Inhibition of human endothelial cell nitric oxide synthesis by advanced glycation end-products but not glucose: relevance to diabetes.

Endothelial dysfunction, with decreased NO (nitric oxide) biosynthesis, may play a pathophysiological role in diabetic vasculopathy. The aim of the present study was to determine the relative contributions of glucose and AGE (advanced glycation end-product) accumulation in suppressing NOS-3 (the endothelial isoform of NO synthase). Cultured HUVECs (human umbilical vein endothelial cells) were incubated with different concentrations of glucose, unmodified albumin or AGE-modified albumin for different times. NOS activity was measured from the conversion of L-[(3)H]arginine into L-[(3)H]citrulline, and the expression, serine phosphorylation and O-glycosylation of NOS-3 were determined by Western blotting. High (25 mmol/l) glucose, for up to 12 days of incubation, had no effect on the activity or expression of NOS-3, nor on its degree of serine phosphorylation or O-glycosylation, compared with physiological (5 mmol/l) glucose. By contrast, AGE-modified albumin exerted a concentration- and time-dependent suppression of NOS-3 expression in HUVECs at a range of concentrations (0-200 mg/l) found in diabetic plasma; this was evident after 24 h, whereas inhibition of NOS activity was seen after only 3 h incubation with AGE-modified albumin, consistent with our previous observations of rapid suppression of NOS-3 serine phosphorylation and NOS-3 activity by AGE-modified albumin. In conclusion, AGE-modified albumin suppresses NOS-3 activity in HUVECs through two mechanisms: one rapid, involving suppression of its serine phosphorylation, and another slower, involving a decrease in its expression. We also conclude that, in the context of the chronic hyperglycaemia in diabetes, the effects of AGEs on endothelial NO biosynthesis are considerably more important than those of glucose.