: The long-term objective of this project is to further the understanding of contribution of homocysteine in vascular disease. Previous studies have indicated a decrease in the bioavailability of endothelial nitric oxide and an increase in the concentration of nitrotyrosine in the aortic wall associated with hyperhomocysteinemia. The plasma levels of homocysteine have shown to be inversely related to peroxisome proliferator activated receptor (PPAR), a nuclear receptor, which ameliorates vascular dysfunction. The central hypothesis of this proposal is that increased levels of homocysteine suppress the activity of PPAR by increasing the generation of nitrotyrosine and metalloproteinase activity, and decreasing the endothelial nitric oxide concentration. The central hypothesis will be addressed by the following four specific aims: 1) To determine whether the homocysteine binds to PPAR, the competitive binding of homocysteine and agonist (fibrate) to PPAR will be measured using homocysteine-cellulose affinity chromatography and aortic nuclear extracts. Bound PPAR will be eluted with fibrate and characterized by antibody to PPAR. 2) To determine whether the increase in PPAR expression decreases nitrotyrosine levels and increases endothelial nitric oxide concentration in a murine model of hyperhomocysteinemia, the concentrations of PPAR and nitrotyrosine in the aortas of hyperhomocysteinemic mice treated with and without fibrate will be measured by Western blot analysis. The levels of nitric oxide will be measured by estimating the total nitrate/nitrite concentration. 3) To determine whether the increase in PPAR decreases the levels of metalloproteinase and elastinolysis, the matrix metalloproteinase activity will be measured using specific substrate gel zymography, and the elastinolysis by identifying elastin fragments using anti-elastin antibody. 4) To determine whether an increase in PPAR expression reverses the homocysteine-mediated vascular dysfunction, the aortic contractile function will be measured. The proposed studies will elucidate the molecular, cellular and extracellular mechanism by which homocysteine promotes arterial lesions and should provide new insights to therapeutic ramifications for vessel wall disease.
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