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Ayako Takahashi,
Masanori Asakura,
Shin Ito,
Kyung-Duk Min,
Kazuhiro Shindo,
Yi Yan,
Yulin Liao,
Satoru Yamazaki,
Shoji Sanada,
Yoshihiro Asano,
Hatsue Ishibashi-Ueda, Seiji Takashima,
Tetsuo Minamino,
Hiroshi Asanuma,
Naoki Mochizuki,
Masafumi Kitakaze
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ABSTRACT: Incretin hormones-including glucagon-like peptide-1 (GLP-1), a target for diabetes mellitus (DM) treatment-are associated with cardioprotection. As dipeptidyl peptidase IV (DPP-IV) inhibition increases plasma GLP-1 levels in vivo, we investigated the cardioprotective effects of the DPP-IV inhibitor vildagliptin in a murine heart failure (HF) model. We induced transverse aortic constriction (TAC) in C57BL/6J mice, simulating pressure-overloaded cardiac hypertrophy and HF. TAC or sham-operated mice were treated with or without vildagliptin. An intraperitoneal glucose tolerance test revealed that blood glucose levels were higher in the TAC than in sham-operated mice, and these levels improved with vildagliptin administration in both groups. Vildagliptin increased plasma GLP-1 levels in the TAC mice and ameliorated TAC-induced left ventricular enlargement and dysfunction. Vildagliptin palliated both myocardial apoptosis and fibrosis in TAC mice, demonstrated by histological, gene and protein expression analyses, and improved survival rate on day 28 (TAC with vildagliptin, 67.5%; TAC without vildagliptin, 41.5%; P < 0.05). Vildagliptin improved cardiac dysfunction and overall survival in the TAC mice, both by improving impaired glucose tolerance and by increasing GLP-1 levels. DPP-IV inhibitors represent a candidate treatment for HF patients with or without DM.
AJP Heart and Circulatory Physiology 03/2013; · 3.71 Impact Factor
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Hatasu Kobayashi,
Satoru Yamazaki, Seiji Takashima,
Wanyang Liu,
Hiroko Okuda,
Junxia Yan,
Yukiko Fujii,
Toshiaki Hitomi,
Kouji H Harada,
Toshiyuki Habu,
Akio Koizumi
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ABSTRACT: Moyamoya disease (MMD) and moyamoya syndrome are vasculopathies characterized by progressive stenosis in the circle of Willis and its branches. The RNF213 gene, which encodes a novel class of proteins, characterized by both E3 ligase and AAA+ATPase activities, has been identified as the susceptibility gene for MMD. However, its physiological functions remain unknown. MMD and moyamoya syndrome are often accompanied by diabetes mellitus. In this study, we generated Rnf213 knockout (KO) C57BL/6 mice (Rnf213-/-; Ins2+/+), which were mated with Akita (C57BL/6 Rnf213+/+; Ins2+/C96Y) mice, a strain that develops diabetes spontaneously by 5weeks of age, to obtain mice lacking Rnf213 and carrying the Akita mutation (KO/Akita, Rnf213-/-; Ins2+/C96Y). Body weight and blood glucose concentration were measured from 6 to 20weeks. Glucose tolerance, insulin resistance, plasma insulin and leptin concentrations, food consumption, pancreatic insulin content and histopathology were evaluated at 18weeks of age. We found that glucose tolerance, as indicated by AUC, was 20% lower (p<0.05) and insulin contents in pancreas were 150% higher (p<0.05), in KO/Akita than in Akita mice. The number of CHOP positive β-cells assayed by histopathological examination was 30% lower and food consumption was 34% lower in KO/Akita than in Akita mice (p<0.05 each). These findings indicated that the disruption of Rnf213 improved glucose tolerance by protecting islet β cells.
Biochemical and Biophysical Research Communications 02/2013; · 2.48 Impact Factor
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Shinichiro Suna,
Yasuhiko Sakata,
Daisaku Nakatani,
Keiji Okuda,
Masahiko Shimizu,
Masaya Usami,
Sen Matsumoto,
Masahiko Hara,
Kouichi Ozaki,
Hiroya Mizuno,
Tetsuo Minamino, Seiji Takashima,
Masami Nishino,
Yasushi Matsumura,
Hiroshi Takeda,
Toshihiro Tanaka,
Hiroshi Sato,
Masatsugu Hori,
Issei Komuro
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ABSTRACT: AIMS: We previously reported the association of single nucleotide polymorphisms in the lymphotoxin alpha (LTα) gene with susceptibility to acute myocardial infarction (AMI) and increased mortality after discharge. In the present study, we investigated whether the adverse effect of LTα C804A polymorphism on mortality could be pharmacologically modified by statin treatment after AMI. METHODS AND RESULTS: We conducted a multicenter study that included 3486 post-AMI patients between 1998 and 2008. During a median follow-up period of 1775 days, 247 deaths were recorded. The mortality rate was significantly higher in LTα 804A allele carriers compared to non-804A allele carriers (7.9% vs. 5.7%, p = 0.011). The LTα 804A allele was significantly associated with increased mortality for post-AMI patients not receiving statins (hazard ratio [HR]: 1.48, 95% confidence interval [CI]: 1.03-2.12, p = 0.034), but not for those receiving statins (HR: 1.22, 95% CI: 0.70-2.10, p = 0.486). In-vitro experimental analyses demonstrated that the LTα 804A polymorphic protein, 26Asn-LTα(3), induced monocyte-endothelial interaction and endoplasmic reticulum (ER) stress in cardiomyocytes more strongly than the LTα(3) 804C polymorphic protein 26Thr-LTα(3). However, the effects of both LTα(3) proteins were decreased and became comparable by the pretreatment of cells with pravastatin. CONCLUSION: LTα C804A polymorphism was associated with an increased risk of mortality for AMI patients, although this effect was masked in patients treated with statins. This finding is supported by the observed attenuation of 26Asn-LTα(3)-mediated monocyte-endothelial interaction and ER stress in cardiomyocytes treated with pravastatin. LTα C804A polymorphism may have potential as a novel therapeutic target for secondary prevention after AMI.
Atherosclerosis 01/2013; · 3.79 Impact Factor
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ABSTRACT: The prognoses for patients with certain diseases are estimated by averaging the results of clinical trials. To investigate the possibility of deriving a mathematical formula for the estimation of prognosis, we formulated the equation τ=f(x(1), …, x(p)), where x(1), …, x(p) are clinical features and τ represents the clinical outcome for heart failure (HF). We attempted to determine the function to mathematically formulate the relationship between clinical features and outcomes for these patients. We followed 151 patients (mean age: 68.6±14.6 years; men: 61.6%) who were consecutively hospitalized and discharged as a result of acute decompensated HF (ADHF) between May 2006 and December 2009. The mathematical analysis was performed through a probabilistic modeling of the relational data by assuming a Poisson process for rehospitalization owing to HF and by linearly approximating the relationship between the clinical factors and the mean elapsed time to rehospitalization. The former assumption was validated by a statistical test of the data, and the contribution of each parameter was assessed based on the coefficients of the linear relation. Using a regularization method to analyze 402 clinical parameters, we identified 252 factors that substantially influenced the elapsed time until rehospitalization. With the probability model based on the Poisson process, the actual (X; 388±377 days) and estimated (Y; 398±381 days) elapsed times to rehospitalization were tightly correlated (Y=1.0076X+6.5531, R(2)=0.9879, P<0.0001). We established a mathematical formula that closely predicts the clinical outcomes of patients who are hospitalized with ADHF and discharged after appropriate treatment.Hypertension Research advance online publication, 20 December 2012; doi:10.1038/hr.2012.200.
Hypertension Research 12/2012; · 2.58 Impact Factor
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ABSTRACT: Adenosine monophosphate-activated protein kinase (AMPK), a metabolic protein kinase, and its upstream kinase LKB1 play crucial roles in the establishment and maintenance of cell polarity. Although the shapes of polarized cells display extraordinary diversity, the key molecules involved in cell polarity are relatively well conserved. Here, we review the mechanisms and factors responsible for organizing cell polarity and the role of LKB1 and AMPK in cell polarity.
Genes to Cells 08/2012; 17(9):737-47. · 2.68 Impact Factor
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ABSTRACT: BACKGROUND: The endocannabinoid system is known to play a role in regulating myocardial contractility, but the influence of cannabinoid receptor 1 (CB1) deficiency on chronic heart failure (CHF) remains unclear. In this study we attempted to investigate the effect of CB1 deficiency on CHF induced by pressure overload and the possible mechanisms involved. METHODS AND RESULTS: A CHF model was created by transverse aortic constriction (TAC) in both CB1 knockout mice and wild-type mice. CB1 knockout mice showed a marked increase of mortality due to CHF from 4 to 8weeks after TAC (p=0.021). Five weeks after TAC, in contrast to wild-type mice, CB1 knockout mice had a higher left ventricular (LV) end-diastolic pressure, lower rate of LV pressure change (±dp/dt max), lower LV contractility index, and a larger heart weight to body weight ratio and lung weight to body weight ratio compared with wild-type mice (all p<0.05-0.001). Phosphorylation of the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (P38 and ERK) was higher in CB1 knockout mice than that in wild-type mice. In cultured neonatal rat cardiomyocytes, a CB1 agonist reduced cAMP production stimulated by isoproterenol or forskolin, and suppressed phosphorylation of the EGFR, P38, and ERK, while the inhibitory effect of a CB1 agonist on EGFR phosphorylation was abrogated by CB1 knockdown. CONCLUSION: These findings indicate that cannabinoid receptor 1 inactivation promotes cardiac remodeling by enhancing the activity of the epidermal growth factor receptor and mitogen-activated protein kinases.
International journal of cardiology 05/2012; · 7.08 Impact Factor
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Akemi Yoshida,
Hiroshi Asanuma,
Hideyuki Sasaki,
Shoji Sanada,
Satoru Yamazaki,
Yoshihiro Asano,
Yoshiro Shinozaki,
Hidezo Mori,
Akito Shimouchi,
Motoaki Sano,
Masanori Asakura,
Tetsuo Minamino, Seiji Takashima,
Masaru Sugimachi,
Naoki Mochizuki,
Masafumi Kitakaze
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ABSTRACT: Inhalation of hydrogen (H(2)) gas has been shown to limit infarct size following ischemia-reperfusion injury in rat hearts. However, H(2) gas-induced cardioprotection has not been tested in large animals and the precise cellular mechanism of protection has not been elucidated. We investigated whether opening of mitochondrial ATP-sensitive K+ channels (mK(ATP)) and subsequent inhibition of mitochondrial permeability transition pores (mPTP) mediates the infarct size-limiting effect of H(2) gas in canine hearts.
The left anterior descending coronary artery of beagle dogs was occluded for 90 min followed by reperfusion for 6 h. Either 1.3% H(2) or control gas was inhaled from 10 min prior to start of reperfusion until 1 h of reperfusion, in the presence or absence of either 5-hydroxydecanoate (5-HD; a selective mK(ATP) blocker), or atractyloside (Atr; a mPTP opener).
Systemic hemodynamic parameters did not differ among the groups. Nevertheless, H(2) gas inhalation reduced infarct size normalized by risk area (20.6±2.8% vs. control gas 44.0±2.0%; p<0.001), and administration of either 5-HD or Atr abolished the infarct size-limiting effect of H(2) gas (42.0±2.2% with 5-HD and 45.1±2.7% with Atr; both p<0.001 vs. H(2) group). Neither Atr nor 5-HD affected infarct size per se. Among all groups, NAD content and the number of apoptotic and 8-OHdG positive cells was not significantly different, indicating that the cardioprotection afforded by H(2) was not due to anti-oxidative actions or effects on the NADH dehydrogenase pathway.
Inhalation of H(2) gas reduces infarct size in canine hearts via opening of mitochondrial K(ATP) channels followed by inhibition of mPTP. H(2) gas may provide an effective adjunct strategy in patients with acute myocardial infarction receiving reperfusion therapy.
Cardiovascular Drugs and Therapy 04/2012; 26(3):217-26. · 3.13 Impact Factor
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ABSTRACT: Increased expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) is frequently observed in certain cancers such as ovarian and breast cancers, and this protein is a desirable target for drug delivery by a drug delivery system (DDS). In the present study, we developed novel immunoliposomes targeting HB-EGF for cancer therapy. The immunoliposomes significantly associated with Vero-H cells overexpressing HB-EGF compared with their binding to wild-type Vero cells, whereas liposomes without modification by the antibody did not associate with either type of cells. Moreover, enhanced uptake of the immunoliposomes into Vero-H cells was observed as well as that into MDA-MB-231 human breast cancer cells, which are known to highly express HB-EGF. These results suggest that HB-EGF mediates the binding and uptake of the immunoliposomes in HB-EGF-expressing cells. Next, we determined the therapeutic effect of these immunoliposomes encapsulating an anticancer drug on tumor-bearing mice. For this purpose, we prepared doxorubicin (DOX)-encapsulated immunoliposomes and injected them intravenously into mice bearing MDA-MB-231 cancer cells. As a result, these DOX-encapsulated immunoliposomes suppressed not only tumor progression but also tumor regression. In conclusion, our results indicate that anti-HB-EGF antibody-modified liposomes could be a useful DDS carrier for the treatment of HB-EGF-expressing cancers.
Journal of Controlled Release 10/2011; 160(2):274-80. · 5.73 Impact Factor
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ABSTRACT: Aims We investigated the influence of type one cannabinoid receptor (CB1) deficiency on acute heart failure (AHF) and the underlying mechanism. Acute heart failure syndrome is an important clinical problem because of its high morbidity and mortality rates. Activation of CB1 induces vascular dilation and reinforces the properties of morphine, long-standing therapies for AHF syndrome, but the effect of endogenous CB1 activation on AHF is largely unknown. Methods and results Acute heart failure mouse model characterized by hypertension and pulmonary oedema was created by using transverse aortic constriction (TAC). Mortality, echocardiography, haemodynamic, morphology, and circulatory catecholamine levels in response to TAC were evaluated in CB1 knockout (KO) and wild-type mice. Type one cannabinoid receptor KO mice had a much higher mortality rate at 1 week after TAC attributable to AHF (65 vs. 11%, P< 0.001). One hour after TAC, CB1 KO mice had significant larger lung weight to body weight ratio (LW/BW, 14.53 + 1.09 mg/g in KO vs. 10.42 + 0.36 mg/g in WT, P < 0.01) and higher plasma epinephrine levels (9720 + 1226 pg/mL vs. 6378 + 832 pg/mL, P < 0.05). Pharmacological activation of CB1 reduced LW/BW in wild-type mice. Administration of epinephrine to wild-type TAC mice significantly increased left ventricular end-diastolic pressure and LW/BW, while CB1 agonists reduced the LW/BW and the plasma levels of catecholamine and increased myocardial activity of AMP-activated protein kinase. Conclusion Endogenous activation of CB1 in mice has cardiac protection in AHF, which is attributable to the inhibition of excessive sympathetic activation.
European Heart Journal 07/2011; · 10.48 Impact Factor
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Wanyang Liu,
Daisuke Morito, Seiji Takashima,
Yohei Mineharu,
Hatasu Kobayashi,
Toshiaki Hitomi,
Hirokuni Hashikata,
Norio Matsuura,
Satoru Yamazaki,
Atsushi Toyoda, [......],
Asao Fujiyama,
Roman Herzig,
Boris Krischek,
Liping Zou,
Jeong Eun Kim,
Masafumi Kitakaze,
Susumu Miyamoto,
Kazuhiro Nagata,
Nobuo Hashimoto,
Akio Koizumi
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ABSTRACT: Moyamoya disease is an idiopathic vascular disorder of intracranial arteries. Its susceptibility locus has been mapped to 17q25.3 in Japanese families, but the susceptibility gene is unknown.
Genome-wide linkage analysis in eight three-generation families with moyamoya disease revealed linkage to 17q25.3 (P<10(-4)). Fine mapping demonstrated a 1.5-Mb disease locus bounded by D17S1806 and rs2280147. We conducted exome analysis of the eight index cases in these families, with results filtered through Ng criteria. There was a variant of p.N321S in PCMTD1 and p.R4810K in RNF213 in the 1.5-Mb locus of the eight index cases. The p.N321S variant in PCMTD1 could not be confirmed by the Sanger method. Sequencing RNF213 in 42 index cases confirmed p.R4810K and revealed it to be the only unregistered variant. Genotyping 39 SNPs around RNF213 revealed a founder haplotype transmitted in 42 families. Sequencing the 260-kb region covering the founder haplotype in one index case did not show any coding variants except p.R4810K. A case-control study demonstrated strong association of p.R4810K with moyamoya disease in East Asian populations (251 cases and 707 controls) with an odds ratio of 111.8 (P = 10(-119)). Sequencing of RNF213 in East Asian cases revealed additional novel variants: p.D4863N, p.E4950D, p.A5021V, p.D5160E, and p.E5176G. Among Caucasian cases, variants p.N3962D, p.D4013N, p.R4062Q and p.P4608S were identified. RNF213 encodes a 591-kDa cytosolic protein that possesses two functional domains: a Walker motif and a RING finger domain. These exhibit ATPase and ubiquitin ligase activities. Although the mutant alleles (p.R4810K or p.D4013N in the RING domain) did not affect transcription levels or ubiquitination activity, knockdown of RNF213 in zebrafish caused irregular wall formation in trunk arteries and abnormal sprouting vessels.
We provide evidence suggesting, for the first time, the involvement of RNF213 in genetic susceptibility to moyamoya disease.
PLoS ONE 01/2011; 6(7):e22542. · 4.09 Impact Factor
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Hiroyuki Takahama,
Hiroshi Asanuma,
Shoji Sanada,
Masashi Fujita,
Hideyuki Sasaki,
Masakatsu Wakeno,
Jiyoong Kim,
Masanori Asakura, Seiji Takashima,
Tetsuo Minamino,
Kazuo Komamura,
Masaru Sugimachi,
Masafumi Kitakaze
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ABSTRACT: Histamine has a positive inotropic effect on ventricular myocardium and stimulation of histamine H₂ receptors increases the intracellular cAMP level via Gs protein, as dose stimulation of β-adrenergic receptors, and worsens heart failure. To test whether a histamine H₂ receptor blocker had a beneficial effect in addition to β-adrenergic receptor blockade, we investigated the cardioprotective effect of famotidine, a histamine H₂ receptor blocker, in dogs receiving a β-blocker. We induced heart failure in dogs by rapid ventricular pacing (230 beats/min). Animals received no drugs (control group), famotidine (1 mg/kg daily), carvedilol (0.1 mg/kg daily), or carvedilol plus famotidine. Both cardiac catheterization and echocardiography were performed before and 4 weeks after the initiation of pacing. Immunohistochemical studies showed the appearance of mast cells and histamine in the myocardium after 4 weeks of pacing. In the control group, the left ventricular ejection fraction (LVEF) was decreased after 4 weeks compared with before pacing (71 ± 2 vs. 27 ± 2%, p < 0.05) and mean pulmonary capillary wedge pressure (PCWP) was increased (8 ± 1 vs. 19 ± 3 mmHg). Famotidine ameliorated the decrease of LVEF and increase of PCWP, while the combination of carvedilol plus famotidine further improved both parameters compared with the carvedilol groups. These beneficial effects of famotidine were associated with a decrease of the myocardial cAMP level. Histamine H₂ receptor blockade preserves cardiac systolic function in dogs with pacing-induced heart failure, even in the presence of β-adrenergic receptor blockade. This finding strengthens the rationale for using histamine H₂ blockers in the treatment of heart failure.
Archiv für Kreislaufforschung 11/2010; 105(6):787-94. · 7.35 Impact Factor
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Shuichiro Higo,
Yoshihiro Asano,
Hisakazu Kato,
Satoru Yamazaki,
Atsushi Nakano,
Osamu Tsukamoto,
Osamu Seguchi,
Mitsutoshi Asai,
Masanori Asakura,
Hiroshi Asanuma,
Shoji Sanada,
Tetsuo Minamino,
Issei Komuro,
Masafumi Kitakaze, Seiji Takashima
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ABSTRACT: Three mammalian isoforms of heterochromatin protein 1 (HP1), α, β, and γ, play diverse roles in gene regulation. Despite their structural similarity, the diverse functions of these isoforms imply that they are additionally regulated by post-translational modifications. Here, we have identified intermolecular disulfide bond formation of HP1 cysteines in an isoform-specific manner. Cysteine 133 in HP1α and cysteine 177 in HP1γ were involved in intermolecular homodimerization. Although both HP1α and HP1γ contain reactive cysteine residues, only HP1γ readily and reversibly formed disulfide homodimers under oxidative conditions. Oxidatively dimerized HP1γ strongly and transiently interacted with TIF1β, a universal transcriptional co-repressor. Under oxidative conditions, HP1γ dimerized and held TIF1β in a chromatin component and inhibited its repression ability. Our results highlight a novel, isoform-specific role for HP1 as a sensor of the cellular redox state.
Journal of Biological Chemistry 10/2010; 285(41):31337-47. · 4.77 Impact Factor
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Hai Ying Fu,
Ken-ichiro Okada,
Yulin Liao,
Osamu Tsukamoto,
Tadashi Isomura,
Mitsutoshi Asai,
Tamaki Sawada,
Keiji Okuda,
Yoshihiro Asano,
Shoji Sanada,
Hiroshi Asanuma,
Masanori Asakura, Seiji Takashima,
Issei Komuro,
Masafumi Kitakaze,
Tetsuo Minamino
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ABSTRACT: Apoptosis may contribute to the development of heart failure, but the role of apoptotic signaling initiated by the endoplasmic reticulum in this condition has not been well clarified.
In myocardial samples from patients with heart failure, quantitative real-time polymerase chain reaction revealed an increase in messenger RNA for C/EBP homologous protein (CHOP), a transcriptional factor that mediates endoplasmic reticulum-initiated apoptotic cell death. We performed transverse aortic constriction or sham operation on wild-type (WT) and CHOP-deficient mice. The CHOP-deficient mice showed less cardiac hypertrophy, fibrosis, and cardiac dysfunction compared with WT mice at 4 weeks after transverse aortic constriction, although the contractility of isolated cardiomyocytes from CHOP-deficient mice was not significantly different from that in the WT mice. In the hearts of CHOP-deficient mice, phosphorylation of eukaryotic translation initiation factor 2alpha, which may reduce protein translation, was enhanced compared with WT mice. In the hearts of WT mice, CHOP-increased apoptotic cell death with activation of caspase-3 was observed at 4 weeks after transverse aortic constriction. In contrast, CHOP-deficient mice had less apoptotic cell death and lower caspase-3 activation at 4 weeks after transverse aortic constriction. Furthermore, the Bcl2/Bax ratio was decreased in WT mice, whereas this change was significantly blunted in CHOP-deficient mice. Real-time polymerase chain reaction microarray analysis revealed that CHOP could regulate several Bcl2 family members in failing hearts.
We propose the novel concept that CHOP, which may modify protein translation and mediate endoplasmic reticulum-initiated apoptotic cell death, contributes to development of cardiac hypertrophy and failure induced by pressure overload.
Circulation 07/2010; 122(4):361-9. · 14.74 Impact Factor
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Atsushi Nakano,
Hisakazu Kato,
Takashi Watanabe,
Kyung-Duk Min,
Satoru Yamazaki,
Yoshihiro Asano,
Osamu Seguchi,
Shuichiro Higo,
Yasunori Shintani,
Hiroshi Asanuma,
Masanori Asakura,
Tetsuo Minamino,
Kozo Kaibuchi,
Naoki Mochizuki,
Masafumi Kitakaze, Seiji Takashima
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ABSTRACT: AMP-activated protein kinase (AMPK) is an energy-sensing Ser/Thr protein kinase originally shown to be regulated by AMP. AMPK is activated by various cellular stresses that inhibit ATP production or stimulate ATP consumption. In addition to its role in metabolism, AMPK has recently been reported to reshape cells by regulating cell polarity and division. However, the downstream targets of AMPK that participate in these functions have not been fully identified. Here, we show that phosphorylation of the microtubule plus end protein CLIP-170 by AMPK is required for microtubule dynamics and the regulation of directional cell migration. Both inhibition of AMPK and expression of a non-phosphorylatable CLIP-170 mutant resulted in prolonged and enhanced accumulation of CLIP-170 at microtubule tips, and slower tubulin polymerization. Furthermore, inhibition of AMPK impaired microtubule stabilization and perturbed directional cell migration. All of these phenotypes were rescued by expression of a phosphomimetic CLIP-170 mutant. Our results demonstrate, therefore, that AMPK controls basic cellular functions by regulating microtubule dynamics through CLIP-170 phosphorylation.
Nature Cell Biology 06/2010; 12(6):583-90. · 19.49 Impact Factor
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ABSTRACT: HB-EGF is a member of the EGF family of growth factors that bind and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. ProHB-EGF functions as the receptor for the diphtheria toxin (DT). HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Monoclonal antibodies (mAbs) specific for HB-EGF could be an important tool in HB-EGF research. However, few such mAbs have been established to date. In this study, we newly generated seven clones of hybridoma-derived mAbs by immunizing HB-EGF null mice with recombinant human HB-EGF protein. All mAbs specifically bound to human HB-EGF but not to mouse HB-EGF. Epitope mapping analysis showed that most of the mAbs recognized the EGF-like domain. Although none of the newly isolated mAbs directly inhibited the mitogenic activity of HB-EGF for EGFR-expressing cells, some strongly inhibited DT-binding. Interestingly, some of the mAbs efficiently inhibited ectodomain shedding of proHB-EGF, and consequently prevented the cell growth of the EGFR-expressing cells in a co-culture system with proHB-EGF-expressing cells. Hence, these new anti-HB-EGF mAbs may advance clinical as well as basic research on HB-EGF.
Journal of biochemistry 03/2010; 148(1):55-69. · 1.95 Impact Factor
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Kyung-Duk Min,
Masanori Asakura,
Yulin Liao,
Kenji Nakamaru,
Hidetoshi Okazaki,
Tomoko Takahashi,
Kazunori Fujimoto,
Shin Ito,
Ayako Takahashi,
Hiroshi Asanuma, [......],
Shoji Sanada,
Osamu Seguchi,
Atsushi Nakano,
Yosuke Ando,
Toshiaki Otsuka,
Hidehiko Furukawa,
Tadashi Isomura, Seiji Takashima,
Naoki Mochizuki,
Masafumi Kitakaze
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ABSTRACT: Although various management methods have been developed for heart failure, it is necessary to investigate the diagnostic or therapeutic targets of heart failure. Accordingly, we have developed different approaches for managing heart failure by using conventional microarray analyses. We analyzed gene expression profiles of myocardial samples from 12 patients with heart failure and constructed datasets of heart failure-associated genes using clinical parameters such as pulmonary artery pressure (PAP) and ejection fraction (EF). From these 12 genes, we selected four genes with high expression levels in the heart, and examined their novelty by performing a literature-based search. In addition, we included four G-protein-coupled receptor (GPCR)-encoding genes, three enzyme-encoding genes, and one ion-channel protein-encoding gene to identify a drug target for heart failure using in silico microarray database. After the in vitro functional screening using adenovirus transfections of 12 genes into rat cardiomyocytes, we generated gene-targeting mice of five candidate genes, namely, MYLK3, GPR37L1, GPR35, MMP23, and NBC1. The results revealed that systolic blood pressure differed significantly between GPR35-KO and GPR35-WT mice as well as between GPR37L1-Tg and GPR37L1-KO mice. Further, the heart weight/body weight ratio between MYLK3-Tg and MYLK3-WT mice and between GPR37L1-Tg and GPR37L1-KO mice differed significantly. Hence, microarray analysis combined with clinical parameters can be an effective method to identify novel therapeutic targets for the prevention or management of heart failure.
Biochemical and Biophysical Research Communications 02/2010; 393(1):55-60. · 2.48 Impact Factor
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ABSTRACT: This study was aimed to investigate whether the antihypertrophic effects of adiponectin in murine hearts are associated with the modulation of HB-EGF signaling. We determined the myocardial expressions of adiponectin and adiponectin receptors, brain natriuretic peptide (BNP), and HB-EGF in normal and hypertrophied hearts of adiponectin knockout mice or wild-type mice with transverse aortic constriction (TAC). Then, we observed the effects of adiponectin on cardiac hypertrophy and HB-EGF signaling in cultured neonatal rat cardiomyocytes and whole hearts of adiponectin-null mice. The myocardial mRNA and protein expressions of adiponectin in the hypertrophied hearts were significantly downregulated, and the mRNA expression of adiponectin was inversely correlated with the heart-to-body weight ratio, BNP, and HB-EGF. The TAC-induced cardiac hypertrophy and EGF receptor (EGFR) activation in the adiponectin knockout mice were significantly greater than those in the wild-type mice. Furthermore, in vitro experiments revealed that adiponectin inhibited HB-EGF-stimulated protein synthesis, HB-EGF shedding, and EGFR phosphorylation. We conclude that the inhibition of HB-EGF mediated EGFR activation is one of the alternative mechanisms for the antihypertrophic action of adiponectin.
Biochemical and Biophysical Research Communications 02/2010; 393(3):519-25. · 2.48 Impact Factor
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Tamaki Sawada,
Tetsuo Minamino,
Hai Ying Fu,
Mitsutoshi Asai,
Keiji Okuda,
Tadashi Isomura,
Satoru Yamazaki,
Yoshihiro Asano,
Ken-ichiro Okada,
Osamu Tsukamoto,
Shoji Sanada,
Hiroshi Asanuma,
Masanori Asakura, Seiji Takashima,
Masafumi Kitakaze,
Issei Komuro
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ABSTRACT: The unfolded protein response (UPR) is triggered to assist protein folding when endoplasmic reticulum (ER) function is impaired. Recent studies demonstrated that ER stress can also induce cell-specific genes. In this study, we examined whether X-box binding protein 1 (XBP1), a major UPR-linked transcriptional factor, regulates the expression of brain natriuretic peptide (BNP) in cardiomyocytes. In samples from failing human hearts, extensive splicing of XBP1 was observed along with increased expression of glucose-regulated protein of 78 kDa (GRP78), a target of spliced XBP1 (sXBP1), suggesting that the UPR was induced in heart failure in humans. Interestingly, quantitative real-time PCR revealed a positive correlation between cardiac expression of GRP78 and BNP, leading us to test the hypothesis that sXBP1 regulates BNP as well as GRP78 in cardiomyocytes. A pharmacological ER stressor caused a dose-dependent increase in the expression of sXBP1 and BNP by cultured cardiomyocytes. Short interfering RNA targeting XBP1 suppressed the induction of BNP expression by a pharmacological ER stressor or norepinephrine, which was rescued by the adenovirus-mediated overexpression of sXBP1. The promoter assay with overexpression of sXBP1 or norepinephrine showed that the proximal AP1/CRE-like element in the promoter region of BNP was critical for transcriptional regulation of BNP by sXBP1. Direct binding of sXBP1 to this element was confirmed by the chromatin immunoprecipitation assay. These findings suggest that ER stress observed in failing hearts regulates cardiac BNP expression through a novel promoter region of the AP1/CRE-like element.
Journal of Molecular and Cellular Cardiology 02/2010; 48(6):1280-9. · 5.17 Impact Factor
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ABSTRACT: Neuropilin-1 (NRP1) is a multifunctional transmembrane protein which has a short cytoplasmic region with no particular functional domain, and is considered to act as a co-receptor for both VEGFs and semaphorins. However, the molecular mechanisms by which NRP1 carries out such versatile functions are still poorly understood. Here we identified protein kinase CK2 holoenzyme as a novel NRP1 binding protein by our combined purification strategy using epitope-tag immunoprecipitation followed by reverse-phase column chromatography. Further we showed that CK2 binds to the extracellular domain of NRP1 which is also phosphorylated by CK2 both in vitro and in vivo. Our findings of novel molecular interactions and modification of NRP1 may provide a new clue to understand the diverse functions of NRP1.
Biochemical and Biophysical Research Communications 09/2009; 385(4):618-23. · 2.48 Impact Factor
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Hideyuki Sasaki,
Hiroshi Asanuma,
Masashi Fujita,
Hiroyuki Takahama,
Masakatsu Wakeno,
Shin Ito,
Akiko Ogai,
Masanori Asakura,
Jiyoong Kim,
Tetsuo Minamino, Seiji Takashima,
Shoji Sanada,
Masaru Sugimachi,
Kazuo Komamura,
Naoki Mochizuki,
Masafumi Kitakaze
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ABSTRACT: Some studies have shown that metformin activates AMP-activated protein kinase (AMPK) and has a potent cardioprotective effect against ischemia/reperfusion injury. Because AMPK also is activated in animal models of heart failure, we investigated whether metformin decreases cardiomyocyte apoptosis and attenuates the progression of heart failure in dogs.
Treatment with metformin (10 micromol/L) protected cultured cardiomyocytes from cell death during exposure to H2O2 (50 micromol/L) via AMPK activation, as shown by the MTT assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and flow cytometry. Continuous rapid ventricular pacing (230 bpm for 4 weeks) caused typical heart failure in dogs. Both left ventricular fractional shortening and left ventricular end-diastolic pressure were significantly improved in dogs treated with oral metformin at 100 mg x kg(-1) x d(-1) (n=8) (18.6+/-1.8% and 11.8+/-1.1 mm Hg, respectively) compared with dogs receiving vehicle (n=8) (9.6+/-0.7% and 22+/-0.9 mm Hg, respectively). Metformin also promoted phosphorylation of both AMPK and endothelial nitric oxide synthase, increased plasma nitric oxide levels, and improved insulin resistance. As a result of these effects, metformin decreased apoptosis and improved cardiac function in failing canine hearts. Interestingly, another AMPK activator (AICAR) had effects equivalent to those of metformin, suggesting the primary role of AMPK activation in reducing apoptosis and preventing heart failure.
Metformin attenuated oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with activation of AMPK. Therefore, metformin may be a potential new therapy for heart failure.
Circulation 06/2009; 119(19):2568-77. · 14.74 Impact Factor
