Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection.
OBJECTIVE
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 micromol/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 micromol/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.
Rikitake
Yoshiyuki
Y
Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
Kim
Hyung-Hwan
HH
Huang
Zhihong
Z
Seto
Minoru
M
Yano
Kazuo
K
Asano
Toshio
T
Moskowitz
Michael A
MA
Liao
James K
JK
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D001011:Aorta / Q000166:cytology; D015152:Blotting, Northern; D015153:Blotting, Western; D002545:Brain Ischemia / Q000473:pathology; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D000860:Hypoxia; D020244:Infarction, Middle Cerebral Artery; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018696:Neuroprotective Agents / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012039:Regional Blood Flow; D020521:Stroke / Q000517:prevention & control*; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation; D054460:rho-Associated Kinases
D000577:Amides; D000959:Antihypertensive Agents; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018696:Neuroprotective Agents; D011725:Pyridines; D012333:RNA, Messenger; C431085:hydroxyfasudil; C108830:Y 27632; D009569:Nitric Oxide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; C049347:fasudil