Cellular Mechanisms of Retinal Angiogenesis

描述（由申请人提供）：本申请建议继续一个旨在阐明控制缺血性视网膜病变机制的项目。我们一直在研究超氧化物生成酶NADPH氧化酶在触发血管内皮生长因子（VEGF）过度表达导致血管炎症反应和病理性血管生成中的作用。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NOX 2亚型在诱导活性氧（ROS）和血管生成细胞因子的产生以及引起血管炎症反应和玻璃体视网膜新生血管形成中具有关键作用。我们现在建议确定尿素/鸟氨酸途径酶的具体作用，在这些影响。我们在糖尿病模型中的研究表明，精氨酸酶活性的增加通过降低L-精氨酸对EC一氧化氮合酶（eNOS）的可用性而引起血管内皮细胞（EC）功能障碍，所述eNOS降低NO并增加ROS。增加酪氨酸酶活性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/神经胶质增生，多胺和脯氨酸分别诱导细胞生长和胶原蛋白产生。我们的初步数据显示，在OIR、DR或EIU模型中，腺苷酸酶活性和腺苷酸酶I（AI）表达增加，并且AI的增加在缺乏NOX 2的小鼠中被消除。我们的研究还表明，视网膜色素酶活性和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基因的一个拷贝的缺失与AII缺失的组合降低了DR和EIU中的细胞因子产生。此外，在OIR模型中的初步研究表明，用胰蛋白酶抑制剂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。基于我们先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NADPH途径的激活在视网膜病变期间引起视网膜血管功能障碍和诱导病理性血管生成和纤维化/神经胶质增生中具有关键作用。我们的具体目标如下：1.确定视网膜病变期间NADPH氧化酶衍生的ROS在增加NOS表达/活性和解偶联NOS中的作用。2.定义视网膜病变过程中酪氨酸酶激活的分子机制。3.测试糖尿病是否通过激活视网膜血管酶导致视网膜血管功能障碍。4.通过增加视网膜坏死酶检测视网膜缺血是否会导致病理性血管生成和纤维化/神经胶质增生。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工作年龄成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进展，开始于血管炎症反应和功能障碍以及血管内皮细胞衬里的损伤，并进展为病理性血管生成，随后是纤维化/神经胶质增生和视网膜脱离。了解引发这一过程的因素和事件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，这种酶会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，这为减少这种致盲疾病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.