Age-related macular degeneration (AMD) is a prominent cause of blindness in the Western world. To date, its molecular pathogenesis as well as the sequence of events leadi... More
Age-related macular degeneration (AMD) is a prominent cause of blindness in the Western world. To date, its molecular pathogenesis as well as the sequence of events leading to retinal degeneration remain largely ill-defined. While the invasion of choroidal neovasculature in the retina is the primary mechanism that precipitates loss of sight, an earlier dry form may accompany it. Here we provide the first evidence for the protective role of the Retinal Pigment Epithelium (RPE)-resident metabolite receptor, succinate receptor 1 (SUCNR1; G-Protein coupled Receptor-91 (GPR91), in preventing dry AMD-like lesions of the outer retina. Genetic analysis of 925 patients with geographic atrophy and 1199 AMD-free peers revealed an increased risk of developing geographic atrophy associated with intronic variants in the SUCNR1 gene. In mice, outer retinal expression of SUCNR1 is observed in the RPE as well as microglial cells and decreases progressively with age. Accordingly, Sucnr1−/− mice show signs of premature sub-retinal dystrophy with accumulation of oxidized-LDL, abnormal thickening of Bruch's membrane and a buildup of subretinal microglia. The accumulation of microglia in Sucnr1-deficient mice is likely triggered by the inefficient clearance of oxidized lipids by the RPE as bone marrow transfer of wild-type microglia into Sucnr1−/− mice did not salvage the patho-phenotype and systemic lipolysis was equivalent between wild-type and control mice. Our findings suggest that deficiency in SUCNR1 is a possible contributing factor to the pathogenesis of dry AMD and thus broaden our understanding of this clinically unmet need. Keywords: AMD, GPR91, geographic atrophy, microglia, succinate, metabolite receptor. Less
Purpose.: Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Act... More
Purpose.: Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)–transformed retinal pigment epithelial ARPE-19/HPV-16 cells. Methods.: Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined. Results.: The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H2O2, and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5′ untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress. Conclusions.: These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies. Less
Purpose: The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood–retinal barrier. Factors that r... More
Purpose: The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood–retinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the blood–retinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1. Methods: Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca2+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels. Results: Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca2+]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion. Conclusions: Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells. Less
Cigarette smoke is the most important environmental risk factor for developing age-related macular degeneration (AMD). Damage to the retinal pigment epithelium (RPE) caus... More
Cigarette smoke is the most important environmental risk factor for developing age-related macular degeneration (AMD). Damage to the retinal pigment epithelium (RPE) caused by cigarette smoke may underlie the etiology of AMD. This study investigated the molecular and cellular effects of cigarette smoke exposure on human RPE cells. ARPE-19 or primary human RPE cells were exposed to cigarette smoke extract (CSE) or hydroquinone (HQ), a component of cigarette smoke. The effect of this exposure on key aspects of RPE vitality including viability, cell size, mitochondrial membrane potential (DeltaPsi(m)), superoxide production, 4-hydroxy-2-nonenal (4-HNE), vascular endothelial growth factor (VEGF), and heme oxygenase-1 (HO-1) expression was determined. Exposure of RPE cells to CSE or HQ caused oxidative damage and apoptosis, characterized by a reduction in cell size and nuclear condensation. Evidence of oxidative damage also included increased lipid peroxidation (4-HNE) and mitochondrial superoxide production, as well as a decrease in intracellular glutathione (GSH). Exogenous administration of antioxidants (GSH and N-acetyl-cysteine) prevented oxidative damage to the RPE cells caused by CSE. Cigarette smoke also induced expression of VEGF, HO-1, and the transcription factor nuclear factor erythroid-derived 2, like 2 (NRF2). However, NRF2 was only modestly involved in CSE-induced HO-1 expression, as shown by the NRF2 small interfering RNA studies. These new findings demonstrate that cigarette smoke is a potent inducer of oxidative damage and cell death in human RPE cells. These data support the hypothesis that cigarette smoke contributes to AMD pathogenesis by causing oxidative damage and cell death to RPE cells. Less
Purpose: The objective of this study was to characterize efflux proteins (P-glycoprotein (P-gp), multidrug resistance proteins (MRP1-6) and breast cancer resistance prote... More
Purpose: The objective of this study was to characterize efflux proteins (P-glycoprotein (P-gp), multidrug resistance proteins (MRP1-6) and breast cancer resistance protein (BCRP)) of retinal pigment epithelium (RPE) cell lines. Methods: Expression of efflux proteins in two secondary (ARPE-19, D407) and two primary (HRPEpiC and bovine) RPE cell lines was measured by quantitative RT-PCR and western blotting. Furthermore, activity of MRP1 and MRP5 of ARPE-19 cell line was assessed with calcein-AM and carboxydichlorofluorescein (CDCF) probes. Results: Similar efflux protein profile was shared between ARPE-19 and primary RPE cells, whereas D407 cell line was notably different. D407 cells expressed MRP2 and BCRP, which were absent in other cell lines and furthermore higher MRP3 transcript expression was found. MRP1, MRP4 and MRP5 were identified from all human RPE cell lines and MRP6 was not expressed in any cell lines. The pattern of efflux protein expression did not change when ARPE-19 cells were differentiated on filters. The calcein-AM and CDCF efflux tests provided evidence supporting MRP1 and MRP5 activity in ARPE-19 cells. Conclusions: MRP1, MRP4 and MRP5 are the main efflux transporters in RPE cell lines. There are differences in efflux protein expression between RPE cell lines. Less
beta-Blockers are a class of agents that have been used extensively in topical preparations for the treatment of glaucoma. Recent evidence indicates that they may also be... More
beta-Blockers are a class of agents that have been used extensively in topical preparations for the treatment of glaucoma. Recent evidence indicates that they may also be useful in a number of retinal diseases. Because biocompatibility is of utmost importance in the treatment of ocular-related diseases, we compared the in vitro cytotoxicity, using the MTT assay, of eight clinically available beta-blockers (propranolol, alprenolol, atenolol, labetalol, metoprolol, pindolol, timolol, and bisoprolol) on human corneal epithelial and retinal pigment epithelial cell lines. Primary and immortalized corneal and retinal cell lines were compared for their susceptibility to the cytotoxic effect of the drugs. The cytotoxicity of beta-blockers was also evaluated on human skin keratinocytes and fibroblasts in order to investigate susceptibility differences as a function of the tissue of origin. Results demonstrated large differences in cytotoxicity (about 60-fold) for these closely related drugs on the same cell line. Conversely, only relatively small differences in cytotoxicity were observed between the different cell lines for the same drug, indicating that the mechanism of cytotoxicity is not cell-specific. Calculation of the ratio between the cytotoxicity of beta-blockers and their beta-blocking constant is presented as a potential tool to help identify the least irritating, most potent drug. Less
Sphingosine-1-phosphate (S1P) is a pleiotropic lysolipid that has recently been implicated in the regulation of tissue fibrosis. However, the fibrogenic potential of S1P ... More
Sphingosine-1-phosphate (S1P) is a pleiotropic lysolipid that has recently been implicated in the regulation of tissue fibrosis. However, the fibrogenic potential of S1P in the eye has not previously been investigated. In the current study, we evaluated cells from the anterior and posterior segments of the eye for the presence of S1P and their potential ability to produce and respond to S1P. In addition, we investigated the regulatory role of S1P as a mediator of proliferation, cellular transformation and pro-fibrotic protein expression in human retinal pigmented epithelial cells. Expression of S1P receptors and sphingosine kinases (the enzymes that produce S1P) was examined using RT-PCR, and intracellular localization of S1P was examined using immunoblotting, immunohistochemistry and ELISA in primary human retinal pigmented epithelial (RPE) cells, primary human conjunctival fibroblasts (ConF), and primary human corneal fibroblasts (CF). RPE cell proliferation was determined using an MTT-based cell proliferation assay, and RPE myofibroblast transformation, collagen type I production and profibrotic protein expression were assessed using immunofluorescence, ELISA and immunoblot. S1P(1-3, 5) receptors and sphingosine kinases 1 and 2 were expressed and intracellular pools of S1P were detected in RPE cells, ConF and CF. S1P stimulated RPE cell proliferation in a dose- and time-dependent manner. S1P induced myofibroblast transformation of RPE cells, as indicated by increased alpha-smooth muscle actin (alpha-SMA) expression and its incorporation into prominent stress fibers, and promoted collagen type I production. S1P stimulated the expression of plasminogen activator inhibitor-1 (PAI-1) and heat shock protein 47 (HSP47), two proteins that are linked to increased tissue fibrosis. Combined, these data demonstrate that RPE cells, ConF and CF from the human eye not only have the molecular ability to produce and respond to S1P, but also contain S1P. Furthermore, S1P promotes proliferation, myofibroblast transformation, collagen production and pro-fibrotic protein expression by human RPE cells. These data suggest that S1P is a previously unrecognized mediator of profibrotic cellular function and signaling in the eye. Less
The proinflammatory cytokine interleukin-20 (IL-20) may exert the majority of its activity in the skin. We examined the effect of various treatments including several for... More
The proinflammatory cytokine interleukin-20 (IL-20) may exert the majority of its activity in the skin. We examined the effect of various treatments including several forms of phototherapy on IL-20 expression using cultured normal human epithelial keratinocytes (NHEK). Broadband UVB light, recombinant (r) IL-1 and rIL-8 increased, while hydrocortisone reduced, NHEK supernatant IL-20 levels. Elevation of NHEK IL-20 mRNA and maximal supernatant IL-20 levels occurred with a UVB light dose (40 mJ cm(-2)) that reduced cell viability by approximately 50%. While this UVB light dose also elevated supernatant IL-1 alpha and IL-8 levels, antibody neutralization studies indicated that neither of these cytokines was directly responsible for this increase in IL-20 expression. However, the elevation in IL-20 levels was fully inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB-203580, suggesting involvement of this stress signaling pathway in this UVB light response. Photodynamic therapy (PDT) with the photosensitizer lemuteporfin, UVA light, cisplatin, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) or recombinant interferon-gamma (rIFN-gamma) either had little effect or decreased NHEK supernatant IL-20 levels. Reduced IL-20 levels paralleled the cytotoxic actions of PDT, UVA light or cisplatin and the antiproliferative effect of rIFN-gamma. Neither rIL-20 supplementation nor anti-IL-20 antibody treatments affected cell viability indicating that soluble IL-20 did not affect the short-term survival of UVB light-irradiated NHEK. Stimulation of IL-20 expression in keratinocytes by UVB light suggests that this cytokine might participate in skin responses to this ever-present environmental factor and potentially has a role in UV light-associated dermatoses. Less
