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. ? ?
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