The death of macrophages within an atherosclerotic plaque may play a fundamental role in conversion of the plaque to an unstable plaque, one that is vulnerable to rupture and hemorrhage. It is currently unknown precisely what kills macrophages within the plaque. One likely possibility is accumulation of oxidized lipids and free cholesterol derived from lipoproteins that have been trapped and retained by the extra-cellular matrix. Accumulation of oxidized LDL by macrophages in vitro is accompanied by depletion of glutathione, the major endogenous antioxidant for most cell types. Pharmacological stimulation of glutathione synthesis protects macrophages from the cytotoxic effects of oxidized LDL. Our preliminary data suggests that overexpression of the catalytic subunit of glutamate cysteine ligase, the rate-limiting enzyme for glutathione synthesis also protects macrophages from death due to oxidized LDL, oxidized lipid moieties and other prooxidants. Oxidized LDL is also a potent inducer of the expression of both the catalytic and regulatory subunits of glutamate cysteine ligase. Thus, we hypothesize that increased stable expression of glutamate cysteine ligase in macrophages will protect the cells from pro-oxidant induced death and increase the stability of atherosclerotic plaques. To test this hypothesis and to further investigate how oxidized lipid moieties contribute to the regulation of expression of the glutamate cysteine ligase genes in macrophages, we propose the following three specific aims. 1.To determine the role of oxidized lipid components of oxidized LDL in the regulation of macrophage expression of the glutamate cysteine ligase subunit genes. 2.To determine whether ad how increased expression of GCL-c by RAW cells inhibits pro-oxidant and free cholesterol induced death. 3.To determine the effects of bone marrow transplantation of cells over-expressing GCL-c (increased capacity to make glutathione) or deficient in GCL-m (decreased capacity to make glutathione) on macrophage death and atherosclerosis in older apo E-l- mice with established lesions. Strategies such as those included in the present proposal that are designed to prevent macrophage death have a high probability of successfully stabilizing atherosclerotic plaques and should help reduce plaque rupture, occlusive thrombosis, myocardial infarction and stroke.
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