Rho-Kinase Inhibition Acutely Augments Blood Flow in Focal Cerebral Ischemia via Endothelial Mechanisms

Abstract

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 À/À ) 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 À/À 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 À/À 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.

Cerebral Blood Flow Regulation by Nitric Oxide: Recent Advances

“…Bolus infusion of L-3,5,3Ј-triiodothyronine to mice undergoing transient focal cerebral ischemia increased Akt activity in the brain, reduced cerebral infarct volume, and improved neurological deficit scores; these effects were attenuated or absent in eNOS-knockout and thyroid hormone receptor (TR ␣1 , ␤ )-knockout mice and were abolished in wild-type mice 82 treated with a triiodothyronine antagonist, indicating that the activation of Akt and eNOS seems to contribute to the vasodilatory and neuroprotective effects of thyroid hormone (Hiroi et al, 2006). Rho-kinase inhibitors reduced the area of ischemic cortex in wild-type mice but not in eNOS-knockout mice (Shin et al, 2007). The effect of Rhokinase inhibitors on cerebral blood flow in the ischemic cortex is probably endothelium-dependent, and Rho-kinase may negatively regulate eNOS activity in ischemic brain.…”

Fasudil and Ozagrel in Combination Show Neuroprotective Effects on Cerebral Infarction after Murine Middle Cerebral Artery Occlusion

“…The C max and AUC values of hydroxyfasudil were approximately 134 and 396% of the parent drug, respectively. Hydroxyfasudil and fasudil strongly inhibit Rho kinase with almost equally potency (Yano et al, 2008), and both can improve cerebral infarct in mice (Shin et al, 2007;Yamashita et al, 2007). Thus, hydroxyfasudil contributes to the potency of fasudil.…”

Rho Kinases in Health and Disease: From Basic Science to Translational Research

“…Ischemic stroke is a common complication of atherosclerosis, leading to brain damage associated with a high rate of death or severe disability. In animal models of acute cerebral ischemia, ROCK inhibitors increase cerebral blood flow and reduce the area of infarct, likely through stimulation of eNOS activity (Shin et al, 2007). Inhibition of ROCK also prevents ischemia/reperfusion-induced ROS-dependent blood-brain barrier damage (Kahles et al, 2007).…”