Tohru Yamawaki

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.

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.

Intramural delivery of a specific tyrosine kinase inhibitor with biodegradable stent suppresses the restenotic changes of the coronary artery in pigs in vivo

OBJECTIVES This study was designed to examine whether or not intramural delivery of ST638 (a specific tyrosine kinase inhibitor) with biodegradable stent can suppress the restenotic changes of the coronary artery in vivo. BACKGROUND Clinical and animal studies demonstrated that restenosis after coronary intervention results from a combined effect of neointimal formation and geometric remodeling (decrease in total cross-sectional area). Thus, the most effective strategy to prevent the restenosis appears to inhibit both the neointimal formation and geometric remodeling by antiproliferative agent and stent, respectively. We have previously shown that ST638 markedly suppresses the restenotic changes of the porcine coronary artery when applied from the adventitial site. METHODS A poly-L-lactic acid biodegradable stent was coated with either ST638 (0.8 mg) or equimolar of its inactive metabolite, ST494. A pair of these stents were implanted alternatively in the left anterior descending or circumflex coronary artery in pigs (n=6). Three weeks after the procedure, coronary stenosis was assessed by angiography followed by histological examination. RESULTS Coronary stenosis was significantly less at the ST638 stent site than at the ST494 stent site (47+/-5% vs. 25+/-4%, p < 0.01). Histological examination also showed that the extent of neointimal formation and that of geometric remodeling were significantly less at the ST638 stent site than at the ST494 stent site (p < 0.05). CONCLUSIONS These results indicate that intramural delivery of a specific tyrosine kinase inhibitor with biodegradable stent overcomes the proliferative stimuli caused by balloon injury, the stent itself, and the drug coating on the stent, resulting in the suppression of the restenotic changes of the coronary artery in vivo. This strategy might also be useful in the clinical setting in humans.

Sarpogrelate, a selective 5-HT2A serotonergic receptor antagonist, inhibits serotonin-induced coronary artery spasm in a porcine model.

Serotonin is one of the most important vasoactive substances and has been implicated in the pathogenesis of coronary artery spasm and of acute coronary syndrome. We have recently demonstrated that local and long-term treatment with interleukin-1beta(IL-1beta) causes coronary arteriosclerotic changes and hyperconstrictive responses to serotonin in pigs in vivo. However, it remains to be examined which serotonergic (5-HT) receptor subtype mediates coronary spasm and whether alterations in serotonergic receptors are involved in the abnormality. In this study, we examined the inhibitory effect of sarpogrelate, a selective 5-HT2A serotonergic receptor antagonist, on the serotonin-induced coronary spasm as well as the possible alterations of serotonergic receptors in our porcine model. A segment of the porcine coronary artery was carefully dissected and aseptically wrapped with cotton mesh absorbing IL-1beta-bound microbeads from the adventitia. Two weeks after the procedure, angiographic study was performed, followed by binding assay for 5-HT1B and 5-HT2A serotonergic receptors and reverse transcription-polymerase chain reaction (RT-PCR) analysis for mRNA of those receptors. Angiographic study showed coronary vasospastic responses to serotonin at the IL-1beta-treated site. Sarpogrelate dose-dependently inhibited the serotonin-induced coronary spasm, but it did not affect the prostaglandin F2alpha-induced vasoconstriction. Radiolabeled receptor-binding assay showed that receptor affinity or receptor number of the 5-HT1B, or 5-HT2A receptors did not differ significantly between the spastic and the control sites. Furthermore, RT-PCR analysis showed that the expression of neither 5-HT2A nor 5-HT1B receptor mRNA was significantly altered at the spastic site. These results indicate that serotonin-induced coronary spasm is mediated primarily by 5-HT2A receptor in our porcine model, although the 5-HT2A receptor was not up-regulated, suggesting that alteration in the signal-transduction pathway for vascular smooth muscle contraction beyond the 5-HT2A receptor plays a primary role in the pathogenesis of coronary spasm in our porcine model.

Simultaneous assessment of wall motion and coronary flow velocity in the left anterior descending coronary artery during dipyridamole stress echocardiography

OBJECTIVES This study was designed to assess the feasibility and clinical meaning of simultaneous assessment of wall motion and coronary flow velocity (CFV) reserve in the left anterior descending coronary artery (LAD) by transthoracic approach for detecting LAD stenosis during dipyridamole stress echocardiography. BACKGROUND Coronary flow reserve plays an important role, which can be evaluated by transthoracic Doppler echocardiography during vasodilator stress. METHODS Dipyridamole stress test was performed in 110 patients with known or suspected coronary artery disease. CFV in the distal LAD was obtained at baseline and after dipyridamole infusion, and wall motion was also assessed up to the administration of atropine, if required. All patients underwent quantitative coronary angiography within 2 days of the stress test, and significant LAD stenosis was defined as > 50% stenosis. RESULTS The success rate of both measurements was 92%. CFV reserve < 2 had a higher sensitivity (94% vs 72%, P <.01) and a lower specificity (65% vs 95%, P <.01) than wall-motion assessment for detecting significant LAD stenosis, and diagnostic accuracy between 2 methods was comparable (81% vs 82%). A total of 69 patients (73%) showed concordant results of the 2 methods, and diagnostic accuracy for detecting significant LAD stenosis was high (94%) in this subset of patients. CONCLUSIONS The simultaneous assessment of CFV and wall motion was feasible in the majority of cases during dipyridamole stress echocardiography. Although diagnostic accuracy between the 2 tests was comparable, concordant results of the 2 methods provided accurate diagnosis in detecting significant LAD stenosis.

Endothelial vasodilator function is preserved at the spastic/inflammatory coronary lesions in pigs.

BACKGROUND The question of whether or not endothelial vasodilator function in the spastic coronary artery is preserved is still controversial. We recently developed a porcine model in which long-term and local treatment with interleukin-1beta (IL-1beta) from the adventitial site causes coronary arteriosclerotic changes and vasospastic responses to autacoids. The aim of this study was to examine the endothelial vasodilator function in our new porcine model of the spasm both in vivo and in vitro. METHODS AND RESULTS A segment of the porcine coronary artery was aseptically wrapped with cotton mesh that held absorbed IL-1beta-bound microbeads. Two weeks after the procedure, intracoronary administration of serotonin caused coronary vasospasm at the IL-1beta-treated site (n = 10). Coronary vasodilatation to bradykinin, substance P, or an increase in coronary blood flow was preserved at the spastic site. Vasodilator responses to 3-morpholinosydnonimine (an NO donor) and nitroglycerin also were comparable between the 2 sites. The vasoconstricting response to N(G)-monomethyl-L-arginine and the extent of the augmentation of the serotonin-induced vasoconstriction were comparable between the 2 sites. Organ chamber experiments showed that endothelium-dependent relaxations to bradykinin, the calcium ionophore A23187, and even the vasospastic agonist serotonin were preserved at the spastic site, whereas contractions to serotonin were augmented at the spastic site regardless of the presence or absence of the endothelium (n = 6). Endothelium-independent relaxations to sodium nitroprusside were also preserved at the spastic site. CONCLUSIONS These results indicate that endothelial vasodilator function is preserved at the spastic site and that the spasm is caused primarily by smooth muscle hypercontraction in our porcine model.

Platelet activating factor causes hyperconstriction at the inflammatory coronary lesions in pigs in vivo.

Background Although platelet activating factor (PAF) is an important vasoactive substance released from activated leukocytes, platelets and endothelial cells, little is known about its effect at the inflammatory coronary lesions in vivo.Objective To examine the coronary vasomotor responses to PAF at the inflammatory lesions in our swine model with mterleukin-1β (IL-1β) in vivo.Methods Under aseptic conditions, the proximal segment of the porcine left coronary artery was dissected and wrapped with cotton mesh absorbing IL-1β. Two weeks after the operation, coronary vasomotion in response to intracoronary administration of 0.3 and 1 μg/kg PAF, 1, 3, and 10 μg/kg serotonin, 1, 3 and 10 μg/kg histamine, and 5 and 50 μg/kg prostaglandin F2α was examined by coronary artenography.Results At the IL-1β-treated site, PAF, serotonin and histamine, but not prostaglandin F2α1 caused hyper-constriction (n = 8). A synergy of the vasoconstricting effects of PAF and serotonin was also noted (n = 6). Administration of TCV-309, a selective PAF receptor antagonist, abolished the hyperconstrictive responses to PAF but not those to other agonists (n = 6). The PAF-induced coronary hyperconstrictions were significantly inhibited by administrations of the protein kinase C inhibitors staurosponne and sphingosme, but not by administrations of ryanodine, thapsigargin, or indomethacm (n = 4 each).Conclusions These results indicate that PAF causes hyperconstriction at the inflammatory coronary lesions in vivo by itself as well as in a synergistic manner with serotonin and that the PAF-induced hyperconstrictions are substantially mediated by a protein kinase Cdependent pathway in vivo.

Statin Therapy May Prevent Restenosis After Successful Coronary Intervention, Independent of Lipid-lowering Effect and CRP Level

BACKGROUND As statins have the anti-atherosclerotic pleiotropic effects, we retrospectively examined the effects of statins on restenosis after percutaneous coronary intervention (PCI). METHODS We reviewed consecutive 341 patients who underwent successful PCI and follow-up angiography six months after the procedure between January 2002 and December 2004. Statins were initiated in 207 patients (statin group), but not in the other 134 (control group). We compared the angiographic findings, low-density lipoprotein cholesterol (LDL-C) and C-reactive protein (CRP) between the two groups. RESULTS LDL-C level in statin group was significantly higher than those in control group at baseline (116.0 +/- 35.8 vs 103.1 +/- 24.5 mg/dL, p < 0.01); however, the values were inverted between the two groups at follow-up (99.9 +/- 29.5 vs 107.6 +/- 26.0 mg/dL, p = 0.015). CRP levels were comparable between these two groups. Statin group showed significantly lower angiographic restenosis (defined as > or = 50% stenosis at the target site) rate (35.3 vs 46.3%, p = 0.042) and target lesion revascularization (TLR) rate (14.5 vs 23.9%, p = 0.018) than control group. Multivariate analysis indicated that the prescription of statin, but not LDL-C level at follow-up and % reduction of LDL-C during the follow-up period, predict the restenosis prevention. CONCLUSIONS Statins can decrease restenosis and TLR rate after PCI, independent of lipid-lowering effect and CRP level in this study.

A Fatal Case of Amiodarone-Induced Acute Respiratory Distress Syndrome in a Patient with Severe Left Ventricular Dysfunction Due to Extensive Anterior Acute Myocardial Infarction

We here report a case of 71-year-old man with acute extensive anterior myocardial infarction, who was complicated with ventricular tachycardia (VT) even after successful percutaneous coronary intervention. As intravenous administration of nifekalant terminated VT, we started oral administration of amiodarone (day 1). We gave 400 mg of amiodarone a day for the first week and 200 mg a day from the second week. The patient was stable with normoxia by day 20, in spite of pulmonary congestion-like infiltrates on chest X-ray. On day 21, he was complicated with acute respiratory distress syndrome. Immediate discontinuance of amiodarone and high-dose pulse glucocorticoid therapy with intubation slightly improved the infiltrations on chest X-ray. However, glucocorticoid therapy induced hyperglycemia with an increase in plasma osmolality, complicated with hypoalbuminemia, and gastrointestinal bleeding. Despite treatment with a large amount of saline, high-doses of catecholamines, and blood transfusion, the patient died on day 28. It is sometimes difficult to diagnose congestive heart failure or amiodarone-induced pulmonary infiltrates in patients with severe left ventricular dysfunction.