Thrombin Suppresses Endothelial Nitric Oxide Synthase and Upregulates Endothelin-Converting Enzyme-1 Expression by Distinct Pathways

Abstract

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-induced Rapid Geranylgeranylation of RhoA as an Essential Process for RhoA Activation in Endothelial Cells

“…This was inhibited by a statin, and GGPP reversed the suppressive effect of a statin on RhoA activation induced by lysophosphatidylcholine (11). Taken together with previous reports (4,7,17,20), our results suggest that, in ECs, receptor agonist-induced rapid activation of unprocessed GDP-RhoA occurs through stimulation of two sequential steps, geranylgeranylation of RhoA and GDP/GTP exchange in RhoA.…”

“…We and others (4,7,20) demonstrated that thrombin activated RhoA in ECs within 0.5-5 min and increased GEF activity within 1 min in HEK-293T cells (20,35). Holinstat et al (20) showed that thrombin phosphorylated p115RhoGEF in human umbilical vein ECs within 1 min.…”

Rho-Kinase Mediates Hypoxia-Induced Downregulation of Endothelial Nitric Oxide Synthase

“…20 However, in contrast to Y27632, which did not affect basal eNOS expression, 20 we found that treatment with hydroxyfasudil alone increased basal eNOS expression without apparently affecting basal Rho-kinase activity. These findings suggest that hydroxyfasudil may have additional Rho-kinase-independent effects on eNOS expression.…”

The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology

“…72 Furthermore, long-term treatment with thrombin was shown to downregulate the expression of eNOS in endothelial cells. 72,73 Alternatively, a dual receptor system for thrombin in the endothelial cells (PAR1 and PAR4) 18 may explain the thrombin-induced Ca 2ϩ -independent and dependent production of NO. The stimulation of PAR4 has been reported to induce NO production without a concomitant elevation of [Ca 2ϩ ] i in endothelial cells.…”