永井 良三

Although it has been established that myocyte enhancer factor 2 (MEF2) plays pivotal roles in the development of the cardiovascular system as well as skeletal muscle cells, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To investigate the role of MEF2 in vascular inflammation and that of p38 in the activation of MEF2, we infected cultured rat vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a dominant-negative mutant of MEF2A (MEF2ASA) or mitogen-activated protein kinase kinase 6 (MEK6AA), and examined their effects on the expression of monocyte chemoattractant protein-1 (MCP-1), which is known to play important roles in vascular inflammation. We also examined the role of MEF2 in vivo using a rat model of transluminal wire-induced injury of the femoral artery. Angiotensin II (Ang II)-induced expression of MCP-1 mRNA was significantly inhibited by infection with adenoviruses encoding MEF2ASA (AdMEF2ASA) or MEK6AA. Ang II-induced increase of MCP-1 promoter activity was also significantly suppressed by overexpression of MEF2ASA or MEK6AA. Ang II stimulated the transactivating function of MEF2A and this activation was inhibited by overexpression of MEK6AA. Infection with AdMEF2ASA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. AdMEF2ASA infection also inhibited macrophages infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that MEF2 activation via the p38-dependent pathway mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophages infiltration.

One of the most salient characteristics of the heart is its ability to adjust work output to external load. To examine whether a single cardiomyocyte preparation retains this property, we measured the contractile function of a single rat cardiomyocyte under a wide range of loading conditions using a force-length measurement system implemented with adaptive control. A pair of carbon fibers was used to clamp the cardiomyocyte, attached to each end under a microscope. One fiber was stiff, serving as a mechanical anchor, while the bending motion of the compliant fiber was monitored for force-length measurement. Furthermore, by controlling the position of the compliant fiber using a piezoelectric translator based on adaptive control, we could change load dynamically during contractions. Under unloaded conditions, maximal shortening velocity was 106 ± 8.9 μm/s (n = 13 cells), and, under isometric conditions, peak developed force reached 5,720 nN (41.6 ± 5.6 mN/mm 2 n = 17 cells). When we simulated physiological working conditions consisting of an isometric contraction, followed by shortening and relaxation, the average work output was 828 ± 123 J/m3 (n = 20 cells). The top left corners of tension-length loops obtained under all of these conditions approximate a line, analogous to the end-systolic pressure-volume relation of the ventricle. All of the functional characteristics described were analogous to those established by studies using papillary muscle or trabeculae preparations. In conclusion, the present results confirmed the fact that each myocyte forms the functional basis for ventricular function and that single cell mechanics can be a link between subcellular events and ventricular mechanics.

Recent studies have shown the relationship between general inflammatory markers and ischemic heart and cerebrovascular diseases. Here we have investigated the potential association between the circulating white blood cell count and carotid arteriosclerosis in apparently healthy individuals. Between 1994 and 1998, 3455 subjects who had undergone general health screening tests including carotid ultrasonography were enrolled in this study. The intertertile cutoff points for the white blood cell count were 5.1 X 10(3) and 6.4 x 10(3) muL(-1) in the male Subjects and 4.6 x 10(3) and 5.7 x 10(3) muL(-1) in the female subjects. The prevalence of carotid plaque in the first (lowest), the second, and the third tertiles was 19, 28, and 28% in the male subjects, respectively (P < 0.0001), and 10, 15, and 14% in the female subjects, respectively (n.s.). The multivariate analysis showed that the male subjects in the second and third tertiles had increased risk for carotid plaque with odds ratios of 1.54 (95% CI 1.18-2.01) and 1.47 (95% CI 1.11-1.95), respectively, compared to those in the first tertile. When male subjects were subdivided according, to their smoking, status, the association between white blood cell count and carotid plaque was significant in those who smoked, but not in those who had never smoked. These data suggested the possible association between the circulating white blood cell Count and formation of carotid plaque in male smokers, but not in male never smokers or in females, in an apparently healthy Japanese population. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

We previously reported that insulin receptor substrate-2 (IRS-2)-deficient mice develop diabetes as a result of insulin resistance in the liver and failure of beta-cell hyperplasia. In this study we introduced the IRS-2 gene specifically into the liver of Irs2(-/-) mice with adenovirus vectors. Glucose tolerance tests revealed that the IRS-2 restoration in the liver ameliorated the hyperglycemia, but the improvement in hyperinsulinemia was only partial. Endogenous glucose production (EGP) and the rate of glucose disappearance (Rd) were measured during hyperinsulinemic-euglycemic clamp studies: EGP was increased 2-fold in the Irs2(-/-) mice, while Rd decreased by 50%. Restoration of IRS-2 in the liver suppressed EGP to a level similar to that in wild-type mice, but Rd remained decreased in the Adeno-IRS-2-infected Irs2(-/-) mice. Irs2(-/-) mice also exhibit obesity and hyperleptinemia associated with impairment of hypothalamic phosphatidylinositol 3-kinase activation. Continuous intracerebroventricular leptin infusion or caloric restriction yielded Irs2(-/-) mice whose adiposity was comparable to that of Irs2(+/+) mice, and both the hyperglycemia and the hyperinsulinemia of these mice improved with increased Rd albeit partially. Finally combination treatment consisting of adenovirus-mediated gene transfer of IRS-2 and continuous intracerebroventricular leptin infusion completely reversed the hyperglycemia and hyperinsulinemia in Irs2(-/-) mice. EGP and Rd also became normal in these mice as well as in mice treated by caloric restriction plus adenoviral gene transfer. We therefore concluded that a combination of increased EGP due to insulin signaling defects in the liver and reduced Rd due to obesity accounts for the systemic insulin resistance in Irs2(-/-) mice.

Voltage-gated Na(+) channel (I(Na)) is expressed under culture conditions in human smooth muscle cells (hSMCs) such as coronary myocytes. The aim of this study is to clarify the physiological, pharmacological and molecular characteristics of I(Na) expressed in cultured hSMCs obtained from bronchus, main pulmonary and coronary artery. I(Na), was recorded in these hSMCs and inhibited by tetrodotoxin (TTX) with an IC(50) value of approximately 10 nM. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of mRNA showed the prominent expression of transcripts for SCN9A, which was consistent with the results of real-time quantitative RT-PCR. These results provide novel evidence that TTX-sensitive Na(+) channel expressed in cultured hSMCs is mainly composed of Na(v)1.7.

3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, are widely prescribed to lower cholesterol. Recent reports suggest that statins may promote angiogenesis in ischemic tissues. It remains to be elucidated whether statins potentially enhance unfavorable angiogenesis associated with tumor and atherosclerosis. Here, we induced hind limb ischemia in wild-type mice by resecting the right femoral artery and subsequently inoculated cancer cells in the same animal. Cerivastatin enhanced blood flow recovery in the ischemic hind limb as determined by laser Doppler imaging, whereas tumor growth was significantly retarded. Cerivastatin did not affect capillary density in tumors. Cerivastatin, pitavastatin, and fluvastatin inhibited atherosclerotic lesion progression in apolipoprotein E-deficient mice, whereas they augmented blood flow recovery and capillary formation in ischemic hind limb. Low-dose statins were more effective than high-dose statins in both augmentation of collateral flow recovery and inhibition of atherosclerosis. These results suggest that statins may not promote the development of cancer and atherosclerosis at the doses that augment collateral flow growth in ischemic tissues.

Obesity is a major health problem in industrialized societies, and fatty liver disease (hepatic steatosis) is common in obese individuals. Oxidative stress originating from increased intracellular levels of fatty acids has been implicated as a cause of hepatocellular injury in steatosis, although the precise mechanisms remain to be elucidated. p53, widely known as a tumor suppressor, has been shown often to be activated in stressed cells, inducing cell cycle arrest or death. Here we demonstrate that p53 is involved in the molecular mechanisms of hepatocellular injury associated with steatosis. We found that p53 in the nucleus is induced in the liver from two mouse models of fatty liver disease, ob/ob and a transgenic mouse model that overexpresses an active form of sterol regulatory element-binding protein-1 in the liver (TgSREBP-1), the one with obesity and the other without obesity. This activation of the p53 pathway leads to the elevation of p21 mRNA expression, which can be considered an indicator of p53 activity, because ob/ob mice lacking p53 generated by targeting gene disruption exhibited the complete restoration of the p21 elevation to wild type levels. Consistent with these results, the amelioration of hepatic steatosis caused by Srebp-1 gene disruption in ob/ob mice lowered the p21 expression in a triglyceride content-dependent manner. Moreover, p53 deficiency in ob/ob mice resulted in a marked improvement of plasma alanine aminotransferase levels, demonstrating that p53 is involved in the mechanisms of hepatocellular injury. In conclusion, we revealed that p53 plays an important role in the pathogenesis of fatty liver disease.

Phenotypic modulation of smooth muscle from the contractile to the synthetic type underlies the pathogenesis of atherosclerosis. We previously isolated a transcription factor, KLF5 (also known as BTEB2 and IKLF), to activate various gene promoters that are activated in phenotypically modulated smooth muscle cells, such as embryonic smooth muscle myosin heavy chain gene (SMemb), plasminogen activator inhibitor-1 (PAI-1), iNOS, PDGF-A, Egr-1 and VEGF receptors. KLF5 is downregulated with vascular development but upregulated in neointima. Chromatin immunoprecipitation assay showed KLF5 to be induced and to bind the promoter of the PDGF-A gene in response to angiotensin II stimulation. To define the role of KLF5 in cardiovascular remodeling, we targeted the KLF5 gene in mice. In response to external stress, arteries of heterozygotes exhibited diminished levels of smooth muscle and adventitial cell activation. We also found that RARα binds KLF5, and that Am80, a potent synthetic RAR agonist, inhibits angiotensin II-induced cardiac hypertrophy. These results indicate that KLF5 is an essential transcription factor that causes not only smooth muscle phenotypic modulation but also cardiac hypertrophy and fibrosis. © 2004, Elsevier Science B.V. All rights reserved.

Angiogenesis is controlled by anti-angiogenic factors as well as by angiogenic factors, such as VEGF (vascular endothelial growth factor) and HGF (hepatocyte growth factor). Endostatin, a potent endogenous angiogenesis inhibitor, is known to inhibit endothelial proliferation and suppress tumour growth. However, to date, little is known about the pathophysiology of endostatin in ischaemia/reperfusion. To investigate the mechanisms of angiogenesis induced by myocardial ischaemia/reperfusion in more detail, we studied the circulating levels of endostatin, VEGF and HGF in 17 patients with acute myocardial infarction, who underwent early reperfusion therapy. In all patients, serum endostatin, VEGF and HGF levels before reperfusion were increased significantly compared with those in 17 control subjects (endostatin, 49.2 ± 11.7 ng/ml, but not detectable in controls VEGF, 685.6 ± 150.3 pg/ml compared with 173.7 ± 33.6 pg/ml HGF, 3638 ± 1285 pg/ml compared with 59 ± 13 pg/ml values are means ± S.E.M.). After reperfusion, the serum endostatin and VEGF levels decreased significantly, but still remained higher than those in control subjects (endostatin, 19.6 ± 7.0 ng/ml VEGF, 284.2 ± 90.2 pg/ml). In contrast, serum HGF levels increased significantly (15 146 ± 2230 pg/ml) after reperfusion. These data indicated that serum levels of endostatin changed in parallel with those of VEGF in response to myocardial ischaemia/ reperfusion, and the marked increase in serum HGF levels after reperfusion seemed to be, at least in part, due to heparin administration. Our data offer a possible anti-endostatin therapy in patients with acute myocardial infarction to facilitate collateral vessel formation.

BACKGROUND: Cardiac myxomas are generally considered benign, but malignant tumors have been reported. Vascular endothelial growth factor (VEGF), an angiogenic factor, plays a role in the growth, progression, and metastasis of solid tumors and it has been reported that VEGF expression is upregulated in cardiac myxomas that have a high microvessel density. The purpose of this study was to determine whether cardiac myxoma cells possess a VEGF-autocrine system that regulates tumor growth. METHODS AND RESULTS: Immunohistochemical analyses revealed the presence of VEGF and its receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1), in the cytoplasm of tumor cells from 18 of 18 myxoma tissue specimens examined. Two different myxoma cell lines were established and constitutively secreted large amounts of VEGF as determined by enzyme-linked immunosorbent assay. The expression of VEGF, VEGFR-1, and VEGFR-2 mRNA was detected in both cell lines by reverse-transcriptase polymerase chain reaction. Myxoma cell proliferation, as determined by thymidine incorporation, was enhanced by the addition of VEGF in a dose-dependent manner, and cell proliferation was inhibited in a dose-dependent manner by the addition of a neutralizing VEGF antibody. CONCLUSIONS: These results indicate that cardiac myxoma cells possess a VEGF-autocrine system, which could contribute to the malignant potential of histologically benign myxomas through direct stimulation of tumor cell growth as well as through induction of angiogenesis.

We previously reported that hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly activated intracellular signaling such as mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated protein kinase (ERK) 1/2, p38MAPK, and stress-activated protein kinases (SAPKs). To investigate the humoral factors which mediate cardiac response to hypoxia/reoxygenation, we analyzed the conditioned media from cardiac myocytes subjected to hypoxia/reoxygenation by two-dimensional electrophoresis and mass spectrometry. We identified cyclophilin A (CyPA) as one of the proteins secreted from cardiac myocytes in response to hypoxia/reoxygenation. Hypoxia/reoxygenation induced the expression of CyPA and its cell surface receptor CD147 on cardiac myocytes in vitro. This was also confirmed by ischemia/reperfusion in vivo. Recombinant human (rh) CyPA activated ERK1/2, p38MAPK, SAPKs, and Akt in cultured cardiac myocytes. Furthermore, CyPA significantly increased Bcl-2 in cardiac myocytes. These data strongly suggested that CyPA is released from cardiac myocytes in response to hypoxia/reoxygenation and may protect cardiac myocytes from oxidative stress-induced apoptosis. © 2004 Elsevier Inc. All rights reserved.

BACKGROUND: Adrenomedullin (AM) is a novel vasodilating peptide thought to have important effects on cardiovascular function. The aim of this study was to assess the activity of endogenous AM in the cardiovascular system using AM knockout mice. METHODS AND RESULTS: Mice heterozygous for an AM-null mutation (AM+/-) and their wild-type littermates were subjected to aortic constriction or angiotensin II (Ang II) infusion. The resultant cardiovascular stress led to increases in heart weight/body weight ratios, left ventricular wall thickness, and perivascular fibrosis, as well as expression of genes encoding angiotensinogen, ACE, transforming growth factor-beta, collagen type I, brain natriuretic peptide, and c-fos. In addition, renal damage characterized by decreased creatinine clearance with glomerular sclerosis was noted. In all cases, the effects were significantly more pronounced in AM+/- mice. Hearts from adult mice subjected to aortic constriction showed enhanced extracellular signal-regulated kinase (ERK) activation, as did cardiac myocytes from neonates treated acutely with Ang II. Again the effect was more pronounced in AM+/- mice, which showed increases in cardiac myocyte size, protein synthesis, and fibroblast proliferation. ERK activation was suppressed by protein kinase C inhibition to a greater degree in AM+/- myocytes. In addition, treatment of cardiac myocytes with recombinant AM suppressed Ang II-induced ERK activation via a protein kinase A-dependent pathway. CONCLUSIONS: Endogenous AM exerts a protective effect against stress-induced cardiac hypertrophy via protein kinase C- and protein kinase A-dependent regulation of ERK activation. AM may thus represent a useful new tool for the treatment of cardiovascular disease.

Hormone-sensitive lipase (HSL) plays a crucial role in the hydrolysis of triacylglycerol and cholesteryl ester in various tissues including adipose tissues. To explore the role of HSL in the metabolism of fat and carbohydrate, we have generated mice lacking both leptin and HSL (Lepob/ob/HSL -/-) by cross-breeding HSL-/- mice with genetically obese Lepob/ob mice. Unexpectedly, Lepob/ob/HSL-/- mice ate less food, gained less weight, and had lower adiposity than Lep ob/ob/HSL+/+ mice. Lepob/ob/HSL-/- mice had massive accumulation of preadipocytes in white adipose tissues with increased expression of preadipocyte-specific genes (CAAT/enhancer-binding protein β and adipose differentiation-related protein) and decreased expression of genes characteristic of mature adipocytes (CCAAT/enhancer-binding protein α, peroxisome proliferator activator receptor γ, and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1). Consistent with the reduced food intake, hypothalamic expression of neuropeptide Y and agouti-related peptide was decreased. Since HSL is expressed in hypothalamus, we speculate that defective generation of free fatty acids in the hypothalamus due to the absence of HSL mediates the altered expression of these orexigenic neuropeptides. Thus, deficiency of both leptin and HSL has unmasked novel roles of HSL in adipogenesis as well as in feeding behavior.

The human oncoprotein SET/TAF-1beta has been crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystal belongs to space group C2, with unit-cell parameters a=119.6, b=62.8, c=61.0 Angstrom, beta=89.7degrees, and contains two molecules in the asymmetric unit. A complete data set was collected to 2.8 Angstrom resolution using synchrotron radiation.

The human oncoprotein SET/TAF-1beta has been crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as a precipitant. The crystal belongs to space group C2, with unit-cell parameters a = 119.6, b = 62.8, c = 61.0 A, beta = 89.7 degrees, and contains two molecules in the asymmetric unit. A complete data set was collected to 2.8 A resolution using synchrotron radiation.

Although the role of the calcineurin-dependent pathway in the development of cardiac hypertrophy has been intensively studied, little is known of its role in vascular inflammatory diseases such as atherosclerosis and restenosis after angioplasty. To help elucidate the role of calcineurin in vascular inflammation, we infected cultured vascular smooth muscle cells (VSMCs) with an adenovirus construct expressing a constitutively active mutant of calcineurin, and examined its effect on the expression of monocyte chemoattractant protein-1 (MCP-1). We also examined the role of calcineurin in vivo using a transluminal wire injury model of the rat femoral artery. Forced activation of calcineurin significantly increased the expression of MCP-1 both at the transcriptional and protein levels. Angiotensin II (Ang II) also significantly stimulated MCP-1 expression, and this increase was significantly inhibited by cyclosporin A (CyA). Constitutive activation of calcineurin stabilized MCP-1 mRNA without enhancing MCP-1 promoter activity. In accordance with the results, Ang II-induced increase of MCP-1 promoter activity was not suppressed by CyA. Ang II stabilized MCP-1 mRNA, and this effect of Ang II was diminished by CyA. CyA suppressed MCP-1 expression in the femoral artery after the transluminal mechanical injury. CyA also inhibited macrophage infiltration and neointimal formation in the wire-injured femoral arteries. These results suggested that calcineurin mediates vascular inflammation via stimulation of MCP-1 expression in VSMCs and macrophage infiltration.

Resistin is an adipocytokine which plays a role in the development of insulin resistance. In this study, we investigated the direct effect of resistin on vascular endothelial cells. Resistin induced the expression of adhesion molecules such as VCAM-1 and ICAM-1, and long pentraxin 3, a marker of inflammation. The induction of VCAM-1 by resistin was inhibited partially by pitavastatin. Moreover, the induction of VCAM-1 and ICAM-1 by resistin was inhibited by adiponectin, an adipocytokine that improves insulin resistance. Taken together, these results suggest that the balance in the concentrations of adipocytokines such as resistin and adiponectin determines the inflammation status of vasculature, and in turn the progress of atherosclerosis.

Background: The common carotid intima-media thickness (IMT) is correlated with the angiographically determined coronary artery stenosis. However, their correlation is weak, which limits the clinical application of the IMT as a predictor of coronary artery stenosis. The IMT reflects diffuse early-phase atherosclerosis, whereas the angiographically determined coronary artery stenosis is a late-phase phenomenon. The latter is localized and rapidly progressive with plaque rupture and acute thrombosis. Instead of the angiographically determined coronary artery stenosis, we employed myocardial flow reserve (MFR) that reflects diffuse early-phase coronary atherosclerosis and impaired coronary vasodilatation function. We evaluated the relationship between the IMT and the MFR. Methods: Twenty-three patients with angiographically diagnosed coronary artery disease (CAD) underwent B-mode ultrasound examination to measure their common carotid IMT and positron emission tomography (PET) with dipyridamole intervention to obtain their MFR. We also performed B-mode ultrasound examination in 21 patients with hypertension without CAD and in 15 control subjects. Results: The common carotid IMT in patients with CAD was thickened (0.92 +/- 0.15 vs. 0.81 +/- 0.14 mm in patients with hypertension (P < 0.05) and 0.69 +/- 0.13 mm in control subjects (P < 0.01)), The IMT was inversely correlated with the MFR (r = 0.51, P < 0.01). The correlations between the MFR and most of the coronary risk factors (age, blood pressure, serum cholesterol level and triglyceride level, HbAlc level, smoking index) did not reach statistical significance. Conclusions: Thickened common carotid IMT is also an indicator of reduced MFR or early-phase coronary atherosclerosis. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Although abnormal sarcoplasmic reticulum (SR) Ca2+ handling may cause heart failure, there has been no method to directly measure Ca2+ concentration in SR ([Ca2+](SR)) of living cardiomyocytes. We have measured [Ca2+](SR) by expressing novel fluorescent Ca2+ indicators yellow cameleon (YC) 2.1, YC3er, and YC4er in cultured neonatal rat cardiomyocytes. The distribution of YC2.1 was uniform in the cytoplasm, while that of YC3er/YC4er, containing the signal sequence which recruits them to SR, showed reticular pattern and was co-localized with SERCA2a. The treatment with caffeine reversibly decreased the emission ratio (R) in YC3er/YC4er-expressing myocytes, and the treatment with ryanodine and thapsigargin decreased R irreversibly. During the contraction-relaxation cycle, R was changed periodically in the YC2.1- and Mer-expressing myocytes, but its direction of the change was opposite. These results suggest that YC3er/YC4er were specifically localized and functioned in SR as a [Ca2+](SR) indicator. This technique would be useful to understand the function of SR in failing myocardium. (C) 2004 Elsevier Inc. All rights reserved.

The transcription factor Krüppel-like factor 5 (KLF5) and its genetically downstream target gene platelet-derived growth factor-A (PDGF-A) chain are key factors in regulation of cardiovascular remodeling in response to stress. We show that KLF5 mediates a novel distinct delayed persistent induction of PDGF-A chain in response to the model agonist, phorbol ester, through a cis-element previously shown to mediate phorbol ester induction on to PDGF-A chain through the early growth response factor (Egr-1). Interestingly, the nuclear factor-kappaB (NF-kappaB) p50 subunit further cooperatively activates PDGF-A chain through protein-protein interaction with KLF5 but not Egr-1. RNA interference analysis confirmed that KLF5 and p50 are important for induction of PDGF-A chain. Collectively, we identify a novel regulatory pathway in which PDGF-A chain gene expression, under the control of KLF5, is cooperatively activated by the NF-kappaB p50 subunit and a pathophysiological stimulus.

Azelnidipine, a newly-developed Class III dihydropyridine Ca 2+ channel blocker, has cardiovascular protective effects such as antioxidant and antiatherosclerotic effects. The effects of azelnidipine on NO production were investigated in cultured rat aortic smooth muscle cells (rat ASMCs) isolated from Wistar rats and human umbilical vein endothelial cells (HUVECs). The amounts of NO released and stable metabolite nitrite were measured by oxyhemoglobin and the Griess reaction, respectively. The release of LDH from cells was measured to evaluate cell damage. Immunocytochemical analysis was used to evaluate the expression of iNOS and nitrotyrosine protein. Azelnidipine treatment inhibited lipopolysaccaride (LPS) plus interferon-ganma (INF)-induced NO production in a concentration-dependent manner in ASMCs. Azelnidipine had the most potent inhibitory effects among the various Ca 2+-blocking drugs tested (nifedipine, nicardipine, amlodipine, verapamil and diltiazem). Azelnidipine caused less inhibition of NO production already induced by LPS plus INF. Long-term treatment with LPS plus INF induced the expression of iNOS and nitrotyrosine with increased LDH release from cells. Azelnidipine and N-nitro-L-arginine methyl ester hydrochloride, a NOS inhibitor, suppressed such expression. On the other hand, azelnidipine potentiated NO production induced by eNOS in HUVECs. These results suggest that azelnidipine is a potent inhibitor of the induction of iNOS and reduces NO production in vascular smooth muscle cells. The cardiovascular protective effect of azelnidipine is probably mediated by suppression of peroxynitrite production and subsequent cell damage caused by excessive NO production.

骨髄由来血管前駆細胞が傷害後の血管に定着して新規病変形成に関与していることを報告した.今回,進行した高度血管病変のリモデリングに関与する細胞の起源とその動態の制御機構を検討した.骨髄由来前駆細胞は,高度に進行した血管や弁の病変リモデリングに関与していると思われた.よって,薬物療法の前駆細胞への効果が示唆された

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes(e.g. arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, short lifespan). We have shown that mice deficient for the klotho gene show endothelial dysfunction as manifested by an attenuated response of aortic relaxation in response to acetylcholine stimulation. Nitric oxide production was also significantly reduced in klotho deficient mice. A decrease in klotho gene expression in animals under sustained circulatory and metabolic stress(e.g. atherosclerosis). The klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function(e.g. hypertension, vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease.

Background: Angiotensin II infusion into rats causes iron deposition in the kidney, which may augment the proproteinuric effects of this octapeptide. We have investigated whether administration of iron mimics the renal damage induced by angiotensin II. Methods: Rats were treated with iron dextran at a total dose of 960 mg/kg either with or without angiotensin II treatment at a dose of 0.7 mg/kg/day for 7 days. Protein expression of ferritin and heme oxygenase-1, an oxidative stress-sensitive gene, was determined by Western blot analysis and immunohistochemistry. Results: Administration of iron dextran did not significantly increase proteinuria or decrease creatinine clearance in the rats with or without angiotensin II treatment. Prussian blue staining showed that iron deposition was observed mainly in the glomerular and medullar regions in the iron dextran-treated rats, but in the tubular epithelial cells in angiotensin IIinfused rats. Administration of iron dextran upregulated ferritin, but not heme oxygenase-1. Conclusion: Iron dextran did not enhance or cause the renal dysfunction in the angiotensin II-treated or untreated rats, respectively. The distribution of deposited iron and presumably the type of iron compound administered may be important determinants of the development of renal injury. Copyright © 2004 S. Karger AG, Basel.

In order to study the mechanical activity of a single cardiac myocyte under a wide range of load, we have developed a novel force measurement system using carbon fibers. Newly fabricated Graphite Reinforced by Carbon (GRC) fibers greatly facilitate the firm attachment of cell membrane to the fibers. A pair of fibers was attached to both ends of the cell the rigid fiber as a mechanical ground and the compliant fiber for the strain gauge. By connecting the compliant fiber to the piezoelectric translator and applying the position signal to the driver, we could make the myocyte contract under isometric condition. Feedback control of the system also enabled us to study the relation between work output and the load. This system can be a useful tool in studying the mechanical activity of the cardiac myocyte under genetic as well as pharmacological interventions.

An asymptomatic 35 year-old man was referred to our hospital because of abnormal ECG findings. The ECG showed complete right bundle branch block and left anterior hemiblock. Echocardiography revealed a moderately enlarged right ventricle (RV) and an apical aneurysm. RV wall motion showed diffusely moderate impairment, while the systolic function of the left ventricle (LV) was slightly decreased. The ejection fractions (EF) of the RV and LV were calculated as 28.1% and 41.9% by Simpson's method using multiple cardiac computed tomography (CT) scans. A 24 hour ambulatory ECG showed only 372 single premature ventricular contractions (PVC). Cardiac catheterizaion revealed that the RV was enlarged with prominent trabeculation and decreased motion. In an electrophysiologic study, neither electrical stimulation of the RV nor electrical stimulation plus isoproterenol infusion could induce ventricular tachycardia. Pathological examination of a biopsy from the interventricular septum of the RV revealed fibrofatty change in the myocardium. Based on these results, we made a diagnosis of arrhythmogenic right ventriclular cardiomyopathy (ARVC) and administered 5 mg of carvedilol. Sixty days after the initiation of carvedilol therapy, we performed repeat cardiac CT. The EF of the LV was markedly improved from 41.9% to 62.0%, although the EF of the RV was not changed. The number of PVCs showed no change. This case suggests that carvedilol is not only useful for controlling arrhythmia but also for improving left ventriclular function in some patients with ARVC. Sympathetic overactivity is reported to cause sudden death, so carvedilol may be a first-line drug for some patients with ARVC.

Nowadays, evidence-based medicine has entered the mainstream of clinical judgement and the human genome has been completely decoded. Even the concept of individually designed medicine, that is, tailor-made medicine, is now being discussed. Due to their complexity, however, management methods for clinical information have yet to be established. We have conducted a study on a universal technique which enables one to select or produce by employing information processing technology clinical findings from various clinical information generated in vast quantity in day-to-day clinical practice, and to share such information and/or the results of analysis between two or more institutions. In this study, clinically useful findings have been successfully obtained by systematizing actual clinical information and genomic information obtained by an appropriate collecting and management method of information with due consideration to ethical issues. We report here these medical achievements as well as technological ones which will play a role in propagating such medical achievements. Copyright © 2004 by the Japanese Heart Journal.

To further investigate the immunological mechanisms involved, we analyzed the expression of costimulatory molecules in aortic tissue and their counterpart molecules on infiltrating cells of patients with Takayasu's arteritis. We also examined the expression of major histocompatibility complex (MHC) class I chain-related (MIC) A in aortic tissue, which is known to be induced by external stress, and its counterpart NKG2D receptors on infiltrating cells. Among these costimulatory molecules, strong expression of 4-1BBL and Fas was induced in the aortic tissue, and most of the infiltrating cells expressed 4-1BB and FasL, suggesting these pathways play critical roles in T-cell-mediated vascular injury. We also found that MICA was strongly induced in the aortic tissue and that at least part of the infiltrating cells expressed NKG2D receptors. Some infiltrating cells - but not vascular smooth muscle cells - seemed to have undergone apoptosis. Our findings strongly suggest that 4-1BB/4-1BBL and Fas/FasL pathways play important roles in vascular injury in Takayasu's arteritis. We assume that γδ T cells infiltrated aortic tissue recognizing MICA, resulting in the induction of MHC antigens and costimulatory molecules, and then αβ T-cells infiltrated recognizing some autoantigens presented by MHC antigens, leading to chronic inflammation. Copyright © 2004 S. Karger AG, Basel.

Genome-wide scanning is a powerful tool to identify susceptible chromosome loci, however, individual chromosomal regions still have many candidate genes. Although cDNA microarray analysis provides valuable information for identifying genes involved in pathogenesis, expression levels of many genes are changed. A novel approach for identification of therapeutic targets is the combination of genome-wide scanning and the use of DNA chips, as shown in Fig. (1). Using DNA chips, we screened for secreted molecules, the expressions of which were changed in adipose tissues from mice rendered insulin resistance. Decreased expression of one of these molecules, adiponectin/Acrp30, correlates strongly with insulin resistance. Interestingly, recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where adiponectin gene is located. Decreasing serum adiponectin levels are associated with increased risk for type 2 diabetes. Interestingly, adiponectin was decreased in insulin resistant rodent models both of obesity and lipoatrophy, and replenishment of adiponectin ameliorated their insulin resistance. Moreover, adiponectin transgenic mice ameliorated insulin resistance and diabetes Adiponectin knockout mice showed insulin resistance and glucose intolerance. In muscle and liver, adiponectin activated AMP kinase and PPARalpha pathways thereby increasing beta-oxidation of lipids, leading to decreased TG content, which ameliorated insulin resistance under a high-fat diet. Despite similar plasma glucose and lipid levels on an apoE deficient background, adiponectin transgenic apoE deficient mice showed amelioration of atherosclerosis, which was associated with decreased expressions of class A scavenger receptor and tumor necrosis factor alpha. Finally, cDNA encoding adiponectin receptors (AdipoR1 and R2) have been identified by expression cloning, which facilitates the understanding of molecular mechanisms of adiponectin actions and obesity-linked diseases such as diabetes and atherosclerosis and the designing of novel antidiabetic and anti-atherogenic drugs with AdipoR1 and R2 as molecular targets.

Objective: To investigate the functional role of myosin light chain (MLC) isoforms in cardiac muscles, we examined the motor function of two different myosins the structure of which differed only in the MLC. Methods: We purified myosin from atria (A-myosin) and ventricles (V-myosin) of young rats, which contained atrial-type and ventricular-type MLCs, respectively, but having identical α-heavy chain isoform. Actin filament velocity (Vel) was determined in the in vitro motility assay. Average force of myosin molecules (F) was estimated and single events of actin-myosin interaction were recorded with the laser trap technique. Results: Vel was slightly higher in A-myosin than in V-myosin, while actin-activated ATPase activity was not different. F, determined from force versus actin filament length relation, was ∼60% higher in V-myosin (3.3 vs. 2.1 pN/μm). The mean duration of isometric force events was longer in V-myosin than in A-myosin (323±13 vs. 294±30 ms, p&lt 0.05), while the amplitudes of unitary displacement and force of a single myosin molecule did not differ between them. Conclusion: The MLC isoform can be a determinant of force-generating ability of cardiac myosin by modulating crossbridge kinetics without affecting the catalytic activity. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Here we show a novel pathway of transcriptional regulation of a DNA-binding transcription factor by coupled interaction and modification (e.g., acetylation) through the DNA-binding domain (DBD). The oncogenic regulator SET was isolated by affinity purification of factors interacting with the DBD of the cardiovascular transcription factor KLF5. SET negatively regulated KLF5 DNA binding, transactivation, and cell-proliferative activities. Down-regulation of the negative regulator SET was seen in response to KLF5-mediated gene activation. The coactivator/acetylase p300, on the other hand, interacted with and acetylated KLF5 DBD, and activated its transcription. Interestingly, SET inhibited KLF5 acetylation, and a nonacetylated mutant of KLF5 showed reduced transcriptional activation and cell growth complementary to the actions of SET. These findings suggest a new pathway for regulation of a DNA-binding transcription factor on the DBD through interaction and coupled acetylation by two opposing regulatory factors of a coactivator/acetylase and a negative cofactor harboring activity to inhibit acetylation.

Matrix metalloproteinases (MMPs) are involved in plaque rupture, which is the main pathological cause of myocardial infarction (MI). Recently, several genetic studies have demonstrated that MMP-1 1G/2G polymorphism and MMP-3 5A/6A polymorphism modify each transcriptional activity in allele specific manners. Within this context, we conducted case-control studies to examine whether these genetic polymorphisms are associated with susceptibility to MI. Two groups comprising patients with MI (group-1 164 patients, group-2 302 patients) were compared with control group comprising 335 patients without cardiovascular diseases. The MMP-3 5A allele was more frequent in patients with MI than in the control subjects (P=0.018 MI group-1, P=0.0059 MI group-2), whereas there was no disease association for MMP-1 genotypes. Logistic regression analyses revealed that MMP-3 5A/6A polymorphism was associated with susceptibility to MI [odds ratio(OR) (95% confidential interval) 1.67 (1.02-2.74); P=0.042, MI group-1; 1.61 (1.12-2.23); P=0.0095, MI group-2]. Other important findings were that there was strong linkage disequilibrium between these polymorphisms, which are located closely on chromosome 11q.22, and that the 5A-1G haplotype was a genetic risk factor for MI (OR 1.97 P=0.0082, MI group-1 OR 1.51 P=0.017, MI group-2). Taken together, the present findings suggest that genetic variations in these MMP genes and especially their haplotype may be useful genetic markers for determining susceptibility to MI in Japanese.

Carvedilol has hypotensive effects and inhibits agonist-induced cell proliferation of vascular smooth muscle and then prevents vascular remodeling. However, the basic mechanisms have not been clarified. We examined the effects of carvedilol on [Ca2+]i mobilization and voltage-dependent L-type Ca2+ current (ICa.L) in vascular smooth muscle cells, and compared them with metoprolol. [Ca2+]i was measured using fura-2 AM and patch clamp techniques in rat embryonic aortic smooth muscle cells (A7r5). In the presence of extracellular Ca2+, vasopressin and endothelin-1 increased [Ca2+]i due first to the Ca2+ release from store sites, and subsequently Ca2+ entry. Carvedilol did not inhibit the Ca2+ release, but significantly suppressed the sustained rise due to Ca2+ entry concentration-dependently. Nilfedipine and nicardipine (10 microM) partly inhibited the sustained rise, but carvedilol inhibited it more effectively than the Ca2+ channel blockers. Under voltage clamp conditions, carvedilol (0.2-10 microM) reversibly inhibited the ICa.L concentration-dependently without any changes in the current-voltage relationships of ICa.L. Carvedilol shifted the steady-state inactivation for ICa.L to more negative potentials and inhibited ICa.L in a voltage-dependent manner. In addition, carvedilol did not inhibit Ca2+ release from store sites induced by thapsigargin, but significantly inhibited the sustained rise due to capacitative Ca2+ entry unrelated to ICa.L. In contrast, metoprolol did not mimic these effects of carvedilol. These results provide evidence that carvedilol inhibits ICa.L and may also inhibit the channels for agonist (vasopressin and endothelin-1)-induced Ca2+ entry in vascular smooth muscle cells, which might contribute to the vasorelaxing and antiproliferative effects of carvedilol.

Obesity and insulin resistance have been recognized as leading causes of major health issues. We have endeavored to depict the molecular mechanism of insulin resistance, focusing on the function of adipocyte. We have investigated a role of PPARgamma on the pathogenesis of Type II diabetes. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. Moreover, a Pro12Ala polymorphism in the human PPARgamma2 gene was associated with decreased risk of Type II diabetes in Japanese. Taken together with these results, PPARgamma is proved to be a thrifty gene mediating Type II diabetes. Pharmacological inhibitors of PPARgamma/RXR ameliorate high-fat diet-induced insulin resistance in animal models of Type II diabetes. We have performed a genome-wide scan of Japanese Type 2 diabetic families using affected sib pair analysis. Our genome scan reveals at least 9 chromosomal regions potentially harbor susceptibility genes of Type II diabetes in Japanese. Among these regions, 3q26-q28 appeared to be very attractive one, because of the gene encoding adiponectin, the expression of which we had found enhanced in insulin-sensitive PPARgamma-deficient mice. Indeed, the subjects with the G/G genotype of SNP276 in the adiponectin gene were at increased risk for Type II diabetes compared with those having the T/T genotype. The plasma adiponectin levels were lower in the subjects with the G allele, suggesting that genetically inherited decrease in adiponectin levels predispose subjects to insulin resistance and Type II diabetes. Our work also confirmed that replenishment of adiponectin represents a novel treatment strategy for insulin resistance and Type II diabetes using animal models. Further investigation will be needed to clarify how adiponectin exerts its effect and to discover the molecular target of therapies.

Objective - Ephrin-B2 plays a key role in vascular development. The purpose of this study was to elucidate the molecular mechanisms of ephrin-B2 signaling through the EphB receptor in endothelial cells and to determine whether ephrin-B2 contributes to in vivo angiogenesis in adult mice. Methods and Results - A chemotaxis assay on a polycarbonate membrane revealed that ephrin-B2/Fc chimeric protein induced migration of human umbilical vein endothelial cells (HUVECs) at a level 98% greater than control (P&lt 0.01). To determine the signaling pathways activated in the HUVECs by Eph stimulation, phosphatidylinositol-3 kinase (PI3 kinase) activity was determined in an immune complex PI3 kinase assay. Serum-starved HUVECs were stimulated with ephrin-B2/ Fc and compared with unstimulated cells. PI3 kinase activity in stimulated cells was higher than that seen in unstimulated cells. In a chemotaxis assay, the PI3 kinase-specific inhibitor LY294002 blocked the migratory response of HUVECs induced by addition of ephrin-B2/Fc. Finally, ephrin-B2/Fc promoted angiogenesis in vivo in corneal neovascularization and Matrigel plug assays in adult mice, whereas LY294002 reduced angiogenesis in Matrigel that was induced by ephrin-B2/Fc. Conclusions - Ephrin-B2/Fc induces the migration of HUVECs through the PI3 kinase signaling pathway. Ephrin-B2/Fc promotes in vivo angiogenesis in adult mice, suggesting that it contributes to adult angiogenesis.

Solid tumors require vascularization for their growth. Bone marrow-derived endothelial progenitor cells participate in tumor angiogenesis. Here, we show that nicotine markedly accelerated growth of colon cancer cells inoculated subcutaneously in mice but had no effect on proliferation of carcinoma cells in vitro. We found that the tumor growth was associated with increased vascularization of the tumor and that bone marrow-derived cells contributed to the formation of the new blood vessels. Our findings show that nicotine promotes tumor growth, at least in part, by stimulating tumor-associated neovascularization.

We previously demonstrated that Irs2-/- mice develop diabetes due to beta-cell growth failure and insulin resistance; however, glucose-induced insulin secretion was increased in islets isolated from Irs2-/- mice. Pdx-1, a transcription factor important for maintenance of the beta-cell function, was recently reported to be severely reduced in Irs2-/- murine beta-cells. We report herein that Pdx-1 expression, including the amount of Pdx-1 localized in the nucleus, is not down-regulated in our Irs2-/- murine beta-cells with a C57BL/6 background. We have also demonstrated the expression of upstream genes of Pdx-1, such as HNF3beta and HNF1alpha, as well as its downstream genes, including insulin, Glut2, and Nkx6.1, to be well preserved. We have further demonstrated Pdx-1 expression to also be preserved in beta-cells of 30-week-old diabetic Irs2-/- mice. In addition, surprisingly, even in Irs2-/- mice on a high fat diet with markedly elevated blood glucose, exceeding 400 mg/dl, Pdx-1 expression was not reduced. Furthermore, we found Pdx-1 to be markedly decreased in certain severely diabetic Irs2-/- mice with a mixed C57BL/6J x 129Sv background. We conclude that 1) Pdx-1 expression in Irs2-/- mice is regulated in a strain-dependent manner, 2) Irs2-/- mice develop diabetes associated with beta-cell growth failure even when Pdx1 expression is preserved, and 3) Pdx-1 expression is preserved in severely hyperglycemic Irs2-/- mice with a C57BL/6 background on a high fat diet.

We and others have suggested that bone marrow-derived progenitor cells may contribute to the pathogenesis of vascular diseases. On the other hand, it was reported that bone marrow cells do not participate substantially in vascular remodeling in other experimental systems. In this study, three distinct types of mechanical vascular injuries were induced in the same mouse whose bone marrow had been reconstituted with that of GFP or LacZ mice. All injuries are known to cause smooth muscle cell (SMC) hyperplasia. At 4 weeks after wire-mediated endovascular injury, a significant number of the neointimal and medial cells derived from bone marrow. In contrast, marker-positive cells were seldom detected in the lesion induced by perivascular cuff replacement. There were only a few bone marrow-derived cells in the neointima after ligation of the common carotid artery. These results indicate that the origin of intimal cells is diverse and that contribution of bone marrow-derived cells to neointimal hyperplasia depends on the type of model.

Therapeutic angiogenesis has emerged as a promising therapy, but some patients are refractory to exogenous growth factors. In order to identify the genetic determinants of post-natal angiogenesis and physiological vessel formation, we investigated the genetic factors that affected ischemia-induced development of collaterals in mice. An ischemic hindlimb model was generated in C57BL/6, C3H/He, and BALB/c mice. Angiogenesis was markedly different among the mice as determined by the restoration of blood perfusion and capillary density of the ischemic muscle. Impaired collateral vessel formation in BALB/c mice was associated with reduced expression of vascular endothelial cell growth factor (VEGF). Intramuscular gene transfer of VEGF promoted collateral formation in C57BL/6J mice, but not in BALB/c mice. Ineffectiveness of VEGF in BALB/c mice was associated with impaired expression of VEGF receptor. Our findings suggest that genetic background may influence spontaneous collateral formation and therapeutic effectiveness of exogenous VEGF. Alternative strategies other than administration of VEGF alone might be needed to attain optimal angiogenesis in some patients.