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。 用于基因表达谱分析的Affymetrix微阵列研究也出人意料地揭示了转化生长因子-β(TGF-b)诱导白细胞介素11(IL-11)的作用。IL-11通过抑制巨噬细胞产生肿瘤坏死因子-α和白介素1而发挥抗炎作用。IL-11还被证明通过抑制称为细胞凋亡的细胞死亡途径而具有细胞保护特性。我们发现，干扰素-γ下调IL-1和TNF-α诱导的RPE和脉络膜细胞分泌IL-11。而转化生长因子-β诱导的IL-11分泌不受干扰素-γ的影响。我们正在评估IL-11表达的调节机制，以及IL-11作为一种保护视网膜免受氧化和炎症伤害的药物的潜在用途。 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. Affymetrix microarray studies for gene expression profiling also surprisingly revealed the induction of interleukin-11 (IL-11) by transforming growth factor-beta(TGF-b). IL-11 acts as an anti-inflammatory agent by inhibiting the macrophage production of TNF-alpha and IL-1. IL-11 was also shown to have cytoprotective properties by inhibiting cellular death pathway known as apoptosis. We found that IFN-gamma down regulates IL-1 and TNF-alpha induced IL-11 secretion by RPE and choroidal cells. In contrast, TGF-b induced IL-11 secretion was not affected by IFN-gamma. We are evaluating the mechanisms of the regulation of expression of IL-11 and potential usefulness of IL-11 as an agent in the protection of retina from oxidative and inflammatory insults. 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.