Satoshi Kunimoto

Conformational switch of angiotensin II type 1 receptor underlying mechanical stress-induced activation

The angiotensin II type 1 (AT1) receptor is a G protein‐coupled receptor that has a crucial role in the development of load‐induced cardiac hypertrophy. Here, we show that cell stretch leads to activation of the AT1 receptor, which undergoes an anticlockwise rotation and a shift of transmembrane (TM) 7 into the ligand‐binding pocket. As an inverse agonist, candesartan suppressed the stretch‐induced helical movement of TM7 through the bindings of the carboxyl group of candesartan to the specific residues of the receptor. A molecular model proposes that the tight binding of candesartan to the AT1 receptor stabilizes the receptor in the inactive conformation, preventing its shift to the active conformation. Our results show that the AT1 receptor undergoes a conformational switch that couples mechanical stress‐induced activation and inverse agonist‐induced inactivation.

EphA4-Mediated Rho Activation via Vsm-RhoGEF Expressed Specifically in Vascular Smooth Muscle Cells

Abstract— Rho-kinase, an effector of Rho GTPase, increases the contractility of vascular smooth muscle by phosphorylating myosin light chain (MLC) and by inactivating MLC phosphatase. A wide variety of extracellular stimuli activate RhoA via G protein–coupled receptors. In the present study, we demonstrate a novel cell-cell interaction–mediated Rho activation signaling pathway in vascular smooth muscle cells (VSMCs). Among many receptor tyrosine kinases, the Eph family receptors are unique in that they require cell-cell interaction to engage their ligands, ephrin. We found that a novel VSMC-specific guanine nucleotide exchange factor (GEF) for Rho (Vsm-RhoGEF/KIAA0915) was expressed specifically in VSMCs of several organs including the heart, aorta, liver, kidney, and spleen, as examined by the immunohistochemical analysis using a specific antibody against Vsm-RhoGEF. Based on the association of Vsm-RhoGEF with EphA4 in quiescent cells, we tested whether EphA4 and Vsm-RhoGEF were expressed in the same tissue and further studied the molecular mechanism of Vsm-RhoGEF regulation by EphA4. Immunohistochemical analysis showed that EphA4 and Vsm-RhoGEF expression overlapped in VSMCs. Additionally, tyrosine phosphorylation of Vsm-RhoGEF induced by EphA4 upon ephrin-A1 stimulation enhanced the Vsm-RhoGEF activity for RhoA. The requirement of Vsm-RhoGEF for ephrin-A1–induced assembly of actin stress fibers in VSMCs was shown by the overexpression of a dominant-negative form of VSM-RhoGEF and by the depletion of Vsm-RhoGEF using RNA interference. These results suggested that ephrin-A1–triggered EphA4-Vsm-RhoGEF-RhoA pathway is involved in the cell-cell interaction–mediated RhoA activation that regulates vascular smooth muscle contractility.

Vascular Endothelial-Cadherin Stabilizes at Cell–Cell Junctions by Anchoring to Circumferential Actin Bundles through α- and β-Catenins in Cyclic AMP-Epac-Rap1 Signal-activated Endothelial Cells

Vascular endothelial (VE)-cadherin is a cell-cell adhesion molecule involved in endothelial barrier function. Here, we show that initial circumferential actin bundling induced by cyclic AMP-Epac-Rap1 signal and its linkage to VE-cadherin through α- and β-catenins lead to the stabilization of VE-cadherin at cell-cell contacts.

Feasibility of landiolol and bisoprolol for prevention of atrial fibrillation after coronary artery bypass grafting: A pilot study

BACKGROUND We previously performed a trial of intravenous landiolol hydrochloride during and after cardiac surgery (the PASCAL trial) and demonstrated a preventive effect on postoperative atrial fibrillation (AF). In the present study, we investigated the efficacy of increasing the dose and administration period of landiolol for prevention of postoperative AF, as well as the effect of oral bisoprolol in the early postoperative period. PATIENTS AND METHODS A total of 105 patients who underwent coronary artery bypass grafting were randomized to 3 groups: a group receiving intravenous landiolol perioperatively at 5 μg/kg/min for 3 days (group L), a group receiving oral bisoprolol postoperatively together with landiolol (group LB), and a control group without beta-blocker therapy (group C). The primary end point was the presence/absence of postoperative AF. Secondary end points were (1) the early clinical outcome, (2) hemodynamics, (3) cardiac enzymes (creatine kinase isoenzyme MB, troponin-I, and human heart fatty acid-binding protein), (4) high-sensitivity C-reactive protein (hs-CRP) and pentraxin-3, (5) asymmetric dimethylarginine (ADMA), and (6) brain natriuretic peptide. RESULTS Postoperative AF occurred in 14.5% of group L, 9.1% of group LB, and 35.3% of group C. A significant difference was observed between groups LB and C. Significantly higher levels of troponin-I, human heart fatty acid-binding protein, hs-CRP, pentraxin-3, and ADMA were noted in group C than in groups L and LB. CONCLUSIONS Landiolol and bisoprolol prevented postoperative AF. The anti-ischemic, anti-inflammatory, and anti-oxidant effects of these beta-blockers presumably inhibited the onset of AF.

Chimeric DNA–RNA hammerhead ribozyme targeting PDGF A-chain mRNA specifically inhibits neointima formation in rat carotid artery after balloon injury

OBJECTIVE Restenosis of the coronary artery after percutaneous transluminal coronary angioplasty (PTCA) occurs in 30-50% of patients and remains a major clinical problem. We developed ribozyme that targets platelet-derived growth factor (PDGF) A-chain mRNA as a gene therapy for restenosis after PTCA. Thus, we examined the effects of a chimeric DNA-RNA ribozyme targeting PDGF A-chain mRNA on neointima formation in rat carotid artery after balloon injury and evaluated its specificity for PDGF A-chain mRNA by microarray analysis. METHODS Rat carotid artery was injured with a 2F Fogarty catheter, and PDGF A-chain specific ribozyme was delivered to the injured artery with polyethylenimine. Two weeks after injury, the artery was removed, and the intima/media (I/M) ratio was evaluated. Six hours after injury, mRNA was extracted with oligo dT cellulose, and expression of PDGF A-chain mRNA was evaluated by reverse transcription-polymerase chain reaction. Expression of PDGF-AA protein was evaluated by Western blot analysis. Expression of 970 genes was evaluated by microarray (GeneChip, Affimetrix Inc). RESULTS FITC-labeled ribozyme was taken up into the midlayer smooth muscle of the carotid artery until 24 h after balloon injury. Two and 5 microg of ribozyme significantly reduced neointima formation by 44 and 55% of control levels, respectively, in a dose-dependent manner. Ribozyme markedly inhibited expression of PDGF A-chain mRNA as well as production of PDGF-AA protein in injured vessels. Microarray analysis revealed that expression of 525 genes was increased after balloon injury. These genes included FLK-1, interleukin-1 receptor, retinoic acid receptor alpha2 isoform, heat shock protein, MAP kinase kinase, Fas antigen, G6Pase, PI-5-P-kinase, p38 MAP kinase, proliferating cell nuclear antigen, transforming growth factor-beta, extracellular signal-related kinase, and fibroblast growth factor receptor. With respect to expression of cytokine and growth factor mRNAs, the best ribozyme specifically inhibited expression of PDGF A-chain mRNA. CONCLUSIONS Our chimeric DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA inhibited neointima formation in rat carotid artery after balloon injury with specific inhibition of expression of PDGF A-chain mRNA, suggesting that this ribozyme may be useful for therapy of restenosis of coronary artery after PTCA.

Risk stratification of cardiovascular events in patients at all stages of chronic kidney disease using myocardial perfusion SPECT

BACKGROUND Cardiovascular disease is the leading cause of death among patients with chronic kidney disease (CKD). Therefore, stratification of the prognostic risk of cardiovascular events is useful for their clinical management. We evaluated the ability of electrocardiogram (ECG)-gated myocardial perfusion single photon emission computed tomography (SPECT) to predict cardiac events among Japanese patients at all stages of CKD including those on hemodialysis. METHODS Patients with CKD undergoing ECG-gated myocardial perfusion SPECT to investigate suspected ischemic heart disease were followed up to monitor retrospectively major cardiac events including cardiac death, non-fatal myocardial infarction, and unstable angina pectoris. Summed stress score, summed rest score, and summed difference score were estimated with a 20 segment 5-point scoring model. The severity of CKD was divided into five levels based on estimated glomerular filtration rate (eGFR) revised for the Japanese population. RESULTS The follow-up period was 25.8 ± 11.0 months. Sixty-two major cardiac events (cardiac death, n=30; non-fatal myocardial infarction, n=13; unstable angina pectoris, n=19) developed in 2243 patients during the first year of follow-up. The findings of multivariate Cox proportional hazards regression analysis indicated that diabetes, eGFR, the summed difference score, and summed stress score were significant independent predictors of cardiac events. The major cardiac event rate at one year increased in proportion to the progression of CKD stage. The major cardiac event-free survival rate decreased steeply over time in patients with CKD stage 5 compared with those who had CKD stages 4 or less. CONCLUSION Myocardial perfusion SPECT can contribute to the prediction of cardiac events and survival in patients at all stages of CKD including those on hemodialysis.

Low invasive angiogenic therapy for myocardial infarction by retrograde transplantation of mononuclear cells expressing the VEGF gene

BACKGROUND Although transplantation of mononuclear cells (MNCs) induces angiogenesis in myocardial infarction, transplantation requires a large amount of bone marrow or peripheral blood cells. We examined the effects of transplantation of peripheral MNCs expressing an exogenous vascular endothelial growth factor (VEGF) gene in a pig model of acute myocardial infarction (AMI). METHODS MNCs were isolated from 20 ml peripheral blood from pigs and transfected with 10 microg of human VEGF165 plasmid (phVEGF). Myocardial infarction was induced by occlusion of the mid portion of the left anterior descending coronary artery (LAD) in anesthetized pigs. At 4 h after total occlusion, 5 x 10(6) VEGF-transfected MNCs were retrogradely transplanted into the pig via the coronary vein. Cardiac function, neovascularization and histology of the ischemic tissue were evaluated 4 weeks after transplantation. RESULTS MNCs expressing hVEGF and infused via the coronary vein were efficiently delivered the heart in pigs with myocardial infarction. Transplantation of MNCs expressing hVEGF significantly increased left ventricular (LV) function, collateral vessels, and capillary density in heart from AMI model pigs. Transplantation of MNCs expressing hVEGF increased the wall thickness of the scar in the heart after AMI. CONCLUSIONS Retrograde transplantation of peripheral blood MNCs expressing hVEGF efficiently induced angiogenesis and improved the impaired LV function in hearts of pigs with AMI. These findings indicate that angiogenic cells and gene therapy may be useful to treat ischemic heart disease.

Development of Human‐Like Advanced Coronary Plaques in Low‐Density Lipoprotein Receptor Knockout Pigs and Justification for Statin Treatment Before Formation of Atherosclerotic Plaques

Background Although clinical trials have proved that statin can be used prophylactically against cardiovascular events, the direct effects of statin on plaque development are not well understood. We generated low‐density lipoprotein receptor knockout (LDLR −/−) pigs to study the effects of early statin administration on development of atherosclerotic plaques, especially advanced plaques. Methods and Results LDLR −/− pigs were generated by targeted deletion of exon 4 of the LDLR gene. Given a standard chow diet, LDLR −/− pigs showed atherosclerotic lesions starting at 6 months of age. When 3‐month‐old LDLR −/− pigs were fed a high‐cholesterol, high‐fat (HCHF) diet for 4 months (HCHF group), human‐like advanced coronary plaques developed. We also fed 3‐month‐old LDLR −/− pigs an HCHF diet with pitavastatin for 4 months (Statin Prophylaxis Group). Although serum cholesterol concentrations did not differ significantly between the 2 groups, intravascular ultrasound revealed 52% reduced plaque volume in statin‐treated pigs. Pathological examination revealed most lesions (87%) in the statin prophylaxis group were early‐stage lesions, versus 45% in the HCHF diet group (P<0.01). Thin‐cap fibroatheroma characterized 40% of the plaques in the HCHF diet group versus 8% in the statin prophylaxis group (P<0.01), intraplaque hemorrhage characterized 11% versus 1% (P<0.01), and calcification characterized 22% versus 1% (P<0.01). Conclusions Results of our large animal experiment support statin prophylaxis before the occurrence of atherosclerosis. Early statin treatment appears to retard development of coronary artery atherosclerosis and ensure lesion stability. In addition, the LDLR −/− pigs we developed represent a large animal model of human‐like advanced coronary plaque suitable for translational research.

Carperitide and Atrial Fibrillation After Coronary Bypass Grafting The Nihon University Working Group Study of Low-Dose HANP Infusion Therapy During Cardiac Surgery Trial for Postoperative Atrial Fibrillation

Background —Occurrence of atrial fibrillation after cardiac surgery is associated with long-term mortality. We investigated whether infusion of human atrial natriuretic peptide (carperitide) could prevent postoperative atrial fibrillation (POAF). Methods and Results —A total of 668 patients who underwent isolated coronary artery bypass grafting were randomized to receive infusion of carperitide or physiological saline from the initiation of cardiopulmonary bypass. Patients were monitored continuously for one week after surgery to detect atrial fibrillation. The risk factors were investigated by Cox proportional hazard model. POAF occurred in 41/335 patients (12.2%) from the carperitide group versus 110/333 patients (32.7%) from the placebo group (p 150ng/ml, preoperative non-use of angiotensin receptor antagonists, preoperative use of calcium antagonists, postoperative non-use of beta blockers, postoperative non-use of aldosterone blockers, and non-use of carperitide. Conclusions —Perioperative carperitide infusion reduced the occurrence of postoperative atrial fibrillation. Accordingly, carperitide could be a useful option for preventing POAF. Clinical Trial Registration —http://www.umin.ac.jp; Unique Identifier: UMIN000003958Background—Occurrence of atrial fibrillation after cardiac surgery is associated with long-term mortality. We investigated whether infusion of human atrial natriuretic peptide (carperitide) could prevent postoperative atrial fibrillation. Methods and Results—A total of 668 patients who underwent isolated coronary artery bypass grafting were randomized to receive infusion of carperitide or physiological saline from the initiation of cardiopulmonary bypass. Patients were monitored continuously for 1 week after surgery to detect atrial fibrillation. The risk factors were investigated by Cox proportional hazard model. Postoperative atrial fibrillation occurred in 41 of 335 patients (12.2%) from the carperitide group versus 110 of 333 patients (32.7%) from the placebo group (P<0.0001). Postoperative levels of angiotensin-II, aldosterone, creatine kinase MB isoenzyme, human heart fatty acid–binding protein, and brain natriuretic peptide were all significantly lower in the carperitide group. The risk factors for postoperative atrial fibrillation by the Cox proportional hazard model were an age ≥70 years, emergency surgery, preoperative aldosterone level >150 ng/mL, preoperative nonuse of angiotensin receptor antagonists, preoperative use of calcium antagonists, postoperative nonuse of &bgr;-blockers, postoperative nonuse of aldosterone blockers, and nonuse of carperitide. Conclusions—Perioperative carperitide infusion reduced the occurrence of postoperative atrial fibrillation. Accordingly, carperitide could be a useful option for preventing postoperative atrial fibrillation. Clinical Trial Registration—URL: http://www.umin.ac.jp. Unique Identifier: UMIN000003958.

Left ventricular aneurysm associated with isolated noncompaction of the ventricular myocardium

A 66-year-old woman was admitted to our hospital because of left ventricular failure and nonsustained ventricular tachycardia. Two-dimensional echocardiography demonstrated prominent trabeculations and deep intertrabecular recesses, findings consistent with noncompaction of the ventricular myocardium. Myocardial perfusion scintigraphy demonstrated a defect in the anterobasal left ventricular segment. Coronary angiogram was normal, but the left ventriculogram showed an aneurysm in the anterior myocardial segments. This is the first reported case with isolated noncompaction of the ventricular myocardium associated with left ventricular aneurysm.