Role of Ras in Retinal Cell Death in Diabetes

描述(申请人提供)：视网膜病变是糖尿病最常见和最令人恐惧的并发症之一。虽然高血糖被认为是其发生的主要原因，但其确切的机制仍不清楚。这项建议的总体目的是阐明对糖尿病视网膜病变发病机制的新见解。在之前的资助期间，我们已经证明了一种小分子G蛋白H-RAS在视网膜毛细血管细胞加速凋亡中的重要和新的作用。据报道，H-RAS上调基质金属蛋白酶-9(MMP9)的表达，MMP9是金属蛋白酶家族的成员，调节包括细胞凋亡、炎症和基质降解在内的主要生物学功能。我们的中心假说是“H-RAS通路的激活激活了基质金属蛋白酶-9，加速了视网膜毛细血管细胞的凋亡，最终导致糖尿病视网膜病变的发生”。作为支持，我们令人兴奋的初步数据显示，在糖尿病患者的视网膜中，基质金属蛋白酶-9被激活。H-RAS功能的抑制剂可阻止葡萄糖诱导的视网膜毛细血管细胞中基质金属蛋白酶-9的激活，而基质金属蛋白酶-9的特异性抑制剂可改善促炎介质和细胞凋亡的增加。目的1研究糖尿病视网膜微血管中基质金属蛋白酶-9被激活的机制。利用分离的视网膜毛细血管细胞和糖尿病视网膜病变的动物模型，我们将检验这一假说，即在糖尿病中，基质金属蛋白酶-9是通过H-RAS介导的信号级联，即Raf-1-MAPK-ERK-NF-kB被激活的。目的2将探讨活化的基质金属蛋白酶-9导致糖尿病视网膜病变的机制；该假说预测，基质金属蛋白酶-9的激活(A)损伤线粒体，引起视网膜微血管细胞的凋亡，(B)增加炎症和通透性，从而导致视网膜病变的发生。这一假说将在体外和体内系统中通过药物或遗传操作基质金属蛋白酶-9基因进行验证。研究表明，糖尿病视网膜病变在重新开始良好的血糖控制后会继续发展一段时间，这表明这是一种代谢记忆现象。在目标3中，我们将使用一个大鼠模型来检验这一假设，即‘由于高血糖逆转后H-RAS的持续激活，在炎症介质升高和毛细血管细胞持续凋亡的情况下，基质金属蛋白酶-9保持激活状态。我们的初步数据显示，糖尿病大鼠的视网膜H-RAS激活在重建正常血糖后未能停止，这一点得到了支持。通过研究基质金属蛋白酶-9在视网膜毛细血管细胞死亡中的作用，了解糖尿病视网膜病变的信号转导机制，将为未来的药物干预提供新的分子靶点，以抑制这一威胁视力的糖尿病并发症。这将有助于为未来的药物干预设计特定的治疗药物(例如，基质金属蛋白酶-9的抑制剂和反义药物)，患者可以使用这些药物来补充他们尽可能好的血糖控制，以治疗这种威胁视力的糖尿病并发症。 公共卫生相关性：这项建议旨在了解基质金属蛋白酶在葡萄糖诱导的视网膜毛细血管细胞代谢功能改变导致其死亡和糖尿病视网膜病变发展中可能的调节作用(S)。这一结果有望阐明糖尿病视网膜病变的发病机制，并为预防视网膜病变的发生和发展提供新的治疗靶点。

DESCRIPTION (provided by applicant): Retinopathy is one of the most prevalent and feared complications of diabetes. Although hyperglycemia is considered as the major cause of its development, the exact mechanism remains unclear. The overall aim of this proposal is to elucidate new insight into the pathogenesis of diabetic retinopathy. During the previous funding period we have demonstrated an important and novel role for a small molecular weight G- protein, H-Ras, in the accelerated apoptosis of retinal capillary cells. H-Ras is reported to upregulate matrix metalloproteinases-9 (MMP-9), a member of the family of metalloproteinases that regulate major biological functions, including apoptosis, inflammation and matrix degradation. Our central hypothesis is that 'activation of H-Ras cascade activates MMP-9, accelerating apoptosis of retinal capillary cells, and this ultimately leads to the development of retinopathy in diabetes'. In support, our exciting preliminary data show that MMP-9 is activated in the retina in diabetes. Inhibitors of H-Ras function prevent glucose-induced activation of MMP-9 in retinal capillary cells, and specific inhibitors of MMP-9 ameliorate increases in proinflammatory mediators and apoptosis. Aim 1 will determine the mechanism by which MMP-9 is activated in retinal microvasculature in diabetes. Using isolated retinal capillary cells and animal models of diabetic retinopathy, we will test the hypothesis that MMP-9 is activated in diabetes via H-Ras mediated signaling cascade, Raf-1-MAPK-ERK-NF-kB. Aim 2 will investigate the mechanism by which activated MMP-9 results in the development of diabetic retinopathy; and the hypothesis predicts that the activation of MMP-9 (a) damages mitochondria provoking apoptosis of retinal microvascular cells, and (b) increases inflammation and permeability, resulting in the development of retinopathy. This hypothesis will be tested in both in vitro and in vivo systems by pharmacologically or genetically manipulating MMP-9 gene. Studies have shown that diabetic retinopathy continues to progress for sometime after good glycemic control is re-instituted, suggesting a metabolic memory phenomenon. In aim 3, using a rat model, we will test the hypothesis that 'due to the continued activation of H-Ras after reversal of hyperglycemia, MMP-9 remains activated with elevated inflammatory mediators and continued capillary cell apoptosis. This is supported by our preliminary data showing the failure of retinal H-Ras activation to halt after re-establishment of normal glucose in diabetic rats. Understanding the signal transduction mechanisms responsible for the pathogenesis of diabetic retinopathy by characterizing the role of MMP-9 in the retina capillary cell death will provide fresh insight into the novel molecular targets for future pharmacological interventions to inhibit this sight-threatening complication of diabetes. This should help design specific therapeutics (e.g. inhibitors of MMP-9 and antisense) for future pharmacological interventions which patients can use to supplement their best possible glycemic control to treat this sight-threatening complication of diabetes. PUBLIC HEALTH RELEVANCE: This proposal is aimed at understanding the putative regulatory role(s) of matrix metalloproteinases in glucose-induced alterations in metabolic functions of retinal capillary cells leading to their demise, and the development of diabetic retinopathy. The results are expected to elucidate new insight into the pathogenesis of diabetic retinopathy, and reveal novel targets for therapies to prevent the development and progression of retinopathy.