Oxidative Stress and Vascular HO in Diabetes

描述（由申请人提供）：大血管和微血管疾病是1型和2型糖尿病患者发病和死亡的主要原因。内皮功能障碍，如自由基释放增加所证明的，增加粘附分子和细胞凋亡。我们和其他人先前的工作已经表明，HO-1基因表达的降低增强了细胞凋亡和血管损伤，这可以通过药理学介导的HO-1上调来预防。我们的初步研究结果表明，HO-1的上调通过降低细胞血红素含量，增加CO和胆红素水平，降低EC-SOD和DNA降解来防止内皮细胞凋亡。该提案的目标是阐明HO-1预防糖尿病介导的内皮功能障碍的机制，并探索使用遗传和/或药理学方法来实现长期的血管保护。我们的假设是，HO-1基因转移将提供强大的血管保护，通过增加血红素降解，随后增加CO和胆红素的合成。CO和胆红素水平的增加将导致EC-SOD和eNOS的增加，O2-的减少和血管反应的改善。此外，线粒体HO-1水平的增加将增加线粒体转运载体，减少线粒体ROS并阻止细胞色素c的释放和半胱天冬酶9的活化。我们计划使用基因探针（逆转录病毒LXSN、慢病毒和腺病毒）和遗传自发性糖尿病小鼠（NOD）来验证这一假设。我们将利用HO-2（-/-）的功能丧失和功能获得，HO-1和HO-2基因转移到HO-2（-/-）来解读每个基因在血管保护中的作用。以下具体目标将检验这一假设。(1)To确定是否遗传干预，使用逆转录病毒载体，选择性增加HO-1提供糖尿病大鼠和NOD小鼠的血管保护，以及这种效果是否是由于CO，胆红素或两者兼而有之。我们将研究HO-1衍生的CO和胆红素对EC-SOD、eNOS和O2-水平的影响。(2)HO-2（-/-）和NOD小鼠将被用于验证HO-1基因转移可以提供血管保护以及CO、胆红素或两者通过EC-SOD、eNOS的增加、O2-和iNOS的减少而对于血管保护是必需的假设。(3)To确定HO-1基因表达（CO和胆红素）作用于促凋亡和抗凋亡蛋白的外在和内在途径的机制。这将检查CO和胆红素产生的HO-1在线粒体和细胞质中的作用。(4)目的：探讨利用细胞特异性启动子（VE-CAD）过表达HO-1基因的内皮细胞靶向慢病毒载体是否足以抵消糖尿病引起的血管损伤。这一建议在方法上是新颖的。它将首次深入分析HO-1在血管保护中的功能，并开发用于治疗I型糖尿病的创新基因靶向疗法。

DESCRIPTION (provided by applicant): Macro and microvascular diseases are the principal causes of morbidity and mortality in patients with type 1 and type 2 diabetes. Endothelial dysfunction, as evidenced by the increased release of free radicals, increases adhesion molecules and apoptosis. Previous work by us and others has shown that a decrease in HO-1 gene expression enhances apoptosis and vascular injury which can be prevented by pharmacological mediated upregulation of HO-1. Our preliminary results indicate that upregulation of HO-1 prevents endothelial apoptosis by decreasing cellular heme content, increasing CO and bilirubin levels and decreasing EC-SOD and DNA degradation. The goal of this proposal is to elucidate the mechanism by which HO-1 prevents diabetes-mediated endothelial dysfunction and to explore the use of genetic and/or pharmacological approaches to achieve long-term vascular protection. Our hypothesis is that HO-1 gene transfer will provide powerful vascular protection by increasing heme degradation and subsequently increasing CO and bilirubin synthesis. The increased levels of CO and bilirubin will result in an increase in EC-SOD, and eNOS, a decrease in O2- and improved vascular response. In addition, increase in mitochondrial HO-1 levels will increase mitochondrial transport carrier, decrease mitochondrial ROS and prevent release of cytochrome c and activation of caspase 9. We plan to test this hypothesis using genetic probes (retroviral LXSN lentiviral, and adenoviral) and in genetically spontaneously diabetic mice, (NOD) mice. We will use the loss-of-function HO-2 (-/-) and gain-of-function, HO-1 and HO-2 gene transfer to HO-2 (-/-) to decipher the role of each gene in vascular protection. The following specific aims will test the hypothesis. (1)To determine whether genetic intervention, using retroviral vectors, to selectively increases HO-1 provides vascular protection in diabetic rats and NOD mice, and whether this effect is due to CO, bilirubin or both. We will examine the effect HO-1 derived CO and bilirubin on EC-SOD, eNOS and O2- levels. (2) HO-2 (-/-) and NOD mice will be used to test the hypothesis that HO-1 gene transfer can provide vascular protection and that CO, bilirubin or both are obligatory for the vascular protection via increase in EC-SOD, eNOS, decrease in O2- and iNOS. (3)To determine the mechanism whereby HO-1 gene expression (CO and bilirubin) act on the extrinsic and intrinsic pathway of pro-apoptosis and anti-apoptotic proteins. This will examine the role of CO and bilirubin produced by HO-1 in both the mitochondria and cytoplasm. (4) To evaluate whether lentiviral vectors targeting endothelial cells using cell specific promoter (VE-CAD) to overexpress HO-1 gene is sufficient to offset diabetes-induced vascular injury. This proposal is novel in its approach. It will allow for the first time, an in-depth analysis of the function of HO-1 in vascular protection and for the development of innovative gene-targeting therapies for the treatment of type I diabetes.