High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells

BACKGROUND: Hyperglycemia is a primary cause of premature vascular disease. Endothelial cell dysfunction characterized by diminished endothelium-dependent relaxations is likely to be involved. Little is known about the molecular mechanisms of hyperglycemia-induced endothelial dysfunction. METHODS AND RESULTS: This study was designed to determine the effect of hyperglycemia on the L-arginine/nitric oxide (NO) pathway. Expression of endothelial nitric oxide synthase (eNOS) mRNA and production of NO were studied in human aortic endothelial cells exposed to control levels (5.5 mmol/L) and high levels (22.2 mmol/L) of glucose for 5 days. We examined the effect of glucose on NO release by measuring changes in nitrite (NO2-) levels by Griess reaction. Superoxide anion (O2-) production was also examined by the ferrocytochrome c assay. NOS mRNA and protein expression, which were evaluated by reverse transcription-polymerase chain reaction and Western blotting, were approximately twofold greater in endothelial cells exposed to high glucose. Elevated glucose levels increased NO2- production by only 40% but increased the release of O2- by more than threefold. CONCLUSIONS: The present study demonstrates that prolonged exposure to high glucose increases eNOS gene expression, protein expression, and NO release. However, upregulation of eNOS and NO release is associated with a marked concomitant increase of O2- production. These results provide the molecular basis for understanding how chronic exposure to elevated glucose leads to an imbalance between NO and O2-. This may explain impaired endothelial function and be important for diabetic vascular disease.