Role of H-Ras in retinal cell death in diabetes

摘要 糖尿病视网膜病变仍然是失明的主要原因，尽管在该领域的前沿研究， 其发病的分子机制尚不清楚。我们的研究已经证明了 基质金属蛋白酶9（MMP-9）在糖尿病视网膜病变中的作用，并已证明细胞溶质MMP-9 活化是早期事件，随后是其线粒体积累、线粒体功能障碍和 线粒体DNA损伤，引发自由基的恶性循环。表观遗传修饰在MMP-9中起关键作用 在糖尿病中，MMP-9启动子DNA经历动态甲基化-羟甲基化， 组蛋白修饰MMP-9也受同型半胱氨酸（一种含巯基的非蛋白质氨基酸）调节， 糖尿病患者血浆同型半胱氨酸水平升高。同型半胱氨酸的增加与 细胞和代谢异常，包括线粒体损伤和表观遗传修饰。 同型半胱氨酸也是硫化氢（H2S）的前体，由于同型半胱氨酸代谢受损， 糖尿病患者中H2S的血浆水平降低。基于这些，我们的中心假设是， 糖尿病，高同型半胱氨酸激活MMP-9，扰乱线粒体动力学， 线粒体加速细胞凋亡，导致糖尿病视网膜病变的发展。 目的1探讨同型半胱氨酸激活MMP-9在糖尿病中的作用机制， 预测高同型半胱氨酸通过（i）破坏MMP-9与其组织之间的相互作用来激活MMP-9 抑制剂Timp 1，和（ii）诱导表观遗传修饰并增加MMP-9-Timp 1的比率。目标2将 确定同型半胱氨酸损害线粒体动力学的机制，并将测试 同型半胱氨酸增加糖尿病患者线粒体碎裂和线粒体自噬功能障碍的假说 无法正确去除破碎的线粒体目的3将检查调节的治疗潜力 同型半胱氨酸-H2S代谢平衡对糖尿病视网膜病变抑制作用。该计划将采用体外（视网膜 内皮细胞）和体内（来自啮齿动物的视网膜微血管）糖尿病视网膜病变模型，并将利用 完全优化的分子生物学和药理学方法。我们的总体目标是确定新的 参与糖尿病视网膜病变发病机制的调节机制，特别是在视网膜水平 同型半胱氨酸-H2S的调节。该建议是基于一个可测试的中心假设，我们的建议是 研究是创新的，并进行重大的翻译影响，因为他们预计将确定新的 糖尿病性视网膜病变的发生和发展。这将为患者提供 预防/阻止这种威胁视力的糖尿病并发症的额外治疗手段。

ABSTRACT Diabetic retinopathy remains a major cause of blindness, and despite cutting edge research in the field, the molecular mechanism of its pathogenesis remains unclear. Our studies have documented a critical role of matrix metalloproteinase 9 (MMP-9) in diabetic retinopathy, and have demonstrated that cytosolic MMP-9 activation is an early event, that is followed by its mitochondrial accumulation, mitochondrial dysfunction and mtDNA damage, initiating a vicious cycle of free radicals. Epigenetic modifications play a critical role in MMP-9 transcription, and in diabetes, MMP-9 promoter DNA undergoes dynamic methylation-hydroxymenthlation and histone modifications. MMP-9 is also regulated by homocysteine, a thiol-containing non-protein amino acid, and diabetic patients have elevated plasma homocysteine levels. Increased homocysteine is implicated in cellular and metabolic abnormalities including mitochondrial damage and epigenetic modifications. Homocysteine is also a precursor of hydrogen sulfide (H2S), and due to impaired homocysteine metabolism, plasma levels of H2S are decreased in diabetic patients. Based on these, our central hypothesis is that in diabetes, high homocysteine activates MMP-9 and disturbs mitochondrial dynamics, and the damaged mitochondria accelerates apoptosis resulting in the development of diabetic retinopathy. Aim 1 will investigate the mechanism(s) by which homocysteine activates MMP-9 in diabetes, and the model predicts that high homocysteine activates MMP-9 by (i) damaging interactions between MMP-9 and its tissue inhibitor, Timp1, and (ii) inducing epigenetic modifications and increasing the ratio of MMP-9-Timp1. Aim 2 will determine the mechanism(s) by which homocysteine impairs mitochondrial dynamics, and will test the hypothesis that homocysteine increases mitochondrial fragmentation, and dysfunctional mitophagy in diabetes fails to properly remove the fragmented mitochondria. Aim 3 will examine the therapeutic potential of regulating homocysteine-H2S metabolic balance on inhibition of diabetic retinopathy. The plan will employ in vitro (retinal endothelial cells) and in vivo (retinal microvessels from rodents) models of diabetic retinopathy, and will utilize fully optimized molecular biological and pharmacological approaches. Our overall goal is to identify novel regulatory mechanisms involved in the pathogenesis of diabetic retinopathy, specifically at the level of regulation of homocysteine-H2S. The proposal is based on a testable central hypothesis, and our proposed studies are innovative and carry a significant translational impact as they are expected to identify novel therapeutic targets to prevent the development and progression of diabetic retinopathy. This will offer patients additional therapeutic means to prevent/halt this sight-threatening complication of diabetes.