Rho-kinase is a key regulator of endothelial and smooth muscle cell function and morphology, and is increasingly implicated in the pathophysiology of cardiovascular diseases such as hypertension, atherosclerosis, and vasospasm (Shimokawa 2002
). Rho-kinase inhibitors are neuroprotective in experimental cerebral ischemia (Rikitake et al, 2005
; Satoh et al, 2001
; Toshima et al, 2000
); however, it is not known whether this is due to a vasodilator effect on cerebral vessels leading to an acute augmentation of CBF in ischemic brain, or related to other mechanisms, such as a direct neuroprotective action, reduced blood viscosity (Hitomi et al, 2000
), or inhibition of neutrophil accumulation (Satoh et al, 2001
). In this study, we showed that two structurally unrelated Rho-kinase inhibitors, hydroxyfasudil (isoquinolinesulfonic acid derivative) and Y-27632 (pyridine derivative) (Uehata et al, 1997
), acutely improve CBF in focal cerebral ischemic core and penumbra, and do so in an eNOS-dependent fashion. In vitro
experiments on systemic and cerebral arteries showed that eNOS function is reversibly impaired under hypoxic conditions, and that Rho-kinase inhibition partially prevents this impairment, providing a mechanism by which Rho-kinase inhibitors may selectively increase CBF in acutely ischemic brain.
Several recent studies have suggested that hypoxia or ischemia may downregulate eNOS activity via Rho-kinase activation (Fagan et al, 2004
; Takemoto et al, 2002
), through both transcriptional and post-transcriptional mechanisms (Liao et al, 1995
). Consistent with this, we found that hypoxia abolished eNOS-dependent ACh-induced relaxations in isolated systemic and cerebral arteries. This was not due to endothelial injury, since relaxations were completely restored on reoxygenation. The inhibition started within 30 mins and was complete after 60 mins of hypoxia, implicating post-translational mechanisms such as altered eNOS phosphorylation. Impaired eNOS-dependent relaxation by severe hypoxia has previously been reported for coronary arteries in response to bradykinin (Vedernikov et al, 1991
). Consistent with the hypothesis that acute hypoxic downregulation of eNOS activity may occur because of Rho-kinase activation, hydroxyfasudil administration restored eNOS-dependent relaxations during severe hypoxia. The partial rather than complete restoration of eNOS-dependent relaxations may be because of incomplete Rho-kinase inhibition by a relatively low concentration of hydroxyfasudil (3 μ
mol/L) chosen to minimize the direct vasodilator effects of the drug. Overall, our data support the notion that Rho-kinase is acutely activated in hypoxic/ischemic vasculature, both in isolated vessels, in vitro
, and in cerebral ischemia, in situ
, and inhibits eNOS activity thereby worsening CBF.
In support of the hypothesis that hypoxia acutely upregulates Rho-kinase, cortical homogenates obtained from ischemic core 60 mins after dMCAO showed an almost three-fold increase in Rho-kinase activity, as determined by Thr853
-MBS phosphorylation. These data are consistent with previous reports showing Rho-kinase upregulation 24 h after filament occlusion of MCA (Rikitake et al, 2005
; Yagita et al, 2005
), and suggest that this upregulation in ischemic cortex takes place acutely within 60 mins. Rho-kinase is widely expressed in most cell types in brain (Noma et al, 2006
); therefore, measurement of Rho-kinase activity in cortical homogenates is not specific for vascular cells. However, Rho-kinase activity, as measured by adducin phosphorylation, was increased in penumbral microvessels after permanent MCAO (Yagita et al, 2005
). Taken together, these results suggest that Rho-kinase is acutely upregulated in ischemic endothelium and worsens the CBF deficit.
Neuroprotection in cerebral ischemia was previously reported for fasudil (Hitomi et al, 2000
; Satoh et al, 1999
), hydroxyfasudil (Satoh et al, 2001
), and Y-27632 (Rikitake et al, 2005
; Satoh et al, 1996
). For example, hydroxyfasudil (10 mg/kg) reduced infarct size and neurological deficits in rat microembolic ischemia when administered 5 mins after ischemia onset; however, intraischemic CBF reduction was not studied, and neuroprotection was in part attributed to the anti-inflammatory effects of hydroxyfasudil leading to a significant reduction in neutrophil infiltration (Satoh et al, 2001
). In a model of endothelial injury and microvascular thrombosis induced by intracarotid injection of sodium laurate, fasudil (10 mg/kg) reduced infarct size and neurological deficits when administered 5 mins after ischemia onset and then repeated daily for 2 days; a lower dose of 1 mg/kg was ineffective, and CBF changes, again, were not reported (Toshima et al, 2000
). Our laboratory has recently shown that Rho-kinase inhibitors fasudil and Y-27632 reduced infarct size in a transient MCA occlusion model in mice, when administered 10 mg/kg/day for 2 days before ischemia (Rikitake et al, 2005
); this protection was associated with increased resting as well as intraischemic CBF in the treatment group. Our data are consistent with previous studies, and extend them by showing that Rho-kinase inhibitors augment CBF in both ischemic core and penumbra, and do that acutely within 60 mins, as demonstrated by LSF with high spatiotemporal resolution.
Rho-kinase inhibitors may augment CBF via their effects on endothelium and/or smooth muscle cells. In vascular smooth muscle, Rho-kinase modulates the sensitivity of contractile apparatus to intracellular Ca2+
by increasing MLC phosphorylation either directly or via phosphorylation and inhibition of myosin binding subunit of MLC phosphatase (MLCP) (Amano et al, 2000
; Kimura et al, 1996
; Shimokawa 2002
); the degree of MLC phosphorylation determines the degree of vasoconstriction by augmenting actin–myosin interaction. In endothelial cells, Rho-kinase is a negative upstream regulator of eNOS via both transcriptional and post-transcriptional mechanisms, including changes in eNOS mRNA stability (Laufs and Liao 1998
), subcellular translocation of eNOS because of reorganization of actin cytoskeleton, and eNOS phosphorylation at serine 1179 via phosphatidylinositol 3-kinase/Akt pathway (Ming et al, 2002
). Therefore, Rho-kinase inhibition may cause vasodilation and acutely augment CBF in ischemic brain by at least two distinct mechanisms (i.e., direct inhibition of smooth muscle contraction and increased eNOS activity). We explored the relative contribution of these two mechanisms to the observed CBF improvement in ischemic core and penumbra by testing hydroxyfasudil in eNOS−/−
mice, and showed that hydroxyfasudil did not augment CBF in eNOS−/−
mice. These data indicate that eNOS plays an obligatory role in this response, and that the direct vascular smooth muscle relaxing effect of Rho-kinase inhibitors does not augment CBF in this model of focal ischemia. Furthermore, cerebral hemodynamic benefit of Rho-kinase inhibitors developed rapidly suggesting that nontranscriptional mechanisms are involved in acute upregulation of eNOS activity, such as increased eNOS S1179 phosphorylation (Fulton et al, 1999
), which occurs within less than an hour in response to high-dose corticosteroids or statins (Amin-Hanjani et al, 2001
; Endres et al, 1998
The endothelial and smooth muscle mechanisms of vasodilation by Rho-kinase inhibitors appear to be differentially active in systemic and cerebral circulation. This is because hydroxyfasudil did not improve ischemic CBF in eNOS−/− mice, but still caused hypotension similar to wild type in magnitude, albeit more gradual in onset. Hence, direct smooth muscle relaxant effect of hydroxyfasudil decreased systemic, but not cerebrovascular resistance in focal ischemia. In light of the potential detrimental effect of systemic vasodilation and hypotension on cerebral perfusion in acute stroke, new Rho-kinase inhibitors with higher selectivity towards cerebral over systemic vasculature, perhaps by targeting endothelium (i.e., eNOS) rather than smooth muscle (i.e., MLC/MLCP), may be more efficacious in stroke therapy.
Hydroxyfasudil, the active metabolite of fasudil, has longer plasma elimination half-life than fasudil (Satoh et al, 2001
), and is 50, 100, and 1000 times more selective towards Rho-kinase than protein kinases A and C, and MLCK, respectively (Rikitake et al, 2005
). It is freely cell-permeable, and peak plasma concentrations are attained rapidly after both intravenous and intraperitoneal administration of the drug (Satoh et al, 2001
), suggesting that pharmacokinetic properties of the drug are favorable for use in acute stroke therapy. Y-27632 is structurally unrelated to hydroxyfasudil, and also shows higher selectivity towards Rho-kinase than protein kinases A and C, as well as MLCK compared with fasudil (Uehata et al, 1997
). Therefore, acute cerebral hemodynamic improvement by hydroxyfasudil and Y-27632 in focal ischemia is likely due to inhibition of Rho-kinase rather than other protein kinases.
Despite a significant CBF augmentation in ischemic cortex, Rho-kinase inhibitors did not increase resting CBF in nonischemic brain, when measured acutely for 1 h after a single dose. Indeed, resting CBF in nonischemic brain decreased by 15% and 25% by hydroxyfasudil and Y-27632, respectively. This appeared to correspond to their hypotensive effect (), and probably accounted for the lower efficacy of Y-27632 in improving ischemic CBF deficit, which caused more severe and longer lasting hypotension compared with hydroxyfasudil. In contrast to our data, both fasudil and hydroxyfasudil have reportedly increased resting CBF within minutes by as much as 20% in canine cortex, although changes in BP were not provided in that study (Satoh et al, 2001
). Rho-kinase inhibitors are potent hypotensive drugs (Shimokawa 2002
; Takahara et al, 2003
; Uehata et al, 1997
), which may limit their use in high doses for the treatment of acute stroke. It is noteworthy, however, that the improvement in ischemic CBF by Rho-kinase inhibitors occurred despite a reduction in cerebral perfusion pressure because of hypotension, and without an effect on resting CBF, suggesting that Rho-kinase activity is selectively upregulated in ischemic vasculature, and worsens the CBF deficit.
In summary, our data suggest that the rapid nontranscriptional upregulation of eNOS activity by Rho-kinase inhibitors, shared by a number of other neuroprotective strategies including statins (Endres 2005
) and high-dose corticosteroids (Limbourg et al, 2002
), can acutely augment blood flow in cerebral ischemic core and penumbra. We speculate that Rho-kinase inhibitors may provide greater CBF benefit in acute stroke in the presence of pathologic activation of vascular Rho/Rho-kinase, such as in hyperlipidemia, diabetes, hypertension, and hyperhomocysteinemia (Rikitake and Liao 2005
; Seko et al, 2003
; Shah and Singh 2006
; Zhu et al, 2003
). Therefore, Rho-kinase inhibition may present a novel therapeutic opportunity during acute stroke.