Role of Retinal Pigment Epithelium In Retinal Disorders

视网膜色素上皮(RPE)是视网膜和眼部脉络膜之间的单层细胞，对视网膜的正常功能至关重要。许多退行性、炎症性和年龄相关的视网膜疾病都与RPE的退化和/或功能障碍有关。我们开发了一种来源于成人供体眼的人RPE细胞培养系统，并将其作为模型来研究RPE在视网膜疾病的病理生理学中的各种作用。我们将注意力集中在生长因子和炎性细胞因子上，因为这些分子与许多视网膜疾病有关，如葡萄膜炎、老年性黄斑变性(AMD)、糖尿病视网膜病变和视网膜脱离。如果不治疗，这些疾病可能会导致视觉功能丧失。 由于炎症与视网膜病变(AMD)的相关性现在被认为是一个关键成分，我们评估了炎症介质对血管渗漏、视网膜和脉络膜新生血管在AMD血管渗漏、视网膜和脉络膜新生血管中表达的作用。炎症细胞因子是由巨噬细胞和其他渗入视网膜和脉络膜的细胞在视网膜微环境中产生的。应用基因芯片技术(Human Genome U133 plus ARRAY，Affymetrix)，通过基因芯片技术研究炎症细胞因子(IC Mix=干扰素-γ、白介素1和肿瘤坏死因子-α)对人RPE细胞的影响。这个系统提供了基因组范围内大多数人类特有基因表达的变化。IC Mix显著增强多种细胞因子和趋化因子以及血管内皮生长因子(VEGFs)等生长因子的表达。值得注意的是，血管内皮生长因子与许多致盲的视网膜疾病有关，如ARMD。基因芯片分析显示，经IC Mix处理的hRPE细胞中，VEGF-A和VEGF-C的mRNA水平增加了约10倍。我们通过研究hRPE分泌VEGFs蛋白来验证微阵列结果。经IC Mix和单独细胞因子处理后，hRPE细胞分泌的VEGF-A和VEGF-C增加了10~20倍。即使在极低浓度的IC(模拟视网膜微环境中的病理生理条件)下，hRPE细胞也会分泌大量的VEGFs。血管生成素、血小板反应素、内皮抑素和色素上皮衍生因子等血管生成调节因子不受IC Mix处理的影响。结果表明，ARMD的炎症事件(IC Mix)与脉络膜新生血管(VEGFs)密切相关。 老年性黄斑变性(AMD)是一种缓慢进行性的疾病，可能需要几年或几十年的时间才能发展成导致视力丧失的全面疾病。血管内皮生长因子(VEGFs)，也被称为血管渗透因子(从血管和淋巴管渗漏液体)与AMD的相关性已被证明是毫无疑问的。目前AMD的治疗方法包括抗血管内皮生长因子药物(抗体、小分子)，这些药物非常昂贵，需要在眼球后部重复注射。 为了早期干预/预防AMD相关的病理，需要探索使用营养食品作为安全的替代品。我们发现，白藜芦醇(3，5，4-三羟基二苯乙烯，RSV)是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，它下调RPE细胞中炎性细胞因子促进的VEGFs的产生。RSV对VEGFs分泌的这种抑制作用是在模拟眼内病理条件的广泛细胞因子浓度(炎症指标)下观察到的。在此条件下，RSV对内皮抑素、色素上皮衍生因子等抗血管生成分子无明显影响。 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VEGFs来发挥抗血管生成分子的作用。目前，由于RSV的许多有益作用，特别是在美国和欧洲，大量的普通人群正在使用RSV和/或其他RSV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、修饰的RSV或它们的组合配方作为非处方产品。我们建议，经常使用RSV或其他高剂量的含有RSV的产品可能在预防/减缓与AMD相关的重大事件-新生血管方面具有有益的效果。 细胞和分子生物学的最新发现表明，microRNAs(MiRNAs)是潜在的基因表达调节因子，也是发展眼科治疗的有价值的工具。MiRNAs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都被各种酶处理以产生成熟的形式。MiRNAs与信使RNA的3‘非翻译区结合，可能导致信使RNA的降解和/或翻译的抑制。目前正在研究miRNAs作为工具在理解和治疗眼病方面的潜在应用。利用microRNA阵列分析，我们研究了炎症介质对人RPE细胞miRNA表达的调节。初步结果显示，mir-155和mir-146的表达显著上调，这些结果已被实时荧光聚合酶链式反应分析所验证。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.