Molecular Basis of Retinal Angiogenesis

描述（由申请人提供）：过氧化物酶体增殖物激活受体（PPARs）是配体激活转录因子的核受体超家族成员。三种亚型，PPAR-a、PPAR-D和PPAR-R，每一个都由一个基因编码，已经被鉴定出来。PPARs可以被多种配体激活，包括天然存在的脂肪酸、贝特类和胡萝卜素。在最后一个项目期间，我们的研究中的作用，在视网膜血管生成中确定的PPARs，特别是PPAR-D，作为潜在的治疗目标。所有三种PPARs均在血管内皮中表达。在这种情况下，PPAR-a和PPAR-？通常表现出抗炎、抗增殖和抗血管生成作用。相比之下，关于PPAR-κ/d的了解要少得多，尽管它已显示出刺激血管内皮细胞的增殖，并且其活化导致某些癌细胞系中的VEGF诱导，但其他细胞系中则不然。以前的工作清楚地表明，所有三种PPARs通过组织和细胞特异性的高度多效性机制影响血管功能。因此，为了理解视网膜血管生成背景下的这些机制，必须在所涉及的特定视网膜细胞中表征PPAR表达和活化。这些细胞是产生血管生成因子的Muller细胞和对血管生成因子作出反应的视网膜微血管内皮细胞（RMEC）。使用这两种细胞类型的原代培养物，可以系统地研究PPAR-d、生长因子产生和血管生成细胞行为之间的复杂相互作用，从而鉴定PPARs的适当治疗操作。我们的初步数据表明，在与早期和晚期糖尿病视网膜病变（DR）相关的条件下，在Muller细胞和RMEC中，PPAR-κ/d被激活。因此，我们建议使用一种工具的组合来表征PPAR-<$/d在视网膜血管疾病中的作用：1）从野生型和PPAR-<$/d-/-小鼠分离的细胞的比较; 2）从野生型小鼠分离的细胞中的高选择性激动剂和拮抗剂; 3）在早期和晚期DR模型中野生型和PPAR-<$/d-/-小鼠的比较;和4）在野生型小鼠和大鼠的DR模型中的高度特异性激动剂和拮抗剂。我们的体内模型已经被选择为与早期（STZ诱导的糖尿病）和晚期（氧诱导的视网膜病变）DR相关。我们相信这种策略是集中和全面的，它将产生对我们对DR的基本理解和合理治疗方法的发展具有重要意义的信息。 公共卫生相关性：我们建议研究过氧化物酶体增殖物激活受体-/d（PPAR-/d）在早期和晚期糖尿病视网膜病变相关视网膜血管病变中的作用。已发表的研究，结合我们的初步数据，强烈暗示PPAR-γ/d在两个重要的病理过程：视网膜生产的血管内皮生长因子，和视网膜血管内皮细胞增殖和小管形成。我们将利用我们对适当的体外和体内模型的熟悉以及我们对PPAR-<$/d-/-小鼠的拥有来深入了解PPAR-<$/d在视网膜血管疾病中的作用。我们的最终目的是确定在视网膜血管疾病的相关动物模型中PPAR-γ/d特异性拮抗作用的治疗效果。

DESCRIPTION (provided by applicant): Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor super family of ligand-activated transcription factors. Three subtypes, PPAR-a, PPAR-¿/d and PPAR-?, each encoded by a single gene, have been identified. PPARs can be activated by a variety of ligands that include naturally occurring fatty acids, fibrates and prostaglandins. During the last project period, our studies of the roles of prostaglandins in retinal angiogenesis identified PPARs, specifically PPAR-¿/d, as potential therapeutic targets. All three PPARs are expressed in vascular endothelium. In this context, PPAR-a and PPAR-? generally demonstrate anti-inflammatory, anti-proliferative, and anti-angiogenic effects. In contrast, much less is known regarding PPAR-¿/d, although it has been shown to stimulate proliferation of vascular endothelial cells, and its activation leads to VEGF induction in some cancer cell lines, but not others. Previous work clearly suggests that all three PPARs influence vascular function by highly pleiotropic mechanisms that are tissue- and cell- specific. Thus, in order to understand these mechanisms in the context of retinal angiogenesis, PPAR expression and activation must be characterized in the specific retinal cells that are involved. These cells are the Muller cells, which produce angiogenic factors, and the retinal microvascular endothelial cells (RMEC), which respond to the angiogenic factors. Using primary cultures of these two cell types, the complicated interactions between PPAR-¿/d, growth factor production and angiogenic cell behaviors can be studied systematically, leading to the identification of appropriate therapeutic manipulations of PPARs. Our preliminary data indicate that PPAR-¿/d is activated in Muller cells and RMEC under conditions relevant to both early and late diabetic retinopathy (DR). Thus, we propose to use a combination of tools to characterize the role of PPAR-¿/d in retinal vascular disease: 1) comparisons of cells isolated from wild type and PPAR-¿/d-/- mice; 2) highly selective agonists and antagonists in cells isolated from wild type mice; 3) comparisons of wild type and PPAR-¿/d-/- mice in models of early and late DR; and 4) highly specific agonists and antagonists in models of DR in wild type mice and in rats. Our in vivo models have been selected for their relevance to early (STZ- induced diabetes) and late (oxygen-induced retinopathy) DR. We believe this strategy is focused and comprehensive, and it will yield information of importance to our fundamental understanding of DR and to the development of rational therapeutic approaches. PUBLIC HEALTH RELEVANCE: We propose to investigate the role of peroxisome proliferator-activated receptor- ¿/d (PPAR- ¿/d) in early and late stages of retinal vascular pathology related to diabetic retinopathy. Published studies, combined with our preliminary data, strongly implicate PPAR- ¿/d in two important pathological processes: retinal production of vascular endothelial growth factor, and retinal vascular endothelial cell proliferation and tubulogenesis. We will exploit our familiarity with appropriate in vitro and in vivo models and our possession of PPAR- ¿/d-/- mice to gain insight into the role of PPAR- ¿/d in retinal vascular disease. Our final aim will be to determine the therapeutic efficacy of PPAR- ¿/d -specific antagonism in relevant animal models of retinal vascular disease.