NADPH Oxidase, Mitochondrial Dysfunction and Diabetic Retinopathy

视网膜病变仍然是糖尿病最令人担心的使人衰弱的并发症之一。在发病机制上 糖尿病视网膜病变，超氧化物水平显着升高，线粒体功能障碍及其 DNA 受损，导致超氧化物积累增加的恶性循环。新出现的证据表明 NADPH 氧化酶 (Nox) 也是活性氧 (ROS) 的潜在来源，对人体产生有害影响 细胞存活。我们的初步数据表明高血糖激活视网膜中的 Rac1/Nox2 信号轴 及其在线粒体失调之前的毛细血管细胞，这表明 Nox2 激活代表了早期 糖尿病引起的线粒体功能障碍和细胞凋亡的事件。因此，我们的总体假设是 糖尿病中 Nox2 衍生的 ROS 会损害视网膜线粒体，导致其功能障碍和细胞凋亡 毛细血管细胞的增殖加速，导致糖尿病视网膜病变的发生。 我们建议通过解决下提出的补充问题来系统地检验这一假设 三个具体目标。第一个目标是研究高血糖激活 Nox2 的机制 视网膜，并将测试高血糖中 Rac1 介导的 Nox2 激活和 ROS 的假设 一代引发线粒体损伤和细胞凋亡。由于视网膜病变的严重程度与 与高脂血症直接相关，在第二个目标中，脂毒性条件促进高脂血症的机制 将研究糖尿病视网膜病变的发展。我们的工作模型预测脂毒性情况 促进 Rac1 介导的 Nox2 激活和 ROS 生成，引发线粒体损伤和细胞 细胞凋亡。第三个目标将确定 Nox2 的调节对糖尿病发展的影响 视网膜病变，并将检验抑制 Nox2 会减轻线粒体损伤和 糖尿病视网膜病变的后续发展。 这些拟议的研究基于多学科产生的令人信服的初步数据 两个 PI 之间使用有效的体外和体内模型系统进行协作。我们建议利用 Tiam1/Rac1/Nox2 信号通路的已知选择性抑制剂；这些研究的数据将 通过使用该途径中关键信号蛋白的非活性突变体和 siRNA 证实了这一点。体外 研究结果将在体内模型（STZ 诱导的糖尿病大鼠和小鼠，以及 Zucker 糖尿病脂肪 大鼠），以及患有糖尿病视网膜病变的人类捐赠者的视网膜中。我们期望展示的作用 Rac-1介导的Nox2衍生的ROS是线粒体功能障碍发病机制中的“引发者” 视网膜病变。这应该揭示在其早期阶段预防视网膜病变的治疗新靶点 发展，并为患者提供额外的治疗手段来预防/延缓这种威胁视力的情况 糖尿病的并发症。

Retinopathy remains one of the most feared debilitating complications of diabetes. In the pathogenesis of diabetic retinopathy, superoxide levels are significantly elevated, mitochondria are dysfunctional and their DNA is damaged resulting in a vicious cycle of increased superoxide accumulation. Emerging evidence implicates NADPH oxidase (Nox) also as a potential source of reactive oxygen species (ROS) with detrimental effects on cell survival. Our preliminary data show that hyperglycemia activates Rac1/Nox2 signaling axis in the retina and its capillary cells prior to mitochondrial dysregulation, suggesting that Nox2 activation represents an early event in diabetes-induced mitochondrial dysfunction and cell apoptosis. Thus, our overall hypothesis is that Nox2-derived ROS in diabetes damage retinal mitochondria leading to their dysfunction, and apoptosis of capillary cells is accelerated resulting in the development of diabetic retinopathy. We propose to test this hypothesis methodically by addressing complementary questions proposed under three specific aims. The first aim will investigate the mechanism(s) by which hyperglycemia activates Nox2 in the retina, and will test the hypothesis that in hyperglycemia Rac1-mediated Nox2 activation and ROS generation initiate mitochondrial damage and cellular apoptosis. Since the severity of retinopathy is associated directly with hyperlipidemia, in the second aim, the mechanism(s) by which lipotoxic conditions promote the development of diabetic retinopathy will be investigated. Our working model predicts that lipotoxic conditions promote Rac1-mediated Nox2 activation and ROS generation to initiate mitochondrial damage and cellular apoptosis. The third aim will determine the effect of regulation of Nox2 on the development of diabetic retinopathy, and will test the hypothesis that inhibition of Nox2 will attenuate mitochondrial damage and subsequent development of diabetic retinopathy. These proposed studies are based on compelling preliminary data generated via multi-disciplinary collaborative efforts between two PIs using valid in vitro and in vivo model systems. We propose to utilize known selective inhibitors of the Tiam1/Rac1/Nox2 signaling pathways; data from these studies will be confirmed via the use of inactive mutants and siRNAs for key signaling proteins in this pathway. In vitro findings will be further validated in in vivo models (stz-induced diabetic rats and mice, and Zucker diabetic fatty rats), and also in the retina from human donors with diabetic retinopathy. We expect to demonstrate the role of Rac-1-mediated Nox2 derived ROS as the 'initiator' of mitochondrial dysfunction in the pathogenesis of retinopathy. This should reveal novel targets for therapies to prevent retinopathy in the early stages of its development, and offer patients additional therapeutic means to prevent/retard this sight-threatening complication of diabetes.