Hypochlorite-Mediated Impairment of Endothelial Function

次氯酸盐介导的内皮功能损伤

• 批准号: 6851727
• 负责人: John F. Keaney
• 金额: $ 40.38万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-15 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6851727
• 关键词: antioxidants; atherosclerosis; chemical fingerprinting; clinical research; human subject; laboratory rabbit; myeloperoxidase; nitric oxide synthase; oxidative stress; oxidizing agents; phosphorylation; protein engineering; protein localization; protein protein interaction; superoxide dismutase; superoxides; tetrahydrobiopterin; tissue /cell culture; transfection; vascular endothelium

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.

描述（由申请人提供）：氧化应激有助于动脉粥样硬化，然而，证明自由基清除抗氧化剂改善这种疾病的应激发展已被证明是困难的，这可能是由于目前关注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，来自髓过氧化物酶（MPO）的双电子氧化剂，如HOCl，在动脉粥样硬化中是重要的，这些氧化剂对 “经典”脂溶性抗氧化剂，如维生素E。MPO与内皮结合，我们发现HOCl产生eNOS修饰和截短成100 kDa形式，导致NO生物活性受损。HOCl的这种作用被SOD抑制-与HOCl诱导内皮细胞中超氧化物产生的初步数据一致。因此，作为一个中心的假设，我们提出，在动脉粥样硬化HOCI诱导eNOS的修改和截断导致内皮超氧化物的生产和NO生物活性降低。因此，本项目的目标是确定HOCl在调节内皮功能中的作用，并确定所涉及的机制。为了实现这一目标，我们将首先确定HOCl对HAEC中MPO介导的氧化事件的相对贡献，并定义用于检查HOCl对培养的人主动脉内皮细胞（HAEC）中EDNO生物活性的影响的条件。 然后，我们将研究HOCl介导的超氧化物的产生和四氢生物蝶呤氧化和eNOS修饰在这个过程中的潜在作用。对于后者，我们将使用色谱和质谱结合肽指纹来表征这种修饰的蛋白质。有了这些信息，我们将开发相应于HOCl诱导的截短的突变型eNOS在COS-7和内皮细胞中表达，以研究其对EDNO生物活性的影响。将检查这种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，我们将测试HOC在体内的作用，首先确定eNOS修饰和受损的EDNO生物活性之间的关系，在WHHL模型的动脉粥样硬化。然后，我们将用突变型eNOS对对照兔血管进行转染，并试图将eNOS修饰与受损的EDNO生物活性联系起来。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HOCl介导的eNOS修饰和NO生物活性的影响。使用这种策略，我们应该能够确定MPO诱导的氧化对动脉粥样硬化血管素质的贡献。