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Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation
Aguirre, Geoffrey K
Komáromy, András M
Cideciyan, Artur V
Brainard, David H
Aleman, Tomas S
Roman, Alejandro J
Avants, Brian B
Gee, James C
Korczykowski, Marc
Hauswirth, William W
Acland, Gregory M
Aguirre, Gustavo D
Aguirre, Geoffrey K
PLoS Medicine
RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).
Methods and Findings
RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation.
Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.
The study by Samuel Jacobson and colleagues suggests that retinal gene therapy can improve retinal, visual pathway, and visual cortex responses to light stimulation, even after prolonged periods of blindness and in congenitally blind patients.
Editors' Summary
The eye captures light but the brain is where vision is experienced. Treatments for childhood blindness at the eye level are ready, but it is unknown whether the brain will be receptive to an improved neural message. Normal vision begins as photoreceptor cells in the retina (the light-sensitive tissue lining the inside of the eye) convert visual images into electrical impulses. These impulses are sent along the optic nerve to the visual cortex, the brain region where they are interpreted. The conversion of light into electrical impulses requires the activation of a molecule called retinal, which is subsequently recycled by retinal pigment epithelium (RPE) cells neighboring the retina. One of the key enzymes of the recycling reactions is encoded by a gene called RPE65. Genetic changes (mutations) in RPE65 cause an inherited form of blindness called Leber congenital amaurosis (LCA). In this disease, retinal is not recycled and as a result, the photoreceptor cells cannot work properly and affected individuals have poor or nonexistent vision from birth. Previous studies in dog and mouse models of the human disease have demonstrated that the introduction of a functional copy of RPE65 into the RPE cells using a harmless virus (gene therapy) dramatically restores retinal activity. Very recently, a pioneering gene therapy operation took place in London (UK) where surgeons injected a functional copy of RPE65 into the retina of a man with LCA. Whether this operation results in improved vision is not known at this time.
Why Was This Study Done?
Gene therapy corrects the retinal defects in animal models of LCA but whether the visual pathway from the retina to the visual cortex of the brain can respond normally to the signals sent by the restored retina is not known. Early visual experience is thought to be necessary for the development of a functional visual cortex, so replacing the defective RPE65 gene might not improve the vision of people with LCA. In this study, the researchers have studied the visual cortex of RPE65-deficient dogs before and after gene therapy to see whether the therapy affects the activity of the visual cortex. They have also investigated visual pathway integrity and responsiveness in adults with LCA caused by RPE65 mutations. If the visual pathway is disrupted in these patients, they reasoned, gene therapy might not restore their vision.
What Did the Researchers Do and Find?
The researchers used a technique called functional magnetic resonance imaging (fMRI) to measure light-induced brain activity in RPE65-deficient dogs before and after gene therapy. They also examined the reactions of the dogs' pupils to light (in LCA, the pupils do not contract normally in response to light because there is reduced signal transmission along the visual pathway). Finally, they measured the electrical activity of the dogs' retinas in response to light flashes—the retinas of patients with LCA do not react to light. Gene therapy corrected the defective retinal and visual pathway responses to light in the RPE65-deficient dogs and, whereas before treatment there was no response in the visual cortex to light stimulation in these dogs, after treatment, its activity approached that seen in normal dogs. The recovery of cortical responses was permanent and occurred soon after treatment, even in animals that were 4 years old when treated. Next, using structural MRI, the researchers studied human patients with LCA and found that the optic nerve diameter in young adults was within the normal range and that the structure of the visual cortex was very similar to that of normal individuals. Finally, using fMRI, they found that, although the visual cortex of patients with LCA did not respond to dim light, its reaction to bright light was comparable to that of normal individuals.
What Do These Findings Mean?
The findings from the dog study indicate that retinal gene therapy rapidly improves retinal, visual pathway, and visual cortex responses to light stimulation, even in animals that have been blind for years. In other words, in the dog model of LCA at least, all the components of the visual system remain receptive to visual inputs even after long periods of visual deprivation. The findings from the human study also indicate that the visual pathway remains anatomically intact despite years of disuse and that the visual cortex can be activated in patients with LCA even though these people have very limited visual experience. Taken together, these findings suggest that successful gene therapy of the retina might restore some functional vision to people with LCA but proof will have to await the outcomes of several clinical trials ongoing or being planned in Europe and the USA.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040230.
General information on gene therapy is available from the Oak Ridge National Laboratory
Information is provided by the BBC about gene therapy for Leber congenital amaurosis (includes an audio clip from a doctor about the operation)
The National Institutes of Health/National Eye Institute (US) provides information about an ongoing gene therapy trial of RPE65-Leber congenital amaurosis
ClinicalTrials.gov gives details on treatment trials for Leber congenital amaurosis
The Foundation Fighting Blindness has a fact sheet on Leber congenital amaurosis (site includes Microsoft Webspeak links that read some content aloud)
The Foundation for Retinal Research has a fact sheet on Leber congenital amaurosis
Find more detailed information on Leber congenital amaurosis and the gene mutations that cause it from GeneReviews
WonderBaby, information for parents of babies with Leber congenital amaurosis
Related Articles
CD36 Deficiency Leads to Choroidal Involution via COX2 Down-Regulation in Rodents
Houssier, Marianne
Raoul, William
Lavalette, Sophie
Keller, Nicole
Guillonneau, Xavier
Baragatti, Barbara
Jonet, Laurent
Jeanny, Jean-Claude
Behar-Cohen, Francine
Coceani, Flavio
Scherman, Daniel
Lachapelle, Pierre
Ong, Huy
Chemtob, Sylvain
Sennlaub, Florian
PLoS Medicine
In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the "wet" form of AMD. In contrast, very little is known about the mechanisms of the predominant, "dry" form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD.
Methods and Findings
We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36−/− mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%–300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36−/− mice express reduced levels of COX2 and VEGF in vivo, and COX2−/− mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency.
CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.
Florian Sennelaub and colleagues show that CD36 deficiency leads to choroidal involution, a key feature of "dry" age-related macular degeneration, via COX-2 down-regulation in the retinal pigment epithelium.
Editors' Summary
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in industrialized countries. The macula is the central region of the retina, the tissue at the back of the eye that detects light and converts it into electrical messages that are sent to the brain. In the commonest form of AMD—"dry" AMD—the light-sensitive cells in the retina (the photoreceptors) gradually die. This degeneration might occur because of damage to the retinal pigment epithelium (RPE). This layer of dark cells lies between the photoreceptors and the choroid, the layer of the eye that contains blood vessels and brings oxygen to the retina. The RPE keeps the retina healthy by transferring the right amount of oxygen and nutrients from the choroid to the retina and by removing worn-out photoreceptor outer segments (the part of the photoreceptor that actually absorbs light) in a process called phagocytosis (engulfment and digestion). In addition to photoreceptor degeneration and RPE shrinkage, a layer of the choroid rich in small blood vessels (the choriocapillaris) also shrinks in dry AMD. For affected individuals, all these changes (which experts describe as retinal atrophy and choroidal involution) mean that the sharp central vision that is needed for reading and driving is destroyed, leaving only dim, burred images or a black hole at the center of the vision.
Why Was This Study Done?
Little is known about the molecular mechanisms that underlie dry AMD and, consequently, there is no cure for it. In this study, the researchers have tested whether a molecule called CD36, which is expressed on the surface of RPE cells, is involved in dry AMD. CD36 is a scavenger receptor—which means it binds many potentially harmful molecules including oxidized fats (which are present in the photoreceptor outer segments) and is involved in their phagocytosis. Phagocytosis itself induces the expression of several proteins in the RPE cells, including COX2, a "proangiogenic" protein that stimulates the growth of blood vessels. Putting this information together, the researchers hypothesized that a defect in CD36 might cause the characteristic retinal atrophy (by preventing the phagocytosis of worn-out photoreceptor outer segments) and choroidal involution (by preventing the induction of COX2 expression and consequently the maintenance of the blood vessels in the choroid) of dry AMD.
What Did the Researchers Do and Find?
The researchers first show that retinal degeneration occurs in rats and mice that express no CD36. This degeneration (which included a reduction in the thickness of the retina, the presence of irregularly shaped photoreceptor outer segments, and the detachment of these structures from the RPE) was seen in old but not young animals. Choroidal involution was also seen in these CD36-deficient animals. This change was present in young mice and rats but increased with age so that by one year old, the choriocapillaris looked moth-eaten. Next, the researchers show that although RPE cells taken from normal animals and grown in dishes were able to make COX2 in response to exposure to purified photoreceptor outer segments, RPE cells from CD36-deficient animals did not. The expression of vascular endothelial growth factor (VEGF; a protein that is needed for normal choroidal development and whose expression is controlled by COX2) showed a similar pattern. Finally, the researchers report that COX2 deficiency in mice caused similar age-dependent choroidal involution and similar effects on VEGF expression in RPE cells as CD36 deficiency.
What Do These Findings Mean?
These findings show that CD36 deficiency leads to progressive, age-related degeneration of photoreceptors and choroidal involution in rats and mice. They also show that CD36 deficiency causes this choroidal involution, the key feature of dry AMD, because it leads to down-regulation of COX2 expression (and subsequently reduced VEGF expression) in the RPE. Researchers now need to find out whether this mechanism for the development of dry AMD holds in people—what happens in animals does not necessarily happen in people. If it does, pharmacological activation of CD36 or restoration of CD36 expression in the RPE might eventually provide a way to treat dry AMD.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050039.
MedlinePlus provides links to information on macular degeneration and an encyclopedia page on macular degeneration (in English and Spanish)
Pages on the US National Institutes of Health NIH SeniorHealth site provides text and spoken information about AMD
The US National Eye Institute and the UK Royal National Institute of Blind People also provide information about AMD
Wikipedia has pages on the retina, photoreceptor cells, retinal pigment epithelium, and choroid (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
Related Articles
Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis
Bemelmans, Alexis-Pierre
Kostic, Corinne
Crippa, Sylvain V
Hauswirth, William W
Lem, Janis
Munier, Francis L
Seeliger, Mathias W
Wenzel, Andreas
Arsenijevic, Yvan
PLoS Medicine
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.
Methods and Findings
Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.
By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65−/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.
In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function.
Editors' Summary
Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; "amaurosis" is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves.
Why Was This Study Done?
Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed.
What Did the Researchers Do and Find?
Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice.
What Do These Findings Mean?
These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030347.
The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis
Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other
The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis
This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases
This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
Related Articles
Light Damage in Abca4 and Rpe65rd12 Mice
Wu, Li
Ueda, Keiko
Nagasaki, Taka
Sparrow, Janet R.
Investigative Ophthalmology & Visual Science
Bisretinoids form in photoreceptor cells and accumulate in retinal pigment epithelium (RPE) as lipofuscin. To examine the role of these fluorophores as mediators of retinal light damage, we studied the propensity for light damage in mutant mice having elevated lipofuscin due to deficiency in the ATP-binding cassette (ABC) transporter Abca4 (Abca4−/− mice) and in mice devoid of lipofuscin owing to absence of Rpe65 (Rpe65rd12).
Abca4−/−, Rpe65rd12, and wild-type mice were exposed to 430-nm light to produce a localized lesion in the superior hemisphere of retina. Bisretinoids of RPE lipofuscin were measured by HPLC. In histologic sections, outer nuclear layer (ONL) thickness was measured as an indicator of photoreceptor cell degeneration, and RPE nuclei were counted.
As shown previously, A2E levels were increased in Abca4−/− mice. These mice also sustained light damage–associated ONL thinning that was more pronounced than in age-matched wild-type mice; the ONL thinning was also greater in 5-month versus 2-month-old mice. Numbers of RPE nuclei were reduced in light-stressed mice, with the reduction being greater in the Abca4−/− than wild-type mice. In Rpe65rd12 mice bisretinoid compounds of RPE lipofuscin were not detected chromatographically and light damage–associated ONL thinning was not observed.
Abca4−/− mice that accumulate RPE lipofuscin at increased levels were more susceptible to retinal light damage than wild-type mice. This finding, together with results showing that Rpe65rd12 mice did not accumulate lipofuscin and did not sustain retinal light damage, indicates that the bisretinoids of retinal lipofuscin are contributors to retinal light damage.
Fluorophores of the lipofuscin that accumulates in RPE contribute to mechanisms involved in light damage to retina.
Abca4; bisretinoid; light; lipofuscin; retina; retinal degeneration; retinal pigment epithelium
Related Articles
Methodologies for analysis of patterning in the mouse RPE sheet
Boatright, Jeffrey H.
Dalal, Nupur
Chrenek, Micah A.
Gardner, Christopher
Ziesel, Alison
Jiang, Yi
Grossniklaus, Hans E.
Nickerson, John M.
Molecular Vision
Our goal was to optimize procedures for assessing shapes, sizes, and other quantitative metrics of retinal pigment epithelium (RPE) cells and contact- and noncontact-mediated cell-to-cell interactions across a large series of flatmount RPE images.
The two principal methodological advances of this study were optimization of a mouse RPE flatmount preparation and refinement of open-access software to rapidly analyze large numbers of flatmount images. Mouse eyes were harvested, and extra-orbital fat and muscles were removed. Eyes were fixed for 10 min, and dissected by puncturing the cornea with a sharp needle or a stab knife. Four radial cuts were made with iridectomy scissors from the puncture to near the optic nerve head. The lens, iris, and the neural retina were removed, leaving the RPE sheet exposed. The dissection and outcomes were monitored and evaluated by video recording. The RPE sheet was imaged under fluorescence confocal microscopy after staining for ZO-1 to identify RPE cell boundaries. Photoshop, Java, Perl, and Matlab scripts, as well as CellProfiler, were used to quantify selected parameters. Data were exported into Excel spreadsheets for further analysis.
A simplified dissection procedure afforded a consistent source of images that could be processed by computer. The dissection and flatmounting techniques were illustrated in a video recording. Almost all of the sheet could be routinely imaged, and substantial fractions of the RPE sheet (usually 20–50% of the sheet) could be analyzed. Several common technical problems were noted and workarounds developed. The software-based analysis merged 25 to 36 images into one and adjusted settings to record an image suitable for large-scale identification of cell-to-cell boundaries, and then obtained quantitative descriptors of the shape of each cell, its neighbors, and interactions beyond direct cell–cell contact in the sheet. To validate the software, human- and computer-analyzed results were compared. Whether tallied manually or automatically with software, the resulting cell measurements were in close agreement. We compared normal with diseased RPE cells during aging with quantitative cell size and shape metrics. Subtle differences between the RPE sheet characteristics of young and old mice were identified. The IRBP−/− mouse RPE sheet did not differ from C57BL/6J (wild type, WT), suggesting that IRBP does not play a direct role in maintaining the health of the RPE cell, while the slow loss of photoreceptor (PhR) cells previously established in this knockout does support a role in the maintenance of PhR cells. Rd8 mice exhibited several measurable changes in patterns of RPE cells compared to WT, suggesting a slow degeneration of the RPE sheet that had not been previously noticed in rd8.
An optimized dissection method and a series of programs were used to establish a rapid and hands-off analysis. The software-aided, high-sampling-size approach performed as well as trained human scorers, but was considerably faster and easier. This method allows tens to hundreds of thousands of cells to be analyzed, each with 23 metrics. With this combination of dissection and image analysis of the RPE sheet, we can now analyze cell-to-cell interactions of immediate neighbors. In the future, we may be able to observe interactions of second, third, or higher ring neighbors and analyze tension in sheets, which might be expected to deviate from normal near large bumps in the RPE sheet caused by druse or when large frank holes in the RPE sheet are observed in geographic atrophy. This method and software can be readily applied to other aspects of vision science, neuroscience, and epithelial biology where patterns may exist in a sheet or surface of cells.
Related Articles
Expression profiling of the RPE in zebrafish smarca4 mutant revealed altered signals that potentially affect RPE and retinal differentiation
Zhang, Liyun
Ma, Ping
Collery, Ross
Trowbridge, Sara
Zhang, Mingzhi
Zhong, Wenxuan
Leung, Yuk Fai
Molecular Vision
The purpose of this study was to develop a framework for analyzing retinal pigment epithelium (RPE) expression profiles from zebrafish eye mutants.
The fish model we used was SWI/SNF-related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (smarca4), a retinal dystrophic mutant with a previously described retinal phenotype and expression profiles. Histological and Affymetrix GeneChip analyses were conducted to characterize the RPE defects and underlying differential expression, respectively.
Histological analysis revealed that smarca4 RPE was formed, but its differentiation was abnormal. In particular, ultrastructural analysis of smarca4 RPE by transmission electron microscopy demonstrated several defects in melanogenesis. The nature of these defects also suggests that the cytoskeletal dynamics, which are tightly linked with melanogenesis, were impaired in smarca4 RPE. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, the gene expression profiles of microdissected retinas and RPE-attached retinas were measured with Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples by subtracting the retinal expression values from the expression values of the RPE-attached retinas. A factorial analysis was conducted using the expression values of the RPE, retinal, and whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted models to select for three groups of genes: 1) smarca4-regulated RPE genes, 2) smarca4-regulated retinal genes, and 3) smarca4-regulated RPE genes that are not differentially expressed in the retina. Interestingly, the third group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, and paracrine and intracellular signal transduction.
Our analytical framework provides an experimental approach to identify differentially-regulated genes in the retina and the RPE of zebrafish mutants in which both of these tissues are affected by the underlying mutation. Specifically, we have used the method to identify a group of 39 genes that can potentially explain the melanogenesis defect in the smarca4 RPE. In addition, several genes in this group are secreted signaling molecules. Thus, this observation further implicates that the smarca4 RPE might play a role in the retinal dystrophic phenotype in smarca4.
Related Articles
The expression of retinal cell markers in human retinal pigment epithelial cells and their augmentation by the synthetic retinoid fenretinide
Carr, Amanda-Jayne
Vugler, Anthony A.
Yu, Lu
Semo, Maayan
Coffey, Pete
Moss, Stephen E.
Greenwood, John
Molecular Vision
In several species the retinal pigment epithelium (RPE) has the potential to transdifferentiate into retinal cells to regenerate functional retinal tissue after injury. However, this capacity for regeneration is lost in mammals. The synthetic retinoic acid derivative, fenretinide [N(4-hydroxyphenyl) retinamide], induces a neuronal-like phenotype in the human adult retinal pigment epithelial cell line (ARPE-19). These changes are characterized by the appearance of neural-like processes and the expression of neuronal markers not normally associated with RPE cells. Here we assess whether fenretinide can induce a neuroretinal cell phenotype in ARPE-19 cells, by examining retinal cell marker expression.
ARPE-19 cells were treated daily with culture medium containing either 3 μM fenretinide or dimethyl sulfoxide as a control for 7 days. Cells were processed for immunocytochemistry, western blotting, and for analysis by PCR to examine the expression of a panel of RPE, neural, and retinal-associated cellular markers, including classical and non-canonical opsins.
Treatment with fenretinide for 7 days induced the formation of neuronal-like processes in ARPE-19 cells. Fenretinide induced the expression of the cone long wavelength sensitive opsin (OPN1lw) but not rhodopsin (RHO), while decreasing the expression of RPE cell markers. Many of the neuronal and retinal specific markers examined were expressed in both control and fenretinide treated cells, including those involved in photoreceptor cell development and the multipotency of neural retinal progenitor cells. Interestingly, ARPE-19 cells also expressed both photoreceptor specific and non-specific canonical opsins.
The expression of retinal-associated markers and loss of RPE cell markers in control ARPE-19 cells suggests that these cells might have dedifferentiated from an RPE cell phenotype under standard culture conditions. The expression of molecules, such as the transcription factors paired box 6 gene (PAX6), sex determining region Y-box 2 (SOX2), cone-rod homeobox (CRX), and neural retina leucine zipper (NRL), further implies that in culture these cells are predisposed toward a retinal progenitor-like state. The fenretinide-induced increase in photoreceptor cell markers, accompanied by a decrease in RPE cell markers, suggests that retinoids may play a role in the transdifferentiation of RPE cells. Importantly, our data show for the first time the expression of a vertebrate ciliary opsin (OPN1lw) and rhabdomeric-like opsin, opsin 4 (OPN4 also known as melanopsin) in a clonal cell line. Together these data suggest that ARPE-19 cells are primed for and possess the capacity to differentiate toward a retinal cell-like lineage.
Related Articles
Dynamics of phosphorothioate oligonucleotides in normal and laser photocoagulated retina
Shen, W.
Garrett, K.
da Cruz, L.
Constable, I.
Rakoczy, P.
The British Journal of Ophthalmology
AIMS—To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat. METHODS—Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 µl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV). RESULTS—Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue. CONCLUSIONS—These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV.
Related Articles
Differential neuroglycan C expression during retinal degeneration in Rpe65−/− mice
Escher, Pascal
Cottet, Sandra
Aono, Saichiko
Oohira, Atsuhiko
Schorderet, Daniel F.
Molecular Vision
An increased mRNA expression of the genes coding for the extracellular matrix proteins neuroglycan C (NGC), interphotoreceptor matrix proteoglycan 2 (IMPG2), and CD44 antigen (CD44) has been observed during retinal degeneration in mice with a targeted disruption of the Rpe65 gene (Rpe65−/− mouse). To validate these data, we analyzed this differential expression in more detail by characterizing retinal NGC mRNA isoform and protein expression during disease progression.
Retinas from C57/Bl6 wild-type and Rpe65−/− mice, ranging 2 to 18 months of age, were used. NGC, IMPG2, and CD44 mRNA expression was assessed by oligonucleotide microarray, quantitative PCR, and in situ hybridization. Retinal NGC protein expression was analyzed by western blot and immunohistochemistry.
As measured by quantitative PCR, mRNA expression of NGC and CD44 was induced by about 2 fold to 3 fold at all time points in Rpe65−/− retinas, whereas initially 4 fold elevated IMPG2 mRNA levels progressively declined. NGC and IMPG2 mRNAs were expressed in the ganglion cell layer, the inner nuclear layer, and at the outer limiting membrane. NGC mRNA was also detected in retinal pigment epithelium cells (RPE), where its mRNA expression was not induced during retinal degeneration. NGC-I was the major isoform detected in the retina and the RPE, whereas NGC-III was barely detected and NGC-II could not be assessed. NGC protein expression was at its highest levels on the apical membrane of the RPE. NGC protein levels were induced in retinas from 2- and 4-month-old Rpe65−/− mice, and an increased amount of the activity-cleaved NGC ectodomain containing an epidermal growth factor (EGF)-like domain was detected.
During retinal degeneration in Rpe65−/− mice, NGC expression is induced in the neural retina, but not in the RPE, where NGC is expressed at highest levels.
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Microscopic mammalian retinal pigment epithelium lesions induce widespread proliferation with differences in magnitude between center and periphery
Lundh von Leithner, Peter
Ciurtin, Coziana
Jeffery, Glen
Molecular Vision
The vertebrate retina develops from the center to the periphery. In amphibians and fish the retinal margin continues to proliferate throughout life, resulting in retinal expansion. This does not happen in mammals. However, some mammalian peripheral retinal pigment epithelial (RPE) cells continue to divide, perhaps as a vestige of this mechanism. The RPE cells are adjacent to the ciliary margin, a known stem cell source. Here we test the hypothesis that peripheral RPE is fundamentally different from central RPE by challenging different regions with microscopic laser burns and charting differential responses in terms of levels of proliferation and the regions over which this proliferation occurs.
Microscopic RPE lesions were undertaken in rats at different eccentricities and the tissue stained for proliferative markers Ki67 and bromodeoxyuridine (BrdU) and the remodeling metalloproteinase marker 2 (MMP2).
All lesions produced local RPE proliferation and tissue remodeling. Significantly more mitosis resulted from peripheral than central lesions. Unexpectedly, single lesions also resulted in RPE cells proliferating across the entire retina. Their number did not increase linearly with lesion number, indicating that they may be a specific population. All lesions repaired and formed apparently normal relations with the neural retina. Repaired RPE was albino.
These results highlight regional RPE differences, revealing an enhanced peripheral repair capacity. Further, all lesions have a marked impact on both local and distant RPE cells, demonstrating a pan retinal signaling mechanism triggering proliferation across the tissue plane. The RPE cells may represent a distinct population as their number did not increase with multiple lesions. The fact that repairing cells were hypopigmented is of interest because reduced pigment is associated with enhanced proliferative capacities in the developing neural retina.
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RPE-secreted factors: influence differentiation in human retinal cell line in dose- and density-dependent manner
Dutt, Kamla
Douglas, Paul
Cao, Yang
Journal of Ocular Biology, Diseases, and Informatics
Retinal pigment epithelial (RPE) cells play an important role in normal functioning of retina and photoreceptors, and some retinal degenerations arise due to malfunctioning RPE. Retinal pigment epithelium transplantation is being explored as a strategy to rescue degenerating photoreceptors in diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Additionally, RPE-secreted factors could rescue degenerating photoreceptors by prolonging survival or by their ability to differentiate and give rise to photoreceptors by transdifferentiation. In this study, we have explored what role cell density could play in differentiation induced in a human retinal progenitor cell line, in response to RPE-secreted growth factors. Retinal progenitors plated at low (1 × 104 cells/cm2), medium (2–4 × 104 cells/cm2), and high (1 × 105 cells/cm2) cell density were exposed to various dilutions of RPE-conditioned medium (secreted factors) under conditions of defined medium culture. Progenitor cell differentiation was monitored phenotypically (morphological, biochemical analysis, and immunophenotyping, and western blot analysis were performed). Our data show that differentiation in response to RPE-secreted factors is modulated by cell density and dilutions of conditioned medium. We conclude that before embarking on RPE transplantation as a modality for treatment of RP and AMD, one will have to determine the role that cell density and inhibitory and stimulatory neurotrophins secreted by RPE could play in the efficacy of survival of transplants. We report that RPE-conditioned medium enhances neuronal phenotype (photoreceptors, bipolars) at the lowest cell density in the absence of cell–cell contact. Eighty percent to 90% of progenitor cells differentiate into photoreceptors and bipolars at 50% concentration of conditioned medium, while exposure to 100% conditioned medium might increase multipolar neurons (ganglionic and amacrine phenotypes) to a small degree. However, no clear-cut pattern of differentiation in response to RPE-secreted factors is noted at higher cell densities.
Retinal progenitors; Retinal pigment epithelium; Secreted factors; Differentiation; Density
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Adhesion Failures Determine the Pattern of Choroidal Neovascularization in the Eye: A Computer Simulation Study
Shirinifard, Abbas
Glazier, James Alexander
Swat, Maciej
Gens, J. Scott
Family, Fereydoon
Jiang, Yi
Grossniklaus, Hans E.
PLoS Computational Biology
Choroidal neovascularization (CNV) of the macular area of the retina is the major cause of severe vision loss in adults. In CNV, after choriocapillaries initially penetrate Bruch's membrane (BrM), invading vessels may regress or expand (CNV initiation). Next, during Early and Late CNV, the expanding vasculature usually spreads in one of three distinct patterns: in a layer between BrM and the retinal pigment epithelium (sub-RPE or Type 1 CNV), in a layer between the RPE and the photoreceptors (sub-retinal or Type 2 CNV) or in both loci simultaneously (combined pattern or Type 3 CNV). While most studies hypothesize that CNV primarily results from growth-factor effects or holes in BrM, our three-dimensional simulations of multi-cell model of the normal and pathological maculae recapitulate the three growth patterns, under the hypothesis that CNV results from combinations of impairment of: 1) RPE-RPE epithelial junctional adhesion, 2) Adhesion of the RPE basement membrane complex to BrM (RPE-BrM adhesion), and 3) Adhesion of the RPE to the photoreceptor outer segments (RPE-POS adhesion). Our key findings are that when an endothelial tip cell penetrates BrM: 1) RPE with normal epithelial junctions, basal attachment to BrM and apical attachment to POS resists CNV. 2) Small holes in BrM do not, by themselves, initiate CNV. 3) RPE with normal epithelial junctions and normal apical RPE-POS adhesion, but weak adhesion to BrM (e.g. due to lipid accumulation in BrM) results in Early sub-RPE CNV. 4) Normal adhesion of RBaM to BrM, but reduced apical RPE-POS or epithelial RPE-RPE adhesion (e.g. due to inflammation) results in Early sub-retinal CNV. 5) Simultaneous reduction in RPE-RPE epithelial binding and RPE-BrM adhesion results in either sub-RPE or sub-retinal CNV which often progresses to combined pattern CNV. These findings suggest that defects in adhesion dominate CNV initiation and progression.
Author Summary
This paper tests hypotheses for the mechanisms of choroidal neovascularization (CNV), the pathological growth of capillaries in response to physical defects in a structured tissue, the retina, showing that previously neglected cell-cell, cell-ECM and ECM-ECM adhesion failures suffice to determine the loci and progression of neovascularization. Surprisingly, a simple theory based on classes of adhesion failures, which involve variation of only five parameters, can coherently explain the heterogeneous range of CNV growth patterns and dynamics. Our results are generally applicable to other types of tissues where capillaries are close to an epithelium, e.g., lung and gut.
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mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice
Zhao, Chen
Yasumura, Douglas
Li, Xiyan
Matthes, Michael
Lloyd, Marcia
Nielsen, Gregory
Ahern, Kelly
Snyder, Michael
Bok, Dean
Dunaief, Joshua L.
LaVail, Matthew M.
Vollrath, Douglas
The Journal of Clinical Investigation
Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress.
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Quantitative Autofluorescence and Cell Density Maps of the Human Retinal Pigment Epithelium
Ach, Thomas
Huisingh, Carrie
McGwin, Gerald
Messinger, Jeffrey D.
Zhang, Tianjiao
Bentley, Mark J.
Gutierrez, Danielle B.
Ablonczy, Zsolt
Smith, R. Theodore
Sloan, Kenneth R.
Curcio, Christine A.
Investigative Ophthalmology & Visual Science
Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF.
Retinal pigment epithelium–Bruch's membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined.
Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss.
Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LF's role in AMD.
Simultaneous RPE cell density and autofluorescence maps of human donor eyes exhibit a significant increase in lipofuscin autofluorescence with age while RPE cell density remains stable, questioning lipofuscin's role in aging and AMD.
retinal pigment epithelium; autofluorescence; photoreceptor; lipofuscin; cytoskeleton
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Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay
Carr, Amanda-Jayne
Vugler, Anthony
Lawrence, Jean
Li Chen, Li
Ahmado, Ahmed
Chen, Fred K.
Semo, Ma'ayan
Gias, Carlos
da Cruz, Lyndon
Moore, Harry D.
Walsh, James
Coffey, Peter J.
Molecular Vision
To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells.
HESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel™ matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT–PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology.
HESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures.
The ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.
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Altered Expression of Retinal Molecular Markers in the Canine RPE65 Model of Leber Congenital Amaurosis
Hernández, Maria
Pearce-Kelling, Susan E.
Rodriguez, F. David
Aguirre, Gustavo D.
Vecino, Elena
Investigative Ophthalmology & Visual Science
The effect of the functional deletion of the RPE65 gene on the expression of molecular markers in the mutant dog retina was investigated. Some markers changed in the inner and outer retina, but the retinal structure was well preserved.
Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed.
Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies.
Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas.
The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans.
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Osteonectin/SPARC Secreted by RPE and Localized to the Outer Plexiform Layer of the Monkey Retina
Rodríguez, Ignacio R.
Moreira, Ernesto F.
Bok, Dean
Kantorow, Marc
Investigative ophthalmology & visual science
Osteonectin/SPARC is a secreted protein that has been implicated in ocular disease. Deletion of osteonectin/SPARC causes age-onset cataract in mice and the cataractous human lens has increased expression of osteonectin/SPARC. In this study, the expression and localization of osteonectin/SPARC in the monkey retina were determined as was secretion by cultured human retinal pigment epithelial (RPE) cells.
Adult Rhesus monkey eyes (Macaca mulatta) were dissected, and 5-mm macula and peripheral retina punches were obtained. Supernatants were collected from cultured human RPE cells. Subcellular fractionation of whole monkey retina was also performed. Osteonectin/SPARC expression and/or secretion was monitored by Northern and Western blot analyses, and localization was determined by immunocytochemistry.
Outside of the retina osteonectin/SPARC mRNA is broadly expressed in many human tissues. Northern blot analysis shows that in the retina osteonectin/SPARC is expressed almost exclusively by the macular RPE/choroid. Western blot analysis revealed osteonectin/SPARC in both the macula and the peripheral neural retina but only in trace amounts in the RPE/choroid. In subcellular fractions of the whole retina, osteonectin/SPARC was detected, mainly in the soluble fraction but also in the membrane and nuclear fractions. Immunohistochemical analysis localized osteonectin/SPARC specifically to the outer plexiform layer. Western blot analysis of conditioned medium from human RPE cells cultured on porous substrates indicated that osteonectin/SPARC is secreted in large amounts from both the apical and basal sides of the RPE.
Collectively these data provide evidence that osteonectin/SPARC is synthesized in the macular RPE, secreted, and subsequently transported to the outer plexiform layer. The expression pattern of osteonectin/SPARC in the subcellular retinal fractions is consistent with a soluble protein that is transported and internalized.
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Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo
Al-Hussaini, Heba
Kam, Jaimie Hoh
Vugler, Anthony
Semo, Ma'ayan
Jeffery, Glen
Molecular Vision
To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats.
Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67.
In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells).
Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.
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Engineering a Blood-Retinal Barrier With Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium: Transcriptome and Functional Analysis
Peng, Shaomin
Gan, Geliang
Qiu, Caihong
Zhong, Mei
An, Hongyan
Adelman, Ron A.
Rizzolo, Lawrence J.
Stem Cells Translational Medicine
To develop a culture model for drug development and tissue-engineering human retina, retinal pigment epithelia (RPE) were derived from human embryonic stem cells (hESCs), and their barrier properties were compared with those of a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). It was found that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study also identified a panel of genes to monitor further maturation of RPE.
Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood-retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue-engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well-regarded model of RPE function, human fetal RPE isolated from 16-week-gestation fetuses (hfRPE). A serum-free medium (SFM-1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC-derived RPE. In SFM-1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription-polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC-derived RPE is highly differentiated but may be less mature than RPE isolated from 16-week fetuses. The study identified a panel of genes to monitor the maturation of RPE.
Retinal pigmented epithelium; Blood-retinal barrier; Claudins; Embryonic stem cells
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Changes in Adeno-Associated Virus-Mediated Gene Delivery in Retinal Degeneration
Kolstad, Kathleen D.
Dalkara, Deniz
Guerin, Karen
Visel, Meike
Hoffmann, Natalie
Schaffer, David V.
Flannery, John G.
Human Gene Therapy
Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state.
Kolstad et al. evaluate the distribution of vector particles and transduction of AAV administered intravitreally in diseased versus healthy retinas. Whereas healthy retinas are not very receptive to vector penetration and transduction following intravitreal injection, in retinal degenerations the authors show improved and more extensive gene transfer.
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The Aged Retinal Pigment Epithelium/Choroid: A Potential Substratum for the Pathogenesis of Age-Related Macular Degeneration
Chen, Huiyi
Liu, Bin
Lukas, Thomas J.
Neufeld, Arthur H.
PLoS ONE
Although the statement that age is the greatest risk factor for Age-related macular degeneration (AMD) is widely accepted, the cellular and molecular explanations for that clinical statement are not generally known. A major focus of AMD research is the retinal pigment epithelium (RPE)/choroid. The purpose of this study was to characterize the changes in the RPE/choroid with age that may provide a background for the development of AMD.
Methodology/Principal Findings
We compared the transcriptional profiles, key protein levels and histology of the RPE/choroid from young and old mice. Using three statistical methods, microarray data demonstrated marked changes in the old mouse. There were 315 genes differentially expressed with age; most of these genes were related to immune responses and inflammatory activity. Canonical pathways having significant numbers of upregulated genes in aged RPE/choroid included leukocyte extravasation, complement cascades, natural killer cell signaling and IL-10 signaling. By contrast, the adjacent neural retina showed completely different age-related changes. The levels of proteins that participate in leukocyte extravasation and complement pathways were consistently increased in the normal, aged RPE/choroid. Furthermore, there was increased gene expression and protein levels of leukocyte attracting signal, chemokine ligand 2 (Ccl2) in aged RPE/choroid. In old animals, there was marked extravasation and accumulation of leukocytes from the choroidal circulation onto Bruch's membrane and into the RPE.
These phenotypic changes indicate that the RPE/choroid in the normal, old mouse has become an immunologically active tissue. There are signals from the normal, aged RPE/choroid which recruit leukocytes from the circulation and activate the complement cascade. These age-related changes that occur in the RPE/choroid with age, to the extent that they occur in the human retina, may provide the background for an error in regulation of immunological activity to cause AMD to appear in an elderly individual.
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L-DOPA Is an Endogenous Ligand for OA1
Lopez, Vanessa M
Decatur, Christina L
Stamer, W. Daniel
Lynch, Ronald M
McKay, Brian S
PLoS Biology
Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.
Author Summary
Albinism is the loss of pigmentation caused by mutations in one of several different genes that alter pigment synthesis by different mechanisms. In the eye, albinism impairs sensory retina development and causes significant vision problems. Regardless of the genetic mutation that causes albinism, the associated vision problems are the same. Interestingly, none of the pigmentation genes are expressed by the sensory retinal cells affected by albinism but by neighboring, retinal pigment epithelial cells (RPE). Furthermore, loss of pigmentation in RPE somehow leads to imprecise retinal development. To investigate this cellular relationship, we studied OA1, which is encoded by a gene in which mutations cause ocular albinism. OA1 is unique among proteins involved with albinism because OA1 is a potential receptor that could participate in signal transduction rather than being a direct member of the pigment synthesis machinery. We show that the ligand for OA1 is L-DOPA, thus removing OA1 from orphan G-protein coupled receptor (GPCR) status. L-DOPA is a by-product of pigment synthesis, indicating that pigment synthesis and OA1 signaling are intertwined. OA1 signaling is highly selective for L-DOPA, and we show that two closely related molecules, dopamine and tyrosine, bind to OA1 but fail to stimulate signaling. We also show that OA1 signaling controls secretion of a potent neuron survival factor. Taken together, our data suggest that all forms of albinism produce the same retinal defects because of a final common pathway through OA1 signaling with downstream effects on RPE neurotrophic factor secretion.
Albinism produces retinal defects, and OA1 is an orphan G-protein-coupled receptor that leads to albinism without acting directly on melanin synthesis. Here the ligand is identified and a mechanism is proposed by which the various forms of albinism signal through OA1, resulting in the same retinal phenotype.
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A Regulatory Loop Involving PAX6, MITF, and WNT Signaling Controls Retinal Pigment Epithelium Development
Bharti, Kapil
Gasper, Melanie
Ou, Jingxing
Brucato, Martha
Clore-Gronenborn, Katharina
Pickel, James
Arnheiter, Heinz
PLoS Genetics
The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE) is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE–specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells.
Author Summary
The retinal pigment epithelium or RPE in the back of the eye is critical for the normal function of the retina, and its abnormalities can lead to retinal disorders such as adult-onset macular degeneration. Insights into the pathogenesis of such disorders, and potential therapies, may come from using RPE cells generated in vitro from induced pluripotent stem cells. To obtain authentic RPE cells in vitro, we need to thoroughly understand the normal process of their development in vivo. Here we find that the potent retina-inducing transcription factor PAX6 plays a critical anti-retinogenic role in the RPE of mice. But how can PAX6 be pro-retinogenic in the retina and anti-retinogenic in the RPE? To address this question, we used gene expression studies and combined them with chromatin immunoprecipitation assays, which analyze the interaction of transcription factors with chromatin in vivo. Our findings show that, in the RPE, PAX6 cooperates with either one (or both) of two related RPE transcription factors, MITF and TFEC, to suppress extracellular signals that in the normal retina induce a signaling cascade promoting retina formation. Hence, this study provides mechanistic insights into RPE development that may become important for the efficient generation of retina and RPE from induced pluripotent stem cells.
Related Articles
Novel strategy for subretinal delivery in Xenopus
Gonzalez-Fernandez, Federico
Dann, Cheryl A.
Garlipp, Mary Alice
Molecular Vision
The subretinal space, which borders the retinal pigment epithelium (RPE), photoreceptors, and Müller cells, is an ideal location to deliver genetic vectors, morpholino oligos, and nanopharmaceuticals. Unfortunately, materials injected into the space tend to stay localized, and degenerative changes secondary to retinal detachment limit its usefulness. Furthermore, such injection requires penetration of the sclera, RPE/choroid, or the retina itself. Here, we developed a strategy in Xenopus to utilize the continuity of the brain ventricle and optic vesicle lumen during embryogenesis as a means to access the subretinal space.
Wild-type and transgenic embryos expressing green fluorescent protein under the rod-opsin promoter were used for optic vesicle and brain ventricle injections. For injection directly into the optic vesicle, embryos were laid on one side in clay troughs. For brain ventricle injections, embryos were placed standing in foxholes cored from agarose dishes. Linear arrays with each embryo positioned dorsal side toward the micromanipulator facilitated high throughput injections. Twenty-five micrometer micropipettes, which were positioned with a micromanipulator or by hand, were used to pressure inject ~1.0 nl of test solution (brilliant blue, India ink, fluorescein isothiocyanate dextran, or 0.04 µm of latex polystyrene microspheres [FluoSpheres®]). FluroSpheres® were particularly useful in confirming successful injections in living embryos. Anesthetized embryos and tadpoles were fixed in 4% paraformaldehyde and cryoprotected for frozen sections, or dehydrated in ethanol and embedded in methacrylate resin compatible with the microspheres.
Direct optic vesicle injections resulted in filling of the brain ventricle, contralateral optic vesicle, and central canal. Stages 24 and 25 gave the most consistent results. However, even with experience, the success rate was only ~25%. Targeting the vesicle was even more difficult beyond stage 26 due to the flattening of the lumen. In contrast, brain ventricle injections were easier to perform and had a ~90% success rate. The most consistent results were obtained in targeting the diencephalic ventricle, which is located along the midline, and protrudes anteriorly just under the frontal ectoderm and prosencephalon. An anterior midline approach conveniently accessed the ventricle without disturbing the optic vesicles. Beyond stage 30, optic vesicle filling did not occur, presumably due to closure of the connection between the ventricular system and the optic vesicles. Securing the embryos in an upright position in the agarose foxholes allowed convenient access to the frontal cephalic region. On methacrylate sections, the RPE-neural retina interphase was intact and labeled with the microspheres. As development continued, no distortion or malformation of the orbital structures was detected. In green fluorescent protein (GFP), transgenic embryos allowed to develop to stage 41, retinal FluoSpheres® labeling and photoreceptor GFP expression could be observed through the pupil. On cryosections, it was found that the FluoSpheres® extended from the diencephalon along the embryonic optic nerve to the ventral subretinal area. GFP expression was restricted to rod photoreceptors. The microspheres were restricted to the subretinal region, except focally at the lip of the optic cup, where they were present within the retina; this was presumably due to incomplete formation of the peripheral zonulae adherens. Embryos showed normal anatomic relationships, and formation of eye and lens appeared to take place normally with lamination of the retina into its ganglion cell and the inner and outer nuclear layers.
Diencephalic ventricular injection before stage 31 provides an efficient strategy to introduce molecules into the embryonic Xenopus subretinal space with minimal to the developing eye or retina.
Related Articles
Light-induced photoreceptor and RPE degeneration involve zinc toxicity and are attenuated by pyruvate, nicotinamide, or cyclic light
Sheline, Christian T.
Zhou, Yongdong
Bai, Shi
Molecular Vision
Light-induced damage can be a problem after surgery or sun exposure. Short-duration, intense light causes preferential photoreceptor death in the superior central retina of albino mice and rats and serves as a model of oxidation-induced neurodegeneration. Previous work on retinal ischemia-induced neuronal death suggests the involvement of zinc (Zn2+) toxicity in the death and collapse of many retinal cell layers and demonstrates the protective efficacy of pyruvate. Retinal pigment epithelial (RPE) cells were shown to be sensitive to oxidative stress, and zinc, causing loss of nicotinamide adenine dinucleotide (NAD+) and adenine triphosphate (ATP), which was prevented by pyruvate and nicotinamide. We previously showed similar results in cortical neurons exposed to oxidative stress or Zn2+. In vivo, Zn2+ is normally present in the inner and outer segments (associated with rhodopsin), Bruch's membrane and sclera (elastin), RPE, and the outer plexiform layer of the eye (synaptic). In this study, we examine the role of Zn2+ in oxidative stress and light-induced damage in vitro and in vivo.
We modeled retinal toxicity in cell-culture lines derived from retinal tissue: Müller and human retinal pigment epithelial (ARPE-19) cells and a cone photoreceptor-derived line (661W). These cultures were exposed to Zn2+ and OS, and the therapeutic efficacy of pyruvate, nicotinamide, and NAD+ was determined. Sprague Dawley albino rats were exposed to 18 kLux of white fluorescent light for 1–4 h in the presence and absence of pyruvate, nicotinamide, lactate, and cyclic light. The intracellular free zinc concentration ([Zn2+]i) and cell damage were assessed 0.5 and 7 days later, respectively.
We show that Zn2+ and oxidative stress results in increased [Zn2+]i and that Zn2+ therapeutic compounds (pyruvate, nicotinamide, and NAD+) and inhibitors of previously implicated pathways (sirtuin) are efficacious in vitro. Exposure to 18 kLux of cool white fluorescent light for 1 h induced a large increase in Zn2+ staining 4–14 h later, particularly in the superior outer nuclear layer and RPE of dark-maintained Sprague Dawley albino rats; 4 h of light was required to induce similar damage in cyclic light-maintained rats. Photoreceptors and RPE cells died in untreated animals at 3–7 days. However, nicotinamide and pyruvate (intraperitoneal), but not lactate, attenuated this death in treated animals, as measured using optical coherence tomography and confirmed by counting photoreceptor nuclei.
Zn2+ plays a role in this injury, as suggested by the increased Zn2+ staining and the efficacy of Zn2+ therapeutics. These results suggest that cyclic light maintenance, Zn2+ chelation, pyruvate, and nicotinamide promote RPE and photoreceptor survival after injury and could be effective for various forms of retinal neurodegeneration. These results could have immediate clinical applications in surgery- or sun exposure- induced light damage to the retina.
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text:
Journal of Comparative Physiology B
, Volume 159, Issue 2, pp 183-193
First online:
Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis
- D. C. DeemingAffiliated withDepartment of Cell and Structural Biology, University of Manchester
- , M. W. J. FergusonAffiliated withDepartment of Cell and Structural Biology, University of Manchester
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Eggs ofAlligator mississippiensis were incubated at 30 °C and 33 °C throughout incubation up to hatching. Every four days several eggs were opened and the albumen, yolk and extra-embryonic fluids removed and weighed. The embryo was removed and fixed prior to being staged, weighted and measured for various morphometric criteria. Development at 33 °C was accelerated compared with 30 °C in terms of yolk and albumen utilization and embryo growth. Significant losses in yolk mass did not occur until stage 22 at 33 °C but occurred at stage 18 at 30 °C. Different patterns in growth were observed in embryos at the two temperatures at similar morphological stages: between stages 18 and 22 embryos at 33 °C were smaller (in mass and length) compared with embryos at 30 °C despite being morphologically similar. The differences in growth and physiology between embryos at 30 °C (females) and 33 °C (males) were dependent on incubation temperature but not sex. Incubation at 33 °C accelerated both growth and development inAlligator; initially morphogenesis was accelerated by the higher temperature but later, growth rate was accelerated.
Key wordsCrocodilians Morphometrics Extra-embryonic fluids Growth sex determination
Reference tools
- Title
- Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis
- Journal
Journal of Comparative Physiology B
Volume 159, Issue 2 , pp 183-193
- Cover Date
- 1989-03
- Print ISSN
- 0174-1578
- Online ISSN
- Publisher
- Springer-Verlag
- Additional Links
- Topics
- Keywords
- Crocodilians
- Morphometrics
- Extra-embryonic fluids
- Growth sex determination
- Authors
- D. C. Deeming (1)
- M. W. J. Ferguson (1)
- Author Affiliations
- 1. Department of Cell and Structural Biology, University of Manchester, Coupland III Building, Coupland Street, M13 9PL, Manchester, UK

subset: commoncrawl


text:
Drifter Sentenced to Death in Fla. Beheading Case
Posted: 02/21/2011 - A man found guilty in the 2007 slaying and decapitation of a Florida nurse has been sentenced to death.
Posted: 02/21/2011 - A man found guilty in the 2007 slaying and decapitation of a Florida nurse has been sentenced to death.
Posted: 02/21/2011 - Study finds many graphic YouTube self-harm videos
Posted: 02/20/2011 - With a celebrity-studded crowd looking on, Rihanna rocked the halftime show at the NBA All-Star game on her 23rd birthday.
Posted: 02/20/2011 - Democratic senators from several states are calling on Republican Gov. Rick Scott of Florida to enact a program they say would better track the growing trade of abused prescription drugs to other states.
Posted: 02/20/2011 - The head of Wisconsin's powerful teachers' union is calling on educators to return to classrooms Monday and Tuesday rather than continue being absent to protest an anti-union bill at the state Capitol.
Posted: 02/20/2011 - Legislators in the Mexican border state of Chihuahua have asked federal authorities to ban a violent video game based on the drug cartel shootouts in Ciudad Juarez.
Updated: 02/20/2011 - Jurors could soon hear the case against two Florala men accused of taping customers while they tanned at a video store.
Posted: 02/20/2011 - An on-duty DeKalb County police officer collided with another vehicle while driving her patrol car in a wreck that officials say left two people dead.
Updated: 02/20/2011 - Twenty-year-old Trevor Bayne won the Daytona 500 in only his second Sprint Cup start on Sunday.
Updated: 02/20/2011 - MONTGOMERY, Ala. (AP) - Country Crossing casino developer Ronnie Gilley used to spend his days as a high-powered executive, building Gulf Coast condominiums, starting record companies and hobnobbing with country music stars.
Posted: 02/19/2011 - Downtown Atlanta will be full of clowns, cheerleaders and hairdressers this weekend as multiple events are planned for the Georgia World Congress Center and Philips Arena.
Posted: 02/19/2011 - A co-producer of "The Chronicles of Narnia" films and author has died in New York City. He was 39.
Posted: 02/19/2011 - The National Christmas Tree is no more.
Posted: 02/19/2011 - A 78-year-old retired doctor was handcuffed and hauled away on a toboggan for skiing uphill in Wyoming, but he won't face charges.
Posted: 02/19/2011 - An Iowa prison inmate has been indicted for making threats against the president.
Posted: 02/19/2011 - Authorities say a Lexington man is dead after his motorcycle hit a vehicle head-on on Alabama 207.
Posted: 02/19/2011 - Albany police have arrested a man who authorities say held his girlfriend and her two children hostage for nearly three hours in a standoff with officers.
Updated: 02/19/2011 - Alabama Gov. Robert Bentley has acknowledged that sometimes he speaks without considering the consequences.
Posted: 02/19/2011 - Mardi Gras parades in Mobile are in full swing.
Posted: 02/19/2011 - A Florida boy who police say was critically injured when he was doused in chemicals by his father is improving in condition.
Posted: 02/19/2011 - BOSTON (AP) - A spokeswoman for Boston's Logan International Airport says a flight from Tampa, Fla., landed safely after being struck by lightning.
Updated: 02/19/2011 - WASHINGTON (AP) - A University of Georgia scientist has gone public with video and slides showing how oil from the BP spill remains stuck on the bottom of the Gulf of Mexico.
Posted: 02/18/2011 - First the bad news,- postage rates are going up April 17. Now the good news, the basic 44-cent rate for first-class mail stays the same.
Posted: 02/18/2011 - Authorities say a Decatur city court clerk has been arrested and charged with felony theft.
Posted: 02/18/2011 - The administrator of BP's $20 billion Gulf oil spill fund announced new rules Friday in the highly criticized claims process, but the central payment formula remained unchanged from a proposal released earlier this month.


text:
The 76ers hold seven picks in Thursday night's NBA draft, or, analytically speaking, 11.67 percent of all the selections available to the 30 teams. The first two of those picks - the third and the 10th in the opening round - are the most intriguing, of course, and will have the most to say about the ongoing construction of the Sixers roster, but those five second-round picks say a lot more about general manager Sam Hinkie.
Maybe the idea of Andrew Wiggins or Jabari Parker or Joel Embiid wearing a Sixers uniform is what has you glued to what will happen soon after the curtain goes up, but there's something about how Hinkie slalomed through the gates of last year's draft that really makes him the most interesting guy to watch.


text:
Watch an Awesome Dad and His 3-Year-Old, Both in Spider-Man Costumes, Visit an Indoor Trampoline Park
Lucky Oliver!By Eliot Glazer
Lucky Oliver!By Eliot Glazer
Just a reminder: Kids write great stories.By Eliot Glazer
Let's have a tiki!By Eliot Glazer
Plus: Tom Waits took absurdly cool pleasure in showing off his new, grisly medieval rat trap, and more, on our daily late-night roundup.By Caroline Shin
What would we say? "Stop picking your nose. People can see."By Eliot Glazer
Kids pop the darndest booties, yada yada yada.By Eliot Glazer
We'd see it.By Eliot Glazer
How bad does Mr. Avina's class just make you want to run out and get pregnant?By Eliot Glazer
Feel the pain.By Eliot Glazer
"I just farted. You know what I'm sayin'?"By Eliot Glazer
No "ill" will, Boys.By Eliot Glazer
The race for Song of Summer just became adorable!By Eliot Glazer
You're never too young to cook with style.By Kara Baskin
It's called "riding in style."By Eliot Glazer
Lil Kanye gets around, huh?By Eliot Glazer
Austen wants to show you how to make peanut-butter-chocolate-chip cookies. Take it away, kid!By Eliot Glazer
And we could barely bring ourselves to dissect that frog.By Eliot Glazer
Well, they are the future, so we might as well listen.By Eliot Glazer
It's official: No one wants to see The Tree of Life.By Eliot Glazer
Although we still don't really know what it means to be "on the edge of glory" (you're dead? you're alive?), we're always happy to hear P.S. 22 give a song their best shot.By Eliot Glazer


text:
Thats awesome. I'm going to have to try that. I use an aluminum arrow i found in the woods.
Type: Posts; User: daveocelott
Thats awesome. I'm going to have to try that. I use an aluminum arrow i found in the woods.
401Hunter. Thank you for Ivy's first hammock. It only took 10 months for me to get time to get her in it. http://images.tapatalk-cdn.com/15/07/02/1c0939deb848ed7fe93906a9ca60932d.jpg
She is a true...
Too funny. Im definitely on both sides
Count me in too! I've seen some of this hkgear in the wild. Nice.
Mrs. Ocelot and I miss you all. Wishing we could come this weekend. Hope you all have a wonderful time. Hope to see you all soon at the next hang.
Beautiful job
Welcome to the forum!
I agree. Merican ingenuity
Welcome to the addiction (?) Obsession (?). Well anyway welcome to the forums. All the information you desire is here. And some of the best people to answer any questions. Enjoy.
Yep snoring enough to keep the wildlife away. I dont usually snore either.
Hey all. I'm not sure I'm going to be able to make this one. Good news for anyone in the raffle. Maybe I'll be able to catch the winter one. Btw Ivy arrived a healthy 8lbs 1oz 21inches long last...
Thank you so very much. She is really neat. Me and the missus are still trying to find some time to sleep. I can't wait to teach her all about camping and hiking and hammocks and fishing and stuff. I...
Say Hello to Ivy! 8lbs 1 0z 20 inches long. Arrived on monday night at 8:11....
Oh boy! The baby will have just barely arrived but I will see if I can make it. Would be great to see reasons place.
Own back yard. Yes you read correctly. I have napped in the day time I have hiked miles and slept in camps but I haven't spent an entire night out "alone". It wasn't bad. I heard "stuff". There was...
Very nice. Lavish really.
Hurray! Dutch! It will be awesome. Wish we could go. Best of luck!
Bam! And done! Packed in a moment gone in a flash! What hammock?
These beauties have the hole in the bottom and slide neatly into my compactor bag in...
I got it! In fact I bought a second one for my wife.
So very nice.
Welcome to hammock forums. Looks like the addiction has taken hold.
Superfly! No question. I love mine so much I got the wife one.
I 2nd this
It is very good. I like the extra pepper too. It does last a min. Good and hearty. Thank you.
I am looking forward to mine. Cant wait.
I actually had one. It did not work well.
Thank you. I'm sure it will be quite an adventure.
Its a girl! She will be arriving in August. Gonna have to diy a baby hammock to hang from my ridgeline. So exciting!
Who made the awesome deer jerky? Thank you for donating it! I really wish we were able to stay sat night. Tha baby got cold. We find out later today what we r having. Gonna be a hanger in any case....
Your waistline might not. Lol
Thank you Hammock Gear for donating a 20° incubator for the NEHHA Spring Swing raffle so I could win it! I am so excited. I have only heard great things about your gear and I can't wait to find out...
That is a great writeup. I can see myself there while reading it. My wife and I have done a few miles on the bft the last 2 years with her father, uncle, and grandfather. We have been averaging about...
Here you are Gastar, Becky's peanut butter choc chip
Pb cookie recipe:
1 cup creamy pb
1 cup sugar
1 egg
1 tsp vanilla extract
1/2 bag of choc chips
set oven to 375
Mix together all...
Very nice job on the video. Made it look like a great time. It was! Thank you.
I use a table cloth one for car camping and hiking.
Absolutely great time! Awesome! Cant wait for the next one. Yes a huge thank you to Gastar and Sky painter for all their hard work. And an enormous thank you to all the vendors and members who...
And the NEHHA! I won a brand new ultralight explorer at this years spring swing. Thank you so very much Hennessey for donating it so I could win it! You rock! I can't wait to try it!
Thank you guys. I will be sure to send a thank you to hennesy. Again we had an awesome time. Cant wait for the next one.
Hey we had a wonderful time with you all. We apologize for having to leave early. Pregnancy and cold dont always mix. Thank you for everything. I also wanted to ask who donated the hennesy hammock?
We should be there later tonight.
I told my wife too. Lol. So looking forward to seeing you all.
That's not wierd its encouraged. ..is wierd but completely normal somehow. I am really looking forward to this. The wife should be coming this hang. Will be her first nehha. Can't wait to check out...
The stuff sacks are the only thing I use from the jackets now. My hammock is in one and my tarp in the other. Ive been able to fit 4 puffer jackets in one bag. Pretty sure I could have put more
I'm in! Very nice job. Thank you
That's too funny. Cosmic was framed man.
I second that on the fancy feast stove made with the cloth. Boiled quick.
I am so going to this hang. I will bring the wife and the pooch also. Looking forward to all the classes.
Btw, I won one...
I am not in. But thank you for being so generous. There is nothing like a hammock.
I used hot hands in my hammock this last weekend at the nehha hang. Worked very well. Had four in my sleeping bag two for the rear and one for the feet and a floater that ended up on my chest.
I had an awesome time for my first winter hang. Thank you NEHHA! A nicer group of people one would be hard pressed to find. I can't wait for the spring hang. A special thanks to Tim and Pete for all...
Oh good. Ive been reading all these posts about really really warm equipment. No worries we will freeze with you.
The r the crinkle taffeta. They come in so many colors I was able to get thier favorites. And yes I was the awesomest uncle. For a few minutes anyway.


text:
Dr. Vuttarapally.Srinivas
More information about Dr. Vuttarapally.Srinivas
- Worked as a Associate Professor at Deccan Medical College.
- Worked as Consultant Diabetologist at sparsh diabetic center.
- Critical Care
- Diabetes
Doctor is Good, he suggested to go under the test.Ayesha saw Dr. Vuttarapally.Srinivas
Doctor was good and she suggested some test.Muneer saw Dr. Vuttarapally.Srinivas
Doctor is good.suggested medicine for 4 days & told that if it is not cured so,told to consult her(Yes)Reddy Padmalatha saw Dr. Vuttarapally.Srinivas
Doctor is good.he spoke well but what exact i thought he is not that suggested medicine for 1 week.(Yes)Yasmeen Banu. saw Dr. Vuttarapally.Srinivas
Doctor was Ok.suggested test & told to admit at the hsptl but we are not frm here.doctor left frm there again down we consulted with another doctor he saw the reports & suggested medicine till 14th so,again on 14th we have to visit the hsptal but we are not satisfied with doctor behavior its not good leave the patient & going.(Yes)kotamma perumalla saw Dr. Vuttarapally.Srinivas
Doctor was good.we are satisfied with his treatment suggested medicine she is diabetic patient so,told to continue the medicine(Yes).N.Padmavathi saw Dr. Vuttarapally.Srinivas