Cultured Retinal Pigment Epithelial Cells Research Articles

Artemisinin Confers Cytoprotection toward Hydrogen Peroxide-Induced Cell Apoptosis in Retinal Pigment Epithelial Cells in Correlation with the Increased Acetylation of Histone H4 at Lysine 8.

Increased oxidative stress is one of the critical pathologies inducing age-related macular degeneration (AMD), characterized by retinal pigment epithelial (RPE) cell damage and death. The unbalanced acetylation and deacetylation of histones have been implicated in AMD pathogenesis or hydrogen peroxide (H2O2)-induced cell damage. Therefore, strategies aimed at controlling the balance between acetylation and deacetylation may effectively protect RPE cells from oxidative damage. Artemisinin is an antimalarial lactone drug derived from Artemisia annua, with antioxidant activity known to modulate histone acetylation in the brain, but its effect on the retina is unknown. In this study, we aimed to investigate whether Artemisinin exerts a cytoprotective effect on oxidative stress-induced apoptosis in RPE cells by regulating histone acetylation. We hypothesized that Artemisinin confers cytoprotection toward H2O2-induced apoptosis in RPE cells through this mechanism. In the present study, we found that Artemisinin at a sub-clinic dosage of 20 μM inhibited the H2O2-induced cell viability decrease and B-cell lymphoma 2 (Bcl-2) protein level decrease and attenuated the H2O2-induced decrease in the histone H4 lysine (Lys) 8 acetylation [Acetyl-H4 (Lys 8)] level in the retinal RPE cell line D407. As expected, histone deacetylase inhibitor Trichostatin A at the concentration of 250 nM increased the Acetyl-H4 (Lys 8) level in D407 cells and attenuated the H2O2-induced cell viability decrease and apoptosis. Similar findings were obtained using adult RPE (ARPE)19 cells, another human RPE cell line, and primary human RPE cell cultures. In conclusion, these results confirmed our hypothesis and indicated that Artemisinin attenuated H2O2-induced apoptosis in apparent correlation with the increase in the Acetyl-H4 (Lys 8) level, which is associated with gene transcription and cell survival. By modulating histone acetylation, Artemisinin may restore the balance between acetylation and deacetylation and enhance the resistance and survival of RPE cells under oxidative stress. Our study provides novel mechanistic insights into the effect of Artemisinin on histone acetylation and apoptosis in RPE cells and supports the potential application of Artemisinin in the prevention and/or treatment of AMD.

Study of the metallome and transcriptome of a degenerating cellular model of the retinal pigment epithelium

Aims/Purpose: To study the changes on the metallome and transcriptome of a cellular model of the retinal pigment epithelium (RPE) during its in vitro degeneration after 4 months in culture.Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts and cultured for 4 months. Cell pigmentation and barrier function was followed up using optical microscopy and measurement of the transepithelial electrical resistance (TEER). Samples were collected at 21, 91 and 133 days in culture and analysed for gene expression, metal homeostasis of Ca, Na, P, Mg and P, and extracellular deposit formation through RNA sequencing (RNA‐Seq), elemental mass spectrometry (ICP‐MS) and immunocytochemistry assays, respectively.Results: RPE cells formed a differentiated monolayer at 21 days in culture, expressing specific genes and proteins of RPE and giving increasing TEER values. However, after 133 days in culture, loss of tight junction proteins (i.e. CLDN19 and BEST1) and electrical resistance was seen, together with loss of pigmentation and increase of apolipoprotein E deposition in the extracellular space. Gene expression analysis showed an enrichment on apoptotic processes, biomolecule phosphorylation, and signs of epithelial‐to‐mesenchymal transition (EMT). Degeneration of RPE cells concurred with an increase of Na and P content in the cytosol and decrease of Mg, while a decrease of Mg, P and Cu was determined in the insoluble fraction of cells. Time‐dependent changes of metals were accompanied with the alteration of metal transporters and antioxidant enzymes using metals as cofactors.Conclusions: Degeneration of RPE cells in culture was characterized by depigmentation of cells, improper barrier function, downregulation of antioxidant protection, loss of epithelial phenotype, and changes on the levels of Na, P, Mg and Cu, as well as of the expression of their homeostatic proteins.

Multi‐omic study of a cellular model of the retinal pigment epithelium producing extracellular deposits

Aims/Purpose: To study transcriptomic and metabolomic changes during a 6‐month follow‐up of an in vitro model of the retinal pigment epithelium (RPE) producing extracellular deposits.Methods: Primary cultures of human fetal RPE cells were established in P12 transwell inserts for 6 months and harvested at 4, 12, 17 and 25 weeks. Progressive accumulation of extracellular material was determined by apolipoprotein E (APOE) and hydroxyapatite (HAP) localization. Transcriptome was analysed by RNA sequencing (RNAseq) and differential expression analysis was conducted using the platform Dr. Tom. Untargeted metabolomics was carried out by high‐resolution mass spectrometry (LC‐IM‐QTOF) and bioinformatic analysis was performed using Mass Professional Profiler software.Results: RPE cells formed a fully differentiated monolayer at 4 weeks in culture, but tight junctions and electrical resistance decreased from 12 weeks onwards, coinciding with the formation of extracellular deposits containing APOE and HAP. RNAseq showed alterations of cell migration, adhesion and differentiation, and oxidation–reduction, lipid metabolic and apoptotic processes during RPE ageing (4 vs 25 weeks, Q‐value < 0.001). Untargeted metabolomics provided 350 non‐redundant compounds, and principal component analysis showed a clear separation between time points and good reliability within each group (55% of cumulative variance). Moderated t‐test (Benjamin‐Hochberg correction) provided 103 compounds differentially altered during RPE follow‐up (p‐value < 0.001, fold‐change > 2), belonging to trans‐sulfuration, one carbon metabolism, glutathione metabolism, Kennedy pathway for sphingolipids and cysteine and methionine catabolism.Conclusions: Time‐dependent changes were observed in the cell culture model, including progressive sub‐RPE accumulation of APOE and HAP, and alterations in cell adhesion and migration, lipid metabolism and sphingolipids pathway contributing to photoreceptor protection and calcium mobilization.

The Effect of Anti-Autotaxin Aptamers on the Development of Proliferative Vitreoretinopathy.

This study investigated the effect of anti-autotaxin (ATX) aptamers on the development of proliferative vitreoretinopathy (PVR) in both in vivo and in vitro PVR swine models. For the in vitro study, primary retinal pigment epithelial (RPE) cells were obtained from porcine eyes and cultured for cell proliferation and migration assays. For the in vivo study, a swine PVR model was established by inducing retinal detachment and injecting cultured RPE cells (2.0 × 106). Concurrently, 1 week after RPE cell injection, the anti-ATX aptamer, RBM-006 (10 mg/mL, 0.1 mL), was injected twice into the vitreous cavity. Post-injection effects of the anti-ATX aptamer on PVR development in the in vivo swine PVR model were investigated. For the in vitro evaluation, the cultured RPE cell proliferation and migration were significantly reduced at anti-ATX aptamer concentrations of 0.5-0.05 mg and at only 0.5 mg, respectively. Intravitreal administration of the anti-ATX aptamer also prevented tractional retinal detachment caused by PVR in the in vivo PVR model. We observed that the anti-ATX aptamer, RBM-006, inhibited PVR-related RPE cell proliferation and migration in vitro and inhibited the progression of PVR in the in vivo model, suggesting that the anti-ATX aptamer may be effective in preventing PVR.

Inhibiting autophagy by miR-19a-3p/PTEN regulation protected retinal pigment epithelial cells from hyperglycemic damage.

The catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear. An in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition. Autophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition. Our findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR.

Engineering a Biomimetic In Vitro Model of Bruch's Membrane Using Hagfish Slime Intermediate Filament Proteins.

Bruch's membrane resides in the subretinal tissue and regulates the flow of nutrients and waste between the retinal pigment epithelial (RPE) and vascular layers of the eye. With age, Bruch's membrane becomes thicker, stiffer, and less permeable, which impedes its function as a boundary layer in the subretina. These changes contribute to pathologies such as age-related macular degeneration (AMD). To better understand how aging in Bruch's membrane affects surrounding tissues and to determine the relationship between aging and disease, an in vitro model of Bruch's membrane is needed. An accurate model of Bruch's membrane must be a proteinaceous, semipermeable, and nonporous biomaterial with similar mechanical properties to in vivo conditions. Additionally, this model must support RPE cell growth. While models of subretinal tissue exist, they typically differ from in vivo Bruch's membrane in one or more of these properties. This study evaluates the capability of membranes created from recombinant hagfish intermediate filament (rHIF) proteins to accurately replicate Bruch's membrane in an in vitro model of the subretinal tissue. The physical characteristics of these rHIF membranes were evaluated using mechanical testing, permeability assays, brightfield microscopy, and scanning electron microscopy. The capacity of the membranes to support RPE cell culture was determined using brightfield and fluorescent microscopy, as well as immunocytochemical staining. This study demonstrates that rHIF protein membranes are an appropriate biomaterial to accurately mimic both healthy and aged Bruch's membrane for in vitro modeling of the subretinal tissue.

Investigating the effects of 7-ketocholesterol on retinal pigment epithelium bioenergetics.

Age-related macular degeneration (AMD) is associated with formation of drusen, clusters of lipids, and oxidized lipid products under the retinal pigment epithelium (RPE). 7-Ketocholesterol (7KC) is a form of oxidized cholesterol present in drusen and is hypothesized to play a role in AMD pathogenesis. The association of 7KC with cellular toxicity and inflammation, key elements of AMD pathology, has been demonstrated. However, the effects of 7KC on altering RPE bioenergetics, a potentially important pathologic process in AMD, are unclear. Herein, we describe the effects of non-lethal doses of 7KC on the bioenergetics and phenotype of RPE cells in culture. Metabolic analysis demonstrated a significant dose-dependent increase in total ATP production rates that was driven primarily by an increase in glycolysis. The increase in glycolysis was accompanied by an increase in glucose uptake and increased expression of hexokinase 1. Increased levels of Translocase of Outer Mitochondrial Membrane 20and NADH:Ubiquinone Oxidoreductase Core Subunit S1, Succinate dehydrogenase, Ubiquinol-Cytochrome C Reductase Core Protein 2, Cytochrome C Oxidase II, and ATP synthase subunit beta, proteins involved in oxidative phosphorylation (OXPHOS), were also seen. However, specific electron transport chain activity remained unchanged. 7KC-treated cells also demonstrated a change in cellular morphology with decreased expression of epithelial markers. In summary, 7KC has significant effects on the bioenergetics and morphology of RPE cells reflective of findings seen in clinical AMD.

Altered zinc homeostasis in a primary cell culture model of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is progressively degenerated during age-related macular degeneration (AMD), one of the leading causes of irreversible blindness, which clinical hallmark is the buildup of sub-RPE extracellular material. Clinical observations indicate that Zn dyshomeostasis can initiate detrimental intracellular events in the RPE. In this study, we used a primary human fetal RPE cell culture model producing sub-RPE deposits accumulation that recapitulates features of early AMD to study Zn homeostasis and metalloproteins changes. RPE cell derived samples were collected at 10, 21 and 59 days in culture and processed for RNA sequencing, elemental mass spectrometry and the abundance and cellular localization of specific proteins. RPE cells developed processes normal to RPE, including intercellular unions formation and expression of RPE proteins. Punctate deposition of apolipoprotein E, marker of sub-RPE material accumulation, was observed from 3 weeks with profusion after 2 months in culture. Zn cytoplasmic concentrations significantly decreased 0.2 times at 59 days, from 0.264 ± 0.119 ng·μg-1 at 10 days to 0.062 ± 0.043 ng·μg-1 at 59 days (p < 0.05). Conversely, increased levels of Cu (1.5-fold in cytoplasm, 5.0-fold in cell nuclei and membranes), Na (3.5-fold in cytoplasm, 14.0-fold in cell nuclei and membranes) and K (6.8-fold in cytoplasm) were detected after 59-days long culture. The Zn-regulating proteins metallothioneins showed significant changes in gene expression over time, with a potent down-regulation at RNA and protein level of the most abundant isoform in primary RPE cells, from 0.141 ± 0.016 ng·mL-1 at 10 days to 0.056 ± 0.023 ng·mL-1 at 59 days (0.4-fold change, p < 0.05). Zn influx and efflux transporters were also deregulated, along with an increase in oxidative stress and alterations in the expression of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. The RPE cell model producing early accumulation of extracellular deposits provided evidences on an altered Zn homeostasis, exacerbated by changes in cytosolic Zn-binding proteins and Zn transporters, along with variations in other metals and metalloproteins, suggesting a potential role of altered Zn homeostasis during AMD development.

Granzyme B Contributes to Choroidal Neovascularization and Age-Related Macular Degeneration Through Proteolysis of Thrombospondin-1

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. The pathology of neovascular age-related macular degeneration (nAMD), also known as wet AMD, is associated with an abnormal blood vessel growth in the eye and involves an imbalance of proangiogenic and antiangiogenic factors. Thrombospondin (TSP)-1 and TSP-2 are endogenous matricellular proteins that inhibit angiogenesis. TSP-1 is significantly diminished in eyes with AMD, although the mechanisms involved in its reduction are unknown. Granzyme B (GzmB) is a serine protease with an increased extracellular activity in the outer retina and choroid of human eyes with nAMD-related choroidal neovascularization (CNV). This study investigated whether TSP-1 and TSP-2 are GzmB substrates using in silico and cell-free cleavage assays and explored the relationship between GzmB and TSP-1 in human eyes with nAMD-related CNV and the effect of GzmB on TSP-1 in retinal pigment epithelial culture and an explant choroid sprouting assay (CSA). In this study, TSP-1 and TSP-2 were identified as GzmB substrates. Cell-free cleavage assays substantiated the GzmB proteolysis of TSP-1 and TSP-2 by showing dose-dependent and time-dependent cleavage products. TSP-1 and TSP-2 proteolysis were hindered by the inhibition of GzmB. In the retinal pigment epithelium and choroid of human eyes with CNV, we observed a significant inverse correlation between TSP-1 and GzmB, as indicated by lower TSP-1 and higher GzmB immunoreactivity. In CSA, the vascular sprouting area increased significantly with GzmB treatment and reduced significantly with TSP-1 treatment. Western blot showed significantly reduced expression of TSP-1 in GzmB-treated retinal pigment epithelial cell culture and CSA supernatant compared with that in controls. Together, our findings suggest that the proteolysis of antiangiogenic factors such as TSP-1 by extracellular GzmB might represent a mechanism through which GzmB may contribute to nAMD-related CNV. Future studies are needed to investigate whether pharmacologic inhibition of extracellular GzmB can mitigate nAMD-related CNV by preserving intact TSP-1.

Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture.

Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration

BackgroundForkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood–retina barrier of the immune privileged eye.MethodsWe investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1β and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing.ResultsRPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1β to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells.ConclusionOur data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

Vitamin B3, nicotinamide, enhances mitochondrial metabolism to promote differentiation of the retinal pigment epithelium

In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception.

Inflammasome Activation in Retinal Pigment Epithelium from Human Donors with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. One of the risk factors associated with developing AMD is the single nucleotide polymorphism (SNP) found within the gene encoding complement factor H (CFH). Part of the innate immune system, CFH inhibits alternative complement pathway activation. Multi-protein complexes called inflammasomes also play a role in the innate immune response. Previous studies reported that inflammasome activation may contribute to AMD pathology. In this study, we used primary human adult RPE cell cultures from multiple donors, with and without AMD, that were genotyped for the Y402H CFH risk allele. We found complement and inflammasome-related genes and proteins at basal levels in RPE tissue and cell cultures. Additionally, treatment with rotenone, bafilomycin A, and ATP led to inflammasome activation. Overall, the response to priming and activation was similar, irrespective of disease state or CFH genotype. While these data show that the inflammasome is present and active in RPE, our results suggest that inflammasome activation may not contribute to early AMD pathology.

Suppression of EZH2 inhibits TGF-β1-induced EMT in human retinal pigment epithelial cells

Epithelial–mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is critically involved in the occurrence of subretinal fibrosis. This study aimed to investigate the role of enhancer of zeste homolog 2 (EZH2) in EMT of human primary RPE cells and the underlying mechanisms of the anti-fibrotic effect of EZH2 suppression. Primary cultures of human RPE cells were treated with TGF-β1 for EMT induction. EZH2 was silenced by siRNA to assess the expression levels of epithelial and fibrotic markers using qRT-PCR, Western blot, and immunofluorescence staining assay. Furthermore, the cellular migration, proliferation and barrier function of RPE cells were evaluated. RNA-sequencing analyses were performed to investigate the underlying mechanisms of EZH2 inhibition. Herein, EZH2 silencing up-regulated epithelial marker ZO-1 and downregulated fibrotic ones including α-SMA, fibronectin, and collagen 1, alleviating EMT induced by TGF-β1 in RPE cells. Moreover, silencing EZH2 inhibited cellular migration and proliferation, but didn't affect cell apoptosis. Additionally, EZH2 suppression contributed to improved barrier functions after TGF-β1 stimulation. The results from RNA sequencing suggested that the anti-fibrotic effect of EZH2 inhibition was associated with the MAPK signaling pathway, cytokine-cytokine receptor interaction, and the TGF-beta signaling pathway. Our findings provide evidence that the suppression of EZH2 might reverse EMT and maintain the functions of RPE cells. EZH2 could be a potential therapeutic avenue for subretinal fibrosis.

Kir4.2 Potassium Channels in Retinal Pigment Epithelial Cells In Vitro: Contribution to Cell Viability and Proliferation, and Down-Regulation by Vascular Endothelial Growth Factor.

Dedifferentiation and proliferation of retinal pigment epithelial (RPE) cells are characteristics of retinal diseases. Dedifferentiation is likely associated with changes of inwardly rectifying potassium (Kir) channels. The roles of Kir4.2 channels in viability, and proliferation of cultured RPE cells were investigated. Gene expression levels were determined using qRT-PCR. RPE cells expressed Kir2.1, 2.2, 2.4, 3.2, 4.1, 4.2, 6.1, and 7.1 mRNA. Kir4.2 protein was verified by immunocytochemistry and Western blotting. Kir4.2 mRNA in cultured cells was upregulated by hypoxia (hypoxia mimetic CoCl2 or 0.2% O2) and extracellular hyperosmolarity (addition of high NaCl or sucrose). Kir4.2 mRNA was suppressed by vascular endothelial growth factor (VEGF), blood serum, and thrombin whereas platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor-β1 (TGF-β1) increased it. Hyperosmotic Kir4.2 gene expression was mediated by TGF-β1 receptor signaling while hypoxic gene transcription was dependent on PDGF receptor signaling. VEGF receptor-2 blockade increased Kir4.2 mRNA level under control, hyperosmotic, and hypoxic conditions. SiRNA-mediated knockdown of Kir4.2 decreased the cell viability and proliferation under control and hyperosmotic conditions. Kir4.2 channels play functional roles in maintaining the viability and proliferation of RPE cells. Downregulation of Kir4.2 by VEGF, via activation of VEGF receptor-2 and induction of blood-retinal barrier breakdown, may contribute to decreased viability of RPE cells under pathological conditions.

Citrulline protects human retinal pigment epithelium from hydrogen peroxide and iron/ascorbate induced damages.

Oxidative stress plays an important role in the ageing of the retina and in the pathogenesis of retinal diseases such as age‐related macular degeneration (ARMD). Hydrogen peroxide is a reactive oxygen species generated by the photo‐excited lipofuscin that accumulates during ageing in the retinal pigment epithelium (RPE), and the age‐related accumulation of lipofuscin is associated with ARMD. Iron also accumulates with age in the RPE that may contribute to ARMD as an important source of oxidative stress. The aim of this work was to investigate the effects of L‐Citrulline (CIT), a naturally occurring amino acid with known antioxidant properties, on oxidative stressed cultured RPE cells. Human RPE (ARPE‐19) cells were exposed to hydrogen peroxide (H2O2) or iron/ascorbate (I/A) for 4 h, either in the presence of CIT or after 24 h of pretreatment. Here, we show that supplementation with CIT protects ARPE‐19 cells against H2O2 and I/A. CIT improves cell metabolic activity, decreases ROS production, limits lipid peroxidation, reduces cell death and attenuates IL‐8 secretion. Our study evidences that CIT is able to protect human RPE cells from oxidative damage and suggests potential protective effect for the treatment of retinal diseases associated with oxidative stress.

Electrohydrodynamic Jet-Printed Ultrathin Polycaprolactone Scaffolds Mimicking Bruch's Membrane for Retinal Pigment Epithelial Tissue Engineering.

Age-related macular degeneration (AMD) is the leading cause of visual loss and affects millions of people worldwide. Dysfunction of the retinal pigment epithelium (RPE) is associated with the pathogenesis of AMD. The purpose of this work is to build and evaluate the performance of ultrathin scaffolds with an electrohydrodynamic jet (EHDJ) printing method for RPE cell culture. We printed two types of ultrathin (around 7 μm) polycaprolactone scaffolds with 20 μm and 50 μm pores, which possess mechanical properties resembling that of native human Bruch’s membrane and are biodegradable. Light microscopy and cell proliferation assay showed that adult human retinal pigment epithelial (ARPE-19) cells adhered and proliferated to form a monolayer on the scaffolds. The progress of culture matured on the scaffolds was demonstrated by immunofluorescence (actin, ZO-1, and Na+/K+-ATPase) and Western blot analysis of the respective proteins. The RPE cells cultured on EHDJ-printed scaffolds with 20 μm pores presented higher permeability, higher transepithelial potential difference, and higher expression level of Na+/K+-ATPase than those cultured on Transwell inserts. These findings suggest that the EHDJ printing can fabricate scaffolds that mimic Bruch’s membrane by promoting maturation of RPE cells to form a polarized and functional monolayered epithelium with potential as an in vitro model for studying retinal diseases and treatment methods.

Continuous Flow Synthesis of A2E Guided by Design of Experiments and High-Throughput Studies.

N-Retinylidene-N-retinylethanolamine (A2E) is the most studied lipid bisretinoid. It forms lipofuscin deposits in the retinal pigment epithelium (RPE), causing vision impairment and blindness in eye conditions, such as Stargardt's disease, cone-rod dystrophy, Best's macular dystrophy, and potentially age-related macular degeneration. Synthetic A2E is often used for inducing the accumulation of lipofuscins within the lysosomes of RPE cells in culture as an in vitro surrogate of retinal lipofuscin buildup, providing insights into the mechanisms of these eye conditions. Many reports describing the use of synthetic A2E employ material that has been prepared using a one-pot reaction of all-trans-retinal (ATR) and ethanolamine at room temperature for 48 h. We have revisited this synthesis by performing a design of experiments (DoE) and high-throughput experimentation workflow that was tailored to identify the most productive combination of the variables (temperature, solvent, and reagent equivalences) for optimization of A2E yield. Our DoE findings revealed that the interaction of ethanolamine with acetic acid and ATR was pivotal for the formation of A2E in high yield, indicating that imine formation is the critical step in the reaction. Armed with these results, we were able to optimize the method using a microfluidic reactor system before upscaling those conditions for continuous flow synthesis of A2E. This revised method enabled a more efficient production of material, from a reaction time of 48 h to a residence time of 33 min, with an accompanying yield improvement from 49 to 78%. Furthermore, we implemented a simple method to evaluate the quality of the A2E produced using optical spectroscopy and LC-MS characteristics to assure that the biological properties observed with A2E samples are not confounded by the presence of oxidized impurities that are commonly present in conventional A2E samples.