Background and Purpose-Endothelium-derived nitric oxide (NO) plays a pivotal role in vascular protection. The Rho kinase (ROCK) inhibitor, hydroxyfasudil, prevents the downregulation of endothelial NO synthase (eNOS) under hypoxic conditions. However, it is unknown whether inhibition of ROCK can attenuate ischemia-induced endothelial dysfunction and tissue damage in vivo. Methods-Human vascular endothelial cells were treated with increasing concentrations of hydroxyfasudil (0.1 to 100 mol/L) and eNOS expression and activity were measured. To determine the physiological relevance of eNOS regulation by ROCK, we administered fasudil, which is metabolized to hydroxyfasudil in vivo, to mice for 2 days before subjecting them to middle cerebral artery occlusion. Cerebral blood flow, cerebral infarct size, and neurologic deficit were measured. Results-In a concentration-dependent manner, hydroxyfasudil increased eNOS mRNA and protein expression, resulting in a 1.9-and 1.6-fold increase, respectively, at 10 mol/L (PϽ0.05 for both). This correlated with a 1.5-and 2.3-fold increase in eNOS activity and NO production, respectively (PϽ0.05 for both). Fasudil increased cerebral blood flow to both ischemic and nonischemic brain areas, reduced cerebral infarct size by 33%, and improved neurologic deficit score by 37% (PϽ0.05). This correlated with inhibition of brain and vascular ROCK activity and increased eNOS expression and activity. Another ROCK inhibitor, Y-27632, also showed similar effects. The neuroprotective effects of fasudil were absent in eNOS-deficient mice. Conclusions-These findings indicate that the neuroprotective effect of ROCK inhibition is mediated by endotheliumderived NO and suggest that ROCK may be an important therapeutic target for ischemic stroke.

The Rho kinase (ROCK) isoforms, ROCK1 and ROCK2, were initially discovered as downstream targets of the small GTP-binding protein Rho. Because ROCKs mediate various important cellular functions such as cell shape, motility, secretion, proliferation, and gene expression, it is likely that this pathway will intersect with other signaling pathways known to contribute to cardiovascular disease. Indeed, ROCKs have already been implicated in the regulation of vascular tone, proliferation, inflammation, and oxidative stress. However, it is not entirely clear how ROCKs are regulated, what some of their downstream targets are, and whether ROCK1 and ROCK2 mediate different cellular functions. Clinically, inhibition of ROCK pathway is believed to contribute to some of the cardiovascular benefits of statin therapy that are independent of lipid lowering (ie, pleiotropic effects). To what extent ROCK activity is inhibited in patients on statin therapy is not known, but it may have important clinical implications. Indeed, several pharmaceutical companies are already actively engaged in the development of ROCK inhibitors as the next generation of therapeutic agents for cardiovascular disease because evidence from animal studies suggests the potential involvement of ROCK in hypertension and atherosclerosis.