Ryuichiro Murakami

Implantation of Adipose-Derived Regenerative Cells Enhances Ischemia-Induced Angiogenesis

Objective—Therapeutic angiogenesis using autologous stem/progenitor cells represents a novel strategy for severe ischemic diseases. Recent reports indicated that adipose tissues could supply adipose-derived regenerative cells (ADRCs). Accordingly, we examined whether implantation of ADRCs would augment ischemia-induced angiogenesis. Method and Results—Adipose tissue was obtained from C57BL/6J mice, and ADRCs were isolated using standard methods. ADRCs expressed stromal cell–derived factor 1 (SDF-1) mRNA and proteins. Hind limb ischemia was induced and culture-expanded ADRCs, PBS, or mature adipocytes (MAs) as control cells were injected into the ischemic muscles. At 3 weeks, the ADRC group had a greater laser Doppler blood perfusion index and a higher capillary density compared to the controls. Implantation of ADRCs increased circulating endothelial progenitor cells (EPCs). SDF-1 mRNA abundance at ischemic tissues and serum SDF-1 levels were greater in the ADRC group than in the control group. Finally, intraperitoneal injection of an anti–SDF-1 neutralizing antibody reduced the number of circulating EPCs and therapeutic efficacies of ADRCs. Conclusions—Adipose tissue would be a valuable source for cell-based therapeutic angiogenesis. Moreover, chemokine SDF-1 may play a pivotal role in the ADRCs-mediated angiogenesis at least in part by facilitating mobilization of EPCs.

Impact of the high-molecular-weight form of adiponectin on endothelial function in healthy young men

Objective  Adiponectin is an adipocyte‐derived, antiatherogenic protein that is present in plasma as a large multimeric structure of high molecular weight (HMW) and in a trimer or hexamer form (non‐HMW). The biological activities of these isoforms have not yet been elucidated. We therefore examined the effect of these isoforms on endothelial function in healthy young men.

Impact of renal function on coronary plaque composition

BACKGROUND Recent studies have demonstrated that patients with chronic kidney disease are at high risk of atherosclerosis. Recently it has been found that coronary plaque components can be evaluated by integrated backscatter intravascular ultrasound (IB-IVUS), and lipid-rich plaque is associated with vulnerable plaque. The aim of the study was to investigate the relationship between renal function and tissue characterization of coronary plaque composition at the target stenotic site for percutaneous coronary intervention (PCI). METHODS We prospectively performed IB-IVUS before elective PCI in 89 consecutive patients with stable angina. According to estimated glomerular filtration rate (eGFR), they were divided into two groups (eGFR <60 ml/min/ 1.73 m(2) or eGFR > or =60 ml/min/1.73 m(2)). The tissue characteristics of the coronary plaque at each target stenotic site were evaluated by three-dimensional (3D) IB-IVUS just before PCI procedure. RESULTS The patients with eGFR <60 ml/min/1.73 m(2) had higher percentage of lipid volume and lower percentage of fibrous volume compared to the patients with eGFR > or = 60 ml/min/1.73 m(2) on the 3D IB-IVUS images (36.7 +/- 10.6% versus 28.7 +/- 9.3%, P < 0.001 and 59.1 +/- 8.7% versus 66.3 +/- 8.3%, P < 0.001, respectively). eGFR showed a significant negative correlation with lipid volume and had a significant positive correlation with fibrous volume in coronary plaques (r = -0.44, P < 0.0001, and r = 0.46, P < 0.0001, respectively). CONCLUSIONS Impaired renal function was related to higher percentage of lipid volume and lower percentage of fibrous volume in coronary plaque. Our findings may explain the increasing risk of cardiovascular events in patients with renal dysfunction.

The effects of endothelial nitric oxide synthase gene polymorphisms on endothelial function and metabolic risk factors in healthy subjects: the significance of plasma adiponectin levels

OBJECTIVE Genetic variants of the endothelial nitric oxide synthase (eNOS) gene, Glu298Asp and T-786C, have been reported to be associated with cardiovascular disease. Adiponectin is an adipocyte-derived plasma protein with insulin-sensitizing and vascular protective effects; its levels are typically low in metabolic syndrome. Therefore, eNOS gene polymorphisms may also be associated with specific metabolic profiles, including plasma adiponectin levels and atherogenic lipids. METHODS We evaluated the functional significance of eNOS gene Glu298Asp and T-786C polymorphisms on endothelial function and metabolic profiles in 101 healthy young men (mean age 30.3 years) before the progression of atherosclerotic lesions. RESULTS No linkage disequilibrium was found between the two genotypes. The Asp298 allele carriers of the eNOS gene presented significantly higher plasma low density lipoprotein (LDL) cholesterol, LDL particle size, malondialdehyde-modified LDL (MDA-LDL), and fasting insulin levels and lower plasma high density lipoprotein (HDL) cholesterol, apolipoprotein A-I levels, and endothelium-dependent vasodilation when compared with noncarriers. In spite of higher MDA-LDL levels, Asp298 carriers had significantly larger LDL particle size. By contrast, in C-786 allele carriers, systolic blood pressure was significantly higher, and plasma high-molecular-weight adiponectin levels and endothelium-dependent vasodilation were significantly lower than those in non-carriers. CONCLUSIONS Although both eNOS polymorphisms induced endothelial dysfunction, the eNOS T-786C polymorphism may be associated with adiponectin levels, whereas the Glu298Asp polymorphism may be associated with atherogenic lipid levels.

Inhibitory effects of cyclosporin A on calcium mobilization-dependent interleukin-8 expression and invasive potential of human glioblastoma U251MG cells.

Interleukin (IL)-8 produced from glioblastoma is suggested to contribute to its own proliferation and progression. Since various external stimuli have been shown to increase intracellular Ca2+ in glioma cells, we investigated Ca2+ mobilization-dependent IL-8 expression and effect of cyclosporin A (CsA), an inhibitor of calcineurin (Cn), on the expression and invasive potential of human glioblastoma U251MG cells. Combined treatment with Ca2+-ionophore and phorbol-myristate-acetate (A23187/PMA) increased IL-8 mRNA and protein levels. This increase was suppressed by CsA and by another Cn inhibitor FK506. Luciferase reporter gene assay and electrophoretic mobility shift assay revealed that activation of p65-containing nuclear factor-κB was essential for A23187/PMA-dependent activation of IL-8 promoter. CsA suppressed the promoter activity by attenuating IκB-α degradation. U251MG cells expressed IL-8 receptors CXCR-1 and -2, and Matrigel invasion assay revealed that CsA attenuated A23187/PMA-dependent stimulation of invasive potential, probably by inhibiting IL-8 production. In addition, IL-8-dependent proliferation was also suppressed by CsA. Taken together, these results demonstrate the novel inhibitory effects of CsA on glioblastoma cell functions, suggesting CsA as a potential therapeutic adjuvant for glioma treatment.

Pressure Overload-Induced Cardiomyopathy in Heterozygous Carrier Mice of Carnitine Transporter Gene Mutation

Primary systemic carnitine deficiency is an autosomal recessive disorder caused by a decreased renal reabsorption of carnitine because of mutations of the carnitine transporter OCTN2 gene, and hypertrophic cardiomyopathy is a common clinical feature of homozygotes. Although heterozygotes for OCTN2 mutations are generally healthy with normal cardiac performance, heterozygotes may be at risk for cardiomyopathy in the presence of additional risk factors, such as hypertension. To test this hypothesis, we investigated the effects of surgically induced pressure overload on the hearts of heterozygous mutants of a murine model of OCTN2 mutation, juvenile visceral steatosis mouse (jvs/+). Eleven-week-old jvs/+ mice and age-matched wild-type mice were used. At baseline, there were no differences in physical characteristics between wild-type and jvs/+ mice. However, plasma and myocardial total carnitine levels in jvs/+ mice were lower than in wild-type mice. Both wild-type and jvs/+ mice were subjected to ascending aortic constriction with or without 1% l-carnitine supplementation for 4 weeks. At 4 weeks after ascending aortic constriction, jvs/+ mice showed an exaggeration of cardiac hypertrophy and pulmonary congestion, further increased gene expression of atrial natriuretic peptide in the left ventricles, further deterioration of left ventricular fractional shortening, reduced myocardial phosphocreatine:adenosine triphosphate ratio, and increased mortality compared with wild-type mice; l-carnitine supplementation prevented these changes in jvs/+ mice subjected to ascending aortic constriction. In conclusion, cardiomyopathy and heart failure with energy depletion may be induced by pressure overload in heterozygotes for OCTN2 mutations and could be prevented by l-carnitine supplementation.

Risk stratification of patients with prior myocardial infarction and advanced left ventricular dysfunction by gated myocardial perfusion SPECT imaging

AbstractBackground. The Multicenter Automatic Defibrillator Implantation Trial II (MADIT-II) has shown that the prophylactic implantable cardiac defibrillator improves the survival rate of patients with prior myocardial infarction and advanced left ventricular (LV) dysfunction. However, a more accurate noninvasive predictor should be found to identify subgroups at high risk, one that would allow implantable cardiac defibrillator therapy to be directed specifically to the patients who would benefit most. Methods and Results. To elucidate whether technetium 99m tetrofosmin electrocardiogram-gated single photon emission computed tomography (SPECT) imaging at rest can determine the risk of arrhythmic death, 106 patients who met the MADIT-II criteria (LV ejection fraction ≤0.3, myocardial infarction τ;1 month earlier, and no sustained ventricular tachyarrhythmia) were recruited from a pool of 4628 consecutive patients who had undergone resting Tc-99m tetrofosmin SPECT imaging. By use of the endpoints of lethal arrhythmic events, which included documentation of sustained ventricular tachycardia, ventricular fibrillation, or diagnosis of sudden cardiac death, we performed follow-up for a mean of 30 months. Lethal arrhythmic events occurred in 14 patients. Patients with lethal arrhythmic events had a lower LV ejection fraction, greater LV end-systolic and end-diastolic volume indices, and a greater perfusion defect volume than the remaining patients. By receiver operating characteristic curve analysis, myocardial defect volume was the strongest predictor for the development of lethal arrhythmic events. Conclusion. Our results confirm that perfusion defect volume by Tc-99m tetrofosmin scintigraphy is the most pivotal predictor of the future occurrence of lethal arrhythmic events and of sudden cardiac death. Tc-99m tetrofosmin SPECT images may assist in identifying subsets of patients with a greater likelihood of arrhythmic death among patients with LV dysfunction.

Angiotensin II type 1 receptor blockers prevent tumor necrosis factor-α-mediated endothelial nitric oxide synthase reduction and superoxide production in human umbilical vein endothelial cells

Decrease in endothelial nitric oxide synthase (eNOS) expression is one of the adverse outcomes of endothelial dysfunction. Tumor necrosis factor-alpha (TNF-alpha) is known to decrease eNOS expression and is an important mediator of endothelial dysfunction. We hypothesized that an angiotensin II type 1 (AT1) receptor blocker would improve endothelial function via not only inhibition of the angiotensin II signaling but also inhibition of the TNF-alpha-mediated signaling. Therefore we investigated whether an AT1 receptor blocker would restore the TNF-alpha-induced decrease in eNOS expression in cultured human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with an antioxidant (superoxide dismutase, alpha-tocopherol) or AT1 receptor blockers (olmesartan or candesartan) restored the TNF-alpha-dependent reduction of eNOS. The AT1 receptor blocker decreased the TNF-alpha-dependent increase of 8-isoprostane. The superoxide dismutase activities in HUVEC were stable during AT1 receptor blocker treatment, and the AT1 receptor blocker did not scavenge superoxide directly. The AT1 receptor blocker also decreased TNF-alpha-induced phosphorylation of I kappaB alpha and cell death. These results suggest that AT1 receptor blockers are able to ameliorate TNF-alpha-dependent eNOS reduction or cell injury by inhibiting superoxide production or nuclear factor-kappaB activation.

Vinegar Intake Enhances Flow-Mediated Vasodilatation via Upregulation of Endothelial Nitric Oxide Synthase Activity

This study examined the effect of acetate on endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) by immunoblotting assay and the ability of acetic acid to upregulate flow-mediated vasodilatation in humans. In HUVECs, acetate induced a biphasic increase in the phosphorylated form of eNOS. The amount of phosphorylated eNOS was significantly increased by exposure to 200 μmol/l acetate for 20 min (early phase) and for 4 h (late phase). The inhibitors of cAMP-dependent protein kinase (PKA) and AMP-activated protein kinase (AMPK) blocked acetate-induced eNOS phosphorylation in the early and the late phase respectively. Furthermore, in postmenopausal women, maximum forearm blood flow (FBF) in response to shear stress increased in the vinegar (acetic acid) administered group compared to the placebo group. These results suggest that acetic acid-induced eNOS phosphorylation contributes to upregulation of flow-mediated vasodilatation in humans.

l-Caldesmon Regulates Proliferation and Migration of Vascular Smooth Muscle Cells and Inhibits Neointimal Formation After Angioplasty

Objective—Light-type caldesmon (l-CaD) is a potent cytostatic and antiangiogenic protein that regulates cell growth and survival via modulation of the cell shape and cytoskeleton. The aim of this study is to explore the potential value of l-CaD for use as a cytostatic agent to inhibit neointimal formation after angioplasty by suppressing vascular smooth muscle cell (VSMC) growth and migration. Methods and Results—We tested the cytostatic function of l-CaD in cultured VSMCs using assays for apoptosis, cell proliferation, and migration, and evaluated the expression pattern of relevant signaling proteins (focal adhesion kinase [FAK] and mitogen-activated protein kinases) in VSMCs. Transfection of adenoviral vector encoding l-CaD (Ad-l-CaD) resulted in progressive loss of actin stress fibers and cell retraction. Enzyme-linked immunosorbent assay demonstrated that Ad-l-CaD transfection increased the apoptosis rate by 75% and reduced BrdU uptake by 49%. Furthermore, transfection of Ad-l-CaD inhibited migration of VSMCs induced by platelet-derived growth factor-BB (PDGF) by 36% (P<0.05). Immunoblotting analysis revealed that l-CaD overexpression reduced PDGF-induced phosphorylation of both FAK and extracellular signal regulated-kinase (ERK). In balloon-injured rat carotid arteries, Ad-l-CaD transfection inhibited neointimal formation by 37% (P<0.05) without delaying re-endothelialization at 14 days. Conclusions—Overexpression of l-CaD suppressed cell growth and survival in VSMCs and inhibited neointimal formation after experimental angioplasty, partly by regulating the cytoskeletal tension-FAK-ERK axis.