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(Circulation. 2004;110:3708-3714.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

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; 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.

Correspondence to Prof Dr Zhihong Yang, Vascular Biology, Department of Medicine, Division of Physiology, University of Fribourg, Rue du Musée 5, CH-1700, Fribourg, Switzerland. E-mail zhihong.yang{at}unifr.ch

Received June 9, 2004; accepted July 21, 2004.

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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