Thrombin stimulates human endothelial arginase enzymatic activity via RhoA/ROCK pathway - Implications for atherosclerotic endothelial dysfunction

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 ( approximate to 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.