos, I; Wischhusen, J; Rabenstein, B; Mayer, P; Axel, D I; Fröhlich, K U; Karsch, K R
Controversy exists about the net effect of alcohol on atherogenesis. A protective effect is assumed, especially from the tannins and phenolic compounds in red wine, owing to their inhibition of low density lipoprotein (LDL) oxidation. However, increased atherogenesis occurs in subjects with moderate to heavy drinking habits. The purpose of this study was to investigate the influence of alcohol in combination with oxysterols on the endothelium. Cultured human arterial endothelial cells (HAECs) served as an in vitro model to test the cellular effects of various oxysterols. Oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and cholesterol-5,6-epoxides), which are assumed to be the most toxic constituents of oxidized LDL, induced apoptosis in HAECs through calcium mobilization followed by activation of caspase-3. Ethanol, methanol, isopropanol, tert-butanol, and red wine all potentiated oxysterol-induced cell death up to 5-fold, paralleled by further induction of caspase-3. The alcohol effect occurred in a dose-dependent manner and reached a plateau at 0.05% concentration. Alcohol itself did not affect endothelial cell viability, nor did other solvents such as dimethyl sulfoxide mimic the alcohol effect. So far as the physiologically occurring oxysterols are concerned, this effect was apparent only for oxysterols oxidized at the steran ring. The possibility of alcohol facilitating the uptake of oxysterols into the cell was not supported by the data from an uptake study with radiolabeled compounds. Finally, alcohol in combination with oxysterols did cause a dramatic increase in cytosolic calcium influx. Blockage of calcium influx by the calcium channel blocker aurintricarboxylic acid or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abrogated the alcohol-mediated enhancement of oxysterol toxicity. We describe for the first time a mechanistic concept explaining possible adverse effects of alcohol in conjunction with
Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime
Directory of Open Access Journals (Sweden)
M. Mathy-Hartert
Full Text Available We investigated the effects of the antibiotic ceftazidime (CAZ on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−. In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC were capable of taking up active MPO. In presence of H2O2 (10−4 M, this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC. Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2− generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2 was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon. So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.
The ADMA/DDAH/NO pathway in human vein endothelial cells exposed to arsenite.
Osorio-Yáñez, Citlalli; Chin-Chan, Miguel; Sánchez-Peña, Luz C; Atzatzi-Aguilar, Octavio G; Olivares-Reyes, Jesus A; Segovia, José; Del Razo, Luz M
Inorganic arsenic (iAs) exposure is related to cardiovascular disease, which is characterized by endothelial dysfunction and nitric oxide (NO) depletion. The mechanisms underlying NO depletion as related to iAs exposure are not fully understood. The endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), might be a molecular target of iAs. ADMA concentrations are regulated by proteins involved in its synthesis (arginine methyl transferase 1 [PRMT-1]) and degradation (dimethylarginine dimethylaminohydrolase [DDAH]). Both, ADMA and NO are susceptible to oxidative stress. We aimed to determine the ADMA/DDAH/NO pathway in human vein endothelial cells (HUVEC-CS) exposed to arsenite. We exposed HUVEC-CS cells to 1, 2.5 and 5μM of arsenite for 24h. We proved that arsenite at 5μM was able to decrease NO levels with an associated increase in ADMA and depletion of l-arginine in HUVEC-CS cells. We also found a decrease in DDAH-1 protein expression with 5μM of arsenite compared to the control group. However, we did not observe significant differences in PRMT-1 protein expression at any of the concentrations of arsenite employed. Finally, arsenite (2.5 and 5μM) increased NADPH oxidase 4 protein levels compared with the control group. We conclude that ADMA, l-arginine and DDAH are involved in NO depletion produced by arsenite, and that the mechanism is related to oxidative stress. Copyright © 2017 Elsevier Ltd. All rights reserved.
Biodegradable and biocompatible poly(ethylene glycol)-based hydrogel films for the regeneration of corneal endothelium.
Ozcelik, Berkay; Brown, Karl D; Blencowe, Anton; Ladewig, Katharina; Stevens, Geoffrey W; Scheerlinck, Jean-Pierre Y; Abberton, Keren; Daniell, Mark; Qiao, Greg G
Corneal endothelial cells (CECs) are responsible for maintaining the transparency of the human cornea. Loss of CECs results in blindness, requiring corneal transplantation. In this study, fabrication of biocompatible and biodegradable poly(ethylene glycol) (PEG)-based hydrogel films (PHFs) for the regeneration and transplantation of CECs is described. The 50-μm thin hydrogel films have similar or greater tensile strengths to human corneal tissue. Light transmission studies reveal that the films are >98% optically transparent, while in vitro degradation studies demonstrate their biodegradation characteristics. Cell culture studies demonstrate the regeneration of sheep corneal endothelium on the PHFs. Although sheep CECs do not regenerate in vivo, these cells proliferate on the films with natural morphology and become 100% confluent within 7 d. Implantation of the PHFs into live sheep corneas demonstrates the robustness of the films for surgical purposes. Regular slit lamp examinations and histology of the cornea after 28 d following surgery reveal minimal inflammatory responses and no toxicity, indicating that the films are benign. The results of this study suggest that PHFs are excellent candidates as platforms for the regeneration and transplantation of CECs as a result of their favorable biocompatibility, degradability, mechanical, and optical properties. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.
Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard
Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.
Calcimimetic R-568 and its enantiomer S-568 increase nitric oxide release in human endothelial cells.
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Mario Bonomini
Full Text Available Calcimimetics, such as R-568, are thought to activate G protein-linked Ca(2+-sensing receptor (CaSR by allosterically increasing the affinity of the receptor for Ca(2+ allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects.Focusing on human umbilical vein endothelial cells (HUVECs, we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca(2+ levels (using a single cell approach and FURA-2AM, in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO by DAF-2DA, by Nitric Oxide Synthase (NOS radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting. We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca(2+ levels by mobilization of Ca(2+ from intracellular stores, which in turn augmented NO release by a time- and Ca(2+-dependent increase in eNOS-ser1177 phosphorylation levels.Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional
Expression of Eph receptor tyrosine kinases and their ligands in human Granulosa lutein cells and human umbilical vein endothelial cells.
Xu, Y; Zagoura, D; Keck, C; Pietrowski, D
Corpus luteum development is regulated by gonadotropins and accompanied by extremely rapid vascularization of the avascular granulosa cell compartiment by endothelial cells (EC). The proliferation of Granulosa cells (GC) and EC is a complex interplay and takes place in a spatially and temporarily coordinated manner. The erythropoietin-producing hepatoma amplified sequence (Eph) receptors and their ligands-the ephrins- are a recently detected family of membrane located protein tyrosine kinases which play a crucial role in the growth and development of nerve and blood vessel network. We report about the mRNA expression pattern of Ephs and their ligands in human GC, in human EC, and in carcinoma cell lines OvCar-3 and Hela. The mRNA of EphA4, EphA7, ephrinA4, ephrinB1 and ephrinB2 was detected in GC and EC, while EphA2 was expressed only in GC. The expression of various Ephs and ephrins did not change in GC after stimulation with human chorion gonadotropin. Our study analyzes for the first time the expression of the complete human Eph/ephriny-system in GC and in EC. The remarkable similarity between these two cell types supports the theory of a functional relationship of EC and GC. In addition, it was shown that hCG is not a major determinant of Eph/ephrin regulation in GC.
Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells.
Ugusman, Azizah; Zakaria, Zaiton; Hui, Chua Kien; Nordin, Nor Anita Megat Mohd
Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). HUVECS WERE DIVIDED INTO FOUR GROUPS: control, treatment with 180 microM hydrogen peroxide (H(2)O(2)), treatment with 150 microg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H(2)O(2) for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.
Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells
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Azizah Ugusman
Full Text Available OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs. METHODS: HUVECs were divided into four groups: control, treatment with 180 μM hydrogen peroxide (H2O2, treatment with 150 μg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.
Cultured human astrocytes secrete large cholesteryl ester- andtriglyceride-rich lipoproteins along with endothelial lipase
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Yang, Lin; Liu, Yanzhu; Forte, Trudy M.; Chisholm, Jeffrey W.; Parks, John S.; Shachter, Neil S.
We cultured normal human astrocytes and characterized their secreted lipoproteins. Human astrocytes secreted lipoproteins in the size range of plasma VLDL (Peak 1), LDL (Peak 2), HDL (Peak 3) and a smaller peak (Peak 4), as determined by gel filtration chromatography, nondenaturing gradient gel electrophoresis and transmission electron microscopy. Cholesterol enrichment of astrocytes led to a particular increase in Peak 1. Almost all Peak 2, 3 and 4 cholesterol and most Peak 1 cholesterol was esterified (unlike mouse astrocyte lipoproteins, which exhibited similar peaks but where cholesterol was predominantly non-esterified). Triglycerides were present at about 2/3 the level of cholesterol. LCAT was detected along with two of its activators, apolipoprotein (apo) A-IV and apoC-I. ApoA-I and apoA-II mRNA and protein were absent. ApoJ was present equally in all peaks but apoE was present predominantly in peaks 3 and 4. ApoB was not detected. The electron microscopic appearance of Peak 1 lipoproteins suggested partial lipolysis leading to the detection of a heparin-releasable triglyceride lipase consistent with endothelial lipase. The increased neuronal delivery of lipids from large lipoprotein particles, for which apoE4 has greater affinity than does apoE3, may be a mechanism whereby the apoE {var_epsilon}4 allele contributes to neurodegenerative risk.
Analysis of Drug Effects on Primary Human Coronary Artery Endothelial Cells Activated by Serum Amyloid A
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K. Lakota
Full Text Available Background. RA patients have a higher incidence of cardiovascular diseases compared to the general population. Serum amyloid A (SAA is an acute-phase protein, upregulated in sera of RA patients. Aim. To determine the effects of medications on SAA-stimulated human coronary artery endothelial cells (HCAEC. Methods. HCAEC were preincubated for 2 h with medications from sterile ampules (dexamethasone, methotrexate, certolizumab pegol, and etanercept, dissolved in medium (captopril or DMSO (etoricoxib, rosiglitazone, meloxicam, fluvastatin, and diclofenac. Human recombinant apo-SAA was used to stimulate HCAEC at a final 1000 nM concentration for 24 hours. IL-6, IL-8, sVCAM-1, and PAI-1 were measured by ELISA. The number of viable cells was determined colorimetrically. Results. SAA-stimulated levels of released IL-6, IL-8, and sVCAM-1 from HCAEC were significantly attenuated by methotrexate, fluvastatin, and etoricoxib. Both certolizumab pegol and etanercept significantly decreased PAI-1 by an average of 43%. Rosiglitazone significantly inhibited sVCAM-1 by 58%. Conclusion. We observed marked influence of fluvastatin on lowering cytokine production in SAA-activated HCAEC. Methotrexate showed strong beneficial effects for lowering released Il-6, IL-8, and sVCAM-1. Interesting duality was observed for NSAIDs, with meloxicam exhibiting opposite-trend effects from diclofenac and etoricoxib. This represents unique insight into specific responsiveness of inflammatory-driven HCAEC relevant to atherosclerosis.
Tyrosol and its metabolites as antioxidative and anti-inflammatory molecules in human endothelial cells.
Muriana, Francisco J G; Montserrat-de la Paz, Sergio; Lucas, Ricardo; Bermudez, Beatriz; Jaramillo, Sara; Morales, Juan C; Abia, Rocio; Lopez, Sergio
Tyrosol (Tyr) is a phenolic compound found in virgin olive oil. After ingestion, Tyr undergoes extensive first pass intestinal/hepatic metabolism. However, knowledge about the biological effects of Tyr metabolites is scarce. We chemically synthesized Tyr glucuronate (Tyr-GLU) and sulphate (Tyr-SUL) metabolites and explored their properties against oxidative stress and inflammation in TNF-α-treated human umbilical vein endothelial cells (hECs). Tyr and Tyr-SUL prevented the rise of reactive oxygen species, the depletion of glutathione, and the down-regulation of glutathione peroxidase 1, glutamate-cysteine ligase catalytic subunit, and heme oxygenase-1 genes. Tyr-SUL and to a lower extent Tyr and Tyr-GLU prevented the phosphorylation of NF-κB signaling proteins. Tyr-GLU and Tyr-SUL also prevented the over-expression of adhesion molecules at gene, protein, and secretory levels, and the adhesion (Tyr-SUL > Tyr-GLU) of human monocytes to hECs. In vivo, Tyr, and most notably Tyr-SUL in a dose-dependent manner, ameliorated plantar and ear edemas in mice models of acute and chronic inflammation. This study demonstrates the antioxidant and/or anti-inflammatory properties of Tyr metabolites, with Tyr-SUL being the most effective.
Corneal collagen crosslinking for keratoconus. A review
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M. M. Bikbov
Full Text Available Photochemical crosslinking is widely applied in ophthalmology. Its biochemical effect is due to the release of singlet oxygen that promotes anaerobic photochemical reaction. Keratoconus is one of the most common corneal ectasia affecting 1 in 250 to 250 000 persons. Currently, the rate of iatrogenic ectasia following eximer laser refractive surgery increases due to biomechanical weakening of the cornea. Morphologically and biochemically, ectasia is characterized by corneal layers thinning, contact between the stroma and epithelium resulting from Bowman's membrane rupture, chromatin fragmentation in keratocyte nuclei, phagocytosis, abnormal staining and arrangement of collagen fibers, enzyme system disorders, and keratocyte apoptosis. In corneal ectasia, altered enzymatic processes result in the synthesis of abnormal collagen. Collagen packing is determined by the activity of various extracellular matrix enzymes which bind amines and aldehydes of collagen fiber amino acids. In the late stage, morphological changes of Descemet's membrane (i.e., rupture and detachment develop. Abnormal hexagonal-shaped keratocytes and their apoptosis are the signs of endothelial dystrophy. The lack of analogs in domestic ophthalmology encouraged the scientists of Ufa Eye Research Institute to develop a device for corneal collagen crosslinking. The parameters of ultraviolet (i.e., wavelength, exposure time, power to achieve the desired effect were identified. The specifics of some photosensitizers in the course of the procedure were studied. UFalink, a device for UV irradiation of cornea, and photosensitizer Dextralink were developed and adopted. Due to the high risk of endothelial damage, this treatment is contraindicated in severe keratoconus (CCT less than 400 microns. Major effects of corneal collagen crosslinking are the following: Young's modulus (modulus of elasticity increase by 328.9 % (on average, temperature tolerance increase by 5�
Functional activities of receptors for tumor necrosis factor-alpha on human vascular endothelial cells.
Paleolog, E.M.; Delasalle, S.A.; Buurman, W.A.; Feldmann, M.
Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the control of endothelial cell function and hence in regulating traffic of circulating cells into tissues in vivo. Stimulation of endothelial cells in vitro by TNF-alpha increases the surface expression of leukocyte adhesion
Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells
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Beese Michaela
Full Text Available Abstract Background Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer in vitro. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC which are widely used as model for endothelial cell-related studies. Results We here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na and for longer incubation periods 8-(4-chlorophenylthio-cAMP (pCPT-cAMP exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin. Conclusions pCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible in vitro model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins
Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.
Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo
Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. Copyright © 2015 Elsevier Inc. All rights reserved.
Sex differences in vascular endothelial function and health in humans: impacts of exercise.
Green, Daniel J; Hopkins, Nicola D; Jones, Helen; Thijssen, Dick H J; Eijsvogels, Thijs M H; Yeap, Bu B
What is the topic of this review? This brief review discusses potential sex differences in arterial function across the age span, with special emphasis on the effects of oestrogen and testosterone on the vascular endothelium. What advances does it highlight? We discuss the relationship between the impacts of sex hormones on arterial function and health in the context of epidemiological evidence pertaining to the menopause and ageing. Studies performed in humans are emphasized, alongside insights from animal studies. Findings suggest that the combination of exercise and hormone administration should be potentially synergistic or additive in humans. This brief review presents historical evidence for the purported impacts of male and female sex hormones on the vasculature in humans, including effects on macro- and microvascular function and health. Impacts of ageing on hormonal changes and arterial function are considered in the context of the menopause. Physiological data are presented alongside clinical outcomes from large trials, in an attempt to rationalize disparate findings along the bench-to-bedside continuum. Finally, the theoretical likelihood that exercise and hormone treatment may induce synergistic and/or additive vascular adaptations is developed in the context of recent laboratory studies that have compared male and female responses to training. Differences between men and women in terms of the impact of age and cardiorespiratory fitness on endothelial function are addressed. Ultimately, this review highlights the paucity of high-quality and compelling evidence regarding the fundamental impact, in humans, of sex differences on arterial function and the moderating impacts of exercise on arterial function, adaptation and health at different ages in either sex. © 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.
Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL.
Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-ichiro
Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test. Copyright © 2013 Elsevier Ltd. All rights reserved.
Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity
Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.
Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.
Flavonoids from artichoke (Cynara scolymus L.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells.
Li, Huige; Xia, Ning; Brausch, Isolde; Yao, Ying; Förstermann, Ulrich
Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipid-lowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.
Thick keratoconic cornea associated with posterior polymorphous corneal dystrophy.
Zaarour, K; Slim, E; Antoun, J; Waked, N
We herein report a case of bilateral unusually thick non-edematous keratoconic corneas with associated endothelial features of posterior polymorphous corneal dystrophy (PPCD). We report the case of a 27-year-old myopic woman who presented for refractive surgery. Slit lamp exam showed bilateral corneal protrusion with diffuse deep stromal and endothelial vesicular opacities and small paracentral bands. Topography showed generalized advanced corneal steepening in both eyes with increased anterior and posterior central corneal elevations in comparison to the best fit sphere. Ultrasound pachymetry showed central corneal thickness of 605μm (RE) and 612μm (LE). On specular biomicroscopy, cell density of 2503 cells/mm 2 RE and 1526 cells/mm 2 LE with significant cellular pleomorphism and polymegathism were noted. Clinical and paraclinical findings together suggest the presence of simultaneous keratoconus and PPCD. The literature has suggested an association between PPCD and steep cornea. Moreover, many reports have also described cases of associated PPCD and keratoconus with characteristic thinning and ectasia, in comparison to the unusual thick corneas noted in our patient, despite the absence of edema. Identification of genetics factors is further needed to clarify this association. This case describes a patient whose corneas present features of both keratoconus and PPCD and is unique due to the presence of increased corneal thickness despite the absence of edema. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
The hematopoietic chemokine CXCL12 promotes integration of human endothelial colony forming cell-derived cells into immature vessel networks.
Newey, Sarah E; Tsaknakis, Grigorios; Khoo, Cheen P; Athanassopoulos, Thanassi; Camicia, Rosalba; Zhang, Youyi; Grabowska, Rita; Harris, Adrian L; Roubelakis, Maria G; Watt, Suzanne M
Proangiogenic factors, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 (FGF-2) prime endothelial cells to respond to "hematopoietic" chemokines and cytokines by inducing/upregulating expression of the respective chemokine/cytokine receptors. Coculture of human endothelial colony forming cell (ECFC)-derived cells with human stromal cells in the presence of VEGF and FGF-2 for 14 days resulted in upregulation of the "hematopoietic" chemokine CXCL12 and its CXCR4 receptor by day 3 of coculture. Chronic exposure to the CXCR4 antagonist AMD3100 in this vasculo/angiogenesis assay significantly reduced vascular tubule formation, an observation recapitulated by delayed AMD3100 addition. While AMD3100 did not affect ECFC-derived cell proliferation, it did demonstrate a dual action. First, over the later stages of the 14-day cocultures, AMD3100 delayed tubule organization into maturing vessel networks, resulting in enhanced endothelial cell retraction and loss of complexity as defined by live cell imaging. Second, at earlier stages of cocultures, we observed that AMD3100 significantly inhibited the integration of exogenous ECFC-derived cells into established, but immature, vascular networks. Comparative proteome profiler array analyses of ECFC-derived cells treated with AMD3100 identified changes in expression of potential candidate molecules involved in adhesion and/or migration. Blocking antibodies to CD31, but not CD146 or CD166, reduced the ECFC-derived cell integration into these extant vascular networks. Thus, CXCL12 plays a key role not only in endothelial cell sensing and guidance, but also in promoting the integration of ECFC-derived cells into developing vascular networks.
Pharmacologic strategies in the prevention and treatment of corneal transplant rejection.
Tabbara, Khalid F
Corneal transplantation remains one of the most successful organ transplantation procedures in humans. The unique structure of the cornea, with its absence of blood vessels and corneal lymphatic, allows the survival of corneal allograft. Recent advances in sutures, storage media, microsurgical instrumentation, and new pharmacological strategies have greatly improved the success of corneal transplantation and the prevention of corneal allograft rejection. Our strategies in the management and prevention of corneal graft rejection can modify and improve the survival of corneal allografts. Preoperative evaluation, understanding the risk factors, and management of ocular surface disorders may greatly improve the survival of the corneal transplant. Early recognition of corneal allograft rejection and aggressive treatment may improve the survival of the corneal graft. Furthermore, patients who undergo corneal transplantation should be maintained under close ophthalmic surveillance and patients should be informed to report immediately whenever symptoms of corneal graft rejection occur. The mainstay of therapy is topical corticosteroids. In severe cases, periocular, intravenous, and oral corticosteroids therapy can be rendered. New therapeutic modalities such as cyclosporine, tacrolimus, daclizumab, mycophenolate mofetil, leflunomide, rapamycin, and others may prove to be of help in the prevention and treatment of corneal graft rejection. Early recognition of corneal graft rejection and prompt treatment are mandatory for the successful survival of the corneal allograft.
Effect of silica nanoparticles with variable size and surface functionalization on human endothelial cell viability and angiogenic activity
Guarnieri, Daniela; Malvindi, Maria Ada; Belli, Valentina; Pompa, Pier Paolo; Netti, Paolo
Silica nanoparticles could be promising delivery vehicles for drug targeting or gene therapy. However, few studies have been undertaken to determine the biological behavior effects of silica nanoparticles on primary endothelial cells. Here we investigated uptake, cytotoxicity and angiogenic properties of silica nanoparticle with positive and negative surface charge and sizes ranging from 25 to 115 nm in primary human umbilical vein endothelial cells. Dynamic light scattering measurements and nanoparticle tracking analysis were used to estimate the dispersion status of nanoparticles in cell culture media, which was a key aspect to understand the results of the in vitro cellular uptake experiments. Nanoparticles were taken up by primary endothelial cells in a size-dependent manner according to their degree of agglomeration occurring after transfer in cell culture media. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake, compared to negatively charged nanoparticles. However, this effect was contrasted by the tendency of particles to form agglomerates, leading to lower internalization efficiency. Silica nanoparticle uptake did not affect cell viability and cell membrane integrity. More interestingly, positively and negatively charged 25 nm nanoparticles did not influence capillary-like tube formation and angiogenic sprouting, compared to controls. Considering the increasing interest in nanomaterials for several biomedical applications, a careful study of nanoparticle-endothelial cells interactions is of high relevance to assess possible risks associated to silica nanoparticle exposure and their possible applications in nanomedicine as safe and effective nanocarriers for vascular transport of therapeutic agents.
Ganoderma atrum polysaccharide ameliorates anoxia/reoxygenation-mediated oxidative stress and apoptosis in human umbilical vein endothelial cells.
Zhang, Yan-Song; Li, Wen-Juan; Zhang, Xian-Yi; Yan, Yu-Xin; Nie, Shao-Ping; Gong, De-Ming; Tang, Xiao-Fang; He, Ming; Xie, Ming-Yong
Ganoderma atrum polysaccharide (PSG-1), a main polysaccharide from Ganoderma atrum, possesses potent antioxidant