Induction of heme oxygenase-1 (HO-l) is an adaptive and cytoprotective response in cells and tissues exposed to oxidative stress of a diverse nature. Recent studies have demonstrated the importance of HO-l expression in atherogenesis. Oxidized LDL (oxLDL) is implicated in the pathogenesis of atherosclerosis, a major cause of morbidity and mortality, specifically in patients with predisposing factors such as diabetes mellitus, hypertension, hyperlipidemia, renal disease, and obesity. Our previous studies have demonstrated that exposure of human aortic endothelial cells to oxLDL results in the induction of HO-l. OxLDL is a complex structure consisting of several chemically distinct components. Our preliminary studies have identified linoleyl hydroperoxide (13-HPODE, LAox), an oxidized C: 18 containing fatty acid, as the major component of oxLDL responsible for HO- 1 induction. Most importantly, such induction occurs via increased HO-l gene transcription through molecular mechanisms different from known inducers of the gene. The studies in this proposal will evaluate the biological role of HO-l induction in response to oxLDL both in vitro and in vivo using HO-l knock out mice as well as delineate the regulatory elements that control oxLDL- mediated HO-1 gene expression in human aortic endothelial cells.
Aim I A will evaluate the effects of an atherogenic diet in HO-l deficient mice in vivo.
Aim I B will involve in vitro experiments in a model of oxLDL-induced injury in endothelial cells derived from the HO-l deficient mice.
Aim II A will evaluate specific regions of oxLDL-inducible altered chromatin structure of the human HO-l gene.
Aim II B will involve studies to characterize the oxLDL-responsive element using luciferase and human growth hormone reporter genes. The studies outlined in Aim IIIA will evaluate DNA-protein interactions at the single nucleotide resolution by in vivo footprinting and Aim IIIB will evaluate the functional significance of potential DNA-protein binding regions by site-directed mutagenesis. These studies have a potential application for the development of novel molecular approaches to manipulate expression of the human HO-l gene and thus exploit its cytoprotective effects in atherosclerotic cardiovascular diseases, wherein oxLDL is an important mediator.
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