Background-Prostaglandins generated by cyclooxygenase (COX) have been implicated in hyperglycemia-induced endothelial dysfunction. However, the role of individual COX isoenzymes as well as the molecular mechanisms linking oxidative stress and endothelial dysfunction in diabetes remains to be clarified. Methods and Results-Human aortic endothelial cells were exposed to normal (5.5 mmol/L) and high (22.2 mmol/L) glucose. Glucose selectively increased mRNA and protein expression of COX-2. Its upregulation was associated with an increase of thromboxane A 2 and a reduction of prostacyclin (PGI 2 ) release. Glucose-induced activation of PKC resulted in the formation of peroxynitrite and tyrosine nitration of PGI 2 synthase. NO release was reduced despite 2-fold increase of endothelial NO synthase expression. Phorbol ester caused an increase of COX-2 and endothelial NO synthase expression similar to that elicited by glucose. These effects were prevented by the PKC inhibitor calphostin C. N-acetylcysteine, vitamin C, and calphostin C prevented ROS formation, restored NO release, and reduced colocalization of nitrotyrosine and PGI 2 synthase. Expression of p22 phox , a subunit of NAD(P)H oxidase, was increased, and diphenyleneiodonium inhibited ROS formation. By contrast, indomethacin did not affect glucose-induced ROS generation. Conclusions-Thus, high glucose, via PKC signaling, induces oxidative stress and upregulation of COX-2, resulting in reduced NO availability and altered prostanoid profile.

Background-Enhanced expression of adhesion molecules by the endothelium may account for vascular damage in diabetics and nondiabetic patients who develop stress hyperglycemia during acute myocardial infarction. We analyzed the phosphorylation of protein kinase C␤ 2 (PKC␤ 2 ) at serine/threonine residues, which may contribute to the endothelial dysfunction during acute hyperglycemia. Furthermore, this study was designed to investigate whether selective blockade of this regulatory mechanism may prevent the development of endothelial hyperadhesiveness. Methods and Results-Incubation of the human aortic endothelial cells with high glucose (22.2 mmol/L) resulted in significant increase of vascular cell adhesion molecule (VCAM)-1 protein expression (172Ϯ15% versus control; PϽ0.01). Phorbol 12-myristate 13-acetate, a potent activator of PKC, mimicked the effect of high glucose on VCAM-1 expression. High glucose led to a rapid increase (181Ϯ22% versus control; PϽ0.01) of membrane-bound PKC␤, reflecting activation of this enzyme.The nonselective inhibitor of PKC␤ 1 and PKC␤ 2 isoforms LY379196, as well as CGP53353, a highly selective inhibitor of PKC␤ 2 , prevented in a dose-dependent manner upregulation of VCAM-1. Incubation with high glucose was associated with increased PKC␤ 2 phosphorylation at the Ser-660 residue, and both LY379196 and CGP53353 prevented this event. Exposure of the cells to high glucose also reduced the protein level of the inhibitory subunit of nuclear factor-B, IB␣, leading to its enhanced binding activity. Selective inhibition of PKC␤ abolished IB␣ degradation. Conclusions-Our findings demonstrate for the first time that phosphorylation of Ser-660 represents a selective regulatory mechanism for glucose-induced upregulation of VCAM-1. Therefore, PKC␤ 2 -selective inhibitors may be promising drugs for treatment of endothelial dysfunction during acute hyperglycemia and possibly in diabetes. Key Words: diabetes mellitus Ⅲ cell adhesion molecules Ⅲ endothelium Ⅲ inhibitors S tress hyperglycemia during myocardial infarction is associated with an increased risk of in-hospital mortality in patients with and without diabetes. 1,2 Furthermore, after stroke, the severity of hyperglycemia correlates strongly with mortality and morbidity in diabetic and nondiabetic patients. 3 One of the possible mechanisms that could explain the worse outcomes in acute hyperglycemia is glucose-induced upregulation of adhesion molecules in endothelial cells. 4,5 Expression of adhesion molecules leads to rolling and migration of activated white blood cells into the vessel wall. Under such conditions, endothelial cells become a target for cytokines secreted by adherent leukocytes that in turn result in inflammation and activation of the coagulation cascade. Subsequent induction of tissue factor expression is one of the crucial steps in thrombus formation. However, the precise molecular mechanism by which hyperglycemia regulates adhesion molecule transcription is still unclear. See p 7Glucose upregulates protein kinase C (PKC). ...