Oxidant stress is widely recognized to play a role in functional alterations that develop in cells exposed to hyperglycemia and hyperlipidemia in the diabetic milieu, and has been implicated as a mechanism that accelerates atherosclerosis in diabetes. The mechanisms by which diabetes increases oxidant stress, and those by which oxidant stress modifies endothelial function are poorly understood. Our preliminary studies establish new insights into how elevated glucose increases oxidant stress and the mechanisms by which its effects on cell function are mediated. Exposure of cultured human endothelial cells to elevated glucose for 7-10 days increases the production of both NO and superoxide anion (O2-), and consequently decreases the bioactivity of NO as indicated by decreased levels of cyclic GMP. Further evidence that NO is inactivated by reacting with O2- to form the reaction product, peroxynitrite (00N0-) is found in the increased levels of its reaction product with tyrosine, 3-o-nitrotyrosine, found in the cells. While the function of many proteins may be affected, we have found that prostacyclin synthase (PGIS) is particularly susceptible to tyrosine nitration; the levels of nitrated PGIS increase and its activity decreases in endothelial cells grown in elevated glucose. This may not only explain why diabetes decreases levels of PGI2, but also why increases have been noted in its precursor PGH2 that activates thromboxane A2 receptors (TPr). Our studies have shown that activation of TPr can modulate ICAM-1 and VCAM-l expression in human endothelial cells. The expression of adhesion molecules is enhanced by 02-, and indeed, exposure to elevated glucose enhances adhesion molecule expression. Thus, oxidant stress induced by elevated glucose may modulate the activity of PGIS and stimulation of TPr, thereby modulating adhesion molecule expression. Indeed, we have found that blockade of TPr inhibits atherosclerosis in the Apo E deficient mouse, a model in which diabetes enhances atherogenesis. There are three specific aims: 1) to determine the mechanism by which elevated glucose and fatty acids increases production of NO and 02- and causes tyrosine nitration and inactivation of PGI synthase, 2) to determine the role of TPr stimulation by eicosanoid products due to endothelial cell oxidant stress and PGI synthase inactivation in causing the increased leukocyte adhesion and apoptosis caused by high glucose and fatty acids, and 3) to determine if oxidant stress and PGI synthase inactivation contributes to the increased atherogenesis caused by diabetes in transgenic mouse models.