Published Online
on September 1, 2005

A more recent version of this article appeared on October 1, 2005

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Submitted on April 1, 2005
Revised on June 17, 2005
Accepted on July 5, 2005

Inhibition of Rho Kinase (ROCK) Leads to Increased Cerebral Blood Flow and Stroke Protection

Yoshiyuki Rikitake MD, PhD; Hyung-Hwan Kim PhD; Zhihong Huang PhD; Minoru Seto PhD; Kazuo Yano MS; Toshio Asano PhD; Michael A. Moskowitz MD; and James K. Liao MD^*

From the Vascular Medicine Research Unit, Cardiovascular Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass (Y.R., H.-H.K., J.K.L.); the Laboratory of Stroke and Neurovascular Regulation, Massachusetts General Hospital and Harvard Medical School, Boston, Mass (Z.H., M.A.M.); and the Institute for Life Science Research, Asahi Kasei Pharma Corporation, Fuji, Shizuoka, Japan (M.S., K.Y., T.A.).

^* To whom correspondence should be addressed. E-mail: jliao{at}rics.bwh.harvard.edu.

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 µmol/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 µmol/L (P<0.05 for both). This correlated with a 1.5- and 2.3-fold increase in eNOS activity and NO production, respectively (P<0.05 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% (P<0.05). 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 endothelium-derived NO and suggest that ROCK may be an important therapeutic target for ischemic stroke.

Key words: cerebral blood flow • nitric oxide • stroke

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