Ichiro Ikegaki

Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm

OBJECTIVE We recently demonstrated in our swine model of coronary artery spasm that enhanced myosin light chain (MLC) phosphorylations (both MLC mono- and diphosphorylations) play a central role in the pathogenesis of the spasm. However, the molecular mechanism for and the phosphorylation sites for the enhanced MLC phosphorylations were unknown. In the present study, we addressed these points using hydroxyfasudil, a novel inhibitor of protein kinases, which we found preferentially inhibits Rho-kinase. METHODS The specificity of the inhibitory effects of hydroxyfasudil on Rho-kinase, MLCK, MRCK beta and PKC were examined by kinase assay in vitro. The left porcine coronary artery was chronically treated with interleukin-1 beta (IL-1 beta, 2.5 micrograms). Two weeks after the operation, coronary artery vasomotion was examined both in vivo and in vitro. MLC phosphorylations were examined by Western blot analysis and the sites for the phosphorylations by anti-phosphorylated MLC antibodies that identified the monophosphorylation site as Ser19 and diphophorylation sites as Ser19/Thr18 of MLC. RESULTS Inhibitory effects of hydroxyfasudil was at least 100 times more potent for Rho-kinase as compared with other protein kinases tested. Intracoronary serotonin (10 micrograms/kg) caused coronary hyperconstriction at the IL-1 beta-treated site in vivo, which was dose-dependently inhibited by hydroxyfasudil (p < 0.01). The coronary segment taken from the spastic site also showed hypercontractions to serotonin in vitro, which were again dose-dependently inhibited by hydroxyfasudil (p < 0.01). Western blot analysis showed that MLC monophosphorylation was significantly greater in the spastic segment than in the control segment, while MLC diphosphorylation was noted only at the spastic segment (p < 0.01). The sites for the mono- and diphosphorylated MLC were identified as the monophosphorylated site Ser19 and diphosphorylated sites Ser19/Thr18 of MLC, respectively. Both types of MLC phosphorylations at the spastic segment were markedly inhibited by hydroxyfasudil (p < 0.01). CONCLUSION These results indicate that hydroxyfasudil-sensitive Rho-kinase-mediated pathway appears to mediate the enhanced MLC phosphorylations (on Ser19 and Ser19/Thr18 residues) and plays a central role in the pathogenesis of coronary artery spasm.

Endothelin and the production of cerebral vasospasm in dogs

We attempted to determine whether or not endothelin (ET) plays a physiological role in cerebral vasospasm. ET (10(-11) - 3 x 10(-8) M) induced a concentration-dependent contraction in isolated canine basilar arteries. Intracisternal injection of 10 to 1,000 pmol ET into dogs induced a dose-dependent decrease in the basilar artery diameter, as measured angiographically on the 1st day. On the 3rd day after the injection of ET (10 pmol), the diameter of the basilar artery had diminished to 76% of the value seen in the control. There were no effects on blood pressure or heart rate. Thus, ET seems to be a potent constrictor of cerebral arteries in vivo, and these effects are long-lasting.

Pharmacological profile of hydroxy fasudil as a selective rho kinase inhibitor on ischemic brain damage.

The neuroprotective property and the effects on hemodynamics of hydroxy fasudil, an active metabolite of an antispastic drug, fasudil, were examined. In rats, hydroxy fasudil was found following intravenous infusion or intraperitoneal administration of fasudil, and the maximum plasma concentration of hydroxy fasudil was approximately 25 or 40% of the parent drug, respectively. The i.v. administration of hydroxy fasudil produced significant increases in regional cerebral blood flow in dogs. Hydroxy fasudil relaxed the KCl, PGF2alpha or U-46619-induced contraction in canine basilar or middle cerebral arterial strips, concentration-dependently. The neuroprotective property of hydroxy fasudil was examined on delayed neuronal death in gerbils. Hydroxy fasudil (3 mg/kg) significantly protected against the ischemia-induced neuronal loss. To further clarify the effect on neurological impairments, hydroxy fasudil was tested in a rat model of microembolization stroke. Intravenous administration of hydroxy fasudil improved neurological functions, significantly reduced the size of the infarct area and prevented the accumulation of neutrophils. The present findings suggest that hydroxy fasudil has an efficacy to improve the hemodynamic function and to inhibit neutrophil-mediated damage, and contributes to the potency and long duration of the cytoprotective properties of fasudil on ischemic brain damage, and also suggest a critical role for rho kinase in the pathogenesis of cerebral ischemic injury, and the potential utility of rho kinase inhibitor as a therapeutic agent in stroke.

Enhanced Myosin Light Chain Phosphorylations as a Central Mechanism for Coronary Artery Spasm in a Swine Model With Interleukin-1β

BACKGROUND Although coronary artery spasm plays an important role in a wide variety of ischemic heart diseases, the intracellular mechanism for the spasm remains to be clarified. We examined the role of myosin light chain (MLC) phosphorylations, a key mechanism for contraction of vascular smooth muscle, in our swine model with interleukin-1beta (IL-1beta). METHODS AND RESULTS IL-1beta was applied chronically to the porcine coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, intracoronary serotonin repeatedly induced coronary hyperconstrictions at the IL-1beta-treated site both in vivo and in vitro, which were markedly inhibited by fasudil, an inhibitor of protein kinases, including protein kinase C and MLC kinase. Western blot analysis showed that during serotonin-induced contractions, MLC monophosphorylation was significantly increased and sustained in the spastic segment compared with the control segment, whereas MLC diphosphorylation was noted only in the spastic segment. A significant correlation was noted between the serotonin-induced contractions and MLC phosphorylations. Both types of MLC phosphorylation were markedly inhibited by fasudil. In addition, MLC diphosphorylation was never induced by a simple endothelium removal in the normal coronary artery, whereas enhanced MLC phosphorylations in the spastic segment were noted regardless of the presence or absence of the endothelium. CONCLUSIONS These results indicate that enhanced MLC phosphorylations in the vascular smooth muscle play a central role in the pathogenesis of coronary spasm in our swine model.

Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase

1 The in vitro and in vivo vasorelaxant effects of HA1077, 1‐(5‐isoquinolinesulphonyl)‐homopiperazine HCl, a novel vasodilator were examined. 2 The inhibitory effects of HA1077 on contractile responses to various agonists were examined on strips of rabbit aorta. The concentration‐response curves to 5‐hydroxytryptamine, prostaglandin F2α, histamine, angiotensin II, noradrenaline and dopamine were concentration‐dependently shifted to the right in the presence of HA1077 (0.3‐3.0 μm). 3 The in vivo vasodilator effects of HA1077 were examined in the constant‐pressure autoperfused coronary vascular bed of dogs. Intra‐coronary administration of HA1077 (3–30 μg per dog) dose‐dependently increased coronary blood flow (CBF), with no effect on mean blood pressure (MBP) or heart rate (HR). Intra‐coronary infusion of atropine, propranolol or diphenhydramine did not modify the in vivo coronary vasodilator response to HA1077. 4 To determine the flow profile for HA1077 in dogs, blood flow in four vascular beds was measured, by use of noncannulating electromagnetic flow probes. HA1077 (0.01‐0.3 mg kg−1, i.v.) dose‐dependently decreased MBP and increased vertebral blood flow (VBF), CBF, renal blood flow (RBF) and femoral blood flow (FBF). 5 A haemodynamic analysis showed that continuous i.v. infusion of HA1077 (0.01 and 0.033 mg kg−1 min−1) dose‐dependently decreased peripheral vascular resistance and increased cardiac output. There were no significant changes in right atrial pressure, dP/dt or ventricular minute work. 6 The effects of HA1077 on various enzymes considered to be related to the regulation of smooth muscle contraction were examined. HA1077 had little effect on cyclic nucleotide phosphodiesterases, yet it potently inhibited protein kinases such as cyclic nucleotide dependent protein kinases and Ca2+/calmodulin dependent myosin light chain kinase. 7 The present study demonstrates that HA1077 is a novel type of arterial vasodilator.

Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo

OBJECTIVE We recently demonstrated that Rho-kinase/ROK/ROCK is functionally upregulated at the arteriosclerotic coronary lesions and plays a key role for coronary vasospastic responses in our porcine model with interleukin (IL)-1beta. In the present study, we tested our hypothesis that Rho-kinase is involved in the pathogenesis of coronary arteriosclerosis per se in our porcine model. METHODS Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta. Two weeks after the procedure, coronary stenotic lesions with constrictive remodeling and vasospastic response to serotonin were noted at the IL-1beta-treated site, as previously reported. Then, animals were randomly divided into two groups; one group was treated with fasudil for 8 weeks followed by 1 or 4 weeks of washout period and another group served as a control. After oral absorption, fasudil is metabolized to hydroxyfasudil that is a specific inhibitor of Rho-kinase. RESULTS In the fasudil group, coronary stenosis and vasospastic response were progressively reduced in vivo, while the coronary hyperreactivity was abolished both in vivo and in vitro. Furthermore, Western blot analysis showed that in the fasudil group, the Rho-kinase activity (as evaluated by the extent of phosphorylation of myosin binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase) was significantly reduced, while histological examination demonstrated a marked regression of the coronary constrictive remodeling. CONCLUSIONS These results indicate that Rho-kinase is substantially involved in constrictive remodeling and vasospastic activity of the arteriosclerotic coronary artery, both of which could be reversed by long-term inhibition of the molecule in vivo. Thus, Rho-kinase may be regarded as a novel therapeutic target for arteriosclerotic vascular 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.

A New Model of Cerebral Microthrombosis in Rats and the Neuroprotective Effect of a Rho-Kinase Inhibitor

Background and Purpose The aim of this study was to develop a new model of stroke based on endothelial damage and thrombotic occlusion in a perforating artery, leading to small cerebral infarcts and neurological deficits in rats. Moreover, the neuroprotective efficacy of fasudil, a rho-kinase inhibitor, was investigated in this model. Methods Fifty-six male Sprague-Dawley rats were used in the present study. Rats were anesthetized with sodium pentobarbital, and 100 &mgr;g of sodium laurate was injected into the left internal carotid artery on days 1 and 3. The thrombus induction and consequent of ischemic brain damage were examined by histopathological analyses and neurological deficit scoring in a posture reflex test. To investigate the neuroprotective effects of fasudil, 1 or 10 mg/kg was administered intraperitoneally 5 minutes after the first injection of sodium laurate and once daily thereafter on the following 2 days. Results One hour after the injection of sodium laurate, microscopic examination of phosphotungstic acid hematoxylin–stained sections (n=5) revealed that microthrombi containing fibrin strands obstructed the perforating arteries in the ipsilateral hemisphere. Under a transmission electron microscope (n=6), endothelial cells appeared exfoliated and the vascular lumen was obstructed by a thrombus composed of degranulated platelets, fibrin, leukocytes, and erythrocytes. No evidence of endothelial cell damage or thrombus could be found in the ipsilateral side of the pial artery (middle cerebral artery). Twenty-four hours after the second injection of sodium laurate (day 4), 13 of 15 rats (86.6%) showed mild to severe neurological deficits. Multiple small cerebral infarcts were observed in the hippocampus, cortex, and thalamus. Treatment with fasudil (1 and 10 mg/kg, n=15 each) resulted in a significant improvement in neurological deficits. Fasudil also significantly reduced the area of cerebral infarction. Conclusions We present a new model of stroke in rats, in which the perforating arteries are selectively occluded by microthrombi. This model is useful to investigate the pathophysiology and treatment of small cerebral infarction, which is caused by perforating arterial occlusive diseases such as lacunar infarcts. Fasudil may be beneficial in the treatment of acute ischemic stroke.

Endothelin: a potential modulator of cerebral vasospasm

1-(5-Isoquinolinesulfonyl)-homopiperazine, HA1077, is a calcium antagonist with anti-vasospastic properties. This compound blocks intracellular actions of calcium in a variety of experiments. In the present study, we examined the effects of HA1077 on the vascular actions of endothelin, an endothelium-derived vasoactive peptide, in dogs in vitro and in vivo. Intracisternal injections of endothelin (0.01 nmol) produced a significant vasospasm, as measured by angiography, similar to that seen in the canine hemorrhage model. Infusion of HA1077 led to a significant dilatation of the spastic basilar artery in endothelin-treated dogs. The rank order of in vitro contractile activity in canine cerebral arteries was a stable thromboxane A2 analog greater than endothelin greater than 5-hydroxytryptamine greater than prostaglandin F2 alpha greater than histamine greater than noradrenaline. HA1077 effectively antagonized the endothelin-induced contraction of canine basilar arterial strips in both calcium-containing and calcium-free medium. The present results indicate that HA1077 is an effective antagonist for endothelin in vitro and in vivo.

Neuroprotective properties of a protein kinase inhibitor against ischaemia-induced neuronal damage in rats and gerbils.

1 . The neuroprotective properties of fasudil (HA1077), a novel protein kinase inhibitor, were evaluated in two animal models of cerebral ischaemia: transient bilateral carotid artery occlusion in Mongolian gerbils and cerebral microembolization in rats. 2 . The cytoprotective effect of fasudil on delayed neuronal death in gerbils was compared with the effects of nimodipine, a calcium channel antagonist and ozagrel, a thromboxane A2 synthetase inhibitor. The average of the neuronal cell density in the ischaemic control group was 17.8 ± 2.1 cells mm−1, whereas fasudil (30 mg kg−1) significantly diminished the loss of CA1 neurones with the average of the neuronal cell density of 101.0 ± 22.0 cells mm−1; nimodipine (10 mg kg−1) and ozagrel (30 mg kg−1) did not significantly protect against the ischaemia‐induced neuronal loss. 3 . In the rat model, the effects of fasudil on the histological and neurological consequences of cerebral microembolization produced via the injection of microspheres were examined. Twenty‐four hours after the injection of microspheres into the internal carotid artery, all animals in the control group showed typical symptoms of stroke. Neurological function was significantly improved in the fasudil‐treated animals. In the controls, the infarcted area in a cortical slice selected to include the hippocampal area was 0.25 ± 0.01 cm2 (mean ± s.e.mean) (43.9 ± 2.4% of cortical section of the half hemisphere); the difference was significant compared to the mean area of 32.7 ± 2.8 and 21.5 ± 4.8% observed in rats treated with fasudil (3, 10 mg kg−1), respectively. Fasudil (10 mg kg−1) significantly suppressed the increased water content in ischaemic brain tissues (saline‐treated rats, 82.4 ± 0.2% vs fasudil‐treated rats, 81.0 ± 0.4%). 4 . These results suggest that: (i) various protein kinases are involved in the pathogenesis of ischaemic injury; and (ii) the inhibition of protein kinases may be efficacious in preventing neuronal death, thus improving neurological function in the brain damage associated with ischaemic stroke.