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。我们和其他人之前的研究表明，HO-1基因表达的减少可以促进细胞凋亡和血管损伤，而这可以通过药物介导的HO-1上调来预防。我们的初步结果表明，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、减少EC-SOD和DNA降解来阻止内皮细胞凋亡。本提案的目的是阐明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血管保护。我们的假设是，HO-1基因的转移将通过增加血红素降解和随后增加CO和胆红素的合成来提供强大的血管保护。CO和胆红素水平升高会导致EC-SOD升高，eNOS升高，O2-降低，血管反应改善。此外，线粒体HO-1水平升高会增加线粒体运输载体，降低线粒体ROS，阻止细胞色素c的释放和caspase 9的激活。我们计划使用基因探针（逆转录病毒LXSN慢病毒和腺病毒）和遗传性自发糖尿病小鼠（NOD）来验证这一假设。我们将使用功能丧失HO-2（-/-）和功能获得，HO-1和HO-2基因转移到HO-2（-/-）来破译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)利用逆转录病毒载体进行基因干预，选择性地增加HO-1是否对糖尿病大鼠和NOD小鼠的血管有保护作用，这种作用是由于一氧化碳、胆红素还是两者兼有。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小鼠将通过EC-SOD、eNOS升高、O2-和iNOS降低来验证HO-1基因转移对血管的保护作用，以及CO、胆红素或两者均为血管保护必需的假设。(3)探讨HO-1基因表达（CO和胆红素）作用于促凋亡和抗凋亡蛋白外源性和内源性通路的机制。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)评价利用细胞特异性启动子（cell specific promoter, VE-CAD）靶向内皮细胞的慢病毒载体过表达HO-1基因是否足以抵消糖尿病诱导的血管损伤。这个建议在方法上是新颖的。它将首次允许深入分析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.