Retinal Pigment Epithelium Cells Cells Research Articles

Epitranscriptome Analysis of Oxidative Stressed Retinal Epithelial Cells Depicted a Possible RNA Editing Landscape of Retinal Degeneration.

Oxidative stress represents one of the principal causes of inherited retinal dystrophies, with many related molecular mechanisms still unknown. We investigated the posttranscriptional RNA editing landscape of human retinal pigment epithelium cells (RPE) exposed to the oxidant agent N-retinylidene-N-retinyl ethanolamine (A2E) for 1 h, 2 h, 3 h and 6 h. Using a transcriptomic approach, refined with a specific multialgorithm pipeline, 62,880 already annotated and de novo RNA editing sites within about 3000 genes were identified among all samples. Approximately 19% of these RNA editing sites were found within 3′ UTR, including sites common to all time points that were predicted to change the binding capacity of 359 miRNAs towards 9654 target genes. A2E exposure also determined significant gene expression differences in deaminase family ADAR, APOBEC and ADAT members, involved in canonical and tRNA editing events. On GO and KEGG enrichment analyses, genes that showed different RNA editing levels are mainly involved in pathways strongly linked to a possible neovascularization of retinal tissue, with induced apoptosis mediated by the ECM and surface protein altered signaling. Collectively, this work demonstrated dynamic RNA editome profiles in RPE cells for the first time and shed more light on new mechanisms at the basis of retinal degeneration.

MiR-125b attenuates retinal pigment epithelium oxidative damage via targeting Nrf2/HIF-1α signal pathway

The retinal pigment epithelium cells (RPE) are sensitive to oxidative stimuli due to long-term exposure to various environmental stimuli. Thus, the oxidative injury of RPE cells caused by the imbalance of redox homeostasis is one of the main pathogenic factors of age-related macular degeneration (AMD). But the sophisticated mechanisms linking AMD to oxidative stress are not fully elucidated. Activation of Nrf2 signal pathway can protect RPE cells from oxidative damage. The present study investigated the regulating mechanism of miR-125b in Nrf2 cascade and evaluated its antioxidant capacity. The in vitro studies indicated that overexpression of miR-125b substantially inhibited Keap1 expression, enhanced Nrf2 expression and induced Nrf2 nuclear translocation. Importantly, functional studies demonstrated that forced expression of miR-125b could significantly elevate cell proliferation and superoxide dismutase (SOD) levels while reduce reactive oxygen species (ROS) overproduction and malondialdehyde (MDA) formation. Further studies showed that miR-125b had no effect when Nrf2 was silenced in ARPE-19 cells. Additionally, the results identified that Nrf2 silence induced ROS accumulation enhances HIF-1α protein expression, while miR-125b could offset this effect via promoting HIF-1α protein degradation. Subsequent in vivo studies demonstrated that sodium iodate induced outer retina thinner was reversed with exogenous supplementation of miR-125b, which was cancelled in Nrf2 knockout mice. In conclusion, this study illustrated that miR-125b can protect RPE from oxidative damage via targeting Nrf2/HIF-1α signal pathway and potentially may serve as a therapeutic agent of AMD.

Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium.

Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood–retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone—an MR-specific agonist, or cortisol or cortisol + RU486—a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial–mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.

UV-Irradiated RPE Cells Assist Differentiation of Bone Marrow Derived Mesenchymal Stem Cells into RPE Cells Under a Direct Co-Culture Environment

Degeneration of retinal pigment epithelium cells (RPE) or delamination of RPE from Bruch’s membrane causes visual impairment. While regeneration of RPE cells is desirable to treat age-related macular degeneration (AMD) RPE cells of mammals, which are inherently non-renewable. Here, the bone marrow derived mesenchymal stem cells (BMSCs) that have the advantage of rapid growth and ease of extraction, which are adopted in order to help regenerate RPE cell. By co-culture with RPE cell, BMSCs exhibited a tendency to differentiate into RPE cell. By irradiating with the ultraviolet (UV) light, RPE cell was inhibited from growing and the differentiation of BMSCs was induced. The B-27® supplement was used as a differentiation factor, which helps BMSCs to differentiation into RPE cells. Specifically, B-27® supplement was mixed into the growth medium, which was added only to the co-culture and BMSCs groups. In consequence, UV-irradiated RPE cells did not grow, BMSCs in the co-culture group exhibited a tendency to differentiate into RPE cells. This was demonstrated by MTT assay, SEM, confocal imaging of the live/dead cells, RT-PCR and histological staining. Cell proliferation was the highest in the co-culture group, and the differentiation of BMSCs into RPE cells was observed in RT-PCR and histological staining. Our method will allow BMSCs with B-27® supplement to offer a new direction for RPE regeneration.

Inhibition of Proteasome Activity Upregulates IL-6 Expression in RPE Cells through the Activation of P38 MAPKs.

Purpose As far as we know, during the development of age-related macular degeneration (AMD), the activity of proteasome in retinal pigment epithelium cells (RPE) gradually decreases. And a lot of research has shown that age-related macular degeneration is closely related to inflammation and autoimmune. Moreover, there are many cytokines (CKs) involved in the course of inflammation. In this study, we are going to investigate how the decrease of proteasome activity affects the production of interleukin-6 (IL-6) in human retinal pigment epithelium cells (ARPE-19). Methods Cultured ARPE-19 was treated with or without MG132, a proteasome inhibitor, and the levels of IL-6 mRNA (messenger ribonucleic acid) in RPE at 1 h, 4 h, 8 h, and IL-6 protein in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were measured by real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA). The protein levels of MCP-1 (monocyte chemoattractant protein-1) in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were also measured by ELISA. Then we tested which of cell signal pathways regulating the production of IL-6 were activated when we added MG132 into the medium by Western blot and electrophoretic mobility shift assays (EMSA). After that, we put the inhibitors of these activated cell signal pathways into the medium individually to see which inhibitor can counteract the effect of upregulating the levels of IL-6 in the culture medium of RPE. Results MG132 decreased the secretion of MCP-1 in the culture medium of RPE, but it increased the expression of IL-6 mRNA in RPE and IL-6 protein level in the culture medium of RPE. MG132 treatment was also found to enhance the level of phosphorylated p38 mitogen-activated protein kinases (MAPKs) and c-Jun N-terminal Kinase (JNK) by Western blotting. More importantly, the effect of MG132 on upregulating the levels of IL-6 was inhibited by SB203580, an inhibitor of P38 MAP kinases. But the JNK inhibitor, SP600125, cannot prevent the effect of upregulating the levels of IL-6 by MG132 in the RPE culture medium. Conclusions We concluded that the proteasome inhibitor, MG132, upregulates IL-6 production in RPE cells through the activation of P38 MAPKs.

BRUCH MEMBRANE AND THE MECHANISM OF MYOPIZATION

The process of emmetropization is the adjustment of the length of the optical axis to the given optical properties of the cornea and lens after the end of the second year of life. Since its underlying mechanisms have not been fully explored yet, we reviewed findings available in the literature to discuss its potential etiology and the mechanism of myopization as an overshooting of emmetropization. Literature review. The process of emmetropization occurs by axial elongation. Up to the end of the second year of life, the eye grows spherically by active increase in scleral volume. Axial elongation in the process of emmetropization is associated with thinning of the retina and reduced density of retinal pigment epithelium cells (RPE) in the retro-equatorial region, and with thinning more of the choroid than of the sclera, starting at the equator and being most marked at the posterior pole. In contrast, retinal thickness and RPE density in the macular region and thickness of Bruch's membrane (BM) in any region are independent of axial length. It led to the hypothesis that axial elongation occurs by production of BM in the retro-equatorial region leading to a decreased RPE density and retinal thinning in that region and a more tube-like than spherical enlargement of the globe, without compromise in the density of the macular RPE cells and in macular retinal thickness. The increased disc-fovea distance in axially myopic eyes is caused by the development and enlargement of parapapillary, BM free, gamma zone while the length of macular BM, and indirectly macular RPE cell density and macular retinal thickness, remain constant. The target tissue for medical modification of emmetropization/myopization may be the RPE, producing and elongating BM in the retro-equatorial region.

A novel Bruch's membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells

Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3–5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.

The use of silicone oil-RMN3 (Oxane HD) as heavier-than-water internal tamponade in complicated inferior retinal detachment surgery

Conventional silicone oil provides suboptimal support of the inferior retina. In this study we evaluated the efficacy of Oxane HD in the management of complex retinal detachments involving lower quadrants of the retina. A prospective, interventional, comparative study. Eighteen patients were recruited. Treatment outcomes were compared with a historical control group of 14 patients. Patients with grade C3 PVR or greater and inferior retinal breaks, recurrent inferior retinal detachments (with or without PVR) and giant retinal tears were included. In those patients who re-detached under heavy silicone oil (n = 4), retro-oil epiretinal membranes (ERMs) were obtained at the time of subsequent surgery to analyse the immunopathological response to oxane HD. Immunohistochemistry was used to detect glia, retinal pigment epithelium cells (RPE), macrophages, T lymphocytes, or neural elements in the tissue using well-characterised monoclonal antibodies. Retinal attachment of the posterior pole following removal of silicone oil was achieved in 66.6% of the treatment group (n = 12) and 64.3% of controls (n = 9) (p = 1.0). Post-operative PVR developed in five patients in the treatment group (27.8%) and five control patients (35.8%). Following removal of silicone oil, residual oil was observed in 27.8% of the treatment group and 7.1% of controls. Median visual acuity, 3 months following removal of silicone oil, was 2.0 (IQR 0.9-2.0) in the treatment group and 1.0 (IQR 0.6-1.8) in the control group. Complications in the treatment group included, hypotony (n = 3), uveitis (n = 2), glaucoma (n = 1). All ERMs analysed demonstrated microscopic appearances typical of PVR. The membranes were fibrocellular in nature, contained RPE and glial cells, and variable amounts of intracellular and extracellular pigment. In addition, all had a dense infiltrate of vacuolated (presumed oil-filled) macrophages. We failed to observe an advantage following the use of Oxane HD in the treatment of inferior retinal detachments. Moreover, Oxane HD was difficult to remove and was associated with a higher incidence of complications.

INTRAVITREAL VORICONAZOLE

Fungal endophthalmitis is a rare but sight-threatening disease. Despite an expanding range of fungal pathogens, there are only few therapeutic agents for its treatment available. Voriconazole is a second-generation synthetic triazole with a broad action against yeasts and molds. The current study investigates the safety of Voriconazole for intravitreal application in a cell culture model. Primary human retinal pigment epithelium cells (RPE) and primary human optic nerve head astrocytes were treated with concentrations of Voriconazole ranging from 25 microg/mL to 10 mg/mL. Possible toxic effects and IC50 were evaluated after 24 hours and under conditions of oxidative stress. By treating the RPE cell lines with tumor-necrosisfactor alpha (TNF-alpha), lipopolysaccharides (LPS), and interleukin-6 (IL-6) the effects of Voriconazole on cellular viability under conditions of inflammation were investigated. Toxicity was evaluated by colorimetric measuring the inhibition of RPE cell proliferation (MTT). Additionally cell viability was quantified by a microscopic live-dead-assay. Concentrations <250 microg/mL Voriconazole had no influence neither on RPE nor on optic nerve head astrocytes cell proliferation and cell viability when administered for 24 hours and under oxidative stress. When preincubated with tumor-necrosis-factor alpha, lipopolysaccharides and interleukin-6 for 24 hours and subsequently treated with Voriconazole at concentrations up to 250 microg/mL for 24 hours no significant decrease in proliferation and viability was observed. This study showed that no significant toxicity existed for Voriconazole in vitro on primary RPE and optic nerve head astrocytes when administered in therapeutic concentrations up to 250 microg/mL.

Inner limiting membrane as membranous support in RPE sheet-transplantation

To investigate the inner limiting membrane as a scaffold for retinal pigment epithelium cells (RPE). Human donor eyes (n = 10) from the Eye Bank Tübingen were used to collect the inner limiting membrane (ILM). These human donor eyes and additional porcine eyes (n = 11) served to isolate RPE cells. A human RPE cell line (ARPE-19) was used as control. RPE cells were cultured on ILM for 3 and 7 days. Phase-contrast photographs of the cells in culture were obtained. Morphology and ultrastructural changes were evaluated by light and transmission electron microscopy. Porcine RPE cells adhere and proliferate when seeded on human ILM. The cells maintained their cuboidal morphology, were polarized, disclosed microvilli on the apical surface, formed intercellular junctions and did not dedifferentiate. Human RPE cells obtained from cadaver eyes barely adhered to the ILM and did not form an intact monolayer. ARPE-19 cells formed a dense colony and maintained epithelial features. The ILM is an ideal matrix to establish an intact RPE monolayer and has the potential to be used as sheet for subretinal transplantation.

Effects of PDTC on the proliferation and PCNA expression of human retinal pigment epithelial cells

To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on the proliferation and PCNA (proliferating cell nuclear antigen) expression of cultured human retinal pigment epithelium cells, human retinal pigment epithelium cells (RPE) were cultured from normal adults who, died accidentally. The effects of PDTC on the proliferation of RPE cells were examined by using methyl thiazlyl tetrazolium (MTT) assay. The effects of PDTC on the PCNA expression of RPE cells were immunohistochemically examined by employing biological image analysis system (BIAS). After treatment with PDTC of various of concentration ranging from 0.062 to 1 g/L for 24 h, or concentrations ranging from 0.031 to 1 g/L, the proliferation of RPE cells decreased in a dose-dependent manner. After treatment with PDTC of concentration varying from 0.062 to 1 g/L for 24 h, the PCNA expression was also suppressed in a dose-dependent manner. It is concluded that PDTC can inhibit the proliferation of RPE cells in vitro in a dose-and time-dependent manner, at least in part, by down-regulating the expression of PCNA. PDTC may be used to prevent and treat the proliferative vitreoretinopathy (PVR).