This proposal requests funds to support research that is directed to investigate the role of free radical-mediated processes in the etiology of vascular pathologies such as atherosclerosis. This competing renewal for years 05 to 10 is based on the following recent advances, which were made possible through support of the existing grant: 1) discovery that nitric oxide (NO) can inhibit free radical-mediated low-density lipoprotein (LDL) oxidation by acting as a lipid peroxyl radical scavenger and that peroxynitrite, the reaction product of superoxide and NO, causes the oxidative modification of LDL; 2) development of a new methodology for ultra-sensitive and specific determination of different classes of lipid hydroperoxides (LOOHs) in LDL and cell membranes; 3) ability to monitor LOOH transfer from membranes to cells and to track their metabolism in situ in cells; and 4) demonstration that selenoperoxidases protect against LDL-LOOH mediated cytotoxicity. We hypothesize that 1) the oxidizability of LDL is mainly dictated by endogenous LOOH acquired by LDL via transfer from LOOH-enriched cells or membranes, and peroxidation of LDL and cell membranes can be inhibited by NO and that the balance between NO and O2 may modulate the extent of LDL oxidation. These hypotheses will be tested using macrophages, endothelial cells, and lipoproteins. In this proposal, we will: l) investigate the ability of lipoproteins to incorporate LOOH from membranes, cells, or other lipoproteins; 2) investigate the uptake and detoxification of lipoprotein-derived LOOHs by cells; 3) investigate the effect of NO on in vitro oxidation of LDL; and 4) investigate the effect of NO on the kinetics of LDL oxidation by macrophages in the presence of NO donors and in cytokine-stimulated macrophages. Techniques to be used include high performance liquid chromatography with electrochemical detection (HPLC-EC), mass spectrometry, and electron spin resonance (ESR). The proposed research is significant because it would provide new mechanistic information concerning free radical mediated pathology of vascular injury and suggest novel therapeutic approaches for treatment of diseases such as atherosclerosis.
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