Shunsuke Tawara

Long-term inhibition of Rho-kinase ameliorates hypoxia-induced pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells, and migration of inflammatory cells for which no satisfactory treatment has yet been developed. It has been recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis and that long-term inhibition of Rho-kinase markedly ameliorates monocrotaline-induced PH in rats. However, it remains to be examined whether direct inhibition of Rho-kinase also ameliorates PH with a different etiology and whether endothelial nitric oxide synthase (eNOS) is involved in the beneficial effects of Rho-kinase inhibition. This study was designed to address those 2 important issues in a hypoxia-induced PH model using wild-type (WT) and eNOS-deficient (eNOS−/−) mice. Long-term blockade of Rho-kinase with fasudil (100 mg/kg/d) for 3 weeks markedly improved PH and right ventricular hypertrophy in WT mice with a lesser but significant inhibition noted in eNOS−/− mice. Fasudil upregulated eNOS with increased Akt phosphorylation in WT but not in eNOS−/− mice. These results suggest that long-term inhibition of Rho-kinase also ameliorates hypoxia-induced PH in mice, for which eNOS activation may partially be involved.

Acute vasodilator effect of fasudil, a Rho-kinase inhibitor, in monocrotaline-induced pulmonary hypertension in rats

Pulmonary arterial hypertension is a progressive and fatal disease for which Rho-kinase may be substantially involved. In this study, we examined the acute vasodilator effects of fasudil, a Rho-kinase inhibitor, in monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. Three weeks after a single subcutaneous injection of MCT (60 mg/kg), hemodynamic variables were measured under conscious and free-moving conditions before and after oral administration of fasudil. MCT caused a significant elevation of mean pulmonary arterial pressure (mPAP). Although a low dose of fasudil (3 mg/kg) had no effect on mPAP, a middle dose (10 mg/kg) caused a significant reduction in mPAP without change in mean systemic arterial pressure (mSAP), and a high dose (30 mg/kg) significantly reduced both mPAP and mSAP. Rho-kinase activity was significantly increased by MCT injection in pulmonary arteries but not in the aorta. Fasudil (10 mg/kg) inhibited only the Rho-kinase activity in pulmonary arteries without any effect in the aorta. Plasma concentration of hydroxyfasudil, a metabolite of fasudil, was within its clinical range in humans. These results demonstrate that fasudil exerts effective and selective vasodilatation of pulmonary arteries in rats with MCT-induced PH at a given dose, suggesting its usefulness for the treatment of the fatal disorder.

Long-term inhibition of Rho-kinase ameliorates diastolic heart failure in hypertensive rats.

Diastolic heart failure (DHF) is a major cardiovascular disorder with poor prognosis; however, its molecular mechanism still remains to be fully elucidated. We have previously demonstrated the important roles of Rho-kinase pathway in the molecular mechanisms of cardiovascular fibrosis/hypertrophy and oxidative stress, but not examined in the development of heart failure. Therefore, we examined in this study whether Rho-kinase pathway is also involved in the pathogenesis of DHF in Dahl salt-sensitive rats, an established animal model of DHF. They were maintained with or without fasudil, a Rho-kinase inhibitor (30 or 100 mg/kg/day, PO) for 10 weeks. Untreated DHF group exhibited overt heart failure associated with diastolic dysfunction but with preserved systolic function, characterized by increased myocardial stiffness, cardiomyocyte hypertrophy, and enhanced cardiac fibrosis and superoxide production. Fasudil treatment significantly ameliorated those DHF-related myocardial changes. Western blot analysis showed that cardiac Rho-kinase activity was significantly increased in the untreated DHF group and was dose-dependently inhibited by fasudil. Importantly, there was a significant correlation between the extent of myocardial stiffness and that of cardiac Rho-kinase activity. These results indicate that Rho-kinase pathway plays an important role in the pathogenesis of DHF and thus could be an important therapeutic target for the disorder.

Effects of combined therapy with a Rho-kinase inhibitor and prostacyclin on monocrotaline-induced pulmonary hypertension in rats.

Pulmonary hypertension (PH) is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells, and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have previously demonstrated that long-term inhibition of Rho-kinase, an effector of the small GTPase Rho, ameliorates monocrotaline-induced PH in rats and hypoxia-induced PH in mice. We also have reported that prostacyclin and its oral analogue, beraprost sodium (BPS), may lack direct inhibitory effect on Rho-kinase in vitro, suggesting that combination therapy with a Rho-kinase inhibitor and BPS is effective for the treatment of PH. In this study, we addressed this point in monocrotaline-induced PH model in rats. Male Sprague-Dawley rats were given a subcutaneous injection of monocrotaline (60 mg/kg). They were maintained with or without the treatment with a Rho-kinase inhibitor, fasudil (30 mg/kg/day), BPS (200 μg/kg/day), or a combination of both drugs for 3 weeks. The combination therapy, when compared with each monotherapy, showed significantly more improvement in PH, right ventricular hypertrophy, and pulmonary medial thickness without any adverse effects. Plasma concentrations of fasudil were not affected by BPS. These results suggest that combination therapy with a Rho-kinase inhibitor and prostacyclin exerts further beneficial effects on PH.

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.

Thrombomodulin alfa attenuates the procoagulant effect and cytotoxicity of extracellular histones through the promotion of protein C activation.

INTRODUCTION Extracellular histones are reported to increase thrombin generation in the plasma and induce endothelial cell death in vitro. These effects of histones were suggested to involve histone-induced inhibition of TM-dependent activated protein C (APC) generation. Therefore, we hypothesized that TM alfa, a recombinant human soluble TM, attenuates these effects of histones by promoting the generation of APC. In the present study, we investigated the effects of TM alfa on the histone-induced decrease in APC generation, an increase in thrombin generation, and endothelial cell death in vitro. METHODS APC generation was investigated using a chromogenic substrate based assay. Thrombin generation in plasma was studied by using a calibrated automated thrombogram method. Histone cleavage was detected by western blot analysis. Histone-induced endothelial cell death was evaluated by the trypan blue exclusion test. RESULTS Histones decreased APC generation and increased thrombin generation in the presence of endothelial cells. TM alfa increased APC generation and decreased thrombin generation in the presence of histones and endothelial cells. TM alfa with thrombin and protein C cleaved histone H3, and attenuated histone-induced endothelial cell death. Antithrombin, an endogenous thrombin inhibitor, and gabexate mesilate, a synthetic protease inhibitor, inhibited thrombin generation, decreased APC generation, and did not have any effect on histone H3 cleavage or histone-induced endothelial cell death. CONCLUSIONS TM alfa attenuated the histone-induced increase in thrombin generation and endothelial cell death by promoting APC generation in vitro.

Pharmacological Differentiation of Thrombomodulin Alfa and Activated Protein C on Coagulation and Fibrinolysis In Vitro

Although thrombomodulin alfa (TM alfa), recombinant human soluble thrombomodulin, exerts antithrombogenic effects through activated protein C (APC), clinical trials suggested that TM alfa has a lower bleeding risk than does recombinant human APC. To address the mechanism explaining this difference, effects of TM alfa and APC on thrombogenic, coagulation, and fibrinolytic processes were compared in vitro. TM alfa and APC inhibited generation of thrombogenic markers, thrombin, and prothrombin fragment F1+2 and prolonged coagulation parameters, activated clotting time (ACT), and activated partial thromboplastin time (APTT). Concentrations of TM alfa effective for thrombin and F1+2 generation inhibition were comparable to those of APC. However, effects of TM alfa on ACT and APTT were clearly weaker than those of APC. TM alfa significantly prolonged clot lysis time (CLT) and decreased LY30, a parameter of degree of fibrinolysis in thromboelastography, whereas APC significantly shortened CLT and increased LY30. These results suggested that while the antithrombogenic effects of TM alfa were similar to those of APC, its anticoagulant effects were lower. In addition, effects of TM alfa were antifibrinolytic, while those of APC were profibrinolytic.