The regulation of oxidant and antioxidant activity has special significance in the development of cardiovascular disease in general, and atherosclerosis in particular. Recent evidence suggests that low density lipoprotein (LDL) that is modified by oxidation accumulates in atherosclerotic lesions, and in vitro studies suggest that oxidized LDL may be responsible for the dysfunctional behavior of lesion cells. The mechanisms of LDL oxidation are not known, but the cells present in lesions can oxidize LDL in vitro. The role of ceruloplasmin in any of these processes has not been investigated, but numerous reports of a potent antioxidant activity that can block the oxidation of lipids motivated us to begin studies of its role in LDL oxidation and lesion formation. Ceruloplasmin is an abundant 132 kDa acute-phase copper-protein carrying 95% of the copper in plasma. In contrast to previous reports, our preliminary studies show that ceruloplasmin is an extremely potent oxidant; at physiological levels it increases the oxidation of LDL by at least 25-fold. We have found that ceruloplasmin function is extremely sensitive to structural modification since oxidant activity (but not a distinct oxidase activity) is completely suppressed by (1) the removal of a single, specific bound copper or by (2) a single proteolytic event that cleaves ceruloplasmin into 116 and 19 kDa fragments. The latter finding may explain earlier reports of antioxidant activity. In other preliminary studies, we have observed that ceruloplasmin may be involved in cell- mediated oxidation. Ceruloplasmin can substitute for free metal ions in stimulating LDL oxidation by endothelial cells and smooth muscle cells. Ceruloplasmin may also regulate macrophage oxidation of LDL; in preliminary studies, we have found that agents that stimulate LDL oxidation by activated U937 cells (a monocytic line) also stimulate ceruloplasmin gene expression and protein production. Furthermore, anti- ceruloplasmin antibodies suppress much of the oxidant activity of U937 cells. Finally, a role for ceruloplasmin in oxidative processes in lesions is supported by our recent observation that ceruloplasmin is abundant in atherosclerotic lesions, but not in adjacent non-lesioned areas, of human carotid endarterectomy specimens. These new findings have led us to propose that the induced synthesis and secretion of ceruloplasmin by activated monocytes contributes to the oxidation of LDL by these and other vascular cells, and thus plays a critical role in pathological accumulation of oxidized lipoproteins in the vessel wall. The hypothesis will be tested in this proposal by examining the structural features of ceruloplasmin required for oxidant activity, by investigating its interaction with LDL, by determining the role of ceruloplasmin in vascular cell-mediated oxidation of LDL, and finally, by studies on the distribution and activity of ceruloplasmin in atherosclerotic lesions.
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