Role of Retinal Pigment Epithelium In Retinal Disorders

视网膜色素上皮（RPE）是存在于眼睛中的视网膜和脉络膜之间的单层细胞，对于视网膜的正常功能至关重要。视网膜的许多退行性、炎性和年龄相关性疾病与RPE的变性和/或功能障碍有关。我们已经开发了一个人RPE细胞培养系统，来自成人供体的眼睛，并已使用此作为模型，以调查视网膜病变的病理生理学中的各种作用的RPE。我们将注意力集中在生长因子和炎性细胞因子上，因为这些分子参与许多视网膜疾病，例如葡萄膜炎、年龄相关性黄斑变性（AMD）、糖尿病视网膜病变和视网膜脱离。如果不治疗，这些疾病可能导致视觉功能丧失。 由于炎症与视网膜疾病（AMD）的关联现在被认为是一个关键组成部分，我们评估了炎症介质对VEGF表达的作用，VEGF是AMD中血管渗漏、视网膜和脉络膜新生血管形成的已知因子。炎性细胞因子由巨噬细胞和其他浸润细胞在视网膜和脉络膜的视网膜微环境中产生。使用GeneChip（Human Genome U133 plus array，Affyellow），我们通过微阵列分析评估了炎性细胞因子（IC混合物=干扰素-γ、白细胞介素-1和肿瘤坏死因子-α）对人RPE细胞的影响。该系统提供了大多数特征性人类基因表达的全基因组变化。IC混合物显著增强了许多细胞因子和趋化因子以及生长因子如血管内皮生长因子（VEGF）的表达。值得注意的是，VEGF与许多致盲性视网膜疾病如ARMD有关。微阵列分析显示，在用IC混合物处理的HRPE细胞中，VEGF-A和VEGF-C mRNA的水平增加约10倍。我们通过HRPE研究VEGF蛋白的分泌来验证微阵列结果。VEGF-A和VEGF-C的分泌在用IC混合物或用单独的细胞因子处理的HRPE细胞中增加10至20倍。即使在模拟视网膜微环境内的病理生理条件的非常低浓度的IC下，HRPE细胞也分泌显著量的VEGF。其他血管生成调节因子如血管生成素、血小板反应蛋白、内皮抑制素和色素上皮衍生因子不受IC混合物处理的影响。我们的研究结果表明，在ARMD的炎症事件（IC混合）和脉络膜新生血管（VEGF）密切相关。 视网膜相关性黄斑变性（AMD）是一种缓慢进行的疾病，可能需要几年或几十年才能发展成导致视力丧失的完全成熟的疾病。 血管内皮生长因子（VEGF），也称为血管渗透因子（血液和淋巴管中的液体渗漏），与AMD的关联已被证明是毫无疑问的。 目前AMD的治疗方法包括抗VEGF药物（抗体，小分子），这些药物非常昂贵，需要反复注射到眼睛后部。 对于AMD相关病理的早期干预/预防，需要探索使用营养品作为安全的替代品。 我们发现白藜芦醇（3，5，4-三羟基芪，RSV），一种天然存在于葡萄和许多植物产品中的多酚植物抗毒素，下调RPE细胞中炎症细胞因子增强的VEGF的产生。RSV对VEGF分泌的这种抑制作用在模拟眼内病理状况的广泛细胞因子浓度（炎症指标）下观察到。 在这些条件下，RSV对抗血管生成分子如内皮抑素和色素上皮衍生因子没有显著影响。 我们的初步研究表明，RSV通过抑制RPE和可能的其他视网膜细胞分泌VEGF而起抗血管生成分子的作用。 目前，由于RSV的许多有益作用，包括延长寿命，大量普通人群，特别是在美国和欧洲，正在使用RSV和/或RSV的其他来源，如葡萄、葡萄酒、葡萄籽提取物作为膳食补充剂。 一些保健品公司正在销售天然的、合成的、改良的RSV或它们的组合制剂作为非处方产品。 我们建议定期使用RSV或其他含有较高剂量RSV的产品可能对预防/减缓新血管形成（与AMD相关的主要事件）具有有益作用。 最近在细胞和分子生物学中的发现指出microRNA（miRNAs）作为基因表达的潜在调节剂和作为在眼部治疗发展中有价值的工具。miRNA是20-25个碱基的非编码RNA寡核苷酸。到目前为止，只有大约500个miRNA基因被鉴定出来，它们的产物在细胞核和细胞质中被各种酶加工成成熟的形式。miRNA与信使RNA（mRNA）的3 '非翻译区结合，这可能导致mRNA降解和/或翻译抑制。正在研究miRNA作为理解和治疗眼部疾病的工具的潜在应用。利用microRNA阵列分析，我们研究了炎症介质对人RPE细胞中miRNA表达的调控。初始结果显示mir-155和mir-146的显著上调，并且这些结果已经通过真实的时间PCR分析验证。我们目前正在研究mir-155和mir-146在调节与视网膜疾病相关的各种细胞因子、趋化因子和生长因子表达中的作用。

Retinal pigment epithelium (RPE), a single layer of cells present between the retina and choroid in the eye, is vital for the normal functioning of the retina. Many of the degenerative,inflammatory and age-related diseases of the retina are associated with the degeneration and /or dysfunction of the RPE. We have developed a human RPE cell culture system,derived from adult donor eyes, and have used this as a model to investigate the various roles of RPE in the pathophysiology of retinal disorders. We focused our attention on growth factors and inflammatory cytokines, since these molecules are involved in many of the retinal disorders such as uveitis, age related macular degeneration (AMD), diabetic retinopathy and retinal detachments. If untreated, these disorders may lead to loss of visual function. Since the association of inflammation with retinal disorders (AMD) is now recognized as a key component, we evaluated the role of inflammatory mediators on the expression of VEGF, a known agent in vascular leakage, retinal and choroidal neovascularization in AMD. Inflammatory cytokines are produced in the retinal microenvironment by macrophages and other infilterating cells into the retina and choroid. Using GeneChip (Human Genome U133 plus array, Affymetrix),we evaluated the effects of inflammatory cytokines(IC mix=interferon-gamma, interleukin-1 and tumor necrosis factor-alpha)on human RPE cells by microarray analyses. This system provides genome-wide changes in the expresssion of most of the characterized human genes. IC mix significantly enhanced the expression of many of the cytokines and chemokines as well as growth factors such as vascular endothelial growth factors (VEGFs). It is important to note that VEGFs are associated with many of the blinding retinal disorders like ARMD. Microarray analysis revealed about 10 fold increase in the levels of VEGF-A and VEGF-C mRNA in HRPE cells treated with IC mix. We validated microarray results by studying the secretion of VEGFs proteins by HRPE. The secretion of VEGF-A and VEGF-C increased by 10 to 20 fold in HRPE cells treated with IC mix or with individual cytokines. Even at very low concentrations of IC, that mimic the patho-physiological conditions within retinal microenvironment, significant quantities of VEGFs are secreted by HRPE cells. Other regulators of angiogenesis such as angiopoietins, thrombospondins, endostatins and pigment epithelial derived factors were not effected by IC mix treatment. Our results showed close relationship between inflammatory events (IC mix)and choroidal neovascularization (VEGFs) in ARMD. Age-related macular degeneration (AMD) is a slow progressive disorder that may take few years or decades to develop in to full blown disease leading to loss of vision. Association of Vascular Endothelial Growth Factors (VEGFs), also known as vascular permeability factor (leakage of fluids from blood and lymphatic vessels), with AMD is proven beyond doubt. Current therapies for AMD include anti-VEGF agents (antibodies, small molecules), which are very expensive and needs repeated injections into the back of the eye. For early intervention / prevention of AMD related pathologies, use of nutraceuticals as safe alternatives needs to be explored. We found resveratrol (3,5,4-trihydroxystilbene, RSV), a polyphenolic phytoalexin present naturally in grapes and many plant products, down-regulate the production of VEGFs enhanced by inflammatory cytokines in RPE cells. This inhibitory effect of RSV on VEGFs secretion was observed at a wide range of cytokine concentrations (indicators of inflammation) that mimics intraocular pathological conditions. Under these conditions, RSV had no significant effects on anti-angiogenic molecules such as endostatin and pigment epithelial derived factor. Our initial studies show that RSV acts as anti-angiogenic moleule by inhibiting VEGFs secretion by RPE and possibly other retinal cells. Currently, a large number of general population especially in USA and Europe are using RSV and / or other sources of RSV such as grapes, wine, grape seed extract as a dietary supplement because of many beneficial effects of RSV including life extension. Some nutraceutical companies are marketing natural, synthetic, modified RSV or their combination formulations as over the counter products. We suggest regular use of RSV or other products containing RSV at higher doses may have beneficial effects in preventing / slowing down of neovascularization, a major event associated with AMD. Recent discoveries in cellular and molecular biology point to the microRNAs(miRNAs)as potential regulators of gene expression and as valuable tools in the development of ocular therapeutics. miRNAs are noncoding RNA oligonucleotides of 20-25 bases. So far only about 500 miRNA genes have been identified, and their products are processed both in nucleus and cytoplasm by a variety of enzymes to produce mature forms. miRNAs bind to 3'untranslated regions of messengerRNAs(mRNA)that may lead to mRNA degradation and/or inhibition of translation. Potential applications of miRNAs as tools in understanding and treatment of ocular diseases are being investigated. Using microRNA array analysis, we studied the regulation of miRNA expression in human RPE cells by inflammatory mediators. Initial results showed significant upregulation of mir-155 and mir-146 and these results have been validated by Real Time PCR analysis. We are currently investigating the role of mir-155 and mir-146 in the regulation of the expression of various cytokines, chemokines and growth factors associated with retinal disorders.