Hypochlorite-Mediated Impairment of Endothelial Function

描述(由申请人提供)：氧化应激有助于动脉粥样硬化的发生，然而，证明清除自由基的抗氧化剂改善这种疾病的应激发展已被证明是困难的，可能是因为目前的重点是脂质过氧化作为动脉粥样硬化中氧化应激的主要载体。有证据表明，髓过氧化物酶(MPO)的两电子氧化剂(如HOCl)在动脉粥样硬化中起重要作用，这些氧化剂对经典的脂溶抗氧化剂如维生素E不敏感。MPO与内皮细胞结合，我们发现HOCl产生eNOS修饰和截断成100 kDa的形式，导致NO生物活性减弱。HOCl的这种作用被超氧化物歧化酶抑制--这与初步数据一致，即HOCl诱导内皮细胞产生超氧化物。因此，作为一个中心假设，我们认为在动脉粥样硬化中，hoci诱导eNOS修饰和截断，导致内皮超氧化物的产生，并降低NO的生物活性。因此，本项目的目标是确定高氯酸盐在调节内皮功能中的作用并确定其参与的机制(S)。为了实现这一目标，我们将首先确定HOCl对MPO介导的HAECs氧化事件的相对贡献，并确定HOCl对培养的人主动脉内皮细胞(HAECs)EDNO生物活性的影响条件。然后，我们将研究HOCl介导的超氧化物的产生以及四氢生物蝶呤氧化和eNOS修饰在这一过程中的潜在作用。对于后者，我们将使用层析和质谱仪结合多肽指纹图谱来表征这种修饰的蛋白质。有了这些信息，我们将开发对应于HOCl诱导的截短的突变型eNOS在COS-7和内皮细胞中表达，以检测其对EDNO生物活性的影响。我们将研究HOCl修饰的eNOS对eNOS细胞分布、蛋白质-蛋白质相互作用和磷酸化状态的潜在影响。然后，我们将通过首先在WHHL动脉粥样硬化模型中确定eNOS修饰和EDNO生物活性受损之间的关系来测试HOC在体内的作用。然后，我们将用突变的eNOS转染对照兔血管，并试图将eNOS的修饰与EDNO生物活性受损联系起来。为了测试MPO介导的氧化在动脉粥样硬化中的作用，我们将用结构无关的MPO抑制剂治疗WHHL兔，并研究其对HOCl介导的eNOS修饰和NO生物活性的影响。使用这一策略，我们应该能够确定MPO诱导的氧化对动脉粥样硬化的血管素质的贡献。

DESCRIPTION (provided by applicant): Oxidative stress contributes to the of atherosclerosis, however that stress development demonstrating radical-scavenging antioxidants ameliorate this disease has proven difficult, perhaps due to the current focus on lipid peroxidation as the major vehicle for oxidative stress in atherosclerosis. There is evidence that two-electron oxidants from myeloperoxidase (MPO), such as HOCl, are important in atherosclerosis and these oxidants would not be sensitive to "classical" lipid-soluble antioxidants such as vitamin E. MPO binds to the endothelium and we have found that HOCl produces eNOS modification and truncation into a 100 kDa form leading to impaired NO bioactivity. This effect of HOCl is inhibited by SOD -- consistent with preliminary data that HOCl induces superoxide production in endothelial cells. Thus, as a central hypothesis, we submit that in atherosclerosis HOCI induces eNOS modification and truncation leading to endothelial superoxide production and reduced NO bioactivity. The goal of this project, therefore, is to identify the role of HOCl in modulating endothelial function and identify the mechanism(s) involved. To achieve this goal, we will first establish the relative contribution of HOCl to MPO-mediated oxidative events in HAECs and define conditions for examining the effect of HOCl on EDNO bioactivity in cultured human aortic endothelial cells (HAECs). We will then examine HOCl-mediated superoxide production and the potential roles of tetrahydrobiopterin oxidation and eNOS modification in this process. With respect to the latter, we will characterize this modified protein using chromatography and mass spectroscopy coupled with peptide fingerprinting. With this information, we will develop mutant eNOS corresponding to HOCl-induced truncation for expression in both COS-7 and endothelial cells to examine its implications for EDNO bioactivity. This HOCl-modified eNOS will be examined for potential effects on eNOS cellular distribution, protein-protein interactions, and phosphorylation status. We will then test the role of HOC in vivo by first defining the relation between eNOS modification and impaired EDNO bioactivity in the WHHL model of atherosclerosis. We will then transfect control rabbit vessels with mutant eNOS and attempt to link eNOS modification with impaired EDNO bioactivity. To test the role of MPO-mediated oxidation in atherosclerosis, we will treat WHHL rabbits with structurally unrelated inhibitors of MPO and examine the implications for HOCl-mediated eNOS modification and NO bioactivity. Using this strategy, we should be able to define the contribution of MPO-induced oxidation to the vascular diathesis of atherosclerosis.