Rho-kinase mediates hyperglycemia-induced plasminogen activator inhibitor-1 expression in vascular endothelial cells

Background - Elevated levels of plasminogen activator inhibitor- 1 ( PAI- 1) are associated with myocardial infarction and stroke, especially in patients with diabetes. The induction of PAI- 1 expression by hyperglycemia involves oxidative stress and protein kinase C ( PKC). However, the mechanism by which hyperglycemia increases PAI- 1 expression is unknown. Methods and Results - Compared with normoglycemia, exposure of human endothelial cells to hyperglycemia, but not mannitol, increased Rho- kinase activity in a time- and concentration- dependent manner. This increase was inhibited by a PKC inhibitor, GF109203X, and antioxidants N- acetylcysteine ( NAC) and reduced form of glutathione ( GSH). This correlated with inhibition of hyperglycemia- induced PAI- 1 expression by GF109203X, NAC, and GSH. Hyperglycemia- increased PAI- 1 mRNA and protein levels were inhibited by Rho- kinase inhibitors hydroxyfasudil and Y27632 and by a dominant- negative mutant of Rho- kinase. The mechanism for this inhibition occurs at the level of gene transcription because Rho- kinase inhibitors repress hyperglycemia- stimulated PAI- 1 promoter activity without affecting mRNA stability. Hyperglycemia failed to stimulate Rho- kinase activity and PAI- 1 expression in heterozygous ROCK I - knockout murine endothelial cells. Conclusions - Hyperglycemia stimulates Rho- kinase activity via PKC- and oxidative stress - dependent pathways, leading to increased PAI- 1 gene transcription. These results suggest that inhibition of ROCK I may be a novel therapeutic target for preventing thromboembolic complications of diabetes and cardiovascular disease.