Pleiotropic effects of the rho-kinase inhibitor fasudil after subarachnoid hemorrhage: a review of preclinical and clinical studies.

There is growing evidence that Rho-kinase contributes to cardiovascular disease, which has made Rho-kinase a target for the treatment of human diseases. To date, the only Rho-kinase inhibitor employed clinically in humans is fasudil, which has been used for the prevention of cerebral vasospasm and subsequent ischemic injury after surgery for subarachnoid hemorrhage (SAH). A number of pathological processes, in particular hemodynamic dysfunctions and inflammatory reactions, are thought to be related in the pathogenesis of delayed cerebral vasospasm and subsequent ischemic injury after SAH. This review focuses on fasudil's pleiotropic therapeutic effects: amelioration of hemodynamic dysfunction and inflammation, and discusses in detail the clinical studies on fasudil administered after the occurrence of SAH.

A missing link in the evolution of the cumulative recorder.

A recently recovered cumulative recorder provides a missing link in the evolution of the cumulative recorder from a modified kymograph to a reliably operating, scientifically and commercially successful instrument. The recorder, the only physical evidence of such an early precommercial cumulative recorder yet found, was sent to Keio University in Tokyo, Japan, in 1952 at the behest of B. F. Skinner at Harvard University. Last used in research in the late 1960s, the cumulative recorder remained locked in a storage room until 2007, when it was found again. A historical context for the recorder is followed by a description of the recorder and a comparison between it and the commercially successful Gerbrands Model C-1 recorder. Labeled the Keio recorder, it is a testament to Skinner's persistence in developing a reliable means of quantifying the behavior of living organisms in real time.

Evidence of a direct cellular protective effect of Rho-kinase inhibitors on endothelin-induced cardiac myocyte hypertrophy.

Using a cellular approach, the present study examined whether fasudil and active metabolite hydroxyfasudil, Rho-kinase inhibitors, exert a direct protective effect on endothelin-induced cardiac myocyte hypertrophy in vitro. Treatment with endothelin (10nM) caused significant hypertrophy of cultured neonatal rat cardiomyocytes by a 21.2% increase in cell surface area. Fasudil (1-10 µM) and hydroxyfasudil (0.3-10 µM) significantly prevented endothelin-induced cardiomyocyte hypertrophy. The present results suggest that inhibition of cardiac hypertrophy by fasudil is, at least in part, due to direct protection of cardiomyocytes from hypertrophy.

Antivasospastic Effects of Hydroxyfasudil, a Rho-Kinase Inhibitor, After Subarachnoid Hemorrhage.

We investigated the anti-vasospastic potential of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage (SAH) and also its effect on hemorheological abnormalities following cerebral ischemia. Chronic cerebral vasospasm was produced using a two-hemorrhage canine model. On day 7, angiographic vasospasm was observed in all animals, and intravenous administration of hydroxyfasudil (3 mg·kg-1·30 min-1) significantly reversed the vasospasm (predose diameter of the basilar artery, 57.9% ± 2.0% of the baseline before the injection of blood; postdose diameter, 64.5% ± 1.9%). The viscosity of whole blood was significantly increased 24 h after 1 h middle cerebral artery occlusion in rats. Hydroxyfasudil (3 and 10 mg/kg, i.p.) significantly decreased blood viscosity. The specificity of hydroxyfasudil was examined against a panel of 17 protein kinases using ELISA analysis. Hydroxyfasudil inhibited Rho-kinase α and ß at a concentration of 10 µM by 97.6% and 97.7%, respectively. No other protein kinase was inhibited with 10 µM hydroxyfasudil by over 40%. The present results indicate hydroxyfasudil is a selective inhibitor of Rho-kinase. The results also suggest that hydroxyfasudil contributes to the potency of fasudil to prevent cerebral vasospasm and hyperviscosity and suggest the potential utility of hydroxyfasudil as a therapeutic agent for patients with SAH.

Antivasospastic effects of hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage.

We investigated the anti-vasospastic potential of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage (SAH) and also its effect on hemorheological abnormalities following cerebral ischemia. Chronic cerebral vasospasm was produced using a two-hemorrhage canine model. On day 7, angiographic vasospasm was observed in all animals, and intravenous administration of hydroxyfasudil (3 mg·kg(-1)·30 min(-1)) significantly reversed the vasospasm (predose diameter of the basilar artery, 57.9% ± 2.0% of the baseline before the injection of blood; postdose diameter, 64.5% ± 1.9%). The viscosity of whole blood was significantly increased 24 h after 1 h middle cerebral artery occlusion in rats. Hydroxyfasudil (3 and 10 mg/kg, i.p.) significantly decreased blood viscosity. The specificity of hydroxyfasudil was examined against a panel of 17 protein kinases using ELISA analysis. Hydroxyfasudil inhibited Rho-kinase α and ß at a concentration of 10 µM by 97.6% and 97.7%, respectively. No other protein kinase was inhibited with 10 µM hydroxyfasudil by over 40%. The present results indicate hydroxyfasudil is a selective inhibitor of Rho-kinase. The results also suggest that hydroxyfasudil contributes to the potency of fasudil to prevent cerebral vasospasm and hyperviscosity and suggest the potential utility of hydroxyfasudil as a therapeutic agent for patients with SAH.

Fasudil protects cultured N1E-115 cells against lysophosphatidic acid-induced neurite retraction through inhibition of Rho-kinase.

The aim of this study was to investigate the possible effects of the Rho-kinase inhibitor, fasudil, on the lysophosphatidic acid (LPA)-induced neurite retraction in N1E-115 cells. In cultured N1E-115 cells, LPA produced a marked increase in the population of rounded cells. Fasudil or hydroxyfasudil, an active metabolite of fasudil, blocked cell rounding in a concentration-dependent manner at levels between 1 and 10 µM, with IC50 values of 1.7 or 1.6 µM, respectively. Fasudil or hydroxyfasudil concentration-dependently inhibited phosphorylation of the myosin binding subunit of myosin phosphatase in N1E-115 cells. These results indicate that Rho-kinase is essential for LPA-induced neurite retraction in N1E-115 cells and that inactivation of Rho-kinase by a Rho-kinase inhibitor, such as fasudil, eliminates cell rounding and promotes neurite outgrowth, thus improving neurological function in the brain damage.

Amelioration of endothelial damage/dysfunction is a possible mechanism for the neuroprotective effects of Rho-kinase inhibitors against ischemic brain damage.

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.

Increased leukocyte ROCK activity in patients after acute ischemic stroke.

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 >or=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.

Correspondence between neurological deficit, cerebral infarct size, and Rho-kinase activity in a rat cerebral thrombosis model.

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.

Demonstration of elevation and localization of Rho-kinase activity in the brain of a rat model of cerebral infarction.

Evidence that Rho-kinase is involved in cerebral infarction has accumulated. However, it is uncertain whether Rho-kinase is activated in the brain parenchyma in cerebral infarction. To answer this question, we measured Rho-kinase activity in the brain in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere. At 6 h after injection, increase of activating transcription factor 3 (ATF3) and c-Fos was found in the ipsilateral hemisphere, suggesting that neuronal damage occurs. At 0.5, 3, and 6 h after injection of laurate, Rho-kinase activity in extracts of the cerebral hemispheres was measured by an ELISA method. Rho-kinase activity in extracts of the ipsilateral hemisphere was significantly increased compared with that in extracts of the contralateral hemisphere at 3 and 6 h but not 0.5 h after injection of laurate. Next, localization of Rho-kinase activity was evaluated by immunohistochemical analysis in sections of cortex and hippocampus including infarct area 6 h after injection of laurate. Staining for phosphorylation of myosin-binding subunit (phospho-MBS) and myosin light chain (phospho-MLC), substrates of Rho-kinase, was elevated in neuron and blood vessel, respectively, in ipsilateral cerebral sections, compared with those in contralateral cerebral sections. These findings indicate that Rho-kinase is activated in neuronal and vascular cells in a rat cerebral infarction model, and suggest that Rho-kinase could be an important target in the treatment of cerebral infarction.

Historical note on cumulative recorders manufactured in Japan.

The history of cumulative recorders manufactured in Japan between the early 1970s and the present is described. The first such instrument was the Tosoku-Kogyo Company's Model T-45A, followed thereafter by its Model T-45B, built on the same chassis as the T-45A, but with different electrical and mechanical components. When Tosoku-Kogyo closed its recorder operations, the recorder continued to be manufactured and sold by Okubo-Sokkouki as its Model OS 180. The appearance of such commercially manufactured cumulative recorders in countries other than the United States further attests to the internationalization of behavior analysis during the mid- to late-20th century.

Wide therapeutic time window for Rho-kinase inhibition therapy in ischemic brain damage in a rat cerebral thrombosis model.

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.

Wide therapeutic time window for fasudil neuroprotection against ischemia-induced delayed neuronal death in gerbils.

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.

Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection.

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.

Periorbital pain associated with hemifacial spasms that responded to botulinum toxin treatment and microvascular decompression surgery: a case report.

Periorbital pain is unusual in patients with blepharospasms. We report a patient with hemifacial spasms who presented with severe ipsilateral periorbital aching pain. After treatment with botulinum toxin type A, the left hemifacial spasms and left periorbital pain improved temporarily. Microvascular decompression surgery was then performed for the facial spasms. The patient's left hemifacial spasms disappeared immediately after the operation, and his left periorbital pain was also totally resolved.

Prostacyclin does not inhibit rho-kinase: an implication for the treatment of pulmonary hypertension.

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.

[A patient with HTLV I-associated myelopathy (HAM) complaining of burning pain on the bilateral feet: a case report].

A 67-year-old man complained of a burning pain and weakness of bilateral feet after contusion of the left lumbar region. Skin as well as bone dystrophy and disturbance of bladder function were not seen, but low skin temperature was observed in the left lower leg. Glove anesthesia was seen on bilateral feet. Patellar tendon reflex was accentuated but Achilles tendon reflex was diminished, and bilateral Babinski sign was positive. Compression of the spinal cord or spinal root nerve was not noticed by MRI, myelography and myelo-CT (from cervical to lumbar level). We suspected the complex regional pain syndrome type I, and performed sympathetic blockade, but burning pain was not relieved. We looked for spinal tumor, myelitis, collagen disease, vitamin deficiency and malignancy but could not find out any disorder. However, the patient had neuropathic sign in electromyogram, and high anti-HTLV-I antibody titers in blood serum (8192x) and cerebrospinal fluid (256x). We diagnosed this case as HTLV I-associated myelopathy (HAM). He developed, so called, HTLV I-associated pneumonia at 74 years of age. We suggest that HAM may rarely accompany a burning pain and neuropathy (not myelopathy) as main symptoms. The present case suggests that a patient with HAM may develop HTLV I-associated pneumonia during its process; indicating a new concept of this very rare disease.

Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction.

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.

Effects of Rho-kinase inhibitor on vasopressin-induced chronic myocardial damage in rats.

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.