We investigated the neuroprotective effects of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, in a rat stroke model in which endothelial damage and subsequent thrombotic occlusion were selectively induced in perforating arteries. By examining the effects on the endothelial damage/dysfunction, we thought to explore the mechanism of Rho-kinase inhibitors. Hydroxyfasudil (10mg/kg, i.p., once daily for 3 days) significantly improved neurological functions and reduced the size of the infarct area produced by internal carotid artery injection of sodium laurate in a rat cerebral microthrombosis model. Treatment with fasudil or hydroxyfasudil concentration-dependently inhibited tumor necrosis factor alpha induced tissue factor expression on the surface of cultured human umbilical vein endothelial cells. They also inhibited thrombin-induced endothelial hyperpermeability. The present findings suggest that hydroxyfasudil is efficacious in preventing brain damage associated with cerebral ischemia, and is partially responsible for fasudil's cytoprotective potential. The results also suggest that the therapeutic benefits against ischemic injury of Rho-kinase inhibitors are attributed, at least in part, to activity upon endothelial damage/dysfunction.

Whether Rho-kinase activity is really associated with the pathogenesis of cerebral infarction remains unclear. To consider this question, we investigated correspondences between severity of neurological deficit, infarct size, amount of various marker proteins, and Rho-kinase activity in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere in rats. We prepared rats with various severities of neurological deficit (mild to severe) 3 days after injection of laurate, then measured infarct size and amount of various marker proteins, phosphorylation of substrates of Rho-kinase, myosin-binding subunit (MBS), myosin light chain (MLC), ezrin/radixin/moesin (ERM), and adducin using Western blot methods. First, infarct size increased corresponding to the severity of neurological deficit. Second, amounts of activating transcription factor 3, nestin, CD68, proliferating cell nuclear antigen, and heat shock protein 70 were increased, whereas neurofilament and myelin-associated glycoprotein were decreased corresponding to the severity of neurological deficit and infarct size. Finally, Rho-kinase activity (phospho-MBS/MBS, phospho-MLC/MLC, phospho-ERM/ERM, and phospho-adducin/adducin) was increased corresponding to the severity of neurological deficit and infarct size. Rho-kinase thus appears to play a crucial role in the pathogenesis of cerebral infarction.

Whether Rho-kinase activity is really associated with the pathogenesis of cerebral infarction remains unclear. To consider this question, we investigated correspondences between severity of neurological deficit, infarct size, amount of various marker proteins, and Rho-kinase activity in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere in rats. We prepared rats with various severities of neurological deficit (mild to severe) 3 days after injection of laurate, then measured infarct size and amount of various marker proteins, phosphorylation of substrates of Rho-kinase, myosin-binding subunit (MBS), myosin light chain (MLC), ezrin/radixin/moesin (ERM), and adducin using Western blot methods. First, infarct size increased corresponding to the severity of neurological deficit. Second, amounts of activating transcription factor 3, nestin, CD68, proliferating cell nuclear antigen, and heat shock protein 70 were increased, whereas neurofilament and myelin-associated glycoprotein were decreased corresponding to the severity of neurological deficit and infarct size. Finally, Rho-kinase activity (phospho-MBS/MBS, phospho-MLC/MLC, phospho-ERM/ERM, and phospho-adducin/adducin) was increased corresponding to the severity of neurological deficit and infarct size. Rho-kinase thus appears to play a crucial role in the pathogenesis of cerebral infarction.

The aim of this study was to investigate the influence of delayed Rho-kinase inhibition with fasudil on second ischemic injury in a rat cerebral thrombosis model. Cerebral ischemia was induced in rats by injecting 150 mug of sodium laurate into the left internal carotid artery on day 1. In the ischemic group, the regional cerebral blood flow (rCBF) was significantly decreased 6.5 h after the injection. Fasudil (3 mg/kg/30 min i.v. infusion) significantly increased rCBF. The viscosity of whole blood was significantly increased 48 h after the injection of sodium laurate. Fasudil (10 mg/kg, i.p.) significantly decreased blood viscosity. To clarify the therapeutic time window of fasudil, rats received their first i.p. administration of fasudil (10 mg/kg) 6 h after an injection of sodium laurate. Administration of fasudil twice daily was continued until day 4. Fasudil prevented the accumulation of neutrophils within the brain as seen from measurements taken on day 3, and improved neuronal functions and reduced the infarction area as seen on day 5. Fasudil and hydroxyfasudil, an active metabolite of fasudil, concentration-dependently inhibited phosphorylation of myosin binding subunit of myosin phosphatase in neutrophils. The present results indicate that inhibition of Rho-kinase activation with fasudil is effective for the treatment of ischemic brain damage with a wide therapeutic time window by improving hemodynamic function and preventing the inflammatory responses. These results suggest that fasudil will be a novel and efficacious approach for the treatment of acute ischemic stroke.

We investigated the neuroprotective effects of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, in a rat stroke model in which endothelial damage and subsequent thrombotic occlusion were selectively induced in perforating arteries. By examining the effects on the endothelial damage/dysfunction, we thought to explore the mechanism of Rho-kinase inhibitors. Hydroxyfasudil (10mg/kg, i.p., once daily for 3 days) significantly improved neurological functions and reduced the size of the infarct area produced by internal carotid artery injection of sodium laurate in a rat cerebral microthrombosis model. Treatment with fasudil or hydroxyfasudil concentration-dependently inhibited tumor necrosis factor alpha induced tissue factor expression on the surface of cultured human umbilical vein endothelial cells. They also inhibited thrombin-induced endothelial hyperpermeability. The present findings suggest that hydroxyfasudil is efficacious in preventing brain damage associated with cerebral ischemia, and is partially responsible for fasudil's cytoprotective potential. The results also suggest that the therapeutic benefits against ischemic injury of Rho-kinase inhibitors are attributed, at least in part, to activity upon endothelial damage/dysfunction.

BACKGROUND: Rho-kinase (ROCK) is a downstream effector of Rho GTPase that is known to regulate various pathological processes important to the development of ischemic stroke, such as thrombus formation, inflammation, and vasospasm. Inhibition of ROCK leads to decreased infarct size in animal models of ischemic stroke. This study tests the hypothesis that ROCK activity increases during the acute phase of ischemic stroke. METHODS: Serial blood samples were drawn from 10 patients with acute ischemic stroke presenting within 24 h of symptom onset and with NIHSS scores >/=4. Samples were taken at 24, 48, and 72 h. Leukocyte ROCK activity was determined by immunoblotting leukocyte lysates with antibodies to the phosphorylated form of myosin-binding subunit (P-MBS) of myosin light chain phosphatase (MLCP). MBS and P-MBS contents were normalized to alpha-tubulin, and ROCK activity was expressed as the ratio of P-MBS to MBS. ROCK activities in these 10 patients were compared to baseline ROCK activities in 10 control subjects without acute illness and matched for sex, age, and number of vascular risk factors using a two-tailed Student's t-test. RESULTS: The mean NIHSS score in patients with stroke was 15.4. ROCK activity was significantly increased at 24 and 48 h in patients after acute ischemic stroke when compared to control values, with peak elevations at 48 h after stroke onset. There was no apparent correlation between ROCK activity and stroke severity based on NIHSS. CONCLUSIONS: Leukocyte ROCK activity is increased in patients after acute ischemic stroke with maximal activity occurring about 48 h after stroke onset. These findings suggest that activation of ROCK may play a role in the pathogenesis of ischemic stroke in humans.

The neuroprotective potential and therapeutic time window for fasudil, a Rho-kinase inhibitor (RKI), were evaluated for delayed neuronal death in gerbils. A preliminary screening was done on fasudil, ozagrel, and edaravone using a single administration in a delayed neuronal death study. Intraperitoneal (i.p.) administration of edaravone, a free radical scavenger (3, 10 mg/kg) immediately after re-circulation did not reduce neuronal degeneration. We previously reported that ozagrel, a thromboxane A(2) synthetase inhibitor (30 mg/kg) also did not reduce neuronal degeneration, while fasudil (3, 30 mg/kg) significantly protected against the ischemia-induced neuronal loss. To clarify the therapeutic time window of fasudil, which showed a positive effect in a preliminary screening, animals received their first i.p. administration of fasudil (10 mg/kg) 24 or 48 h after ischemia. Administration of fasudil twice daily was continued until day 6. Fasudil significantly protected against the ischemia-induced delayed neuronal death when the treatment was started 24 h after ischemia. In gerbils, hydroxyfasudil, an active metabolite of fasudil, was found following an i.p. administration of fasudil (10 mg/kg), and the value of the area under the plasma level curve of hydroxyfasudil was 7 times higher than that of fasudil. Hydroxyfasudil may contribute to the potency of fasudil. The present findings indicate that the RKI fasudil reduces ischemic neuronal damage with a wide therapeutic time window in gerbil, and may be useful in the treatment of acute ischemic stroke in humans.

Background The pleiotropic effects of HMG-CoA reductase inhibitors (statins) are thought to be mediated through inhibition of small GTP-binding proteins; however, it remains to be examined whether clinical concentrations/doses of statins actually exert them. Methods and Results In vitro studies with cultured human umbilical venous endothelial cells found that statins (atorvastatin, pitavastatin and pravastatin at 10 mumol/L) had no inhibitory effects on RhoA/Rho-kinase or Ras, but atorvastatin and pitavastatin inhibited membrane Rac1 expression. In animal studies of angiotensin II (AngII)-in-fused rats, atorvastatin showed only mild inhibitory effects on AngII-induced cardiovascular hypertrophy, whereas fasudil, a selective Rho-kinase inhibitor, significantly suppressed it. Statins had no inhibitory effects on RhoA/Rho-kinase, but inhibited both membrane and GTP-bound Rac1 in the heart, whereas fasudil only inhibited Rho-kinase activity. Furthermore, the combination of atorvastatin and fasudil showed more effective inhibitory effects than fasudil alone. Finally, in studies of normal healthy volunteers, clinical doses of pravastatin or atorvastatin (20 mg/day for 1 week) significantly inhibited Rac1, but not RhoA/Rho-kinase activity, in circulating leukocytes. Conclusions The pleiotropic effects of statins, if any, at their clinical doses are mediated predominantly through inhibition of the Rac1 signaling pathway.

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 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.

Primary pulmonary hypertension continues to be a fatal disease. We have recently demonstrated that long-term inhibition of Rho-kinase, an effector of the small GTPase Rho, is effective for the treatment of pulmonary hypertension (PH) in rats and humans. Prostacyclin has been clinically used for the treatment of PH with moderate success. However, it remains to be examined whether Rho-kinase inhibition is involved in its beneficial effects on PH. In an ELISA assay, neither prostacyclin nor its oral analogue, beraprost sodium, inhibited Rho-kinase even at higher concentrations (10(-7) to 10(-5) M, 100 to 10,000 times higher than their clinical concentrations), whereas specific Rho-kinase inhibitors, fasudil and hydroxyfasudil, markedly (approximately 95%) inhibited the Rho-kinase activity at 10(-5) M (near their clinical concentrations). Beraprost sodium did not significantly suppress serotonin-induced vascular smooth muscle cell (VSMC) contractions or Rho-kinase activity of the rat aorta without endothelium, as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, whereas hydroxyfasudil markedly suppressed the VSMC contractions and Rho-kinase activity. These results indicate that prostacyclin lacks direct inhibitory effect on Rho-kinase and suggest that combination therapy with prostacyclin and a Rho-kinase inhibitor could exert further beneficial effects on PH.

This study was designed to investigate possible effects of the Rho-kinase inhibitor, fasudil, on the progression of renal failure in rats with unilateral ureteral obstruction. The renal failure markers monitored were the extent of renal interstitial fibrosis and that of macrophage infiltration. In kidneys with unilateral ureteral obstruction, interstitial fibrosis was observed, using Sirius-Red staining, on day 16 after unilateral ureteral obstruction. Macrophage infiltration was observed by immunohistochemistry, using the antibody, ED1. Interstitial fibrosis and macrophage infiltration were significantly attenuated in fasudil-treated animals. The migration of monocytes in vitro elicited by N-formyl-methionyl-leucyl-phenylalanine was potently inhibited by fasudil and its active metabolite, hydroxyfasudil. These results suggest that inhibition of Rho-kinase produces a reduction of macrophage infiltration and represents a new therapeutic strategy for renal fibrosis, a major factor in the progression to end-stage renal failure.

The aim of this study was to develop a new model of vasopressin-induced chronic myocardial damage based on sustained ST-segment depression in electrocardiogram (ECG) with progression of myocardial fibrosis in rats. Furthermore, using this model, we examined the prophylactic potential of fasudil, a Rho-kinase inhibitor, against myocardial damage induced by vasopressin. In 10-week old male Donryu rats, intravenous administration of arginine vasopressin (0.5 iu/kg) induced significant ST-segment depression. Two days and one week after the administration of vasopressin, ST-segment depression was -0.19 +/- 0.02 and -0.14 +/- 0.02 mV, respectively. Fasudil (10 and 30 mg/kg, p.o.) significantly attenuated the ST-segment depression induced by vasopressin. One week after the administration of vasopressin, the percent area of myocardial fibrosis in control animals (0.42 +/- 0.11%, p < 0.01) was significantly greater than that in normal animals (0.05 +/- 0.01%). Fasudil (10 and 30 mg/kg) significantly prevented the development of the fibrosis. We present a new model of chronic myocardial damage based on sustained ST-segment depression with progression of myocardial fibrosis in rats, and suggest that this model may be useful to investigate the treatment of chronic angina. Inhibition of Rho-kinase is efficacious in preventing the ECG change and development of fibrosis induced by vasopressin in this model.