Retinal Pigment Epithelial Cell Function Research Articles

High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression.

Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis.

Microphthalmia-associated transcription factor regulates the visual cycle genes Rlbp1 and Rdh5 in the retinal pigment epithelium.

Regeneration of the visual pigment by cells of the retinal pigment epithelium (RPE) is fundamental to vision. Here we show that the microphthalmia-associated transcription factor, MITF, which plays a central role in the development and function of RPE cells, regulates the expression of two visual cycle genes, Rlbp1 which encodes retinaldehyde binding protein-1 (RLBP1), and Rdh5, which encodes retinol dehydrogenase-5 (RDH5). First, we found that Rlbp1 and Rdh5 are downregulated in optic cups and presumptive RPEs of Mitf-deficient mouse embryos. Second, experimental manipulation of MITF levels in human RPE cells in culture leads to corresponding modulations of the endogenous levels of RLBP1 and RDH5. Third, the retinal degeneration associated with the disruption of the visual cycle in Mitf-deficient mice can be partially corrected both structurally and functionally by an exogenous supply of 9-cis-retinal. We conclude that the expression of Rlbp1 and Rdh5 critically depends on functional Mitf in the RPE and suggest that MITF has an important role in controlling retinoid processing in the RPE.

Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+ /K+ -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd.

Extracellular Matrix Alterations and Deposit Formation in AMD.

Age related macular degeneration (AMD) is the primary cause of vision loss in the western world (Friedman et al., Arch Ophthalmol 122:564-572, 2004). The first clinical indication of AMD is the presence of drusen. However, with age and prior to the formation of drusen, extracellular basal deposits accumulate between the retinal pigment epithelium (RPE) and Bruch's membrane (BrM). Many studies on the molecular composition of the basal deposits and drusen have demonstrated the presence of extracellular matrix (ECM) proteins, complement components and cellular debris. The evidence reviewed here suggests that alteration in RPE cell function might be the primary cause for the accumulation of ECM and cellular debri found in basal deposits. Further studies are obviously needed in order to unravel the specific pathways that lead to abnormal formation of ECM and complement activation.

Retinal pigment epithelium transplantation: concepts, challenges, and future prospects.

The retinal pigment epithelium (RPE) is a single layer of cells that supports the light-sensitive photoreceptor cells that are essential for retinal function. Age-related macular degeneration (AMD) is a leading cause of visual impairment, and the primary pathogenic mechanism is thought to arise in the RPE layer. RPE cell structure and function are well understood, the cells are readily sustainable in laboratory culture and, unlike other cell types within the retina, RPE cells do not require synaptic connections to perform their role. These factors, together with the relative ease of outer retinal imaging, make RPE cells an attractive target for cell transplantation compared with other cell types in the retina or central nervous system. Seminal experiments in rats with an inherited RPE dystrophy have demonstrated that RPE transplantation can prevent photoreceptor loss and maintain visual function. This review provides an update on the progress made so far on RPE transplantation in human eyes, outlines potential sources of donor cells, and describes the technical and surgical challenges faced by the transplanting surgeon. Recent advances in the understanding of pluripotent stem cells, combined with novel surgical instrumentation, hold considerable promise, and support the concept of RPE transplantation as a regenerative strategy in AMD.

Improvement of RPE cells growth and metabolism abilities by mitochondrial transfer of neural stem cells

Background Researches showed that stem cells can rescue damaged cells through mitochondrial transfer.This mode has been used to regenerative cell-based therapy.Retinal pigment degeneration is an eye disease of retinal pigment epithelial (RPE) cell apoptosis as pathogenetic mechanism.Whether stem cells can repair the target cells by above mechanism has not been clarified. Objective This study was to investigate the influence of mitochondrial transfer on the function of RPE cells. Methods The RPE cells of Long-Evans rats were isolated and cultured and the third generation of cells were used in sequential experiment.The cells were identified by detecting the expressions of RPE65 and Bestrophin proteins with immunofluorescence stain.Mouse neural stem cells (NSCs) (C17.2 strain) with green fluorescence protein (GFP) and without GFP were cultured.Mitotracker-green and Mitotracker-red staining were separately used to labeled the mitochondria of RPE cells and NSCs.RPE cells were co-cultured with NSCs, and wheat germ agglutinin (WGA) was used to mark the tunneling nanotubes (TNT) between the two kinds of cells, and then the mitochondrial migration in TNT was exhibited by the laser scanning confocal microscope.The proportion of RPE cells in different cycles was assayed after marked with propidium iodide (PI) by flow cytometry.The contents of ATP, ADP and AMP in RPE cells were detected by high performance liquid chromatography (HPLC). Results The third-generation of RPE cells grew well with the RPE65- and Bestrophin-positive rate >85%.The Mitotracker-red- labeled rates of NSCs and RPE cells were no less than 95%.TNT structure was seen to appear the blue fluorescence between RPE cells and NSCs 24 hours after co-cultured and the red dye mitochondria from NSCs migrated toward red dye mitochondria from RPEs with the lapse of time.The RPE cell proportion reduced in G1phase and increased by 5% and 2% in the S phase and G2/M phase respectively after mitochondrial transfer than before (P=0.016, 0.114, 0.189). The contents of ATP, ADP and AMP in the RPE cells were (8.77±3.68), (2.76±0.92) and (1.07±0.65)μg/mg after cell co-culture, and those before co-culture were (11.29±2.29), (3.12±0.95) and (1.59±1.22)μg/mg, without significant differences between them (P=0.370, 0.668, 0.553). Conclusions NSCs can transfer normal mitochondria to co-cultured RPEs via TNT structure.Mitochondrial exchange might be one of therapeutic mechanisms of NSCs recuing damaged RPE cells. Key words: Stem cells; Pigment epithelium of eye/cytology; Retinal degeneration/therapy; Cells, cultured; Coculture techniques; Mitochondria/metabolism; Nanotubes; Animal

NHE8 is essential for RPE cell polarity and photoreceptor survival.

A new N-ethyl-N-nitrosourea (ENU)-induced mouse recessive mutation, identified by fundus examination of the eye, develops depigmented patches, indicating retinal disorder. Histology data show aberrant retinal pigment epithelium (RPE) and late-onset photoreceptor cell loss in the mutant retina. Chromosomal mapping and DNA sequencing reveal a point mutation (T to A) of the Slc9a8 gene, resulting in mutant sodium/proton exchanger 8 (NHE8)-M120K protein. The lysine substitution decreases the probability of forming the 3rd transmembrane helix, which impairs the pore structure of the Na+/H+ exchanger. Various RPE defects, including mislocalization of the apical marker ezrin, and disrupted apical microvilli and basal infoldings are observed in mutant mice. We have further generated NHE8 knockout mice and confirmed similar phenotypes, including abnormal RPE cells and late-onset photoreceptor cell loss. Both in vivo and in vitro data indicate that NHE8 co-localizes with ER, Golgi and intracellular vesicles in RPE cells. Thus, NHE8 function is necessary for the survival of photoreceptor cells and NHE8 is important for RPE cell polarity and function. Dysfunctional RPE may ultimately lead to photoreceptor cell death in the NHE8 mutants. Further studies will be needed to elucidate whether or not NHE8 regulates pH homeostasis in the protein secretory pathways of RPE.

Senescent retinal pigment epithelial cells are more sensitive to vascular endothelial growth factor: implications for "wet" age-related macular degeneration.

Senescence of the retinal pigment epithelial (RPE) cell layer has been implicated in the occurrence of age-related macular degeneration (AMD). The present study examines whether the ability of vascular endothelial growth factor (VEGF) to decrease the barrier function of RPE cells is enhanced in senescent RPE cells, which could contribute to the pathology of "wet" AMD. Low or high population doubling level (PDL) range ARPE-19 human RPE cells were cultured in 6-well plates on membrane-containing inserts. After 2 weeks, the cells were treated with either VEGF or its vehicle and their transepithelial electrical resistance (TEER) was measured. One week later, the cells were stained for senescence-associated β-galactosidase (SABG) activity. VEGF was significantly more effective in reducing the TEER of the high PDL ARPE-19 cell layers than the low PDL layers (25% decrease vs. 6% decrease; t-test, P=0.0013). The low PDL cell layers had a modest uniform level of SABG staining. In contrast, the high PDL layers displayed darker and more mottled SABG staining indicative of the presence of senescent cells. The present results show that the ability of VEGF to reduce the barrier function of RPE cell layers is greater in high PDL layers, which display signs of senescence, than in low PDL layers. Senescence-induced changes in the responsiveness of RPE cell layers to VEGF could contribute to the pathology of AMD. Agents that strengthen the barrier properties of RPE cells or reduce their responsiveness to VEGF could be effective in treating "wet" AMD.

Decline in cellular clearance systems induces inflammasome signaling in human ARPE-19 cells

Retinal pigment epithelium (RPE) plays a major role in the maintenance of photoreceptors, and degeneration of RPE results in the development of age-related macular degeneration (AMD). Accumulation of intracellular protein aggregates, increased oxidative stress, and chronic inflammation are all factors damaging the functionality of aged RPE cells. Here, we report that inhibition of proteasomal degradation with MG-132 and autophagy with bafilomycin A1 resulted in the release of IL-1β but not that of IL-18 in human ARPE-19 cells. NLRP3 receptor became upregulated, and caspase-1, the functional component of an inflammasome complex, was activated. In addition to accumulating intracellular protein aggregates, inhibition of degradation systems induced oxidative stress which was demonstrated by elevated amounts of intracellular 4-hydroxynonenal (HNE)-protein adducts. Along with IL-1β, exposure to MG-132 and bafilomycin A1 resulted in the secretion of IL-8. A low concentration (1pg/ml) of IL-1β was capable of triggering significant IL-8 production which also became attenuated by treatment with a specific caspase-1 inhibitor. These results suggest that decline in intracellular degradation systems results not only in increased amounts of intracellular protein aggregates and oxidative stress but also in the activation of NLRP3 inflammasomes, arisen as a result of elevated production of biologically active IL-1β.

Diagnosis and complementary examinations.

Development of neuroprotective therapies requires an understanding of the mechanisms of retinal degeneration and a way to monitor response to treatment. With the increasing availability of genetic testing, precise characterization of the retinal degeneration phenotype is essential. This chapter covers standard and innovative diagnostic techniques and complementary examinations needed for the evaluation and treatment of retinal degenerative diseases. It aims to provide an overview of functional and structural diagnostic tools for the evaluation of retinal degenerative diseases, but is not intended as a comprehensive reference. Subjective assessment of visual function includes psychophysical tests, such as perimetry and microperimetry. Electrophysiology tests, such as the electroretinogram and electro-oculogram, are crucial in the assessment of retinal degenerative diseases and provide an objective assessment of global photoreceptor and retinal pigment epithelial cell function. Retinal structural measures are correlated with measures of retinal function to characterize the disease phenotype, including fundus photography using color, near-infrared, and autofluorescence imaging. Ocular perfusion can be assessed using fluorescein, indocyanine green, and noninvasive angiography. Optical coherence tomography provides information about retinal structure. Resolution of all images of retinal structure can be improved using adaptive optics, which permits visualization of individual photoreceptors and retinal pigment epithelial cells in the macula.

Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases, including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells, direct cell reprogramming driven by lineage-determining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter, we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover, they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination, but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling, drug screening, and cell therapy of retinal degeneration.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-013-0011-2) contains supplementary material, which is available to authorized users.

Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases, including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells, direct cell reprogramming driven by lineage-determining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter, we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover, they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination, but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling, drug screening, and cell therapy of retinal degeneration.

Involvement of acid-sensing ion channel 1a in functions of cultured human retinal pigment epithelial cells

In the retina, pH fluctuations may play an important role in adapting retinal responses to different light intensities and are involved in the fine tuning of visual perception. Acidosis occurs in the subretinal space (SRS) under pathological conditions such as age-related macular degeneration (AMD). Although it is well known that many transporters in the retinal pigment epithelium (RPE) cells can maintain pH homeostasis efficiently, other receptors in RPE may also be involved in sensing acidosis, such as acid-sensing ion channels (ASICs). In this study, we investigated whether ASIC1a was expressed in the RPE cells and whether it was involved in the function of these cells. Real-time RT-PCR and Western blotting were used to analyze the ASIC1a expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide (H(2)O(2)). Furthermore, inhibition or over-expression of ASIC1a in RPE cells was obtained using inhibitors (amiloride and PCTx1) or by the transfection of cDNA encoding hASIC1a. Cell viability was determined by using the MTT assay. The real-time RT-PCR and Western blotting results showed that both the mRNA and protein of ASIC1a were expressed in RPE cells. Inhibition of ASICs by amiloride in normal RPE cells resulted in cell death, indicating that ASICs play an important physiological role in RPE cells. Furthermore, over-expression of ASIC1a in RPE cells prolonged cell survival under oxidative stress induced by H(2)O(2). In conclusion, ASIC1a is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells by protecting them from oxidative stress.

Identification of miRNA Signatures during the Differentiation of hESCs into Retinal Pigment Epithelial Cells

Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.

Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses

Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium‐derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.

Age-related maculopathy susceptibility 2 participates in the phagocytosis functions of the retinal pigment epithelium.

Age-related macular degeneration (AMD) is a multifactorial disease and a prevalent cause of visual impairment in developed countries. Many studies suggest that age-related maculopathy susceptibility 2 (ARMS2) is a second major susceptibility gene for AMD. At present, there is no functional information on this gene. Therefore, the purpose of the present study was to detect the expression of ARMS2 in retinal pigment epithelium (RPE) cells and to investigate the effect of ARMS2 on the phagocytosis function of RPE cells. Immunofluorescence and reverse transcriptase PCR were used to demonstrate the presence and location of ARMS2 in ARPE-19 (human retinal pigment epithelial cell line, ATCC, catalog No.CRL-2302) cells. siRNA was used to knock down ARMS2 mRNA, and the effects of the knockdown on the phagocytosis function of the ARPE-19 cells were evaluated via Fluorescence Activated Cell Sorting (FACS). ARMS2 was present in ARPE-19 cells, localized in the cytosol of the perinuclear region. The expression of ARMS2 mRNA (messenger RNA) in ARPE-19 cells transfected with ARMS2-siRNA (small interfering RNA, 0.73±0.08) was decreased compared with normal cells (1.00±0.00) or with cells transfected with scrambled siRNA (0.95±0.13) (P<0.05). After incubation of RPE cells with a latex beads medium for 12, 18, or 24 hours, the fluorescence intensities were 38.04±1.02, 68.92±0.92, and 78.00±0.12 in the ARMS2-siRNA-transfected groups, respectively, and 77.98±5.43, 94.87±0.60, and 98.30±0.11 in the scrambled siRNA-transfected groups, respectively. The fluorescent intensities of the same time points in the two groups were compared using Student's t-test, and the p values were all less than 0.001 at the three different time points. There is endogenous expression of ARMS2 in ARPE-19 cells. ARMS2 plays a role in the phagocytosis function of RPE cells, and this role may be one of the mechanisms that participates in the development of AMD.

Retinal pigment epithelial cells use a MerTK‐dependent mechanism to limit the phagocytic particle binding activity of αvβ5 integrin

αvβ5 integrin and Mer tyrosine kinase (MerTK) receptors reside at the apical surface of the retinal pigment epithelium (RPE) in the eye to promote the diurnal, synchronised phagocytosis of shed photoreceptor outer segment fragments (POS) that is critical for vision. Phagocytosis assays studying RPE cells in culture have defined roles for αvβ5 in POS surface binding and for MerTK in engulfment of bound POS. Both receptors have thus far only been studied separately. It is therefore unknown if αvβ5 integrin activity in POS binding is independent of the engulfment function of RPE cells. This study investigates how increasing αvβ5 receptor levels affect POS binding and internalisation by wild-type (wt), αvβ5- or MerTK-deficient RPE. β5 integrin-green fluorescent protein (β5-GFP) fusion proteins formed heterodimeric receptors with endogenous αv integrin subunits at the apical surface of mouse or rat RPE cells that co-immunoprecipitated focal adhesion kinase and redistributed with bound POS such as endogenous αvβ5 receptors. In β5(-/-) RPE cells, de novo formation of αvβ5-GFP receptors restored POS binding and internalisation up to, but not, above wt POS uptake levels. In wt RPE cells, increasing levels of αvβ5 surface receptors by over-expressing β5-GFP only moderately stimulated POS binding, even if POS internalisation was inhibited pharmacologically or by lowering incubation temperatures. In contrast, the same increase in αvβ5 receptor levels dramatically enhanced POS binding of RPE cells lacking MerTK. Furthermore, decreasing MerTK expression by RNA interference increased POS binding to endogenous αvβ5 receptors of wt RPE cells. Expressing β5-GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from β5(-/-) mice, indicating that these cells do not irreversibly lose other components of the phagocytic machinery. RPE cells expressing the engulfment receptor MerTK control POS binding by limiting activity of endogenous αvβ5 and αvβ5-GFP integrins, although they reside at the apical, phagocytic surface. In contrast, RPE cells permanently or transiently losing MerTK expression lack this regulatory mechanism and bind excess POS via surface αvβ5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface αvβ5 integrin receptors in RPE cells.

Effect of Acid-sensing ion channel 1a in cultural human retinal pigment epithelial cells during oxidative stress

Objective To investigated whether Acid-sensing ion channel 1 a (ASIC1a) is expressed in the retinal pigment epithelium (RPE) cells and whether it is involved in the function of these cells.Methods Real time revere transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to analyze the ASIC1a expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide (H2O2 ).Furthermore,blockage or overexpression of ASIC1a in RPE cells were obtained using inhibitors (amiloride and PCTxl ) or by the transfection of cDNA encoding hASIC1a.Cell viability was determined using the methyl thiazol tetrazolium (MTT) assay.Results The RT-PCR and Western blot results showed that both the mRNA and the protein of ASIC1a are expressed in RPE.Blockade of ASICs by amiloride in normal RPE cells resulted in cell death,indicating that ASICs play an important physiological role in RPE cells.Furthermore,overexpression of ASIC1a in RPE increased cell survival after oxidative stress induced by H2O2.Conclusion ASIC1a is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells in protecting them from oxidative stress. Key words: Acid-sensing ion channel 1a; Retinal pigment epithelium; Oxidative stress

Increased Expression of Tight Junctions in ARPE-19 Cells Under Endoplasmic Reticulum Stress

Purpose: To investigate the effects of endoplasmic reticulum (ER) stress on the tight junctions of the retinal pigment epithelial (RPE) cells in vitro.Materials and Methods: ER stress was induced in cultured ARPE-19 cells, a human RPE cell line, by exposure to tunicamycin (TM) or to thapsigargin (TG). After 6, 12, 24 and 48 hours of exposure, the expressions of GRP78/Bip (Bip), C/EBP-homologous protein (CHOP), vascular endothelial growth factor (VEGF), zonula occludens (ZO)-1, occludin and claudin-1 were determined by real-time RT-PCR. Immunoblot analysis and/or immunohistochemistry for proteins of tight junctions and ER stress markers, viz., Bip, activating transcription factor (ATF) 6, CHOP, and caspase-4, were performed at 48 hours after the exposure. Enzyme-linked immunosorbent assay was used to determine the concentration of VEGF165. Transepithelial electrical resistance (TER) of the ARPE-19 cells was determined to measure the permeability.Results: The expressions of the mRNAs and/or proteins of Bip, CHOP, ATF6 and caspase-4 were significantly increased in ARPE-19 cells under ER stress induced by TM and TG. The mRNAs of VEGF were also increased by both TM and TG. However, the concentration of VEGF165 was not significantly increased after 48 hours exposure to TM and TG compared to that of the control in the apical chamber medium. The proteins and mRNAs of occludin and claudin-1 were significantly increased by TM and TG, and that of ZO-1 was significantly increased by TG. Immunohistochemistry showed that the staining of ZO-1, occludin and claudin-1 under ER stress was stronger than that of the control. A significant increase of TER was observed after exposure to TM and TG.Conclusions: The increased expressions of tight junction molecules by TM− or TG-exposed ARPE-19 cells indicate that ER stress can alter the function of RPE cells and may be involved in the pathogenesis of age-related macular degeneration.