Endothelial dysfunction represents a common pathogenic framework that contributes in both types of diabetes mellitus to the development of vascular disease that affects micro- and macro- blood vessels. Recent evidence indicates that the endothelial dysfunction associated with diabetes is the local formation of oxidants and free radicals. However, the mechanisms by which diabetes increases oxidant stress, and those by which oxidant stress modifies endothelial function are poorly understood. Our preliminary results have established new insights into how hyperglycemia and/or hyperlipidemia increase oxidant stress. Exposure of cultured human aortic endothelial cells to elevated glucose for 3-10 days increases the production of superoxide anion (O2), which reacts with nitric oxide (NO) to generate a potent oxidant, ONOO. Increased levels of its reaction product with tyrosine, 3-nitrotyrosine, are found in the cells. Although the function of many proteins may be affected, we have found that prostacyclin synthase (PGIS) is particularly susceptible to tyrosine nitration in human aortic endothelial cells exposed to elevated glucose. The levels of nitrated PGIS increase and its activity decreases. This may not only explain why diabetes decreases levels of PGI2, but also why increases have been noted in the PGI2 precursor, PGH2, which acts upon thromboxane A2 receptor (termed TP-receptor, TPr). Our preliminary studies have shown that exposure of human aortic endothelial cell to elevated glucose enhances adhesion molecular expression, endothelial cell apoptosis, inhibits Akt and insulin signaling by mechanisms which depend on ONOO- and TPr activation. Our central hypothesis is that diabetes via hyperglycemia/hyperlipidemia increases the generation of O2 and then ONOO, resulting in PGIS nitration and TPr stimulation and insulin resistance.
The aims of the proposed studies are: 1). To determine the role(s) of ONOO-triggered PGIS nitration and TPr activation in enhancing endothelial cell adhesion molecule expression and apoptosis in the HAEC cells exposed to hyperglycemia/FFAs and to determine if PGIS is resistant to nitration caused by the streptozotocin-indued diabetes in the transgenic mice (hSOD +/+) or knockout mice (eNOS -/-, gp91phox-/-). 2). To establish the links between cellular oxidant stress, PGIS nitration and TPr activation, and impaired insulin signaling in cells exposed to hyperglycemia/FFAs. Based on the results, we will assess which of these alterations is likely to be pathogenetic for cell damage. These studies will provide novel information as to how the metabolic stress associated with diabetes cause damage to the endothelium and how endothelial cell attempts to protect itself against these stresses and whether ONOO or TPr are potential targets for therapy.
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