The objective of the proposed studies is to further characterize the cytotoxicity of cholesterol oxidation products from the standpoint of their role in atherogenesis. Accordingly, the experiments put forth in this proposal will address the effects of cholesterol oxides (CO) as the angiotoxic components of oxidatively modified lipoproteins. The studies are focused on a mixture of cholesterol oxidation products representative of that found in oxidized lipoproteins isolated from human and primate plasma. We submit that these cholesterol oxidation products constitute the major cytotoxic components of plasma lipids and that their toxicity is manifested in the arterial wall, particularly under conditions of hypercholesterolemia where these products are also markedly elevated. The angiotoxicity of this mixture of CO will be investigated via the following experiments. 1) The pharmacokinetics of the CO mixture will be characterized following intravenous injection into New Zealand White rabbits. The composition and amounts injected will approximate levels found in hypercholesterolemic rabbits, however, the effects will be ascertained under normo- cholesterolemic conditions. The time course of assimilation into the major lipoproteins (LDL & VLDL) and uptake by the aorta after single and repeated intravenous injections will be determined. 2) Cytotoxicity of LDL/VLDL fractions recovered from rabbits injected with the CO mixture will be studied using cultured endothelial cells, smooth muscle cells, and macrophage/monocytes. Parameters of cell injury and angiotoxicity include: a) Induction of cellular loading of LDL/VLDL cholesterol and accumulation of cholesteryl esters; b) Inhibition HMG-CoA reductase activity and, c) Receptor-mediated rates of LDL uptake by cells, d) Extent to which cytotoxicity and/or DNA damage and perturbation of cholesterol/cholesteryl ester biosynthesis is calcium (Ca2+)-dependent. 3) We will determine if injection of CO enriched lipoproteins induces early atherosclerotic events in normocholesterolemic rabbits. The amounts and intervals of the injected CO mixture will be adjusted to attain level found in hypercholesterolemic rabbits. The following end points will be used to compare cell injury in vivo: a) Permeability changes to radiolabeled autologous LDL. b) Thymidine uptake to measure aortic tissue injury. c) Aortic cell death assessed by cytoplasmic accumulation of IgG. d) Distribution and content of Ca2+in aortic tissue. e) Cholesterol, CO and cholesteryl ester content in the aortic wall. In addition, histological analysis of aortic specimens will be used to evaluate atherosclerotic lesion development after long-term CO administration.
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