Role of H-Ras in Retinal Cell Death in Diabetes

描述(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。我们发现在糖尿病视网膜病变的发病机制中，基质金属蛋白酶-9通过小分子G蛋白H-RAS在视网膜被激活，激活的基质金属蛋白酶-9损伤线粒体并加速视网膜毛细血管细胞的凋亡，从而保护了糖尿病小鼠视网膜病变的发生。糖尿病改变了组蛋白的修饰，而MMP-9被证明是由其启动子区域的组蛋白修饰来调节的。我们的初步数据显示，糖尿病时，视网膜基质金属蛋白酶-9启动子上p65亚单位核因子-kB的募集增加，组蛋白3的二甲基化赖氨酸9(H3K9me2)的募集减少，赖氨酸特异的去甲基酶(LSD1)被激活，组蛋白去乙酰酶(Sirt1)被抑制，并且MnSOD的过表达保护糖尿病诱导的基质金属蛋白酶-9的增加，组蛋白修饰和组蛋白修饰酶的改变。此外，在啮齿动物血糖控制较差的一段时间后，重新建立正常的血糖控制并不能对基质金属蛋白酶-9的激活、组蛋白甲基化和Sirt1抑制提供任何好处。因此，我们的总体假设是，在糖尿病患者中，视网膜基质金属蛋白酶-9受组蛋白修饰的调节，在高血糖刺激终止后，该酶保持升高，视网膜病变继续进展。我们建议通过解决在三个特定目标下提出的互补性问题来系统地检验这一假设。第一个目标将研究糖尿病激活视网膜基质金属蛋白酶-9的分子机制，并将检验这一假设，即由于组蛋白修饰，p65在基质金属蛋白酶-9启动子上的募集增加，从而导致其激活。由于氧化应激被认为是一种 在糖尿病视网膜病变的发生发展中起着重要作用，在第二个目的中，我们将探讨氧化应激在调节基质金属蛋白酶-9中的作用。我们的工作假说预测，由于氧化应激增加，Sirt1被抑制，去乙酰化减少，这有助于p65与基质金属蛋白酶-9启动子的结合。第三个目标将确定基质金属蛋白酶-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，并将检验假设，由于持续的组蛋白修饰，基质金属蛋白酶-9在高血糖逆转后保持活跃，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。中心假说将在分离的细胞中使用活性质粒、siRNAs和药物抑制剂进行验证，体外发现将在使用基因操纵的小鼠的活体模型中得到验证，并在患有糖尿病视网膜病变的人类捐赠者的视网膜微血管中得到验证。这一结果有望证明组蛋白修饰在糖尿病视网膜基质金属蛋白酶-9激活中的作用，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，以防止视网膜病变在其发展的早期阶段，并在高血糖终止后的进展中。这将为患者提供额外的治疗 预防/延缓威胁视力的糖尿病并发症的手段。

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.