Koji Magota

Asubio Pharma Co., Ltd., Kōbe, Hyōgo, Japan

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Publications (26)122.94 Total impact

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    ABSTRACT: Vascular endothelial cells are exposed to an acidic pH, and CXC chemokine receptor type 4 (CXCR4) is a key protective molecule against acidosis. We investigated the effect of coupling factor 6 (CF6), a novel proton import activator, on CXCR4 signaling and its molecular mechanism. CF6 decreased CXCR4 expression in human umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent manner. Pretreatment with small interfering RNA (siRNA) for hypoxia-inducible factor (HIF)-1α or PP1, a specific c-Src inhibitor, attenuated the CF6-induced decrease in CXCR4 without affecting CF6-induced intracellular acidosis. Chromatin immunoprecipitation revealed that CF6 enhanced the interaction between HIF-1α and the CXCR4 promoter at the hypoxia response element. CF6 also enhanced protein-protein interactions between phospho-c-Src and histone deacetylase 3 (HDAC3), but did not affect the binding of HDAC3 to the CXCR4 promoter at the hypoxia response element. Apoptotic cells, as measured by an Annexin-V-FITC Propidium Iodide Kit, were increased by CF6 in normoxia and hypoxia at 24 h; however, this increase was abolished by pretreatment with either siRNA for HIF-1α or the CXCR4 ligand. The coronary arteries and perivascular tissues obtained from CF6-overexpressing transgenic mice showed a lower expression of CXCR4 in the heart, increased wall thickness and infiltration of CD16-positive, CD206-positive or apoptotic cells. CF6 decreases CXCR4 expression through both HIF-1α- and c-Src-mediated mechanisms in vascular endothelial cells. Because CXCR4 has an important role in survival function, CF6 may have a role in the progression of arteriosclerosis via these complex mechanisms.Hypertension Research advance online publication, 20 March 2014; doi:10.1038/hr.2014.65.
    Hypertension Research 03/2014; · 2.79 Impact Factor
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    ABSTRACT: The spontaneous microaggregation of platelets (SMAPs) is a marker for the prognosis of patients with cardiovascular diseases. Coupling factor 6 (CF6) binds to the plasma membrane ATP synthase and functions as a pro-atherogenic molecule in the cardiovascular system. However, the role of CF6 in SMAPs and stroke remains unknown. In 650 consecutive patients, including those with acute-onset stroke, and 20 control subjects, platelet-rich plasma (PRP) was obtained, and SMAP was measured using a laser light-scattering aggregometer. The cytosolic cyclic adenosine monophosphate (cAMP) concentration in platelets was measured using an enzyme-linked immunosorbent assay. CF6 increased SMAPs in patients and control subjects to a similar degree by binding to the α- and β-subunits of ATP synthase and inducing intracellular acidosis. It was abolished by PRP pretreatment with antibodies against CF6, and the α- or β-subunit of the plasma membrane ATP synthase, and the ATP synthase inhibitor efrapeptin. CF6 increased SMAPs in patient groups with and without antiplatelet therapy to a similar degree, and no difference was found among the subgroups taking aspirin, thienopyridine or cilostazol. The cytosolic cAMP concentration in platelets was decreased by CF6 in the presence of the direct adenylate cyclase activator forskolin. Pretreatment of PRP with the G(s) activator cholera toxin blocked the decrease, whereas the G(i) inactivator pertussis toxin and cilostazol had no influence. The CF6-induced acceleration of SMAPs was suppressed by cholera toxin but not by cilostazol or pertussis toxin. CF6 enhanced SMAPs by decreasing cytosolic cAMP. Because it was observed irrespective of antiplatelet agents, CF6 appears to be a novel target for antiplatelet therapy.Hypertension Research advance online publication, 7 February 2013; doi:10.1038/hr.2012.231.
    Hypertension Research 02/2013; · 2.79 Impact Factor
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    ABSTRACT: Regular exercise improves systolic cardiac dysfunction through Akt cascade-mediated physiological hypertrophy in congestive heart failure. Tissue acidosis impairs Akt cascade, and coupling factor 6 induces tissue acidosis via activation of ecto-F(1)F(o) complex. We tested the hypothesis that coupling factor 6 attenuates physiological cardiac hypertrophy induced by exercise and its benefit in mice. Adult wild-type mice (n = 20) and coupling factor 6-overexpressing transgenic mice (n = 20) were divided into two groups with or without 4-week exercise consisting of 90-min swimming twice daily. Left ventricular posterior wall and interventricular septum thicknesses were increased by 0.12 ± 0.1 and 0.16 ± 0.1 mm, respectively, after 4-week swimming in wild-type mice (both P < 0.01), but unchanged in transgenic mice. Fractional shortening was increased from 37 ± 1 to 41 ± 1% after 4-week swimming in wild-type mice (P < 0.05), whereas it was unchanged in transgenic. The insulin-like growth factor 1 (IGF-1) receptor protein and its phosphorylated form in the heart were both increased by 1.83 ± 0.23 and 1.83 ± 0.09 times, respectively, after 4-week swimming in wild-type mice (both P < 0.05), but were unchanged in transgenic. Downstream phosphoinsulin receptor substrate 1, phosphoinositide 3-kinase, and phospho-Akt were increased by 2.22 ± 0.22, 1.78 ± 0.31, and 2.24 ± 0.49 times, respectively, in wild-type mice (all P < 0.05), but were unchanged in transgenic. Restoration of phospho-Akt by IGF-1 injection recovered left ventricular hypertrophy and systolic function after 4-week swimming in transgenic. Overexpression of coupling factor 6 attenuates exercise-induced physiological cardiac hypertrophy by downregulating Akt signaling, thereby cancelling its benefit for cardiac function in mice. Reduction in coupling factor 6 level seems to be useful for drawing the exercising effects on cardiac function.
    Journal of hypertension 02/2012; 30(4):778-86. · 4.02 Impact Factor
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    ABSTRACT: Despite advances in pharmacological treatments, diabetes with hypertension continues to be a major public health problem with high morbidity and mortality rates. We recently identified a circulating peptide coupling factor 6 (CF6), which binds to the plasma membrane ATP synthase (ecto-F(1)F(o) complex), resulting in intracellular acidosis. We investigated whether overexpression of CF6 contributes to diabetes and hypertension by intracellular acidosis. Transgenic mice overexpressing CF6 (also known as ATP5J) were generated, and physiological, biochemical and molecular biology studies were performed. CF6 overexpression elicited a sustained decrease in intracellular pH in tissues (aorta, kidney, skeletal muscle and liver, with the exception of adipose tissue) that express its receptor, the β-subunit of ecto-F(1)F(o) complex. Consistent with the receptor distribution, phospho-insulin receptor β, phosphoinositide 3-kinase activity and the phospho-Akt1:total Akt1 ratio were all decreased in the skeletal muscle and the liver in transgenic compared with wild-type mice, resulting in a decrease of plasma membrane-bound GLUT4 and an increase in hepatic glucose production. Under a high-sucrose diet, transgenic mice had insulin resistance and mild glucose intolerance; under a high-salt diet, they had elevated blood pressure with increased renal RAS-related C3 botulinum substrate 1 (RAC1)-GTP, which is an activator of mineralocorticoid receptor. Through its action on the β-subunit of ecto-F(1)F(o) complex, which results in intracellular acidosis, CF6 plays a crucial role in the development of insulin resistance and hypertension. This finding might advance our understanding of the mechanisms underlying diabetes and hypertension, possibly also providing a novel therapeutic target against cardiovascular disease.
    Diabetologia 02/2012; 55(2):520-9. · 6.49 Impact Factor
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    ABSTRACT: In male coupling factor 6 (CF6)-overexpressing transgenic (TG) mice, a high-salt diet induces hypertension and cardiac systolic dysfunction with excessive reactive oxygen species generation. However, the role of gender in CF6-mediated pathophysiology is unknown. We investigated the effects of ovariectomy and estrogen replacement on hypertension, cardiac dysfunction and Rac1 activity, which activates radical generation and the mineralocorticoid receptor, in female TG mice. Fifteen-week-old male and female TG and wild-type (WT) mice were fed a normal- or high-salt diet for 60 weeks. Systolic and diastolic blood pressures were higher in the TG mice fed a high-salt diet than in those fed a normal-salt diet at 20-60 weeks in males but only at 60 weeks in females. The blood pressure elevation under high-salt diet conditions was concomitant with a decrease in left ventricular fractional shortening. In the WT mice, neither blood pressure nor cardiac systolic function was influenced by a high-salt diet. In the female TG mice, bilateral ovariectomy induced hypertension with cardiac systolic dysfunction 8 weeks after the initiation of a high-salt diet. The ratios of Rac1 bound to guanosine triphosphate (Rac1-GTP) to total Rac1 in the heart and kidneys were increased in the ovariectomized TG mice, and estrogen replacement abolished the CF6-mediated pathophysiology induced under the high-salt diet conditions. The overexpression of CF6 induced salt-sensitive hypertension, complicated by systolic cardiac dysfunction, but its onset was delayed in females. Estrogen has an important role in the regulation of CF6-mediated pathophysiology, presumably via the downregulation of Rac1.
    Hypertension Research 01/2012; 35(5):539-46. · 2.79 Impact Factor
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    ABSTRACT: Reactive oxygen species are involved in the pathogenesis of congestive heart failure. We recently showed that coupling factor 6, a component of adenosine trisphosphate (ATP) synthase, induces hypertension by intracellular acidosis, which is related to reactive oxygen species generation. We investigated the effect of high-salt diet on the cardiac performance and reactive oxygen species generation in coupling factor 6-overexpressing transgenic mice. Baseline echocardiographic findings, reactive oxygen species generation, protein expression of sarcoplasmic/endoplasmic reticulum of Ca-ATPase 2 and phospholamban, and ATP content in the heart were similar between 7-week-old transgenic and wild-type mice. When the mice were fed with 8% salt diet for 20-24 weeks, fractional shortening of the left ventricle was decreased in transgenic mice compared with wild-type mice and was recovered by intraperitoneal administration of anticoupling factor 6 antibody. Nicotinamide adenine dinucleotide phosphate oxidase activity in the heart was increased in transgenic mice after the high-salt diet concomitantly with c-Src activation. The level of 8-iso-prostaglandin F2α was increased in transgenic heart compared with wild-type heart. The protein expression of sarcoplasmic/endoplasmic reticulum of Ca-ATPase 2 was decreased and that of phospholamban was increased in transgenic heart. In cDNA microarray analysis, the genes related to ATP synthesis and glycolysis were decreased in transgenic heart, concomitantly with the decrease in ATP content and the increase in β-myosin heavy chain. These suggest that coupling factor 6 induces the development of systolic dysfunction and upregulation of nicotinamide adenine dinucleotide phosphate oxidase in the heart under the high-salt diet.
    Journal of hypertension 11/2010; 28(11):2243-51. · 4.02 Impact Factor
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    ABSTRACT: Homocysteine (Hcy) is an independent predictor of stroke. Coupling factor 6 (CF6) is regulated by nuclear factor kappa B (NF-kappaB) signaling which is activated by Hcy. We tested the hypothesis that CF6 is elevated with Hcy in stroke. We also examined the effect of vitamin treatment on CF6 and Hcy levels. The 59 Japanese patients with a recent history of stroke were randomly assigned to a group without vitamin treatment (Group 1, n = 29) and to a group with treatment with both folic acid and vitamin B(12) for 2 months (Group 2, n = 30). The CF6 level was elevated in the patients with stroke compared with that in controls (n = 64) at admission. In a multiple regression model, the plasma CF6 level was weakly correlated to the total Hcy (tHcy) level. In Group 1, the plasma tHcy and CF6 levels were unchanged. In Group 2, however, they were both decreased, and there was a weak positive correlation between the decreases in plasma levels of CF6 and tHcy. CF6 is elevated in patients with stroke independently of risk factors. Since Hcy and vitamin treatment affect CF6 levels in stroke, CF6 appears to be a novel molecule for the pathogenesis and treatment of stroke.
    Annals of medicine 01/2010; 42(1):79-86. · 3.52 Impact Factor
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    ABSTRACT: Coupling factor 6 (CF6) is composed of 76 amino acids and is present in the peripheral stalk of mitochondrial ATP synthase. The generation of CF6 is positively regulated by tumor necrosis factor alpha and shear stress via nuclear factor kappaB, and by high glucose via protein kinase C and p38 mitogen-activated protein kinase. CF6 is released outside of the cells from vascular endothelial cells, and binds to the beta-subunit of the plasma membrane-bound ATP synthase in vascular endothelial cells and leads to intracellular acidosis. CF6 produces vasoconstriction, and the biological active site resides at the C-terminal portion. CF6 suppresses prostacyclin generation via inhibition of cytosolic phospholipase A(2). CF6 also suppresses nitric oxide synthase activity via an increase in asymmetric dimethylarginine and a decrease in platelet/endothelial cell adhesion molecule-1. CF6 induces the gene and protein expression of proatherogenic molecules such as endothelin 2, urokinase type plasminogen activator receptor, estrogen receptor beta, a soluble short form of vascular endothelial growth factor receptor-1, and lectin-like oxidized low-density lipoprotein receptor-1. The plasma level of CF6 is elevated in patients with essential hypertension, diabetes mellitus, end-stage renal disease, acute myocardial infarction, and coronary heart disease. It is likely that CF6 contributes to the pathogenesis of cardiovascular diseases, but further intensive investigation is needed.
    Archiv für Experimentelle Pathologie und Pharmakologie 07/2009; 380(3):205-14. · 2.15 Impact Factor
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    ABSTRACT: Coupling factor 6 (CF6) attenuates the endothelial generation of prostacyclin. However, the role of CF6 in the resistance arteriole that is directly related to vascular tone is not determined yet. We investigated the effect of endogenous and exogenous CF6 on prostacyclin generation in cultured vascular smooth muscle cells (VSMCs). We cultured resistance arteriole VSMCs from the mesenteric artery network of spontaneously hypertensive rats (SHRs, n = 8) and Wistar-Kyoto rats (WKY, n = 8) by enzymatic method. The gene expression of CF6 was higher by 76 +/- 24% in SHR-derived VSMCs compared with WKY rat-derived VSMCs (P < 0.05) concomitant with the reduced degradation rate of CF6 mRNA. The release of CF6 in SHRs was higher than that in WKY rats (11.0 +/- 0.8 vs. 3.8 +/- 0.4 pg/microg protein, P < 0.05). Prostacyclin generation was attenuated in mesenteric arteriolar VSMCs from SHRs compared with those from WKY rats, but it was restored by neutralization of CF6 with its antibody. Exogenous administration of CF6 suppressed arachidonic acid release in a dose-dependent manner, and it was greater in SHRs than in WKY rats. Pretreatment with PP1, an inhibitor of tyrosine kinase c-Src, or receptor blockers such as ADP, efrapeptin, and an antibody to beta-subunit of ATP synthase blocked CF6-induced decrease in prostacyclin generation. These data suggest that CF6 suppresses prostacyclin generation in resistance arteriole VSMCs in an autocrine or paracrine fashion, and it is enhanced in SHRs by the overproduction of CF6 and the hyperresponsiveness to CF6.
    Journal of hypertension 06/2009; 27(9):1823-8. · 4.02 Impact Factor
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    ABSTRACT: Vascular endothelial growth factor (VEGF) is a well-known promoter of angiogenesis, but its receptor VEGFR-1 and a soluble short form of VEGFR-1 (sFlt-1) play a negative role in the VEGF signal pathway by trapping VEGF. We recently showed that endogenous prostacyclin inhibitor coupling factor 6 (CF6) forces the clockwise rotation of F(1) motor of plasma membrane adenosine triphosphate synthase and induces intracellular acidosis and c-Src activation. We investigated the role of CF6 in regulation of sFlt-1, and its mechanism in human umbilical vein endothelial cells. The ratio of sFlt-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased at 24 h by 1.59+/-0.29-fold by 10(-7) M CF6 (P<0.05), concomitantly with the increases in intercellular adhesion molecule-1 and lectin-like oxidized low-density lipoprotein receptor-1 and no change in VEGF-A. When the dose of CF6 was increased to 10(-6) M, no further increase in sFlt-1 mRNA was observed. The release of sFlt-1 protein was increased by 1.72+/-0.24-fold (P<0.05) at 48 h after exposure to CF6 at 10(-7) M, and it was blocked by pretreatment with anti-CF6 antibody. The immunoreactive bands for sFlt-1 and VEGFR-1 were both increased by CF6 to similar degrees. Pretreatment with PP1, an inhibitor of c-Src, and 10(-5) Mefrapeptin, an inhibitor of F(1) motor, inhibited CF6-induced increases in expression and release of sFlt-1 (P<0.05). In mice overexpressing CF6, the plasma level of sFlt-1 was increased by 1.36+/-0.29-fold compared with that in wild-type mice (P<0.05). These indicate that CF6 might increase the expression and release of sFlt-1 in the vessels through acidosis-induced c-Src activation.
    Hypertension Research 03/2009; 32(3):182-7. · 2.79 Impact Factor
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    ABSTRACT: Coupling factor 6 (CF6) induces hypertension by attenuating the endothelial generation of prostacyclin. However, intracellular signalling of CF6 in the resistance arteriole vascular smooth muscle cells (VSMCs) that are directly related to vasoconstriction has not been determined. Here we investigated the direct effect of exogenous CF6 on Ca2+ signalling in cultured VSMCs and the in vivo role of endogenous CF6 in the genesis of hypertension using CF6 transgenic (TG) mice. CF6 induced a monophasic increase in the intracellular free Ca2+ concentration ([Ca2+]i) through nifedipine-sensitive Ca2+ channels in A7r5 cells, a cell line of VSMCs, and enhanced the angiotensin II-induced spike phase of [Ca2+]i to a greater degree in VSMCs derived from spontaneously hypertensive rats (SHRs). In the mesenteric arterioles obtained from CF6-TG mice that manifested hypertension, angiotensin II-induced vasoconstriction was enhanced, compared with wild-type mice, and its enhancement was abolished by an anti-CF6 antibody. Pre-treatment with PP1, a tyrosine kinase c-Src inhibitor, blocked CF6-induced increase in Ca2+ signalling in VSMCs and vasoconstriction in TG mice. The receptor of CF6 was F1 motor of adenosine triphosphate (ATP) synthase with a higher affinity in SHRs. CF6 decreased intracellular pH via activation of ATPase activity and led to c-Src activation to a greater degree in SHR-derived VSMCs. CF6 causes hypertension by directly enhancing Ca2+ signalling in VSMCs and vasoconstriction in the mesenteric arteriolar network via c-Src activation.
    Cardiovascular research 01/2009; 81(4):780-7. · 5.80 Impact Factor
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    ABSTRACT: Coupling factor 6 (CF6), a component of ATP synthase, suppresses the generation of prostacyclin and nitric oxide (NO). Platelet endothelial cell adhesion molecule-1 (PECAM-1) is involved in shear-induced NO production. To investigate the linkage between the actions of CF6 and PECAM-1, we examined the effects of CF6 on PECAM-1 expression and shear-mediated NO release, comparatively with those of angiotensin II (AngII). Treatment of human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells (HAEC) with CF6 at 10(-7)M or AngII at 10(-7)M for 24h suppressed PECAM-1 gene and protein expression. CF6 or AngII activated c-Src at 15 min in HUVEC, and blockade of c-Src with PP1, its specific inhibitor, restored them. Efrapeptin, an inhibitor of ATPase, attenuated CF6-induced suppression of PECAM-1 gene expression by blockade of acidification, whereas superoxide dismutase or apocinin, an inhibitor of NADPH oxidase, blocked AngII-induced suppression of PECAM-1. Exposure of the cells to shear stress at 25 dynes/cm(2) for 30 min enhanced phosphorylation of eNOS at Ser(1177) and NO release. Pretreatment with CF6 or AngII for 24h attenuated them in HUVEC and HAEC. These suggest that CF6 downregulates PECAM-1 expression via c-Src activation and attenuates shear-induced NO release presumably by suppressing eNOS phosphorylation.
    Atherosclerosis 03/2008; 200(1):45-50. · 3.71 Impact Factor
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    ABSTRACT: Coupling factor 6 (CF6), a component of ATP synthase, inhibits phospholipase A2 and induces vasoconstriction. However, because arachidonic acid acts in the widespread fields of vascular biology, CF6 might exert profound effects in addition to vasoconstriction. We investigated the effect of CF6 on the gene expression profile in human umbilical vein endothelial cells. The increased gene expression after 24-h exposure to CF6 at 10 mol/l, assessed by cDNA microarray (n = 3), included neuregulin-1 (1.84 +/- 0.07 fold compared with control, P < 0.05) and relaxin-1 (1.74 +/- 0.20, P < 0.05), both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77 +/- 0.24, P = 0.06) and estrogen receptor beta (1.74 +/- 0.36, P = 0.08), both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1; 1.73 +/- 0.20, P < 0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, measured by real-time quantitative reverse transcription-polymerase chain reaction, was increased by 9 +/- 2% (n = 10, P < 0.01) at 48 h after CF6 at 10 mol/l, whereas the ratio of DDAH-2 to GAPDH was decreased by 12 +/- 2% (n = 8, P < 0.01). DDAH-2 protein and activity were decreased by 28 +/- 5% (n = 5, P < 0.01) and 19 +/- 2% (n = 6, P < 0.01) by CF6, respectively. ADMA release was enhanced by 20 +/- 8% and NOS activity was decreased by 13 +/- 1% (both n = 8, P < 0.05) by CF6. CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release by enhancing its synthesis and suppressing its degradation.
    Journal of Hypertension 03/2006; 24(3):489-97. · 3.81 Impact Factor
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    ABSTRACT: Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. We showed a high plasma level of CF6 in human hypertension. The present study focused on the identification and characterization of a receptor for CF6 and its post-receptor signaling pathway. Incubation of human umbilical vein endothelial cells (HUVECs) with an excess of free CF6 reduced by 50% the immunoreactivity for the antibody to beta-subunit of ATP synthase at the cell surface, but unaffected that for the alpha-subunit antibody. A significant displacement of radioligand was observed at 3x10(-9) through 10(-7) M unlabeled CF6, and the Kd was 7.6 nM. Adenosine diphosphate (ADP) at 10(-7) M and beta-subunit antibody suppressed the binding of (125)I-CF6 by 81.3+/-9.7% and 32.0+/-2.0%, respectively, whereas the alpha-subunit antibody unaffected it. The hydrolysis activity of ATP to ADP was increased by 1.6-fold by CF6 at 10(-7) M, and efrapeptin at 10(-5) M, an inhibitor of ATP synthase, blocked it. CF6 at 10(-7) M decreased intracellular pH in 2',7'-bis(carboxyethyl-5 (6))-carboxyfluorescein-loaded HUVEC. Amyloride at 10(-4) M augmented the pH decrease in response to CF6, whereas efrapeptin at 10(-5) M blocked it. Arachidonic acid release was suppressed by CF6, and it was reversed by efrapeptin at 10(-5) M or beta-subunit antibody or ADP at 10(-7) M. The beta-subunit antibody suppressed coupling factor 6-induced increase in blood pressure. These indicate that membrane-bound ATP synthase functions as a receptor for CF6 and may have a previously unsuspected role in the genesis of hypertension by modulating the concentration of intracellular hydrogen.
    Hypertension 12/2005; 46(5):1140-6. · 6.87 Impact Factor
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    ABSTRACT: We previously showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis and a vasoconstrictor, is present on the surface of human umbilical vein endothelial cells (HUVEC) and is released outside of the cells by shear stress. We investigated the intracellular signaling mechanism for shear-induced release of CF6 in HUVEC and the effects of troglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), both peroxisome proliferator-activated receptor (PPAR)-gamma ligands, on it. The release and gene expression of CF6 in HUVEC were enhanced by shear stress at 25 dyn/cm2, measured by radioimmunoassay and real-time RT-PCR, respectively. The intracellular content of CF6 was decreased after exposure to shear stress at 25 dyn/cm2. Transfection experiments with deletional and mutational CF6 promoter constructs, and with dominant negative mutant IkappaB kinase alpha (K44M) demonstrated that shear-induced CF6 transcription was dependent on nuclear factor-kappa B (NF-kappaB) activation. Pretreatment with troglitazone or 15d-PGJ2 inhibited the shear-induced release and gene expression of CF6, whereas fenofibric acid, a PPAR-alpha ligand, had no influence on them. Western blot and immunostaining showed that troglitazone and 15d-PGJ2 inhibited the shear-induced, reactive oxygen species (ROS)-mediated activation of NF-kappaB at the level of IkappaB protein. The shear-induced gene expression and release of CF6 in HUVEC are mediated by the ROS-related activation of NF-kappaB signaling pathway. Troglitazone and 15d-PGJ2 inhibit them at the IkappaB protein level.
    Cardiovascular Research 08/2005; 67(1):134-41. · 5.94 Impact Factor
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    ABSTRACT: We recently showed that mitochondrial coupling factor 6 (CF6) is present as a pressor substance and a prostacyclin inhibitor in systemic circulation. However, the regulation mechanism for circulating CF6 is unknown. We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the generation and release of CF6. We used two kinds of cells, human umbilical vein endothelial cells (HUVEC) and ECV-304. The concentration of CF6 in the medium increased with time in both ECV-304 and HUVEC. Treatment of ECV-304 and HUVEC with TNF-alpha enhanced the release of CF6 in a dose-dependent manner concomitantly with the decrease in CF6 content in the mitochondria at 24 h. The released CF6 was characterized to be an active full-length peptide by Western blot. The ratio of CF6 to GAPDH mRNA, measured by real-time polymerase chain reaction, was 1.7 fold increased at 1 h after exposure to TNF-alpha in ECV-304 and HUVEC. This enhanced gene expression and release was blocked or suppressed by 70% by stable transfection of dominant negative mutant I kappa B kinase alpha whose efficacy was confirmed by blockade of translocation of NF-kappa B p65 protein and of degradation of I kappa B alpha protein. Flow cytometry analysis revealed that the cell surface-associated CF6 was significantly increased at 24 h after TNF-alpha in a dose-dependent manner. TNF-alpha stimulates the gene expression of CF6 via activation of NF-kappa B signaling pathway, and promotes the release of CF6 from ECV-304 and HUVEC.
    Cardiovascular Research 07/2004; 62(3):578-86. · 5.94 Impact Factor
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    ABSTRACT: Plasma asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is an independent predictor of overall mortality and cardiovascular outcome in hemodialysis patients. However, not only ADMA but also traditional risk factors account for only part of the high cardiovascular morbidity and mortality in these patients. We investigated cross-sectionally the association between coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis, and cardiovascular events in 95 hemodialysis patients. Plasma CF6 level was measured by radioimmunoassay, whereas plasma ADMA level by high-performance liquid chromatography (HPLC). Plasma levels of CF6 and ADMA were threefold higher in hemodialysis patients than in control individuals, and there was a positive correlation between these two compounds (r=0.25, P < 0.05). Plasma CF6 level was positively correlated with serum creatinine level (r=0.36, P < 0.01) and was reduced after dialysis (P < 0.05). Plasma CF6 and ADMA levels were both higher in hemodialysis patients complicating ischemic heart disease (myocardial infarction and/or angina) than in those free of cardiovascular events. In a multiple regression model, plasma CF6 level (r=0.24, P=0.023) and ADMA level (r=0.26, P=0.023) were independently related to the occurrence of ischemic heart disease in hemodialysis patients. CF6 is a novel risk factor for ischemic heart disease in end-stage renal disease (ESRD). Synergism of this peptide and ADMA might contribute to its occurrence presumably by inhibition of prostacyclin and nitric oxide production. A prospective study is needed to evaluate this issue more precisely.
    Kidney International 12/2003; 64(6):2291-7. · 7.92 Impact Factor
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    ABSTRACT: Coupling factor 6 is an endogenous inhibitor of prostacyclin synthesis and might function as an endogenous vasoconstrictor in the fashion of a circulating hormone in rats. We investigated the role of coupling factor 6 in human hypertension. The patients with essential hypertension (EH) (n = 30) received a series of normal salt diet (12 g salt/day) for 3 days, low salt diet (2 g salt/day) for 7 days, and high salt diet (20-23 g salt/day) for 7 days. Normotensive control subjects (n = 27) received normal and low salt diets. The plasma level of coupling factor 6, measured by radioimmunoassay, during normal salt diet was higher in patients with EH than in normotensive subjects (17.6 +/- 1.7 versus 12.8 +/- 0.5 ng/ml, P < 0.01). Whereas the plasma level of coupling factor 6 was unchanged after salt restriction in normotensive subjects, it was decreased after salt restriction (from 12 g/day to 2 g/day) and was increased after salt loading (from 2 g/day to 20-23 g/day) in patients with EH. This increase in plasma level of coupling factor 6 was abolished by oral administration of ascorbic acid, but the level of blood pressure was unaffected. The percentage changes in plasma coupling factor 6 level after salt restriction and loading were positively correlated with those in mean blood pressure (r = 0.57, P < 0.01), and negatively correlated with those in plasma nitric oxide level (r = -0.51, P < 0.05). These indicate that circulating coupling factor 6 is elevated in human hypertension and modulated by salt intake presumably via reactive oxygen species.
    Journal of Hypertension 12/2003; 21(12):2323-8. · 3.81 Impact Factor
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    ABSTRACT: We showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis, is present in the systemic circulation as a pressor substance in rats. We investigated the possibility of vascular endothelial cells as a source of circulating CF6. We used 2 cultured endothelial cell lines, human umbilical vein endothelial cells (HUVECs) and ECV 304 cells (transformed HUVECs), for this study. Immunofluorescence microscopy of both ECV 304 and HUVECs confirmed the surface-associated immunoreactivity of anti-CF6 antibody on the plasma membrane. The concentration of CF6 in the medium increased gradually with time in both ECV 304 and HUVECs in static conditions. Exposure of ECV 304 and HUVECs to a fluid shear stress enhanced the release of CF6: In ECV 304, the concentration of CF6 in the medium (ng. well(-1). 6 hours(-1)) was 2.1+/-0.8 at baseline, 4.3+/-0.8 after shear at 15 dynes/cm(2), and 57.7+/-8.4 after shear at 25 dynes/cm(2). CF6 contents in the cell homogenate and mitochondria were both significantly increased after exposure of ECV 304 to 6-hour shear at 15 dynes/cm(2), whereas they were unchanged after shear stress at 25 dynes/cm(2). The ratio of CF6 to GAPDH mRNA was enhanced significantly, by 1.8+/-0.2-fold, after 6-hour shear stress at 25 dynes/cm(2). Flow cytometry analysis revealed that the surface-associated CF6 was significantly increased in a 3-hour static condition after the previous exposure of the cells to shear stress for 3 hours. Vascular endothelial cells are a source of CF6, and shear stress regulates the release of the surface-associated CF6.
    Circulation 01/2002; 104(25):3132-6. · 15.20 Impact Factor
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    ABSTRACT: The possible presence of an unknown prostacyclin-synthesis inhibitory substance has been reported in some strains of rats. We purified the inhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatography and reverse-phase high performance liquid chromatography. The automated gas-phase sequencing indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cleavable fusion protein strategy, attenuated baseline and bradykinin (10−6 M)-induced prostacyclin synthesis and [3H] arachidonic acid (AA) release in human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10−7 M recombinant coupling factor 6. Baseline and bradykinin-induced [3H] AA release were suppressed by arachidonyl trifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 (PLA2), and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine nor bromoenol lactone affected AA release. Intravenous injection of recombinant coupling factor 6 increased arterial blood pressure in rats, whereas a specific antibody to coupling factor 6 decreased systemic blood pressure. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic PLA2 and functions as an endogenous vasoconstrictor.
    International Congress Series 01/2002; 1244:131-141.

Publication Stats

275 Citations
122.94 Total Impact Points

Institutions

  • 2012–2013
    • Asubio Pharma Co., Ltd.
      Kōbe, Hyōgo, Japan
  • 2001–2012
    • Hirosaki University
      Khirosaki, Aomori Prefecture, Japan
  • 2008–2009
    • Pennington Biomedical Research Center
      Baton Rouge, Louisiana, United States
  • 2004
    • Fujitsu Ltd.
      Kawasaki Si, Kanagawa, Japan