Minoru Seto

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.

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 &mgr;mol/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 &mgr;mol/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.

Ca2+-Dependent Activation of Rho and Rho Kinase in Membrane Depolarization–Induced and Receptor Stimulation–Induced Vascular Smooth Muscle Contraction

Abstract— Ca2+ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase–dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca2+-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca2+ or the addition of a dihydropyridine Ca2+ channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N19RhoA suppressed Ca2+-induced Thr695 phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca2+ removal and depletion of the intracellular Ca2+ store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A2 analogue–induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca2+-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.

Rho kinase (ROCK) inhibitors.

The Rho kinase (ROCK) isoforms, ROCK1 and ROCK2, were initially discovered as downstream targets of the small GTP-binding protein Rho. Because ROCKs mediate various important cellular functions such as cell shape, motility, secretion, proliferation, and gene expression, it is likely that this pathway will intersect with other signaling pathways known to contribute to cardiovascular disease. Indeed, ROCKs have already been implicated in the regulation of vascular tone, proliferation, inflammation, and oxidative stress. However, it is not entirely clear how ROCKs are regulated, what some of their downstream targets are, and whether ROCK1 and ROCK2 mediate different cellular functions. Clinically, inhibition of ROCK pathway is believed to contribute to some of the cardiovascular benefits of statin therapy that are independent of lipid lowering (ie, pleiotropic effects). To what extent ROCK activity is inhibited in patients on statin therapy is not known, but it may have important clinical implications. Indeed, several pharmaceutical companies are already actively engaged in the development of ROCK inhibitors as the next generation of therapeutic agents for cardiovascular disease because evidence from animal studies suggests the potential involvement of ROCK in hypertension and atherosclerosis.

Effects of fasudil in acute ischemic stroke: Results of a prospective placebo-controlled double-blind trial

BACKGROUND A multicenter, double-blind, placebo-controlled study was conducted to assess the efficacy and safety of fasudil, a Rho-kinase inhibitor (RKI), in the treatment of acute ischemic stroke. METHODS A total of 160 patients, who were able to receive drug treatment within 48 h of acute ischemic stroke onset were enrolled. Patients received either 60 mg fasudil or a placebo (saline) by intravenous injection over 60 min, twice daily for 14 days. The primary end points were neurological status at 2 weeks after the start of treatment, and clinical outcome at 1 month after the onset of symptoms. RESULTS Fasudil treatment resulted in significantly greater improvements in both neurological functions (p=0.0013), and clinical outcome (p=0.0015). There were no serious adverse events reported in the fasudil group. The average trough value (12 h values) of active metabolite hydroxyfasudil, another RKI, in healthy elderly volunteers receiving 60 mg of fasudil was 0.077 microM-a concentration well above that needed to inhibit Rho-kinase (0.025-0.05 microM). CONCLUSION Treatment with fasudil within 48 h of acute ischemic stroke onset significantly improved the patients clinical outcome. This study found fasudil to be a useful and safe drug for patients with acute ischemic stroke. Further evaluations, for example, 3-month functional outcomes in a larger clinical trial, may help to define the efficacy of fasudil in acute ischemic 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.

Essential role of rho kinase in the ca2+ Sensitization of Prostaglandin F2α‐Induced Contraction of Rabbit Aortae

Inhibition of dephosphorylation of the 20 kDa myosin light chain (MLC20) is an important mechanism for the Ca2+‐induced sensitization of vascular smooth muscle contraction. We investigated whether this mechanism operates in prostaglandin F2α (PGF2α)‐induced contraction of rabbit aortic smooth muscle and, if so, whether protein kinase C (PKC) or rho‐associated kinase (rho kinase) contribute to the inhibition of dephosphorylation. In normal medium, PGF2α (10 μm) increased the phosphorylation of MLC20 and developed tension. The rho‐kinase inhibitors fasudil and hydroxyfasudil inhibited these changes, despite having no effect on a phorbol‐ester‐induced MLC20 phosphorylation. After treatment with verapamil or chelation of external Ca2+ with EGTA, PGF2α increased the MLC20 phosphorylation and tension without an increase in [Ca2+]i, all of which were sensitive to fasudil and hydroxyfasudil. ML‐9, a MLC kinase inhibitor, quickly reversed the KCl‐induced MLC20 phosphorylation and contraction to the resting level. However, fractions of PGF2α‐induced contraction and MLC20 phosphorylation were resistant to ML‐9 but were sensitive to fasudil. Ro31‐8220 (10 μm), a PKC inhibitor, did not affect the phosphorylation of MLC20 and the tension caused by PGF2α, thus excluding the possibility of the involvement of PKC in the PGF2α‐induced MLC20 phosphorylation. PGF2α increased phosphorylation at Thr654 of the myosin binding subunit (MBS) of myosin phosphatase, which is a target of rho kinase, and fasudil decreased the phosphorylation. These data suggest that the PGF2α‐induced contraction is accompanied by the inhibition of MLC20 dephosphorylation through rho kinase‐induced MBS phosphorylation, leading to Ca2+ sensitization of contraction. An actin‐associated mechanism may also be involved in the PGF2α‐induced sensitization.

Dynamics of myosin light chain phosphorylation at Ser19 and Thr18/Ser19in smooth muscle cells in culture

Using the specific antibodies pLC1 and pLC2 for mono- and diphosphorylated 20-kDa myosin light chain (MLC20) at Ser19 and at both Thr18 and Ser19, respectively, we visualized the dynamics of the MLC20 phosphorylation in rabbit aortic smooth muscle cells (cell line SM-3) stimulated with PGF2alpha. In the resting state, the diphosphorylated form was located in the peripheral region of the cell, such as the leading edge or the adhesion plaque, and the monophosphorylated form was located not only in the peripheral region but also on a discontinuous fibrillary structure along the long axis of the cell. After stimulation with 30 microM PGF2alpha, although localization of the monophosphorylated form changed little, the content of the diphosphorylated form increased and the distribution spread along the fibrillary structure to an extent the same as or similar to that of the monophosphorylated form, which colocalized with actin filament bundles. The diphosphorylation of MLC20 was more sensitive to protein kinase inhibitors, HA-1077, HA-1100, staurosporine, wortmannin, and ML-9, than was the monophosphorylation. In light of these observations, we propose that MLC20 diphosphorylation and monophosphorylation are regulated by different mechanisms.

Ca2+–calmodulin‐dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells

Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor‐activated Ca2+ influx in vertebrate cells. We previously reported the involvement of intracellular Ca2+ in the receptor‐mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca2+, and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca2+ entry through TRPC5 channels, induced upon stimulation of the G‐protein‐coupled ATP receptor, was abolished by treatment with W‐13, an inhibitor of calmodulin. ML‐9 and wortmannin, inhibitors of Ca2+–calmodulin‐dependent myosin light chain kinase (MLCK), and the expression of a dominant‐negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML‐9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole‐cell patch‐clamp technique was inhibited by ML‐9, whereas TRPC5 channel activity observed in the cell‐excised, inside‐out patch was unaffected by ML‐9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML‐9 in HEK293 cells. These findings strongly suggest that intracellular Ca2+–calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.

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.