These results indicate that hydroxyfasudil-sensitive Rho-kinase-mediated pathway appears to mediate the enhanced MLC phosphorylations (on Ser19 and Ser19/Thr18 residues) and plays a central role in the pathogenesis of coronary artery spasm.
Background-Tissue factor plays a pivotal role in thrombus formation in acute coronary syndromes. However, the regulatory mechanisms underlying tissue factor expression are poorly understood. Statins are effective in patients with acute coronary syndromes. Hence, the aim of this study was to clarify in human endothelial cells the signaling pathways of thrombin-induced tissue factor expression and potential inhibitory effects of statins. Methods and Results-In human aortic endothelial cells, simvastatin prevented tissue factor induction by thrombin (4 U/mL) in a concentration-dependent manner. The increase in tissue factor activity on the cell surface was also blocked by simvastatin. Simvastatin also prevented the upregulation of tissue factor expression and activity in human aortic smooth muscle cells. Mevalonate (100 mol/L) reversed the inhibitory effect of simvastatin on tissue factor expression. Thrombin induced rapid activation of Rho A and p38 MAP kinase. The Rho-kinase inhibitor Y-27632 and the p38 MAP kinase inhibitor SB203580 prevented tissue factor induction. Akt was dephosphorylated by thrombin; the phosphoinositol 3-kinase inhibitor wortmannin enhanced its dephosphorylation as well as thrombin-induced tissue factor expression. Simvastatin prevented thrombin-induced Rho A activation but not p38 MAP kinase activation. Akt dephosphorylation by thrombin was blocked by both simvastatin and Y-27632. Conclusions-Endothelial tissue factor induction by thrombin is regulated by Rho/Rho-kinase, Akt, and p38 MAP kinase.Simvastatin prevents its induction through inhibition of Rho/Rho-kinase and activation of Akt. These findings provide new insights into the action of statins in acute coronary syndromes.
Abstract-An imbalance of nitric oxide and endothelin plays an important role in cardiovascular disease. Thrombin exerts profound effects on endothelial function. The present study investigated the molecular mechanisms by which thrombin regulates endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE)-1 expression in human endothelial cells. Incubation of human umbilical vein endothelial cells with thrombin (0.01 to 4 U/mL) for 15 to 24 hours markedly downregulated eNOS and increased ECE-1 protein level in a dose-dependent manner. Thrombin also decreased eNOS mRNA and increased ECE-1 mRNA level. In mRNA stability assay, thrombin shortened the half-life of eNOS mRNA but not that of ECE-1 mRNA. Activation of protease-activated receptor 1 by the agonist (SFLLRN, 10 to 100 mol/L) had no effect on eNOS expression but increased ECE-1 level as thrombin. Thrombin activated Rho A and extracellular signal-regulated kinase (ERK)1 and ERK2. Inhibition of Rho A by C3 exoenzyme (20 g/mL) and ROCK by Y-27632 (10 mol/L) prevented the downregulation of eNOS expression by thrombin. Y-27632 also prevented the reduction in NOS activity induced by prolonged incubation with thrombin. On the other hand, inhibition of ERK1 and ERK2 activation by PD98059 (50 mol/L) prevented the upregulation of ECE-1 expression by thrombin as well as the increase in ECE activity and ET-1 accumulation in the medium. Treatment of rat aorta with thrombin overnight impaired endothelium-dependent relaxations but not endothelium-independent relaxations. Thus, thrombin suppresses eNOS and upregulates ECE-1 expression via Rho/ROCK and ERK pathway, respectively. These effects of thrombin may be important for endothelial dysfunction in cardiovascular disease, particularly during acute coronary episodes. (Circ Res. 2001;89:583-590.)Key Words: cell signaling Ⅲ mitogen-activated protein kinase Ⅲ endothelial dysfunction Ⅲ protease-activated receptor A therosclerosis is the leading cause of death and accounts for half of the morbidity and mortality in Western countries. 1 An imbalance of endothelium-derived relaxing and contracting factors is a hallmark of cardiovascular disease. 2 Indeed, in human atherosclerosis, the production of nitric oxide (NO) is decreased because of reduced endothelial NO synthase (eNOS) expression. 3 On the other hand, endothelin-1 (ET-1) production and endothelinconverting enzyme-1 (ECE-1) expression are increased in atherosclerosis and restenosis. 4,5 However, the risk factors or mechanisms that lead to this imbalance of endothelial function in human atherosclerosis have not been completely elucidated.Thrombin, the multifunctional enzyme generated in the context of vascular injury from the circulating zymogen prothrombin, is focused in atherosclerosis and its complications. 6 Thrombin plays an important role in platelet activation, modulation of vasomotion, and vascular smooth muscle proliferation or migration 7-12 and in turn contributes to vasospasm and vascular remodeling. 13,14 Acutely, thrombin stimulates eNOS ac...
These results indicate that enhanced MLC phosphorylations in the vascular smooth muscle play a central role in the pathogenesis of coronary spasm in our swine model.
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