Sakiko Inamoto

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.

Anti-CD26 Monoclonal Antibody–Mediated G1-S Arrest of Human Renal Clear Cell Carcinoma Caki-2 Is Associated with Retinoblastoma Substrate Dephosphorylation, Cyclin-Dependent Kinase 2 Reduction, p27kip1 Enhancement, and Disruption of Binding to the Extracellular Matrix

Purpose: CD26 is a 110-kDa cell surface glycoprotein with a role in tumor development through its association with key intracellular proteins. In this report, we show that binding of soluble anti-CD26 monoclonal antibody (mAb) inhibits the growth of the human renal carcinoma cells in both in vitro and in vivo experiments. Experimental Design: Growth inhibition by anti-CD26 mAb was assessed using proliferation assay and cell cycle analysis. Anti-CD26 mAb, chemical inhibitors, dominant-negative, or constitutively active forms of specific signaling molecules were used to evaluate CD26-associated pathways. The in vivo growth-inhibitory effect of anti-CD26 mAb was also assessed in a human renal carcinoma mouse xenograft model. Results:In vitro experiments show that anti-CD26 mAb induces G1-S cell cycle arrest associated with enhanced p27kip1 expression, down-regulation of cyclin-dependent kinase 2, and dephosphorylation of retinoblastoma substrate. Moreover, our data show that enhanced p27kip1 expression is dependent on the attenuation of Akt activity. Anti-CD26 mAb also internalizes cell surface CD26, leading to decreased binding to collagen and fibronectin. Experiments with a mouse xenograft model involving human renal carcinoma cells show that anti-CD26 mAb treatment drastically inhibits tumor growth in tumor-bearing mice, resulting in enhanced survival. Conclusions: Taken together, our data strongly suggest that anti-CD26 mAb treatment may have potential clinical use for CD26-positive renal cell carcinomas.

Chronic Hypoxia Accelerates the Progression of Atherosclerosis in Apolipoprotein E-Knockout Mice

The aim of this study was to investigate the effect of chronic hypoxia on the development and progression of atherosclerosis in apolipoprotein E-knockout (apoE-KO) mice. Male and female apoE-KO mice (6 weeks old) and age- and sex-matched wild-type mice were kept under hypoxic conditions (10.0±0.5% O2) in a gas chamber or in room air for 3 weeks. Aortic atherosclerotic plaque was not observed in wild-type mice under normoxic or hypoxic conditions. In the apoE-KO mice, however, hypoxia induced proliferation of smooth muscle cells and plaque formation in the aorta, which were not observed under normoxic conditions. Although sexual dimorphism of the response to hypoxia was not observed, these hypoxia-induced atherogenic changes were accompanied by a significant increase of plasma low density lipoprotein (LDL) cholesterol and NADPH-dependent vascular superoxide (O2−) production. Furthermore, matrix metalloproteinase (MMP)-9 was activated in the aorta of apoE-KO mice. In conclusion, chronic hypoxia accelerated the development of atherosclerosis in apoE-KO mice, along with increased O2− production and activated MMP-9 in the aorta.

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.

Nifedipine enhances the cardioprotective effect of an angiotensin-II receptor blocker in an experimental animal model of heart failure

This study was designed to examine the hypothesis that a calcium channel blocker nifedipine (CCB) could enhance the cardioprotective effect of an angiotensin-II receptor blocker candesartan (ARB) in the treatment for heart failure. Isoproterenol (ISP) was injected into male rats at 300 mg/kg to produce progressive heart failure. Three months later, the rats were divided into 4 groups and treated for 4 weeks with 1) vehicle (n=20), 2) ARB at 0.2 mg/kg/day (n=6), 3) CCB at 10 mg/kg/day (n=6), or 4) both drugs (n=8). Rats injected with saline served as controls (n=13). ISP caused severe myocardial degeneration and decreased the capillary density (Dcap) of the left ventricular (LV) myocardium (mean±SD: 2,197±627 vs. 2,847 ±298 N/mm2 for normal controls), while increasing plasma thiobarbituric acid-reactive substances (TBARS; 3.6±1.1 vs. 1.9±0.5 nmol/ml). Although ARB therapy preserved cardiac morphology, it had little effect on Dcap or oxidative stress. On the other hand, CCB decreased plasma TBARS and 4-hydroxy-2-nonenal protein expression in LV myocardium. Furthermore, the combination of CCB and ARB increased Dcap and preserved the ultrastructure of LV myocardium, so this combination may be a useful option for the treatment of heart failure.

Pitavastatin reduces oxidative stress and attenuates intermittent hypoxia-induced left ventricular remodeling in lean mice.

We have reported previously that intermittent hypoxia related to sleep apnea induces cardiovascular remodeling secondary to the oxidative stress. The aim of this study was to examine the effect of pitavastatin as an antioxidant to prevent intermittent hypoxia-induced left ventricular (LV) remodeling in mice without hypercholesterolemia. Eight-week-old male C57BL/6J mice (n=35) were exposed to intermittent hypoxia (30 s exposure to 5% oxygen, followed by 30 s exposure to 21% oxygen) for 8 h per day during the daytime or maintained under normoxic conditions; in addition, they were either treated with pitavastatin (3 mg kg−1 per day) or vehicle for 10 days. After cardiac catheterization and blood sampling, the LV myocardium was examined. The systemic blood pressure and plasma level of total cholesterol were similar among the four groups. Intermittent hypoxia significantly increased the expression levels of 4-hydroxy-2-nonenal (4-HNE) proteins, TNF-α and TGF-β mRNA, and also the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL)-positive myocardial cells in the LV myocardium. In addition, enhanced hypertrophy of the cardiomyocytes, perivascular fibrosis and histological degeneration were observed in the mice exposed to hypoxic stress. Treatment with pitavastatin significantly suppressed the expression levels of the 4-HNE proteins, cytokines, superoxide production and TUNEL-positive myocardial cells in the LV myocardium, consequently attenuating the hypoxia-induced histological changes. Pitavastatin preserved, at least partially, the morphological structure of the LV myocardium in lean mice exposed to intermittent hypoxia, through its antioxidant effect.

Efficacy of edaravone, a free radical scavenger, on left ventricular function and structure in diabetes mellitus.

This study was designed to assess the efficacy of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger that possesses anti-oxidant effects, on cardiac function and fine structure of the left ventricular myocardium in diabetes mellitus. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of spontaneous development of type II diabetes (30 weeks; n = 15) were divided into two groups and treated with edaravone 30 mg/kg/d or vehicle for 2 weeks. OLETF rats showed hyperglycemia (352 ± 71 mg/dl vs normal control; 128 ± 52 mg/dl), increased thiobarbituric acid-reactive substances (TBARS; 6.9 ± 2.5 n M/ml vs 2.8 ± 0.6 n M/ml), and decreased superoxide dismutase activity (21.5 ± 0.9 U/ml vs 25.8 ± 0.7 U/ml). Increased left ventricular end-diastolic pressure (12 ± 3 mm Hg vs 6 ± 2 mm Hg) and hypertrophied cardiocytes (23.1 ± 1.4 vs 17.6 ± 1.0 &mgr;m) were also observed (P < 0.05, respectively). Edaravone could not improve plasma glucose level and hemodynamic parameters but significantly decreased TBARS values (3.8 ± 0.5) and increased superoxide dismutase activity (24.5 ± 0.8) (vs OLETF, P < 0.05, respectively). Moreover, edaravone effectively preserved cardiocyte diameter (18.2 ± 0.9 &mgr;m) and the fine structure of mitochondria. Thus, edaravone exhibits modest cardiac protection in diabetes mellitus independent of blood sugar level.