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。许多炎症性、感染性和其他视网膜疾病都与RPE的退化和/或功能障碍有关。我们已经开发了一个人类RPE细胞培养系统，并将其作为一个模型来研究RPE在视网膜疾病的病理生理学中的各种作用。由于转化生长因子-β与视网膜增生性、退行性、炎症性和感染性疾病有关，我们将注意力集中在转化生长因子-β。老年性黄斑变性(ARMD)、增生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。我们利用基因芯片(Human Genome U133 plus ARRAY，Affymetrix)，通过基因芯片分析，评价了转化生长因子对人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。转化生长因子可显著增强多种细胞外基质蛋白(ECM)的表达，如胶原、纤维连接蛋白、凝血酶敏感蛋白和硫酸软骨素蛋白多糖，以及多种生长因子，如血管内皮生长因子(VEGF)和血小板衍生生长因子(PDGF)。值得注意的是，ECM和生长因子与ARMD、PVR、RD等许多致盲视网膜疾病有关。 为了研究细胞外基质成分的作用及转化生长因子对细胞外基质的调节作用，我们通过将转化生长因子受体I(TGF-R I)与Nestin-Cre小鼠杂交，有条件地删除神经元(CKO)中的转化生长因子信号，建立了一种小鼠模型。几乎所有出生的CKO小鼠的视网膜脱离都发生在神经感觉视网膜(NSR)的RPE/光感受器层交界处。6-硫酸软骨素的免疫测定显示，CKO小鼠的反应非常弱，而野生型小鼠的光感受器层呈强阳性染色。对我们的CKO模型的研究表明，视网膜细胞中缺乏功能性的转化生长因子RI会导致视网膜下间隙和光感受器层中硫酸软骨素蛋白多糖的水平降低。这反过来又会导致视网膜脱离，因为NSR与RPE失去了粘附性。 我们以前曾报道过，转化生长因子是一种强有力的刺激血管内皮生长因子和血小板衍生生长因子表达的人RPE细胞。由于视网膜炎症(ARMD)现在被认为是一个关键成分，我们评估了炎症介质对血管内皮生长因子(VEGF)在ARMD视网膜新生血管中的表达的作用。结果显示，干扰素、白介素1和肿瘤坏死因子可显著促进视网膜色素上皮和脉络膜细胞分泌血管内皮生长因子A和C。在视网膜微环境中，巨噬细胞和其他渗入视网膜和脉络膜的细胞产生干扰素、白介素1、肿瘤坏死因子和其他细胞因子。我们正在评估炎症介质调节血管内皮生长因子A、C和其他血管生成因子的机制，以阐明炎症过程在ARMD和其他视网膜新生血管疾病中的作用。 Affymetrix基因表达谱微阵列研究也出人意料地揭示了转化生长因子诱导白细胞介素11(IL-11)的作用。白介素11通过抑制巨噬细胞产生肿瘤坏死因子和白介素1而起到抗炎作用。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，干扰素可下调RPE和脉络膜细胞分泌IL-1和TNF-11。相反，转化生长因子诱导的IL-11分泌不受干扰素的影响。在角膜上皮细胞和基质细胞中也观察到了类似的现象。我们正在评估IL-11作为一种药物在保护视网膜免受氧化和炎症伤害方面的潜在用途。

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. 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 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 down 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. Similar observations were also made in corneal epithelial and stromal cells. We are evaluating the potential usefulness of IL-11 as an agent in the protection of retina from oxidative and inflammatory insults.