Minoru Yoshiyama

Vascular Endothelial Growth Factor–Expressing Mesenchymal Stem Cell Transplantation for the Treatment of Acute Myocardial Infarction

Objective—Vascular endothelial growth factor (VEGF) plays an important role in inducing angiogenesis. Mesenchymal stem cells (MSCs) may have potential for differentiation to several types of cells, including myocytes. We hypothesized that transplantation of VEGF-expressing MSCs could effectively treat acute myocardial infarction (MI) by providing enhanced cardioprotection, followed by angiogenic effects in salvaging ischemic myocardium. Methods and Results—The human VEGF165 gene was transfected to cultured MSCs of Lewis rats using an adenoviral vector. Six million VEGF-transfected and LacZ-transfected MSCs (VEGF group), LacZ-transfected MSCs (control group), or serum-free medium only (medium group) were injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. At 1 week after MI, MSCs were detected by X-gal staining in infarcted region. High expression of VEGF was immunostained in the VEGF group. At 28 days after MI, infarct size, left ventricular dimensions, ejection fraction, E wave/A wave ratio and capillary density of the infarcted region were most improved in the VEGF group, compared with the medium group. Immunohistochemically, &agr;-smooth muscle actin–positive cells were most increased in the VEGF group. Conclusions—This combined strategy of cell transplantation with gene therapy could be a useful therapy for the treatment of acute MI.

Apoptosis Signal-Regulating Kinase 1 Plays a Pivotal Role in Angiotensin II–Induced Cardiac Hypertrophy and Remodeling

Abstract— Multiple lines of evidence establish that angiotensin II (Ang II) induces not only hypertension but also directly contributes to cardiac diseases. Apoptosis signal-regulating kinase 1 (ASK1), one of mitogen-activated protein kinase kinase kinases, plays a key role in stress-induced cellular responses. However, nothing is known about the role of ASK1 in cardiac hypertrophy and remodeling in vivo. In this study, by using mice deficient in ASK1 (ASK1−/− mice), we investigated the role of ASK1 in cardiac hypertrophy and remodeling induced by Ang II. Left ventricular (LV) ASK1 was activated by Ang II infusion in wild-type mice, which was mediated by angiotensin II type 1 receptor and superoxide. Although Ang II-induced hypertensive effect was comparable to wild-type and ASK1−/− mice, LV ASK1 activation by Ang II was not detectable in ASK1−/− mice, and p38 and c-Jun N-terminal kinase (JNK) activation was lesser in ASK−/− mice than in wild-type mice. Elevation of blood pressure by continuous Ang II infusion was comparable between ASK1−/− and wild-type mice. However, Ang II–induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hypertrophy–related mRNA upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation, was significantly attenuated in ASK1−/− mice compared with wild-type mice. These results provided the first in vivo evidence that ASK1 is the critical signaling molecule for Ang II–induced cardiac hypertrophy and remodeling. Thus, ASK1 is proposed to be a potential therapeutic target for cardiac diseases.

Beneficial effects of combination of ACE inhibitor and angiotensin II type 1 receptor blocker on cardiac remodeling in rat myocardial infarction.

OBJECTIVEnAngiotensin-converting enzyme (ACE) inhibitor and angiotensin II type I receptor blockers (ARB) prevent cardiac remodeling after myocardial infarction (MI). However, it is controversial whether combination therapy of ACE inhibitor and ARB is more effective on cardiac remodeling than each agent alone. In this study, we compared the effects of an ACE inhibitor (temocapril), an ARB (CS-866), and their combination on cardiac remodeling after MI.nnnMETHODSnTemocapril at 3 or 30 mg/kg/day, CS-866 at 1 or 10 mg/kg/day, or combined temocapril and CS-866 at 1.5 and 0.5 mg/kg/day or at 15 and 5 mg/kg/day, respectively, were administered to rats after MI. At 4 weeks after MI, we assessed hemodynamics, cardiac function by Doppler echocardiography and non-infarcted myocardial mRNA expression.nnnRESULTSnAnimals treated with a combination of the two drugs had hemodynamics, heart weights and dimensions similar to the other treated animals. However, the combination of the two drugs suppressed ANP, BNP and other gene expressions related to contractile proteins of fetal type and collagens more effectively than ACE inhibitor or ARB alone.nnnCONCLUSIONnThese data suggest that combination of the two drugs, independent of the hemodynamic effect, may improve left ventricular phenotypic change, collagen accumulation and diastolic function.

Activation of Apoptosis Signal-Regulating Kinase 1 in Injured Artery and Its Critical Role in Neointimal Hyperplasia

Background—Apoptosis signal-regulating kinase 1 (ASK1), recently identified as one of the mitogen-activated protein kinase kinase kinases, is activated by various extracellular stimuli and involved in a variety of cellular function. Therefore, we first examined the role of ASK1 in vascular remodeling. Methods and Results—We used rat balloon injury model and cultured vascular smooth muscle cells (VSMCs). Arterial ASK1 activity was rapidly and dramatically increased after balloon injury. To specifically inhibit endogenous ASK1 activation, dominant-negative mutant of ASK1 (DN-ASK1) was transfected into rat carotid artery before balloon injury. Gene transfer of DN-ASK1 significantly prevented neointimal formation at 14 days after injury. Bromodeoxyuridine labeling index at 7 days after injury showed that DN-ASK1 remarkably suppressed VSMC proliferation in both the intima and the media. We also examined the role of ASK1 in cultured rat VSMCs. Infection with DN-ASK1 significantly attenuated serum-induced VSMC proliferation and migration. We also compared neointimal formation after cuff placement around the femoral artery between mice deficient in ASK1 (ASK1−/− mice) and wild-type (WT) mice. Neointimal formation at 28 days after cuff injury in ASK1−/− mice was significantly attenuated compared with WT mice. Furthermore, we compared the proliferation and migration of VSMCs isolated from ASK1−/− mice with WT mice. Both proliferation and migration of VSMCs from ASK1−/− mice were significantly attenuated compared with VSMCs from WT mice. Conclusions—ASK1 activation plays the key role in vascular intimal hyperplasia. ASK1 may provide the basis for the development of new therapeutic strategy for vascular diseases.

Core Protein of Hepatitis C Virus Induces Cardiomyopathy

Hepatitis C virus (HCV) has been reported to be associated with cardiomyopathy. However, the mechanism of cardiomyopathy in chronic HCV infection is still unclear. Therefore, we investigate the development of cardiomyopathy in mice transgenic for the HCV-core gene. After the age of 12 months, mice developed cardiomyopathy that appeared as left ventricular dilatation, and systolic and diastolic dysfunction assessed by Doppler echocardiography. Histologically, hypertrophy of cardiomyocytes, cardiac fibrosis, disarray and scarcity of myofibrils, vacuolization and deformity of nuclei, myofibrillar lysis, streaming of Z-bands, and an increased number of bizarre-shaped mitochondria were found in HCV-core transgenic mice. These histological changes are just consistent with cardiomyopathy. In conclusion, the HCV-core protein directly plays an important role in the development of cardiomyopathy.

Involvement of Apoptosis Signal-Regulating Kinase-1 on Angiotensin II-Induced Monocyte Chemoattractant Protein-1 Expression

Objective—Monocyte chemoattractant protein 1 (MCP-1) could contribute to enhanced leukocyte recruitment and activation resulting in chronic tissue damage. However, little is known about the molecular mechanisms of cardiac MCP-1 expression. To elucidate these molecular mechanisms, angiotensin II-induced expression of MCP-1 was examined in cultured rat neonatal ventricular cardiomyocytes and fibroblasts by adenovirus gene transfer. Methods and Results—MCP-1 mRNA increased 3.6-fold in cardiac fibroblasts at 3 hours after 100 nmol/L angiotensin-II stimulation (P <0.01), whereas MCP-1 mRNA in cardiomyocytes was unchanged. Angiotensin II significantly enhanced JNK, p38MAPK, and nuclear factor-&kgr;B (NF-&kgr;B) activities of cardiac fibroblasts. Wild-type ASK-1 increased MCP-1 expression of cardiac fibroblasts, whereas dominant negative mutant of ASK-1 (DN-ASK), dominant negative mutant of p38MAPK (DN-p38MAPK), and pyrrolidine dithiocarbamate significantly inhibited such expression. The increased MCP-1 mRNA expression in wild-type ASK-1 transfected fibroblasts was inhibited by cotransfection with adenovirus expressing DN-p38MAPK. On the contrary, the decreased MCP-1 mRNA expression in DN-ASK transfected cells was increased by cotransfection with adenovirus expressing constitutively active MKK6. Conclusion—Angiotensin II induced MCP-1 gene expression in cardiac fibroblasts. The angiotensin II-induced activation of ASK-1 followed by p38MAPK and NF-&kgr;B signaling in cardiac fibroblasts is partially involved in myocardial MCP-1 expression.

Involvement of c-Jun NH2 terminal kinase and p38MAPK in rapamycin-mediated inhibition of neointimal formation in rat carotid arteries.

Objective: Rapamycin-coated stents in coronary artery lesions have recently been shown to be effective in inhibiting neointimal formation. However, little is known about the effects of rapamycin on mitogen-activated protein kinase (MAPK), which is an important signal for neointimal formation. Therefore, we examined the effects of rapamycin on MAPK and transcriptional factors in cultured human coronary artery smooth muscle cells (CASMC) and in balloon-injured rat carotid arteries. Methods and Results: Activation of ERK, JNK, p38MAPK, AP-1, and NF-kB in coronary artery smooth muscle cells was increased by 2% fetal bovine serum. Ten nmol/L of rapamycin prevented the activation of JNK, p38MAPK, AP-1, and NF-kB (65%, 65%, 67%, and 26% respectively, P < 0.01). In an in vivo study, remarkable neointimal formation was observed 14 days after injury. Coating Pluronic gel with 20 and 50 μg rapamycin around the injured artery significantly decreased the intimal area/medial area ratio, compared with vehicle (0.75 vs. 1.2, P < 0.01). Rapamycin prevented the increase in activation of JNK, p38MAPK, AP-1, and NF-kB in injured artery (42%, 70%, 75%, and 60% respectively, P < 0.05). Conclusions: Neointimal formation after balloon injury is inhibited by rapamycin, which is partially mediated by inhibition of JNK and p38MAPK, followed by AP-1 and NF-kB.

Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia

Objective: To examine the effects of microbubble destruction with ultrasound (MB) combined with bone marrow derived mononuclear cell transplantation (BMT) into ischaemic tissues in rat hind limb ischaemia. Methods and results: Unilateral hind limb ischaemia was surgically induced in Lewis rats. At postoperative day 7, rats were randomly divided into three groups: a vehicle treated group, an ultrasound treated group, and an MB treated group. MB treatment increased vascular endothelial growth factor mRNA as assessed by real time polymerase chain reaction (3.0-fold, p < 0.05). At four weeks, the MB group had increases in laser Doppler blood flow index (LDBFI; 1.2-fold, p < 0.05), angiographically detectable collateral vessels (angiographic score: 1.4-fold, p < 0.01), and capillary to muscle fibre ratio (1.4-fold, p < 0.01) in ischaemic limbs compared with the vehicle treated group. No differences were seen between the vehicle and ultrasound treated groups. Secondly, rats were allocated to vehicle treatment, BMT (5 × 106 cells/rat), or a combination of MB and BMT (MB+BMT) at seven days after hind limb ischaemia. BMT treatment significantly increased LDBFI, angiographic score, and capillary to muscle fibre ratio compared with vehicle treatment. Interestingly, MB+BMT treatment produced significantly greater LDBFI (1.2-fold, p < 0.01), angiographic score (1.5-fold, p < 0.01), and capillary to muscle fibre ratio (1.5-fold, p < 0.05) than BMT treatment alone. Conclusions: MB may be a useful technique to enhance BMT induced neovascularisation.

Left ventricular remodeling after myocardial infarction in antecedent hypertensive patients.

Antecedent hypertension adversely affects mortality and heart failure after myocardial infarction (MI). In addition, accelerated ventricular remodeling is a contributor to the increased mortality observed after MI. The purpose of this study was to assess the relationship of antecedent hypertension to ventricular remodeling after MI. Ninety-four patients presenting with a first acute MI who were treated with reperfusion therapy within 12 h of their symptom onset were enrolled in this study. All of them underwent left ventriculography immediately after reperfusion therapy and again at 6 months after the occurrence of MI. Patients were divided into two groups: a hypertensive group and a normotensive group. End-diastolic volume index (EDVI), end-systolic volume index (ESVI), and ejection fraction (EF) values in the acute phase were compared to those at 6 months after acute MI in either group. The hypertensive group showed a significant increase in both EDVI and ESVI after 6 months, whereas the normotensive group did not. In addition, there was no change in EF in the hypertensive group, whereas EF increased significantly after 6 months in the normotensive group. As a result, the percent changes in ESVI and EF were significantly different between the hypertensive group and normotensive group. The results demonstrated that antecedent hypertension interacts with ventricular cavity dilatation after MI.