Evidence for Rho-kinase activation in patients with pulmonary arterial hypertension.

BACKGROUND: Direct evidence for Rho-kinase activation in patients with pulmonary hypertension (PH) is still lacking. METHODS AND RESULTS: Rho-kinase activity in circulating neutrophils was examined by determining the ratio of phosphorylated/total forms of myosin-binding subunit, a substrate of Rho-kinase, in 40 consecutive PH patients and 40 healthy controls. Next, Rho-kinase expression and activity was examined in isolated human lung tissues (5 patients with idiopathic pulmonary arterial hypertension [IPAH], 5 controls) and vascular reactivity of isolated small human pulmonary arteries in vitro (4 IPAH, 4 controls). Rho-kinase activity in circulating neutrophils was significantly increased in the PH patients overall compared with controls (P<0.0001). Significant correlations were noted between Rho-kinase activity and the severity and duration of PAH (all P<0.05). Rho-kinase expression and activity in isolated lung tissues also were significantly increased in the IPAH patients compared with the controls (both P<0.0001). Endothelium-dependent relaxation was markedly impaired and serotonin-induced contraction (in the absence of the endothelium) markedly enhanced in the PAH patients compared with the controls, and the hypercontraction to serotonin was abolished by hydroxyfasudil, a specific Rho-kinase inhibitor. CONCLUSIONS: These results provide the first direct evidence for Rho-kinase activation in patients with PAH, suggesting the therapeutic importance of Rho-kinase in the disorder.

Thrombomodulin alfa prevents the decrease in platelet aggregation in rat models of disseminated intravascular coagulation.

INTRODUCTION: Disseminated intravascular coagulation (DIC), a deadly complication characterized by uncontrolled hypercoagulation, causes a decrease in the platelet count and impairs platelet aggregation. Thrombomodulin (TM) alfa, a recombinant human soluble TM, reduces hypercoagulation in DIC patients. However, the effects of TM alfa on impaired platelet aggregation remain to be determined. In this study, we aim to investigate the effects of TM alfa on platelet aggregation using lipopolysaccharide (LPS)-induced and tissue factor (TF)-induced DIC rat models. MATERIALS AND METHODS: Sprague-Dawley rats were administered TF or LPS intravenously, with or without TM alfa before the injection. Six hours after LPS injection or 1 h after TF infusion, blood samples were obtained, and platelet-rich plasma was prepared. Collagen or adenosine diphosphate-induced platelet aggregation was measured using an aggregometer. In the other experiments, platelets were transfused 1 h after the TF infusion. Five minutes after transfusion, collagen-induced platelet aggregation was also measured. RESULTS: The amplitude of platelet aggregation in platelet-rich plasma was decreased in LPS- and TF-treated rats. TM alfa inhibited the decrease in platelet aggregation in a dose-dependent manner. The washed platelet aggregation amplitude was not decreased in TF-treated rats. Suspension of normal platelets in plasma obtained from TF-treated rats reduced platelet aggregation. Platelet transfusion for TF-treated rats increased the platelet count but was unable to improve platelet aggregation. CONCLUSIONS: TM alfa attenuated impairment of platelet aggregation in LPS- and TF-induced DIC rat models. The changes in plasma composition played a role in the decrease of platelet aggregation in TF-treated rats.

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.

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.

Progress of the study of rho-kinase and future perspective of the inhibitor.

Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho. Accumulating evidence has demonstrated that the Rho/Rho-kinase pathway plays an important role in various cellular functions, not only in vascular smooth muscle cell (VSMC) contraction but also in VSMC proliferation, cell migration, and gene expression. Two isoforms of Rho-kinase encoded by two different genes have been identified: ROCK1 and ROCK2. These isoforms are ubiquitously expressed, but with preferential expression of ROCK2 in the brain and skeletal muscle. The expression of Rho-kinase itself is mediated by the protein kinase C/NF-kappaB pathway with an inhibitory and stimulatory modulation by estrogen and nicotine, respectively. At the cellular level, Rho-kinase mediates VSMC contraction, stimulates VSMC proliferation and migration, and enhances inflammatory cell motility. Rho-kinase also upregulates various molecules that accelerate inflammation/oxidative stress, thrombus formation, and fibrosis, while it downregulates endothelial nitric oxide synthase and inhibits insulin signaling. Rho-kinase activity regulates major morphogenetic events during embryonic development through cell migration, differentiation, and axis formation. In animal and clinical studies, Rho-kinase has been shown to be substantially involved in the pathogenesis of vasospasm, arteriosclerosis, hypertension, pulmonary hypertension, and ischemia/reperfusion injury. Fasudil, a selective Rho-kinase inhibitor developed in Japan, is effective for the treatment of a wide range of cardiovascular diseases, with reasonable safety. Thus Rho-kinase is an important therapeutic target in cardiovascular medicine. This review summarizes the recent progress in the study of Rho-kinase and addresses future perspectives of Rho-kinase inhibitors.

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.

Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin.

BACKGROUND: Thrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro. METHODS: CPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay. RESULTS: CPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor. CONCLUSIONS: TM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro.

Importance of Rac1 signaling pathway inhibition in the pleiotropic effects of HMG-CoA reductase inhibitors.

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 micromol/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)-infused 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.

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.

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 microg/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.

Recent progress in the treatment of pulmonary arterial hypertension: expectation for rho-kinase inhibitors.

Pulmonary arterial hypertension (PAH) is a disease with poor prognosis characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary artery hyperconstriction and remodeling. However, the precise mechanism of PAH still remains to be elucidated. Although anticoagulant agents, vasodilators (e.g., prostaglandins, sildenafil, and bosentan), and lung transplantation are currently used for the treatment of PAH, more effective treatment needs to be developed. Rho-kinase causes vascular smooth muscle hyperconstriction and vascular remodeling through inhibition of myosin phosphatase and activation of its downstream effectors. In a series of experimental and clinical studies, we have demonstrated that Rho-kinase-mediated pathway plays an important role in various cellular functions, not only in vascular smooth muscle hyperconstriction but also in actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expression, all of which may be involved in the pathogenesis of arteriosclerosis. We also have recently demonstrated that Rho-kinase is activated in animal models of PAH with different etiologies (monocrotaline and chronic hypoxia) associated with enhanced pulmonary vasoconstricting and proliferating responses, impaired endothelial vasodilator functions, and pulmonary remodeling. Indeed, we were able to demonstrate that intravenous fasudil, a selective Rho-kinase inhibitor, exerts acute pulmonary vasodilator effects in patients with severe PAH who were refractory to conventional therapies. Taken together, our findings indicate that Rho-kinase is a novel and important therapeutic target of PAH in humans and that Rho-kinase inhibitors are a promising new class of drugs for the fatal disorder.

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 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.