Published Online
on November 29, 2004

A more recent version of this article appeared on December 14, 2004

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Submitted on June 9, 2004
Accepted on July 21, 2004

Thrombin Stimulates Human Endothelial Arginase Enzymatic Activity via RhoA/ROCK Pathway. Implications for Atherosclerotic Endothelial Dysfunction

Xiu-Fen Ming MD, PhD, Christine Barandier PhD, Hema Viswambharan MSc, Brenda R. Kwak PhD, François Mach MD, Lucia Mazzolai MD, Daniel Hayoz MD, Jean Ruffieux BS, Sandro Rusconi PhD, Jean-Pierre Montani MD, and Zhihong Yang MD^*

From Vascular Biology, Department of Medicine, Division of Physiology (X.-F.M., C.B., H.V., J.R., J.-P.M., Z.Y.) and Biochemistry (S.R.), University of Fribourg; Division of Cardiology, University Hospital Geneva (B.R.K., F.M.); and Division of Hypertension and Vascular Medicine, CHUV, Lausanne (L.M., D.H.), Switzerland.

^* To whom correspondence should be addressed. E-mail: zhihong.yang{at}unifr.ch.

Background--Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate L-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis.

Methods and Results--In human endothelial cells isolated from umbilical veins, thrombin concentration- and time-dependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (P<0.001) at 1 U/mL for 24 hours. The effect of thrombin was prevented by C3 exoenzyme or the HMG-CoA reductase inhibitor fluvastatin, which inhibit RhoA, or by the ROCK inhibitors Y-27632 and HA-1077. Adenoviral expression of constitutively active RhoA or ROCK mutants enhanced arginase activity (3-fold, P<0.001), and the effect of active RhoA mutant was inhibited by the ROCK inhibitors. Neither thrombin nor the active RhoA/ROCK mutants affected arginase II protein level, the only isozyme detectable in the cells. Moreover, a significantly higher arginase II activity (1.5-fold, not the protein level) and RhoA protein level (4-fold) were observed in atherosclerotic aortas of apoE^-/- compared with wild-type mice. Interestingly, L-arginine (1 mmol/L), despite a significantly higher eNOS expression in aortas of apoE^-/- mice, evoked a more pronounced contraction, which was reverted to a greater vasodilation by the arginase inhibitor L-norvaline (20 mmol/L) compared with the wild-type animals (n=5, P<0.001).

Conclusions--Thrombin enhances arginase activity via RhoA/ROCK in human endothelial cells. Higher arginase enzymatic activity is involved in atherosclerotic endothelial dysfunction in apoE^-/- mice. Targeting vascular arginase may represent a novel therapeutic possibility for atherosclerosis.

Key words: atherosclerosis • cells • cardiovascular diseases • endothelium-derived factors • signal transduction

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