{'fraction_non_alphanumeric': 0.038120779656146735, 'fraction_numerical': 0.026627077247258272, 'mean_word_length': 5.026551226551226, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 4, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effects of donor age on human adipose-derived adherent stromal cells under oxidative stress conditions

Objective Adipose-derived stromal vascular fractions (SVFs) are heterogeneous complex populations of cells with therapeutic efficacy for tissue generation and vascular stabilization. SVFs have cardiomyogenic potential, and many researchers have examined the possibility of SVF transplantation for heart disease. In cell-based therapies, donor age affects the regenerative capability, cell yield, and differentiation potential of adult tissues; however, opposing or controversial results have been found in humans. We examined whether SVF transplantation into impaired heart tissue shows differential effects according to donor age. Methods We investigated differences in protein expression in human umbilical vein endothelial cells (HUVECs) co-cultured with adipose-derived adherent stromal cells (ADASs) from donors of different ages [>40-year-olds (40s group) and >60-year-olds (60s group)] under oxidative stress conditions. Results Although co-culturing HUVECs with ADASs ameliorated inflammation due to increased oxidative stress conditions, few differences were observed between the ADASs from the 40s and 60s groups. Moreover, the Database for Annotation, Visualization, and Integrated Discovery classification tool revealed differentially expressed genes in the Kyoto Encyclopedia of Genes and Genomes pathway associated with cytokine–cytokine receptor interaction in response to ADASs. Conclusion Protein expression profiles were unchanged in HUVECs induced by isolated ADASs from donors of different ages under oxidative stress conditions.


Introduction
Adipose tissue is a rich source of cells with prospects for tissue engineering and regenerative medicine. 1,2 Adipose-derived stromal vascular fractions (SVFs) contain adipose-derived stem cells (ASCs), endothelial (progenitor) cells, vascular smooth muscle cells, mesenchymal stem cells (MSCs), fibroblasts, macrophages, and pericytes, all of which exhibit mesodermal and ectodermal capacity. 3,4 This heterogeneous complex population has shown efficacy in tissue generation and vascular stabilization 3,4 as well as substantial potential for therapeutic applications in lipotransfer, management of diabetes-related complications, nerve regeneration, burn wound therapy, and other uses. 1,2 In particular, SVFs also have cardiomyogenic potential, 5 and many research groups are studying the possibility of SVF transplantation as a therapy for heart disease. 3,[6][7][8] ASCs improve cardiac function and perfusion after acute and chronic myocardial infarction in the rodent and pig, 3,6,7 and their transplantation has been suggested as a useful therapeutic option for angiogenesis in chronic ischemic heart disease. 8 Despite these attractive advantages of SVFs in clinical interventions, their use in many diseases has not been fully evaluated in clinical studies. 9 Donor age is known to affect the regenerative capability of adult tissues in cellbased therapies; donor age also affects the number of adipose-derived MSCs, cell yield of the SVF, and differentiation potential and angiogenic functional capacity of adipose tissue. [10][11][12] However, controversial results have been shown in humans. [13][14][15][16] Researchers have found that adiposederived MSCs exhibit similar osteogenic paracrine activity in all age groups of donors. 13 No correlation has been shown between the cell yield of ASCs from liposuction aspirates and donor age, 14 and the age of the donor does not affect the proliferation and differentiation capacities of cultured ASCs. 15 In addition, one study showed that the effect of autologous SVF transplantation for rheumatoid arthritis was unrelated to donor age. 16 Therefore, to clarify the above discrepancies in previous research, we performed the present study to determine whether SVF transplantation into impaired heart tissue shows differential effects according to donor age. We investigated the differences in protein expression in human umbilical vein endothelial cells (HUVECs) co-cultured with adipose-derived adherent stromal cells (ADASs) from individuals of different ages under oxidative stress conditions due to an abundant number of endothelial cells in the heart. 17 This study was based on the concept that redox signaling influences many physiological processes in the heart and plays a critical role in pathological cardiac remodeling. 18

Isolation of SVFs and culture of ADASs
SVFs were isolated from human adipose tissue samples by an enzymatic digestion method using the SmartX kit (DongKoo Bio & Pharma Co., Seoul, South Korea) following the manufacturer's instructions. Briefly, adipose tissue was centrifuged at 1,700 Â g for 5 min to remove water, tumescent solution, and oil. Next, 50 ml of fat tissue was digested with 0.075% collagenase type I at 37 C for 30 min under gentle agitation. After the digested tissue was filtered through a 75-mm strainer to remove residual tissue, the cell suspension was centrifuged and washed three times with phosphatebuffered saline (PBS) (HyClone, Logan, UT, USA). Total and live cell counts were performed using the NucleoCounterV R NC-200 TM automated cell counter (ChemoMetec, Allerød, Denmark). The SVFs were cultured in 10% fetal bovine serum (HyClone)-supplemented Dulbecco's Modified Eagle's Media (HyClone) and 1% penicillin/streptomycin at a density of 5 Â 10 4 cells/cm 2 in a 100-mm dish in a humidified atmosphere with 5% CO 2 at 37 C. After plating on culture dishes, nonadherent cells were discarded by changing the culture medium and ADASs passaged three times.

Cell viability assay
Cell viability was measured using Ez-Cytox (Daeillab Service, Seoul, Korea). HUVECs were seeded 24 h prior to H 2 O 2 treatment at a density of 5 Â 10 4 cells/cm 2 onto a 96-well plate. After 24 h, H 2 O 2 (0, 10, 20, 30, 40, or 50 mM) was applied to the HUVECs for 2, 4, or 6 h. After the addition of 10 mL of Ez-Cytox into each well, cell viability was evaluated by measuring the optical density at 450 nm.

Reactive oxygen species detection assay
Reactive oxygen species (ROS) production in H 2 O 2 -treated HUVECs was assessed according to the levels of bright greencolored 2 0 ,7 0 -dichlorofluorescein (DCF) produced by the oxidation of DCF diacetate (DCF-DA) dye. The HUVECs were plated 24 h prior to H 2 O 2 treatment at a density of 5 Â 10 4 cells/cm 2 in a 6-well plate and washed twice with PBS after 24 h. ROS generation was induced in HUVECs after 2, 4, or 6 h of treatment with 0 to 50 mM H 2 O 2 , followed by incubation with 50 mM DCF-DA (Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark at 37 C. The green fluorescence was detected by flow cytometry using a BD Accuri TM C6 Cytometer (BD Biosciences, Piscataway, NJ, USA).

Co-culture of ADASs and HUVECs
HUVECs at passage 5 were plated 24 h prior to H 2 O 2 treatment at a density of 5 Â 10 4 cells/cm 2 in a 6-well plate and then treated with or without 40 mM H 2 O 2 for 4 h. After 4 h, the cells were co-cultured with individual ADASs at passage 3 using trans-well inserts with a 0.4-mm porous translucent polyethylene terephthalate membrane (Falcon; Corning Life Sciences, Pittston, PA, USA) at a cell density identical to that of the HUVECs. Following coculture for 24 or 48 h after the incubation period, the cells in the lower well were then harvested for further analysis. The experimental groups were designed as follows: Group 1 (negative control), HUVECs (monoculture); Group 2 (H 2 O 2 ), HUVECs (monoculture) þ H 2 O 2 ; Groups 3, 4, and 5 (40s group), HUVECs and ADASs (40s group #1, #2 or #3, co-culture) þ H 2 O 2 ; and Groups 6, 7, and 8 (60s group), HUVECs and ADASs (60s group #1, #2, or #3, co-culture) þ H 2 O 2 .

Flow cytometry
For the flow cytometric analysis, cells were collected using Accutase Cell Detachment Solution (Thermo Fisher Scientific, Waltham, MA, USA). The cells (5 Â 10 5 cells/50 mL) were incubated for 1 h with antibodies to CD31, CD34, CD45, CD90, CD105, CD106, or isotype control (Santa Cruz Biotechnology, Santa Cruz, CA, USA) on ice and then washed three times with PBS containing 2.5% fetal bovine serum and 0.1% sodium azide (Sigma-Aldrich; washing solution). The cells were further incubated for 1 h with fluorescein isothiocyanate-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz Biotechnology) on ice in the dark. The cells were then washed three times and analyzed via flow cytometry using a BD Accuri TM C6 Cytometer (BD Biosciences).

Immunoblot analysis
The immunoblot analysis was performed according to the normalized methods of our previous studies. 19 Primary polyclonal antibody (Santa Cruz Biotechnology) and secondary antibodies (horseradish peroxidase-conjugated anti-goat IgG, antimouse IgG or anti-rabbit IgG; Santa Cruz Biotechnology) were used for detection of proteins of interest. The results were visualized using an enhanced chemiluminescence (ECL, Western Blotting Detection kit, GE Healthcare, Sweden) system, and the band intensities were quantified using ImageJ software.

RNA isolation, reverse-transcription polymerase chain reaction (PCR), and quantitative real-time PCR (qPCR)
Total RNA was isolated from HUVECs using TRIzol Reagent Solution (Life Technologies, Frederick, MD, USA) according to the manufacturer's instructions. Oligo dT-primed cDNA was synthesized from total RNA using the Maxime RT PreMix kit (iNtRON Biotechnology, Seongnam, Korea). The level of each gene transcript was quantitatively determined by qPCR using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the SYBR Green Dye system [SYBR Premix Ex Taq (Tli RNase H Plus) and ROX reference dye (Takara Bio Inc., Foster City, CA, USA)]. The transcript level of each gene was normalized to the GAPDH transcript levels. Primers were designed using Primer3 and BLAST, and the primer set sequences are listed in Table 1.

Network analysis
For the gene/protein network analyses, targets identified in this study were analyzed using GeneMANIA (http://www.genema nia.org) and the Database for Annotation, Visualization, and Integrated Discovery (DAVID). 20,21 DAVID provides a comprehensive set of functional annotation tools to interpret the biological relevance of such a large list of genes/proteins, whereas the GeneMANIA interface generates a list of genes with functional similarity based on the available genomics and proteomics information in the database. The GeneMANIA algorithm contains two integral parts: 1) a linear regression-based algorithm for calculating a single, composite functional association network from F, sequence of sense strands; R, sequence of anti-sense strands; IL6, interleukin-6; IL8, interleukin-8; IFNG, interferon gamma; TNF, tumor necrosis factor; FGF1, fibroblast growth factor-1; FGF2, fibroblast growth factor-2; VEGFA, vascular endothelial growth factor A; VEGFB, vascular endothelial growth factor B; PDGFA, platelet-derived growth factor subunit A; PDGFB, platelet-derived growth factor subunit B; ANG, angiopoietin multiple networks derived from different proteomic or genomic data sources and 2) the prediction of gene function.

Statistical analysis
All data were compared via one-way analysis of variance using the Statistical Package for the Social Sciences (SPSS, version 14.0K; SPSS Inc., Chicago, IL, USA). Data are expressed as mean AE standard error of the mean. Group means were considered significantly different at p<0.05, as determined by the protected least significant difference test when analysis of variance indicated an overall significant treatment effect (p<0.05).

Characterization of human ADASs by passage
The 7 donors in this study were divided into 3 groups: >40 years of age (40s group, n=3), >60 years of age (60s group, n=3), and the characterization sample (n=1). The basic characteristics of the donors are shown in Table 2. To investigate the donor ageassociated differences in ADASs isolated from human adipose tissue, ADASs were obtained from each of 3 donors in the 40s and 60s groups, characterized from 1 donor. ADASs isolated from human adipose tissue of donors in the 40s and 60s groups were cultured until the number of cells had sufficiently increased (by passage 3) for coculturing with HUVECs. Therefore, the surface marker expression of ADASs according to the passage number was first investigated using flow cytometry. No differences were observed in the first three passages of ADASs during characterization of ADASs in the subculture steps ( Figure 1 and Table 1). Based on the results obtained during the characterization of the passages, the ADASs used in the current study expressed CD45, CD90, CD105, and CD106 but not CD31 or CD34 at passage 3 ( Figure 1).

Determination of the concentration and treatment time of H 2 O 2 in HUVECs
Analysis of the treatment duration and titration of H 2 O 2 in HUVECs is necessary to evaluate the differential responses of HUVECs to isolated ADASs from donors of different ages under oxidative stress conditions. Accordingly, cell viability and ROS generation of HUVECs were measured using Ez-Cytox and DCF-DA with different concentrations of H 2 O 2 (0-50 mM) and treatment times (0, 2, 4, or 6 h). Figure 2 Table 1.
ADASs, adipose-derived adherent stromal cells; P, passage. (b)), although the cell viability was 87% at this concentration and treatment time (Figure 2(a)). Therefore, HUVECs were treated with 40 mM of H 2 O 2 for 4 h in further studies.

Differential regulation of proteins related to anti-oxidant activity, apoptosis, inflammation, adhesion, and calcium regulation in HUVECs by ADASs
HUVECs pretreated with H 2 O 2 were cultivated with ADASs isolated from human adipose tissue from both donor age groups (40s and 60s groups) for 48 h. The expression levels of proteins related to antioxidant activity, apoptosis, inflammation, adhesion, and calcium regulation in HUVECs were compared between the 40s and 60s groups using western blot analysis.

The expression of anti-oxidant proteins [peroxiredoxin (PRX) and thioredoxin (TRX)]
in HUVECs was down-regulated by H 2 O 2 but showed an upward trend in HUVECs co-cultured with ADASs (40s and 60s groups) (Figure 3). In contrast, the expression of HUVEC protein associated with apoptosis (p53) was increased by H 2 O 2 but decreased in HUVECs co-cultured with ADASs despite variations among individuals ( Figure 3). Moreover, proteins that are important for inflammation (phosphorylated p38), adhesion (intercellular adhesion molecule-1), and calcium regulation (Ca 2þ / calmodulin-dependent protein kinase II and calreticulin) exhibited a tendency to increase in H 2 O 2 -treated HUVECs and were more highly expressed in HUVECs co-cultured with ADASs than in H 2 O 2 -treated HUVECs ( Figure 3). However, p38 was largely unchanged in all groups ( Figure 3). Interestingly, little differences were observed in protein expression related to anti-oxidant activity, apoptosis, inflammation, adhesion,  and calcium regulation between the 40s and 60s donors (Figure 3).

Functional classification of differentially expressed proteins/genes by various bioinformatics resources
To determine the gene interactions for proteins/genes that were differentially altered in H 2 O 2 -treated HUVECs, 15 proteins/ genes associated with anti-oxidation, apoptosis, inflammation, angiogenic factors, or calcium regulation were analyzed using GeneMANIA to determine the interactions of these proteins/genes [ Figure 5(a)]. The 14 query genes (inside circle) and their interacting genes are presented in Figure 5(a). Function-based interactions with identified partners were predicted with genes associated with positive regulation of cell motility, cell migration, and the cellular components required for movement. Furthermore, identification of enriched functional gene groups using DAVID showed that the most strongly affected functional genes in ADASs were found in the Kyoto   Encyclopedia of Genes and Genomes pathway [ Table 3 and Figure 5

Discussion
Adipose-derived SVFs contain pluripotent stem cells that can differentiate into osteogenic, adipogenic, myogenic, neurogenic, chondrogenic, and hematopoietic lineages. 22,23 Specifically, these cells spontaneously show a cardiomyocyte phenotype, which suggests that this phenotype may be required for differentiation in response to specific conditions. 5 Additionally, many reports have been published regarding the possibility of transplantation of adiposederived SVFs as a therapy for heart disease. 3,6-8 Donor age is a critical factor in the regenerative capability of the cells in this cell-based therapy, 10-12 but controversial results have been observed in humans. [13][14][15][16] Our aim was to determine the effects of donor age on adiposederived SVF transplantation into impaired heart tissue. Thus, we examined the effects of ADASs on HUVECs because endothelial cells are abundant in the heart, 17 and oxidative stress conditions were used as an environmental stimulus because of their critical role in many physiological processes in the heart. 18 Therefore, we explored the differential expression of proteins in endothelial cells co-cultured with adiposederived ADASs from donors of different . Interestingly, although co-culturing HUVECs with ADASs ameliorated inflammation caused by an increase in oxidative stress conditions, few differences were observed between the ADASs from the 40s and 60s groups. SVF cells are composed of heterogeneous cell populations including bloodderived cells (CD45 þ ), ASCs (CD31 À , CD34 þ , CD45 À , CD90 þ , CD105 À , and CD106 þ ), MSCs (CD31 À , CD34 À , CD45 À , CD90 þ , CD105 þ , and CD106 þ ), endothelial (progenitor) cells (CD31 þ , CD34 þ , CD45 À , CD90 þ , CD105 À , and CD106 þ ), vascular smooth muscle cells (CD31 À , CD34 þ , CD45 À , CD90 þ , CD105 À , and CD106 -), pericytes (CD31 À , CD34 À , CD45 À , CD90 þ , CD105 À , and CD106 þ ), and other cells. 4,23,24 Differences exist in the phenotype and kinetics of differentiation between freshly isolated SVFs and cultured ADASs. 24,25 However, by passage 3, the differences in surface marker expression of cultured ADASs were negligible ( Figure 1). In addition, the ADASs used in our study expressed CD45, CD90, CD105, and CD106 but neither CD31 nor CD34 (Figure 1). After plastic adherent culture, CD45 in ADASs is usually almost absent. 26 Interestingly, the expanded ADASs used in the present study expressed CD45 by passage 3 (Figure 1). The most likely reasons for this are the method used for isolation of SVFs, 27 the short period of time for adherent culture, and the increased numbers of adherent leukocytes in patients with arthritis 28 (Table 1). These factors may make CD45 expression possible in cultured and expanded ADASs. In this study, we used cultured ADASs from passage 3 to ensure that sufficient numbers of ADASs were available for co-culturing with HUVECs.
Prior to the beginning of this study, we anticipated that anti-oxidant proteins would be down-regulated and that proteins related to apoptosis, inflammation, adhesion, and calcium regulation would be up-regulated in H 2 O 2 -treated HUVECs. We also predicted that these proteins would show opposite expression patterns when exposed to ADASs compared with H 2 O 2 treatment. Consistent with our expectations, the expression levels of most proteins were altered in HUVECs treated with H 2 O 2 (Figure 3 and Figure 4). However, contrary to our expectations, proteins other than anti-oxidant proteins and apoptosis-related proteins were upregulated to a greater extent in cells exposed to ADASs than in the H 2 O 2 -treated HUVECs ( Figure 3).
Oxidative stress is characterized by elevated intracellular ROS that damage lipids, proteins, and DNA, and numerous metabolic, structural, and functional effects due to oxidative stress have been observed in many experimental and clinical investigations. 29 The defense mechanisms against oxidative stress include (enzymatic) antioxidant defenses such as superoxide dismutases, glutathione peroxidases, catalases, PRX, and TRX. 26 Moreover, oxidative stress induces p53 activation and apoptotic induction. 29 Consistent with previous reports, 26,29 anti-oxidant proteins (PRX and TRX) were down-regulated in HUVECs by H 2 O 2 and enhanced by treatment of ADASs, although individual variations were observed. However, p53 was increased by H 2 O 2 and decreased by ADASs (Figure 3). These results imply that proteins and/or extracellular vesicles secreted in response to ADASs may induce enzymatic anti-oxidant defenses and/or anti-apoptotic signals in endothelial cells.
We assumed that proteins showing reverse trends in expression in response to ADASs were affected by pro-inflammatory cytokines through paracrine and/or autocrine reactions 8 ; however, some genes were