Role of H-Ras in Retinal Cell Death in Diabetes

描述（由申请人提供）：糖尿病视网膜病变仍然是年轻人失明的主要原因。我们已经证明在糖尿病视网膜病变的发病机制中，基质金属蛋白酶-9（MMP-9）通过小分子量G蛋白H-Ras在视网膜中被激活，激活的MMP-9损伤线粒体并加速视网膜毛细血管细胞凋亡，并且MMP-9基因敲除的糖尿病小鼠免受视网膜病变的发展。糖尿病改变组蛋白修饰，MMP-9被证明是由其启动子区的组蛋白修饰调节。我们的初步数据表明，在糖尿病中，视网膜MMP-9启动子处NF-κ B p65亚基的募集增加，组蛋白3的二甲基化赖氨酸9（H3 K9 me 2）的募集减少，赖氨酸特异性脱甲基酶（LSD 1）被激活，组蛋白脱乙酰酶（Sirt 1）被抑制，MnSOD的过表达保护了糖尿病诱导的MMP-9的增加，组蛋白修饰和组蛋白修饰酶的改变。此外，在啮齿类动物中，在血糖控制不佳的一段时间后重新建立正常的血糖控制不能为MMP-9活化、组蛋白甲基化和Sirt 1抑制提供任何益处。因此，我们的总体假设是，在糖尿病中，视网膜MMP-9受组蛋白修饰的调节，并且该酶在高血糖损伤终止后仍然升高，并且视网膜病变继续进展。我们建议通过解决三个具体目标下提出的补充问题来系统地测试这一假设。第一个目的将研究糖尿病激活视网膜MMP-9的分子机制，并将测试以下假设：由于组蛋白修饰，MMP-9启动子处p65的募集增加，导致其激活。由于氧化应激被认为是 在糖尿病视网膜病变的发展中起重要作用，在第二个目标中，我们将研究氧化应激在MMP-9调节中的作用。我们的工作假设预测，由于氧化应激增加，Sirt 1被抑制，去乙酰化减少，这有利于MMP-9启动子处的p65结合。第三个目标将确定MMP-9在与糖尿病视网膜病变进展相关的代谢记忆现象中的作用，并将测试以下假设：由于持续的组蛋白修饰，MMP-9在高血糖逆转后保持活性，并且功能障碍的线粒体继续加速毛细血管细胞的凋亡。这些拟议的研究是基于使用有效的体外和体内模型系统产生的令人信服的初步数据。中心假设将在分离的细胞中使用活性质粒、siRNA和药理学抑制剂进行测试，并且将在使用遗传操作的小鼠的体内模型和来自患有糖尿病视网膜病变的人类供体的视网膜微血管中验证体外结果。这些结果有望证明组蛋白修饰在糖尿病视网膜MMP-9活化中的作用，以及与糖尿病视网膜病变进展相关的代谢记忆现象。这将揭示治疗的新靶点，包括组蛋白修饰酶的调节剂，以在其发展的早期阶段预防视网膜病变，并在高血糖症终止后预防其进展。这将为患者提供额外的治疗 预防/延缓糖尿病的视力威胁并发症的手段。

DESCRIPTION (provided by applicant): Diabetic retinopathy remains the leading cause of blindness among young adults. We have shown that in the pathogenesis of diabetic retinopathy, matrixmetalloproteinase-9 (MMP-9) is activated in the retina via a small molecular weight G-protein, H-Ras, activated MMP-9 damages mitochondria and accelerates retinal capillary cell apoptosis, and MMP-9 gene knockout diabetic mice are protected from the development of retinopathy. Diabetes alters histone modifications, and MMP-9 is shown to be regulated by histone modifications in its promoter region. Our preliminary data show that in diabetes, the recruitment of p65 subunit of NF-kB is increased at retinal MMP-9 promoter and that of dimethylated lysine 9 of histone 3 (H3K9me2) is decreased, lysine-specific demethylase (LSD1) is activated and histone deacetylase (Sirt1) is inhibited, and overexpression of MnSOD protects diabetes-induced increase in MMP-9, histone modifications and alterations in the histone modifying enzymes. Furthermore, re-institution of normal glycemic control after a period of poor glycemic control in rodents fails to provide any benefit to MMP-9 activation, histone methylation and Sirt1 inhibition. Thus, our overall hypothesis is that in diabetes, retinal MMP-9 is regulated by histone modifications, and the enzyme remains elevated after termination of hyperglycemic insult and the retinopathy continues to progress. We propose to test this hypothesis methodically by addressing complementary questions proposed under three specific aims. The first aim will investigate the molecular mechanism by which diabetes activates retinal MMP-9, and will test the hypothesis that due to histone modifications, the recruitment of p65 at MMP-9 promoter is increased resulting in its activation'. Since oxidative stress is considered to play an important role in the development of diabetic retinopathy, in the second aim, we will investigate the role of oxidative stress in the regulation of MMP-9. Our working hypothesis predicts that due to increased oxidative stress, Sirt1 is inhibited and deacetylation is decreased, and this facilitates the binding of p65 at the MMP-9 promoter. The third aim will determine the role of MMP-9 in the metabolic memory phenomenon associated with the progression of diabetic retinopathy, and will test the hypothesis that due to continued histone modifications, MMP-9 remains active after reversal of hyperglycemia, and the dysfunctional mitochondria continues to accelerate apoptosis of capillary cells. These proposed studies are based on compelling preliminary data generated using valid in vitro and in vivo model systems. The central hypothesis will be tested using active plasmids, siRNAs and pharmacological inhibitors in isolated cells, and in vitro findings will be validated in in vivo models using genetically manipulated mice and in retinal microvessels from human donors with diabetic retinopathy. The results are expected to demonstrate the role of histone modifications in retinal MMP-9 activation in diabetes, and in the metabolic memory phenomenon associated with the progression of diabetic retinopathy. This should reveal novel targets for therapies, including regulators of histone modifying enzymes, to prevent retinopathy in the early stages of its development, and in its progression after termination of hyperglycemia. This will offer patients additional therapeutic means to prevent/retard the sight-threatening complication of diabetes.