alpha-Crystallin distribution in retinal pigment epithelium and effect of gene knockouts on sensitivity to oxidative stress.

PURPOSE: To investigate the susceptibility of retinal pigment epithelium (RPE) from alphaA (-/-) and alphaB (-/-) mice to oxidative stress, and the subcellular changes of alphaA and alphaB-crystallins under oxidative stress. METHODS: The effect of hydrogen peroxide (H(2)O(2)) on apoptosis in RPE from alphaA (-/-), alphaB (-/-), and wild type (wt) mice was assessed by TUNEL and AnnexinV/Propidium Iodide assays. H(2)O(2)-induced changes in caspase-3 activity and mitochondrial permeability transition (MPT) were determined. Human RPE in early passages (2-4) were starved in 1% FBS-containing Dulbecco's modified Eagle medium (DMEM) and treated with H(2)O(2) for 24 h. Gene expression was quantitated by real time PCR. Confocal microscopy was used to examine alpha-crystallin compartmentalization. Whole cell and mitochondrial alpha-crystallin protein amounts were examined by transmission electron microscopy (TEM) and Western blot analysis. RESULTS: RPE from alphaA (-/-), alphaB (-/-) mice exhibited increased susceptibility to apoptosis induced by H(2)O(2), increased caspase-3 activation, and increased MPT. Treatment of human RPE with H(2)O(2) resulted in a dose-dependent decrease in alphaB-crystallin mRNA expression. Confocal microscopy and subcellular fractionation of RPE showed that H(2)O(2) treatment decreased cytosolic and mitochondrial pools of alphaB-crystallin but caused no change in alphaA-crystallin content. TEM confirmed changes in expression of alphaA and alphaB-crystallins with oxidative stress. CONCLUSIONS: Lack of alpha-crystallins renders RPE cells more susceptible to apoptosis from oxidative stress. Mitochondrial alpha-crystallins may play an important role in the protection from increased susceptibility of RPE in oxidative stress.

Transforming growth factor beta2-induced myofibroblastic differentiation of human retinal pigment epithelial cells: regulation by extracellular matrix proteins and hepatocyte growth factor.

Retinal pigment epithelial (RPE) cells possess the potential to transdifferentiate into myofibroblasts after stimulation with transforming growth factor beta (TGFbeta) and are implicated in the pathogenesis of proliferative vitreoretinopathy. In this study we evaluated how TGFbeta2 and various extracellular matrix (ECM) proteins modulate the transdifferentiation of human fetal retinal pigment epithelial cells (RPE) cells into myofibroblast-like cells. Furthermore, we investigated whether hepatocyte growth factor (HGF) can suppress this transdifferentiation. RPE cells were cultured on ECM coated or uncoated surfaces in the presence or absence of TGFbeta2. HGF was added to certain cultures only once or on a daily basis during the treatment. Transdifferentiation of RPE cells into myofibroblasts was assessed by the quantitation of alpha-smooth muscle actin (alpha-SMA) using immunocytochemistry, flow cytometry, real-time PCR and Western blotting. TGFbeta2 induced a significant increase of alpha-SMA expression in a dose-dependent manner. Compared with growth on uncoated surfaces, RPE cultured on fibronectin (FN)-coated surfaces and stimulated with TGFbeta2 showed a significantly higher alpha-SMA expression than untreated cells. This upregulation of alpha-SMA could be markedly reduced by daily treatment with HGF; however, a single HGF administration did not significantly reduce alpha-SMA. These findings are important for further understanding the interaction of cytokines, RPE cells and their environment in mesenchymal transformation as well as its possible modulation. Continuous or long-term treatment with HGF should be further investigated for its potential to prevent mesenchymal transdifferentiation of RPE cells, and ultimately, PVR in vivo.

Thrombospondin-1 expression in RPE and choroidal neovascular membranes.

PURPOSE: To investigate the expression of thrombospondin 1 (TSP-1) in retinal pigment epithelium (RPE) and choroidal neovascular membranes (CNVMs) from patients with age-related macular degeneration (AMD). METHODS: Tissue sections from normal human fetal and adult eyes and surgically removed CNVMs were immunostained for TSP-1 localization. Polymerase chain reaction and Western blotting were used to analyze TSP-1 mRNA and protein from human RPE cells, respectively. TSP-1 in the supernatant of cultured RPE cells and eye explants were measured using enzyme-linked immunosorbent assay. MTT assay was used to evaluate the RPE survival after TSP-1 treatment. RESULTS: The strongest immunostaining for TSP-1 was observed in the RPE monolayer around drusen in early AMD. The intensity of TSP-1 staining in normal eye sections was much weaker than that of early AMD and CNVM. TSP-1 mRNA was positive in cultured fetal and adult RPE cells. There was increasing secretion of TSP-1 into the supernatant of cultured RPE and eye explants. The specific band of TSP-1 was identified by Western blot. No significant inhibition of RPE survival was found with the exposure to TSP-1. CONCLUSIONS: TSP-1 expression in drusen and CNVM was upregulated and associated with RPE monolayer. TSP-1 may be a natural negative regulator for choroidal neovascularization.

Hepatocyte growth factor protects RPE cells from apoptosis induced by glutathione depletion.

PURPOSE: To study the mechanism of the protective effect of hepatocyte growth factor (HGF) in oxidative injury to RPE cells induced by glutathione (GSH) depletion. METHODS: RPE cells were treated with HGF for 24 hours (20 ng/mL) and then were treated with DL-buthionine-(S,R)-sulfoximine (BSO) for an additional 24 hours. Cell death, apoptosis, and GSH levels were measured. Levels of intracellular reactive oxygen species (ROS) and their cellular localization were assessed by confocal microscopy. Expression of Bcl-2 and release of cytochrome c from mitochondria were quantified. The effect of BSO on caspase-3 activation and expression was determined. Gene expression of key enzymes of GSH metabolism by real-time PCR and regulation and translocation of the transcription factor NF-E2-related factor (Nrf2) by BSO were examined. RESULTS: Treatment with BSO-induced apoptosis in RPE caused a significant decrease in intracellular GSH and in GSH/GSSG ratios. Marked increases in lipid peroxidase (LPO), ROS, and mitochondrial cytochrome c release and a decrease in Bcl-2 expression were observed. Elevated GSH/GSSG ratio (especially in mitochondria), decreased LPO and ROS, attenuation of apoptosis, and partial restoration of Bcl-2 expression were found in the HGF-pretreated cells. BSO activated caspase-3, and this effect was significantly blocked by HGF. Both HGF and BSO induced anti-oxidant gene expression. Nrf2 translocated to the nuclear region after treatment with BSO, whereas HGF did not induce such translocation. CONCLUSIONS: The protective effect of HGF may be attributed in part to the elevation of mitochondrial GSH. BSO and HGF act in concert to enhance GSH-related gene expression in stressed RPE cells.

A selective cyclic integrin antagonist blocks the integrin receptors alphavbeta3 and alphavbeta5 and inhibits retinal pigment epithelium cell attachment, migration and invasion.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors alphavbeta3 and alphavbeta5, was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins alphavbeta3 and alphavbeta5 on RPE cells was examined. METHODS: The effect of a cyclic integrin antagonist and a control peptide (0.01 microg/ml to 300 microg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H3-thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors alphavbeta3 and alphavbeta5 was evaluated by flow cytometry. RESULTS: The integrin antagonist did not inhibit DNA synthesis stimulated by serum, bFGF, or PDGF-BB treatment. RPE attachment onto fibronectin was inhibited in a concentration range of 1-10 microg/ml (p < 0.05). Attachment of the RPE cells onto collagen IV and laminin was inhibited in a range of 3-10 microg/ml (p < 0.05). Serum and PDGF-BB stimulated migration was inhibited by the cyclic integrin antagonist in a concentration range of 1-10 microg/ml (p < 0.05). Furthermore, the cyclic integrin antagonist inhibited PDGF-BB stimulated RPE cell invasion through fibronectin (3 microg/ml: 66% inhibition, p < 0.001). In each of these experiments, the control peptides had no significant effects. PDGF-BB and bFGF pretreatment of RPE cells increased the expression of integrin receptors alphavbeta3 (bFGF: 1.9 fold, PDGF-BB: 2.3 fold) and alphavbeta5 (bFGF: 2.9 fold, PDGF-BB: 1.5 fold). CONCLUSION: A selective inhibition of the integrin receptors alphavbeta3 and alphavbeta5 through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease.

Ceramide-induced apoptosis: role of catalase and hepatocyte growth factor.

The aim of this study was to elucidate cellular mechanisms involved in ceramide-induced apoptosis and its attenuation by hepatocyte growth factor (HGF). Human retinal pigmented epithelial cells (RPE) incubated with C2 ceramide accumulated reactive oxygen species (ROS) in mitochondria and underwent apoptosis in a dose-dependent manner. Ceramide-treated cells showed increased caspase-3 activation and an increase in mitochondrial membrane permeability transition (MPT). Low doses of H2O2 (100 microM) alone induced negligible apoptosis; however, ceramide-induced apoptosis was significantly enhanced by co-incubation with H2O2 (100 microM). Furthermore, ceramide treatment significantly decreased catalase enzymatic activity and protein expression. HGF pretreatment (20 ng/ml) significantly inhibited ceramide-induced apoptosis and reduced the accumulation of ROS, the activation of caspase-3, and the increase in MPT and prevented the reduction in catalase activity and expression. Together, the data suggest that ceramide induces apoptosis in RPE cells by increasing ROS production, MPT, and caspase-3 activation. The ceramide effect is potentiated by H2O2 and associated with a reduction in catalase activity, suggesting that catalase plays a central role in regulating this apoptotic response. The ability of HGF to attenuate these effects demonstrates its effectiveness as an antioxidant growth factor.

Hepatocyte growth factor and its role in the pathogenesis of retinal detachment.

PURPOSE: Hepatocyte growth factor (HGF) regulates barrier function of retinal pigment epithelial (RPE) cells. The purpose of this study was to determine whether overexpression of HGF in the RPE induces retinal detachment (RD). METHODS: E1/E3-deleted adenoviral vectors encoding HGF (Ad CMV.HGF), green fluorescent protein (Ad CMV.GFP), or connective tissue growth factor (AdCMV.CTGF) were injected subretinally in adult pigmented rabbits (5 x 10(4) plaque-forming units [pfu]/eye). Animals were observed for up to 28 days with fundus photography. HGF expression in the retina and vitreous was determined using immunohistochemistry, and ELISA. Histopathologic examinations were performed with light and electron microscopy. RESULTS: Control eyes injected with AdCMV.GFP showed GFP expression almost exclusively in the RPE monolayer. Eyes injected with AdCMV.HGF showed strong HGF immunopositivity in RPE cells at the injection site. Elevated HGF levels were found in the vitreous peaking at postinjection day 7, diminishing to baseline by postinjection day 28. Eyes injected with AdCMV.HGF developed chronic RD and chronic inflammation in the choroid within the time frame of HGF expression. Groups of proliferating RPE cells were seen in the subretinal space in the region of the RD, and in some cases multilayered cellular membranes developed. No RD and minimal morphologic changes were seen in the eyes injected with AdCMV.GFP or AdCMV.CTGF. CONCLUSIONS: Overexpression of HGF in RPE induces chronic, serous RD with subretinal proliferation of RPE. This work provides insight into the pathogenesis of RD and suggests that HGF should be further investigated as a target for therapeutic intervention in RD.

Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor.

PURPOSE: To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight junctions and adherens junctions in the retinal pigment epithelial (RPE) monolayer. METHODS: Fresh bovine eyes were dissected to obtain 2- to 3-mm(2) explants of intact RPE with underlying choroid and sclera. Explants were cultured with or without HGF (20 ng/mL) for various periods (20 minutes to 72 hours). Junction integrity was assessed by transmission and scanning electron microscopy; localization, expression, and phosphorylation of junction proteins; and measurement of transepithelial resistance (TER), diffusion of fluorescent labeling in the plasma membrane, and the migration of RPE cells from the monolayer. RESULTS: Untreated explants consisted of polarized cells with apical microvilli and well-developed tight and adherens junctions. After HGF treatment, the explants showed loss of tight and adherens junctions ultrastructurally, diffusion of fluorescent label from apical to lateral membrane domains, and increased chemotactic migration of RPE cells from the monolayer. Primary cultures of confluent RPE cells showed a progressive decrease in TER. Western blot analysis showed rapid tyrosine phosphorylation of ZO-1, occludin, and beta-catenin within 20 minutes of stimulation. There was a marked loss of ZO-1 protein within 1 hour of HGF treatment. After 6 hours of treatment with HGF, occludin, claudin-1, and beta-catenin were redistributed from the membrane to the cytoplasm. CONCLUSIONS: Treatment of RPE explants with HGF results in rapid disassembly of tight and adherens junctions associated with loss or redistribution of junctional proteins, decreased TER, and increased migration of RPE cells from the monolayer.