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...
Thrombin plays a pivotal role in the pathophysiology of acute coronary syndromes by mediating thrombus formation and endothelium-dependent vasomotor dysfunction. In human endothelial cells, prolonged incubation with thrombin down-regulates endothelial nitric oxide synthase (eNOS) expression via activation of Rho. Statins are effective in patients with acute coronary syndromes. These beneficial effects are attributed to their pleiotropic effects and also to an improved lipid profile. We hypothesized that statins may prevent the down-regulation of eNOS induced by thrombin in human endothelial cells. Human umbilical vein endothelial cells were used. Expression and activity of eNOS protein were evaluated by Western blotting and L-citrulline assay, respectively. Rho A membrane translocation was evaluated by Wesern blotting after fractionation. Stimulation of human umbilical vein endothelial cells with thrombin (4 U/mL, 24 h) significantly decreased eNOS expression. The addition of simvastatin significantly prevented thrombin-induced down-regulation of eNOS expression in a concentration-dependent manner (100 nmol/L to 10 micromol/L). Cerivastatin (10 micromol/L) also reversed the down-regulation of eNOS by thrombin. Both simvastatin and cerivastatin-blocked thrombin-induced decrease in NOS activity. Stimulation with thrombin (4 U/mL, 10 min) significantly increased the membrane translocation of Rho A. Simvastatin (10 micromol/L) and cerivastatin (10 micromol/L) significantly decreased thrombin-induced membrane translocation of Rho A. Therefore, statins blunt thrombin-induced down-regulation of eNOS expression in human endothelial cells. This finding provides a novel mechanism of the pleiotropic effects of statins, which may be beneficial for patients with acute coronary syndromes.
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