ha-1100 and Infarction--Middle-Cerebral-Artery

Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS-/-) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS-/- mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS-/- mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetylcholine; Animals; Brain Ischemia; Cerebrovascular Circulation; Down-Regulation; Endothelium, Vascular; Enzyme Inhibitors; Hypoxia, Brain; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Laser-Doppler Flowmetry; Mice; Mice, Inbred C57BL; Mice, Knockout; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Protein Serine-Threonine Kinases; Rats; rho-Associated Kinases; Stimulation, Chemical; Vasodilator Agents

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.. 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.. 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.. 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.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amides; Analysis of Variance; Animals; Antihypertensive Agents; Aorta; Blotting, Northern; Blotting, Western; Brain Ischemia; Cattle; Cell Line; Cells, Cultured; Cerebrovascular Circulation; Dose-Response Relationship, Drug; Down-Regulation; Endothelium, Vascular; Enzyme Inhibitors; Humans; Hypoxia; Infarction, Middle Cerebral Artery; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Protein Serine-Threonine Kinases; Pyridines; Regional Blood Flow; rho-Associated Kinases; RNA, Messenger; Stroke; Time Factors; Umbilical Veins; Up-Regulation