Losses in vascular relaxation are associated with increased plasma concentrations of native low-density lipoprotein (LDL) cholesterol. The mechanisms by which native LDL cholesterol impairs relaxation remain unclear. Previously, we observed that native LDL increased the cholesterol content of cultured endothelial cells nearly 2-fold. More importantly, the release of superoxide was increased more than 4-fold. As superoxide reacts with nitric oxide at near diffusion rates, increases in endothelial cell superoxide production may play a central role in impairing nitric oxide-mediated vasorelaxation. We hypothesize that prolonged exposure to atherogenic concentrations of native LDL conditions the endothelium to shift the balance of nitric oxide and superoxide, which, in turn impairs relaxation responses. Although ample indirect evidence exists demonstrating that hypercholesterolemia increases superoxide, definitive measurements for identifying and quantifying the reactive oxygen species generated by vascular endothelium have not been performed. Direct links between native LDL, cholesterol and the generation of superoxide and nitric oxide by vascular endothelial cells have not been established. This application examines the mechanisms by which cholesterol shifts the balance of nitric oxide and superoxide and the functional consequences of such shifts on endothelium- and non-endothelium-dependent and receptor-dependent and -independent relaxation of isolated arteries. State-of-the-art Electron Spin Resonance (ESR) and spin-trapping will be employed to identify and quantify radical species from cultured endothelial cells and coronary arterial rings. Arterial rings will be pre-incubated with native LDL or cholesterol-rich liposomes to alter nitric oxide and superoxide balance. Arterial rings will be hung in tissue baths containing spin-traps. Relaxation responses to substance P or A23187 will be recorded. ESR of spin-trapped radical species from the baths will provide the first direct measurements of reactive oxygen species generation during relaxation responses. The mechanisms by which LDL and cholesterol induce eNOS to generate superoxide will be examined with respect to tetrahydropterin metabolisms and post-translation modifications related to disturbances in the interactions between eNOS, caveolin and CaM. These studies will define the physiological relevance of cholesterol to impaired relaxation responses. The research combines the use of cell biology and vascular physiology to understand the biophysical and biochemical mechanisms by which cholesterol enrichment of the endothelium impairs vasorelaxation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Experimental Cardiovascular Sciences Study Section (ECS)
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Medical College of Wisconsin
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