Cellular Mechanisms of Retinal Angiogenesis

描述(由申请人提供)：本申请建议继续一项旨在阐明控制缺血性视网膜病变的机制的项目。我们一直在研究超氧化物产生酶NADPH氧化酶在触发血管内皮生长因子(VEGF)过度表达中的作用，从而导致血管炎症反应和病理性血管生成。我们对氧诱导的视网膜病变(OIR)、糖尿病视网膜病变(DR)和内毒素诱导的葡萄膜炎(EIU)模型的研究表明，NADPH氧化酶NOX2亚型在诱导活性氧(ROS)和血管生成细胞因子的产生、引起血管炎症反应和玻璃体视网膜新生血管中起关键作用。我们现在建议确定尿素/鸟氨酸途径酶精氨酸酶在这些效应中的具体作用。我们在糖尿病模型上的研究表明，精氨酸酶活性的增加通过减少血管内皮细胞(EC)一氧化氮合酶(ENOS)对L精氨酸的可获得性，从而减少NO和增加ROS，从而导致血管内皮细胞功能障碍。精氨酸酶活性的增加也可能通过增加多胺和脯氨酸的形成而促进病理性视网膜血管生成和纤维化/胶质增生，这两种物质分别诱导细胞生长和胶原产生。我们的初步数据显示，在OIR、DR或EIU模型中，精氨酸酶活性和精氨酸酶I(AI)的表达增加，而在缺乏NOX2的小鼠中，AI的增加被抑制。我们的研究还表明，精氨酸酶活性和AI表达的增加与细胞因子表达增加、ROS形成增加和视网膜血管功能障碍有关。此外，AI基因一个拷贝的缺失和所有II基因的缺失都会减少DR和EIU中细胞因子的产生。此外，OIR模型的初步研究表明，精氨酸酶抑制剂的治疗减少了玻璃体-视网膜血管生成，并增强了生理性血管生成。根据我们以前的工作和新的初步数据，我们的总体假设是，NADPH氧化酶诱导的精氨酸酶途径的激活在视网膜病变期间导致视网膜血管功能障碍、诱导病理性血管生成和纤维化/胶质增生中起关键作用。我们的具体目标如下：1.确定NADPH氧化酶来源的ROS在视网膜病变过程中增加精氨酸酶表达/活性和解偶联NOS中的作用。2.明确视网膜病变过程中精氨酸酶激活的分子机制。3.检测糖尿病是否通过激活精氨酸酶导致视网膜血管功能障碍。4.通过增加精氨酸酶活性来检测视网膜缺血是否导致病理性血管生成和纤维化/胶质细胞增生症。公共卫生相关性：视网膜血管疾病和病理性视网膜血管生成是导致工作的老年人和老年人失明的主要原因。涉及病理性血管生成的致盲性视网膜疾病都遵循相同的病理过程，首先是血管炎症反应和血管内皮细胞衬里功能障碍和损伤，然后发展到病理性血管生成，然后是纤维化/胶质增生和视网膜脱离。了解启动这一过程的因素和事件的顺序对于预防和治疗视网膜血管疾病至关重要。限制一种酶的作用，这种酶可以引起炎症反应和血管内皮细胞损伤，并阻止视网膜获得足够的血液，这为减少这种失明情况的发生提供了很大的希望。

DESCRIPTION (provided by applicant): This application proposes continuation of a project designed to elucidate the mechanisms that control ischemic retinopathy. We have been studying the role of the superoxide generating enzyme NADPH oxidase in triggering over-expression of vascular endothelial growth factor (VEGF) resulting in vascular inflammatory reactions and pathological angiogenesis. Our research with models of oxygen-induced retinopathy (OIR), diabetic retinopathy (DR) and endotoxin-induced uveitis (EIU) has revealed that the NADPH oxidase NOX2 isoform has a key role in inducing production of reactive oxygen species (ROS) and angiogenic cytokines and causing vascular inflammatory reactions and vitreo-retinal neovascularization. We now propose to determine the specific role of the urea/ornithine pathway enzyme arginase in these effects. Our studies in models of diabetes have shown that increases in arginase activity cause vascular endothelial cell (EC) dysfunction by decreasing availability of L-arginine to EC nitric oxide synthase (eNOS) which decreases NO and increases ROS. Increasing arginase activity may also contribute to pathological retinal angiogenesis and fibrosis/gliosis by increasing formation of polyamines and proline, which induce cell growth and collagen production, respectively. Our preliminary data show that arginase activity and arginase I (AI) expression are increased in models of OIR, DR or EIU and that the increase in AI is abrogated in mice that lack NOX2. Our studies also indicate that increases in arginase activity and AI expression are correlated with increased cytokine expression increased ROS formation and retinal vascular dysfunction. Furthermore, deletion of one copy of the AI gene in combination with AII deletion decreases cytokine production in both DR and EIU. Moreover, preliminary studies in the OIR model indicate that treatment with an arginase inhibitor reduces vitreoretinal angiogenesis and enhances physiological angiogenesis. Based on our previous work and new preliminary data, our global hypothesis is that NADPH oxidase-induced activation of the arginase pathway has a key role in causing retinal vascular dysfunction and inducing pathological angiogenesis and fibrosis/gliosis during retinopathy. Our specific aims are as follows: 1. Determine the role of NADPH oxidase-derived ROS in increasing arginase expression/activity and uncoupling NOS during retinopathy. 2. Define the molecular mechanisms of arginase activation during retinopathy. 3. Test whether diabetes causes retinal vascular dysfunction by activating arginase. 4. Test whether retinal ischemia causes pathological angiogenesis and fibrosis/gliosis by increasing arginase. PUBLIC HEALTH RELEVANCE: Retinal vascular disease and pathological retinal angiogenesis are a leading cause of blindness in working aged adults and the elderly. Blinding retinal diseases that involve pathological angiogenesis all follow the same pathological progression, beginning with vascular inflammatory reactions and dysfunction and injury of the vascular endothelial cell lining and progressing to pathological angiogenesis, followed by fibrosis/gliosis and retinal detachment. Understanding the factors and sequence of events which initiate this process is critical to preventing and treating retinal vascular diseases. Limiting the actions of an enzyme which can cause inflammatory reactions and vascular endothelial cell damage and prevent the retina from getting enough blood offers much promise for reducing the incidence of this blinding condition.