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。视网膜的许多炎性、感染性和其它疾病与RPE的变性和/或功能障碍有关。我们已经开发了一种人RPE细胞培养系统，并以此为模型来研究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转化生长因子-β（TGF-β）上，因为TGF-β参与视网膜疾病的增殖、变性、炎症和感染病因。年龄相关性黄斑变性（ARMD）、增殖性玻璃体视网膜病变（PVR）和视网膜脱离（RD）是导致视力损害的主要原因。我们使用基因芯片（Human Genome U133 plus array，Affyellow）通过微阵列分析评估TGF-β对人RPE细胞的作用。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。TGF-β显著增强许多细胞外基质蛋白（ECM）如胶原、纤连蛋白、血小板反应蛋白和硫酸肌醇蛋白聚糖以及许多生长因子如血管内皮生长因子（VEGF）和血小板衍生生长因子（PDGF）的表达。值得注意的是，ECM和生长因子与许多致盲性视网膜疾病如ARMD、PVR、RD相关。我们正在使用微阵列分析，以确定细胞外大分子的表达，不知道是目前在视网膜，和他们可能的作用，在视网膜病理生理。 为了研究ECM组分的作用及其通过TGF-β的调节，我们通过将TGF-受体I（TGF-RI）floxed小鼠与nestin-Cre小鼠杂交，通过条件性地删除神经元中的TGF-β信号传导（cKO）来产生小鼠模型。出生时在几乎所有cKO小鼠中观察到的视网膜脱离位于神经感觉视网膜（NSR）的RPE /感光层连接处。 软骨素-6-硫酸盐的免疫测定显示cKO小鼠中的反应非常弱，与野生型小鼠中感光层中的强烈阳性染色相反。用我们的cKO模型进行的研究表明视网膜细胞中功能性TGF-RI的缺乏导致视网膜下腔和光感受器层中硫酸软骨素蛋白聚糖的水平降低。这又会由于NSR与RPE的粘附丧失而导致视网膜脱离。 我们以前曾报道，TGF-β是一种有效的刺激VEGF和PDGF表达的人RPE细胞。由于炎症在视网膜疾病（ARMD）现在被认为是一个关键组成部分，我们评估的作用，炎症介质对VEGF的表达，一个已知的代理人在视网膜新生血管的ARMD。结果表明，干扰素（IFN-）、白细胞介素-1（IL-1）和肿瘤坏死因子（TNF-）可显著促进RPE和脉络膜细胞分泌VEGF-A和VEGF-C。 IFN-γ、IL-1、TNF-α和其他细胞因子在视网膜微环境中由巨噬细胞和其他浸润到视网膜和脉络膜中的细胞产生。我们正在评估炎症介质对VEGF-A、VEGF-C和其他血管生成因子（血管生成素和色素上皮衍生生长因子）的调节机制，以描述ARMD和其他视网膜新生血管疾病中炎症过程的参与。 用于基因表达谱的亲和微阵列研究也令人惊讶地揭示了TGF-β对白细胞介素-11（IL-11）的诱导。 IL-11通过抑制TNF-α和IL-1的巨噬细胞产生而起到抗炎剂的作用。IL-11还显示出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，IFN-γ下调调节RPE和脉络膜细胞分泌IL-1和TNF-α诱导的IL-11。相反，TGF-β诱导的IL-11分泌不受IFN-γ的影响。我们正在评估IL-11表达的调节机制以及IL-11作为保护视网膜免受氧化和炎症损伤的试剂的潜在用途。 最近在细胞和分子生物学中的发现指出microRNA（miRNAs）是基因表达的潜在调节因子。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 inflammatory, infectious and other diseases of the retina are associated with the degeneration and /or dysfunction of the RPE. We have developed a human RPE cell culture system 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 transforming growth factor-beta (TGF-β), since TGF-β is involved in retinal disorders of proliferative, degenerative, inflammatory and infectious etiology. Age related macular degeneration (ARMD), proliferative vitreoretinopathy (PVR) and retinal detachments (RD) are the leading causes of visual impairment. We evaluated the effects of TGF-β on human RPE cells by microarray analyses by using GeneChip (Human Genome U133 plus array, Affymetrix). This system provides genome-wide changes in the expresssion of most of the characterized human genes. TGF-β significantly enhanced the expression of many of the extracellular matrix proteins (ECM) such as collagens, fibronectin, thrombospondin and chondritin sulfate proteoglycans as well as many growth factors such as vascular endothelial growth factors (VEGF) and platelet derived growth factors (PDGF). It is important to note that ECM and growth factors are associated with many of the blinding retinal disorders like ARMD, PVR, RD. We are using microarray analysis to identify the expression of extracellular macromolecules, not known to be present in the retina,and their possible role in retinal pathophysiology. In order to study the role of ECM components and their regulation by TGF-β, we generated a mouse model by conditionally deleting TGF-β signaling in the neurons (cKO) by crossing TGF-β receptor I (TGF-β RI) floxed mice with nestin-Cre mice. The retinal detachments, seen in almost all of the cKO mice at birth, are at the RPE / photoreceptor layer junction of the neurosensory retina (NSR). The immunoassays for chondroitin-6-sulfate showed a very weak reaction in cKO mice in contrast to intense positive staining in the photoreceptor layer in wild-type mice. Studies with our cKO model suggest that the lack of functional TGF-β RI in retinal cells leads to decreased levels of chondroitin sulfate proteoglycans in the subretinal space and photoreceptor layers. This, in turn, causes retinal detachment due to the loss of adhesion of the NSR to RPE. We have previously reported that TGF-β is a potent stimulator of VEGF and PDGF expression by human RPE cells. Since inflammation in retinal disorders (ARMD) is now recognized as a key component, we evaluated the role of inflammatory mediators on the expression of VEGF, a known agent in retinal neovascularization in ARMD. Our results showed that Interferon-γ (IFN-γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) enhance significantly the secretion of VEGF-A and VEGF-C by RPE and choroidal cells. IFN-γ, IL-1, TNF-α and other cytokines are produced in the retinal microenvironment by macrophages and other infilterating cells into the retina and choroid. We are evaluating the mechanisms of the regulation of VEGF-A, VEGF-C and other angiogenic factors(angiogenin and pigment epithelial derived growth factor) by inflammatory mediators to delineate the involvement of inflammatory processes in ARMD and other retinal neovascularization disorders. Affymetrix microarray studies for gene expression profiles also surprisingly revealed the induction of interleukin-11 (IL-11) by TGF-β. IL-11 acts as an anti-inflammatory agent by inhibiting the macrophage production of TNF-α and IL-1. IL-11 was also shown to have cytoprotective properties by inhibiting cellular death pathway known as apoptosis. We found that IFN-γ dowwn regulates IL-1 and TNF-α induced IL-11 secretion by RPE and choroidal cells. In contrast, TGF-β induced IL-11 secretion was not affected by IFN-γ. 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. miRNAs are noncoding RNA oligonucleotides of 20-25 bases. So far only about 500 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.