Cellular Mechanisms of Retinal Angiogenesis

描述（由申请人提供）：本申请建议继续进行旨在阐明缺血性视网膜病变期间控制病理性血管生长的机制的项目。我们一直在研究一氧化氮合酶（NOS）及其产物NO在调节血管内皮生长因子（VEGF）表达和活性中的作用。我们对氧诱导的视网膜病变（OIR）和糖尿病模型的研究表明，NO、超氧化物及其组合产物过氧亚硝酸盐的形成增加有助于缺血性视网膜病变中VEGF的过度表达和相关的血管病理学。我们现在建议确定这些影响的特定分子介质。 最近的临床和实验研究发现，血管紧张素II（Ang II）活性增加在糖尿病和OIR的视网膜VEGF过度表达、血管通透性增高和新生血管形成中起作用。已知Ang II通过增加血管NAD（P）H氧化酶活性和超氧阴离子形成而引起各种形式的心血管疾病中的内皮细胞功能障碍。在大血管内皮细胞中，Ang II引起NAD（P）H的表达和活性增加以产生超氧化物。超氧化物被认为诱导eNOS的“解偶联”以产生超氧化物以及NO，导致过氧亚硝酸盐形成。我们的初步数据表明，视网膜新生血管在OIR与血管的表达和活性增加的NAD（P）H氧化酶亚基gp91 phox，这是与超氧化物和过氧亚硝酸盐的形成增加。这表明通过NAD（P）H氧化酶产生的超氧化物在血管病理学中起作用。使用来自牛视网膜的内皮细胞，我们发现过氧亚硝酸盐的形成与VEGF转录调节因子STAT3的激活和VEGF表达的增加有关。基于这些观察结果，我们假设缺血性视网膜病变期间视网膜新生血管形成主要涉及NAD（P）H氧化酶通过Ang II的激活，导致eNOS“解偶联”、过氧亚硝酸盐形成和VEGF过表达。我们的具体目标是通过使用已建立的细胞生物学方法和体外和体内模型来验证这一假设，实验设计用于回答以下问题：1）视网膜缺血是否通过激活NAD（P）H氧化酶引起VEGF过度表达和新生血管形成？2)缺血时过氧亚硝基阴离子的形成是否通过NAD（P）H氧化酶衍生的超氧化物引起eNOS“解偶联”而增加？3)视网膜缺血是否激活NAD（P）H氧化酶并通过Ang II诱导VEGF过度表达和新生血管形成？4)缺氧和血管紧张素II是否通过过亚硝酸盐介导的STAT3激活增加血管VEGF的表达？

DESCRIPTION (provided by applicant): This application proposes the continuation of a project designed to elucidate the mechanisms controlling pathological vascular growth during ischemic retinopathy. We have been studying the role in this process of nitric oxide synthase (NOS) and its product, NO, in regulating the expression and activity of vascular endothelial growth factor (VEGF). Our research with models of oxygen-induced retinopathy (OIR) and diabetes indicates that increased formation of NO, superoxide and their combination product peroxynitrite contribute to the over expression of VEGF and the associated vascular pathology in ischemic retinopathy. We now propose to identify specific molecular mediators of these effects. Recent clinical and experimental findings have implicated increased angiotensin II (Ang II) activity in retinal VEGF over-expression, vascular hyperpermeability and neovascularization in diabetes and OIR. Ang II is known to cause endothelial cell dysfunction in various forms of cardiovascular disease by increasing vascular NAD(P)H oxidase activity and superoxide anion formation. In macrovascular endothelial cells Ang II causes increases in expression and activity of NAD(P)H to generate superoxide. Superoxide is thought to induce "uncoupling" of eNOS to generate superoxide as:well as NO, resulting in peroxynitrite formation. Our preliminary data suggest that retinal neovascularization during OIR is associated with increased vascular expression and activity of the NAD(P)H oxidase subunit gp91phox which is correlated with increased formation of superoxide and peroxynitrite. This suggests that superoxide production via NAD(P)H oxidase has a role in the vascular pathology. Using endothelial cells from bovine retina we have found that peroxynitrite formation is associated with activation of the VEGF transcription regulator STAT3 and increased expression of VEGF. Based on these observations, we hypothesize that retinal neovascularization during ischemic retinopathy critically involves activation of NAD(P)H oxidase via Ang II, leading to eNOS "uncoupling", peroxynitrite formation and VEGF over-expression. Our specific aims are to test this hypothesis by using established cell biology approaches and both in vitro and in vivo models in experiments designed to answer the following questions: 1) Does retinal ischemia cause over-expression of VEGF and neovascularization via activation of NAD(P)H oxidase? 2) Is peroxynitrite formation increased during ischemia via the action of NAD(P)H oxidase-derived superoxide in causing eNOS "uncoupling"? 3) Does retinal ischemia activate NAD(P)H oxidase and induce over-expression of VEGF and neovascularization via Ang II? 4) Do hypoxia and Ang II increase vascular expression of VEGF via peroxynitrite-mediated activation of STAT3?