Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We previously reported that the heart-specific choline acetyltransferase (ChAT) gene overexpressing mice (ChAT tg) show specific phenotypes including ischemic tolerance and the CNS stress tolerance. In the current study, we focused on molecular mechanisms responsible for systemic and localized anti-inflammatory phenotypes of ChAT tg. ChAT tg were resistant to systemic inflammation induced by lipopolysaccharides due to an attenuated cytokine response. In addition, ChAT tg, originally equipped with less reactive Kupffer cells, were refractory to brain cold injury, with decreased blood brain barrier (BBB) permeability and reduced inflammation. This is because ChAT tg brain endothelial cells expressed more claudin-5, and their astrocytes were less reactive, causing decreased hypertrophy. Moreover, reconstruction of the BBB integrity in vitro confirmed the consolidation of ChAT tg. ChAT tg were also resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neuronal toxicity due to lower mortality rate and neuronal loss of substantia nigra. Additionally, ChAT tg subjected to MPTP showed attenuated BBB disruption, as evident from reduced sodium fluorescein levels in the brain parenchyma. The activated central cholinergic pathway of ChAT tg lead to anti-convulsive effects like vagus nerve stimulation. However, DSP-4, a noradrenergic neuron-selective neurotoxin against the CNS including the locus ceruleus, abrogated the beneficial phenotype and vagotomy attenuated expression of claudin-5, suggesting the link between the cholinergic pathway and BBB function. Altogether, these findings indicate that ChAT tg possess an anti-inflammatory response potential, associated with upregulated claudin-5, leading to the consolidation of BBB integrity. These characteristics protect ChAT tg against systemic and localized inflammatory pathological disorders, which targets the CNS.

DOI： 10.1016/j.bbi.2019.06.005

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND/AIMS: In a previous study, we reported that cardiomyocytes were equipped with non-neuronal cardiac cholinergic system (NNCCS) to synthesize acetylcholine (ACh), which is indispensable for maintaining the basic physiological cardiac functions. The aim of this study was to identify and characterize a pharmacological inducer of NNCCS. METHODS: To identify a pharmacological inducer of NNCCS, we screened several chemical compounds with chemical structures similar to the structure of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Preliminary investigation revealed that SNAP is an inducer of non-neuronal ACh synthesis. We screened potential pharmacological inducers in H9c2 and HEK293 cells using western blot analysis, luciferase assay, and measurements of intracellular cGMP, NO₂ and ACh levels. The effects of the screened compound on cardiac function of male C57BL6 mice were also evaluated using cardiac catheter system. RESULTS: Among the tested compounds, we selected S-nitroso-Npivaloyl-D-penicillamine (SNPiP), which gradually elevated the intracellular cGMP levels and nitric oxide (NO) levels in H9c2 and HEK293 cells. These elevated levels resulted in the gradual transactivation and translation of the choline acetyltransferase gene. Additionally, in vitro and in vivo SNPiP treatment elevated ACh levels for 72 h. SNPiP-treated mice upregulated their cardiac function without tachycardia but with enhanced diastolic function resulting in improved cardiac output. The effect of SNPiP was dependent on SNPiP nitroso group as verified by the ineffectiveness of N-pivaloyl-D-penicillamine (PiP), which lacks the nitroso group. CONCLUSION: SNPiP is identified to be one of the important pharmacological candidates for induction of NNCCS.

DOI： 10.33594/000000064.

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DOI： 10.1016/j.yjmcc.2018.10.013

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ireland Ltd  

Aims: Our previous study revealed that cyclic hindlimb ischaemia–reperfusion (IR) activates cardiac acetylcholine (ACh) synthesis through the cholinergic nervous system and cell-derived ACh accelerates glucose uptake. However, the mechanisms regulating glucose metabolism in vivo remain unknown. We investigated the effects and mechanisms of IR in mice under pathophysiological conditions. Methods: Using IR-subjected male C57BL/6J mice, the effects of IR on blood sugar (BS), glucose uptake, central parasympathetic nervous system (PNS) activity, hepatic gluconeogenic enzyme expression and those of ACh on hepatocellular glucose uptake were assessed. Results: IR decreased BS levels by 20% and increased c-fos immunoreactivity in the center of the PNS (the solitary tract and the dorsal motor vagal nucleus). IR specifically downregulated hepatic gluconeogenic enzyme expression and activities (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) and accelerated hepatic glucose uptake. Transection of a hepatic vagus nerve branch decreased this uptake and reversed BS decrease. Suppressed gluconeogenic enzyme expression was reversed by intra-cerebroventricular administration of a choline acetyltransferase inhibitor. Moreover, IR significantly attenuated hyperglycaemia in murine model of type I and II diabetes mellitus. Conclusions: IR provides another insight into a therapeutic modality for diabetes mellitus due to regulating gluconeogenesis and glucose-uptake and advocates an adjunctive mode rectifying disturbed glucose metabolism.

DOI： 10.1016/j.diabres.2018.03.009

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Authorship：Last author   Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.bbi.2016.12.009

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

We previously developed cardiac ventricle-specific choline acetyltransferase (ChAT) gene-overexpressing transgenic mice (ChAT tgm), i.e. an in vivo model of the cardiac non-neuronal acetylcholine (NNA) system or non-neuronal cardiac cholinergic system (NNCCS). By using this murine model, we determined that this system was responsible for characteristics of resistance to ischaemia, or hypoxia, via the modulation of cellular energy metabolism and angiogenesis. In line with our previous study, neuronal ChAT-immunoreactivity in the ChAT tgm brains was not altered from that in the wild-type (WT) mice brains; in contrast, the ChAT tgm hearts were the organs with the highest expression of the ChAT transgene. ChAT tgm showed specific traits in a central nervous system (CNS) phenotype, including decreased response to restraint stress, less depressive-like and anxiety-like behaviours and anti-convulsive effects, all of which may benefit the heart. These phenotypes, induced by the activation of cardiac NNCCS, were dependent on the vagus nerve, because vagus nerve stimulation (VS) in WT mice also evoked phenotypes similar to those of ChAT tgm, which display higher vagus nerve discharge frequency; in contrast, lateral vagotomy attenuated these traits in ChAT tgm to levels observed in WT mice. Furthermore, ChAT tgm induced several biomarkers of VS responsible for anti-convulsive and anti-depressive-like effects. These results suggest that the augmentation of the NNCCS transduces an effective and beneficial signal to the afferent pathway, which mimics VS. Therefore, the present study supports our hypothesis that activation of the NNCCS modifies CNS to a more stress-resistant state through vagus nerve activity.

DOI： 10.1042/CS20160277

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Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and, to a lesser extent, in the noradrenergic neurons of the locus coeruleus (LC). Most cases of PD are idiopathic and sporadic and are believed to be the result of both environmental and genetic factors. Here, to the best of our knowledge, we report the first evidence that chronic restraint stress (8 h/day, 5 days/week) substantially reduces nigral DA and LC noradrenergic neuronal cell numbers in rats. Loss of DA neurons in the SNpc was evident after 2 weeks of stress and progressed in a time-dependent manner, reaching up to 61% at 16 weeks. This reduction was accompanied by robust microglial activation and oxidative stress and was marked by nitrotyrosine in the SNpc and LC of the midbrain. These results indicate that chronic stress triggers DA and noradrenergic neurodegeneration by increasing oxidative stress, and that activated microglia in the substantia nigra and LC may play an important role in modulating the neurotoxic effects of oxidative stress. Taken together, these data suggest that exposure to chronic stress triggers DA and noradrenergic neurodegeneration, which is a cause of PD. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbi.2015.08.015

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

It has been several years since the function of the non-neuronal cholinergic system was independently reported in cardiomyocytes by several research groups. Although these findings initially seemed to be negligible and insignificant, extraordinary findings about cardiomyocytes were subsequently reported in studies involving the knockdown of the non-neuronal cholinergic system. These studies provide the evidence that this system may be indispensable for maintaining principal cardiac functions. Despite the absence of an appropriate and reliable technology to detect cellular ACh in real time in cardiomyocytes, studies of this system have progressed, albeit very slowly, to gradually consolidate the significance of this system. Based on the many significant findings regarding this system, these will be critical to develop adjunctive intervention therapy against cardiovascular diseases, including peripheral artey disease and heart failure. In this study, previous studies focusing on the non-neuronal cholinergic system are reviewed along with our studies, both indicating the biologically significant roles of the cardiac non-neuronal acetylcholine system from a clinical perspective. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.intimp.2015.05.029

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Background: Saving more limbs of patients with peripheral arterial disease (PAD) from amputation by accelerating angiogenesis in affected limbs has been anticipated for years. We hypothesized that an anti-Alzheimer drug, donepezil (DPZ), can activate angiomyogenic properties of satellite cells, myogenic progenitors, and thus be an additional pharmacological therapy against PAD.
Methods and Results: In a murine hindlimb ischemia model, we investigated the angiogenic effects of a clinical dose of DPZ (0.2mg kg(-1) day(-1)) and its combination with cilostazol, a platelet aggregation inhibitor and a conventional therapeutic drug against PAD. The combination therapy most effectively improved skin coldness and most effectively upregulated vascular endothelial growth factor (VEGF)-producing satellite cells in ischemic hindlimbs. Computed tomography revealed that DPZ remarkably attenuated ischemic muscle atrophy and induced super-restoration in affected hindlimbs. The in vitro study with human aortic endothelial cells showed that DPZ or its combination with cilostazol effectively upregulated the expression of pAkt, hypoxia inducible factor-1 alpha, and VEGF protein. Likewise, in primary cultured satellite cells, DPZ, alone or in combination, upregulated the expression of VEGF, interleukin-1 beta, and fibroblast growth factor 2 protein.
Conclusions: The present results suggest that a clinical dosage of DPZ accelerates angiomyogenesis by directly acting on both endothelial and satellite cells. Therefore, DPZ is a potential additional choice for conventional drug therapy against PAD.

DOI： 10.1253/circj.CJ-14-0095

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Background-Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium.
Methods and Results-To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)-expressing cells and heart-specific ChAT transgenic (ChAT-tg) mice. Compared with cardiomyocytes of wild-type (WT) mice, those of the ChAT-tg mice had high levels of ACh and hypoxia-inducible factor (HIF)-1 alpha protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT-overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT-tg mice showed similar hemodynamics; after MI, however, the ChAT-tg mice had better survival than did the WT mice. In the ChAT-tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post-MI remodeling. The ChAT-tg heart was more resistant to ischemia-reperfusion injury than was the WT heart.
Conclusions-These results suggest that the activated cardiac ACh-HIF-1 alpha cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self-defense against ischemia.

DOI： 10.1161/JAHA.112.004887

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Background: In our previous study, we established the novel concept of a non-neuronal cardiac cholinergic system-cardiomyocytes produce ACh in an autocrine and/or paracrine manner. Subsequently, we determined the biological significance of this system-it played a critical role in modulating mitochondrial oxygen consumption. However, its detailed mechanisms and clinical implications have not been fully investigated.
Aim: We investigated if this non-neuronal cardiac cholinergic system was upregulated by a modality other than drugs and if the activation of the system contributes to favorable outcomes.
Results: Choline acetyltransferase knockout (ChAT KO) cells with the lowest cellular ACh levels consumed more oxygen and had increased MTT activity and lower cellular ATP levels compared with the control cells. Cardiac ChAT KO cells with diminished connexin 43 expression formed poor cell-cell communication, evidenced by the blunted dye transfer. Similarly, the ChAT inhibitor hemicholinium-3 decreased ATP levels and increased MTT activity in cardiomyocytes. In the presence of a hypoxia mimetic, ChAT KO viability was reduced. Norepinephrine dose-dependently caused cardiac ChAT KO cell death associated with increased ROS production. In in vivo studies, protein expression of ChAT and the choline transporter CHT1 in the hindlimb were enhanced after ischemia-reperfusion compared with the contralateral non-treated limb. This local effect also remotely influenced the heart to upregulate ChAT and CHT1 expression as well as ACh and ATP levels in the heart compared with the baseline levels, and more intact cardiomyocytes were spared by this remote effect as evidenced by reduced infarction size. In contrast, the upregulated parameters were abrogated by hemicholinium-3.
Conclusion: The non-neuronal cholinergic system plays a protective role in both myocardial cells and the entire heart by conserving ATP levels and inhibiting oxygen consumption. Activation of this non-neuronal cardiac cholinergic system by a physiotherapeutic modality may underlie cardioprotection through the remote effect of hindlimb ischemia-reperfusion.

DOI： 10.1371/journal.pone.0050761

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DOI： 10.1016/j.ijcard.2011.10.066

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Our recent studies have indicated that acetylcholine (ACh) protects cardiomyocytes from prolonged hypoxia through activation of the PI3K/Akt/HIF-1 alpha/VEGF pathway and that cardiomyocyte-derived VEGF promotes angiogenesis in a paracrine fashion. These results suggest that a cholinergic system plays a role in modulating angiogenesis. Therefore, we assessed the hypothesis that the cholinergic modulator donepezil, an acetylcholinesterase inhibitor utilized in Alzheimer's disease, exhibits beneficial effects, especially on the acceleration of angiogenesis. We evaluated the effects of donepezil on angiogenic properties in vitro and in vivo, using an ischemic hindlimb model of alpha 7 nicotinic receptor-deleted mice (alpha 7 KO) and wild-type mice (WT). Donepezil activated angiogenic signals, i.e., HIF-1 alpha and VEGF expression, and accelerated tube formation in human umbilical vein endothelial cells (HUVECs). ACh and nicotine upregulated signal transduction with acceleration of tube formation, suggesting that donepezil promotes a common angiogenesis pathway. Moreover, donepezil-treated WT exhibited rich capillaries with enhanced VEGF and PCNA endothelial expression, recovery from impaired tissue perfusion, prevention of ischemia-induced muscular atrophy with sustained surface skin temperature in the limb, and inhibition of apoptosis independent of the alpha 7 receptor. Donepezil exerted comparably more effects in alpha 7 KO in terms of angiogenesis, tissue perfusion, biochemical markers, and surface skin temperature. Donepezil concomitantly elevated VEGF expression in intracardiac endothelial cells of WT and alpha 7 KO and further increased choline acetyltransferase (ChAT) protein expression, which is critical for ACh synthesis in endothelial cells. The present study concludes that donepezil can act as a therapeutic tool to accelerate angiogenesis in cardiovascular disease patients. (c) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.yjmcc.2009.11.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Our recent studies have shown that, as indicated by vagal stimulation, an acetylcholinesterase inhibitor donepezil, an anti-Alzheimer's disease drug, prevents progression of heart failure in rats with myocardial infarction, and activates a common cell survival signal shared by acetylcholine (ACh) in vitro. On the basis of this and evidence that vagal innervation is extremely poor in the left ventricle, we assessed the hypothesis that ACh is produced by cardiomyocytes, which promotes its synthesis via a positive feedback mechanism. Rat cardiomyocytes expressed choline acetyltransferase (ChAT) in the cytoplasm and vesicular acetylcholine transporter with the vesicular structure identified by immunogold electron microscopy, suggesting that cardiomyocytes possess components for ACh synthesis. Intracellular ACh in rat cardiomyocytes was identified with physostigmine or donepezil. However, with atropine, the basal ACh content was reduced. In response to exogenous ACh or pilocarpine, cardiomyocytes increased the transcriptional activity of the ChAT gene through a muscarinic receptor and ChAT protein expression, and, finally, the intracellular ACh level was upregulated by pilocarpine. Knockdown of ChAT by small interfering RNA accelerated cellular energy metabolism, which is suppressed by ACh. Although physostigmine had a minimal effect on the ChAT promoter activity by inhibiting acetylcholinesterase, donepezil resulted in elevation of the activity, protein expression and intracellular ACh level even in the presence of sufficient physostigmine. Orally administered donepezil in mice increased the ChAT promoter activity in a reporter gene-transferred quadriceps femoris muscle and the amount of cardiac ChAT protein. These findings suggest that cardiomyocytes possess an ACh synthesis system, which is positively modulated by cholinergic stimuli. Such an amplification system in cardiomyocytes may contribute to the beneficial effects of vagal stimulation on the ventricles.

DOI： 10.1111/j.1742-4658.2009.07208.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

In a recent study, we demonstrated that vagal stimulation increases the survival of rats with myocardial infarction by inhibiting lethal arrhythmia through regulation of connexin43 (Cx43). However, the precise mechanisms for this effect remain to be elucidated. To investigate these mechanisms and the signal transduction for gap junction regulation, we investigated the effect of acetylcholine (ACh), a parasympathetic nerve system neurotransmitter, on the gap junction component Cx43 using H9c2 cells. When cells were subjected to hypoxia, the total Cx43 protein level was decreased. In contrast, pretreatment with ACh inhibited this effect. To investigate the signal transduction, cells were pretreated with L-NAME, a nitric oxide synthase inhibitor, followed by ACh and hypoxia. L-NAME was found to suppress the ACh effect. However, a NO donor, SNAP, partially inhibited the hypoxia-induced reduction in Cx43. To delineate the mechanisms of the decrease in Cx43 under hypoxia, cells were pretreated with MG132, a proteasome inhibitor. Proteasome inhibition produced a striking recovery of the decrease in the total Cx43 protein level under hypoxia. However, cotreatment with MG132 and ACh did not produce any further increase in the total Cx43 protein level. Functional studies using ACh or okadaic acid, a phosphatase inhibitor, revealed that both reagents inhibited the decrease in the dye transfer induced by hypoxia. These results suggest that ACh is responsible for restoring the decrease in the Cx43 protein level, resulting in functional activation of gap junctions.

DOI： 10.1254/jphs.FP0051023

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Doxorubicin (DOX) is widely used to treat patients suffering from cancer, but the usage for patients is limited because of the dose-dependent cardiotoxicity. We hypothesized that DOX induces apoptosis through caspase activation in cardiomyocytes, and we examined this hypothesis using both rat primary cultured cardiomyocytes and rat hearts from an animal model. Cardiomyocytes were treated with DOX for 24 h. The activity of caspase-3 was significantly increased by DOX treatment. In rats with DOX injected intravenously once a week for 5 weeks, left ventricular fractional shortening evaluated by echocardiography was significantly decreased at age 14 weeks, 2 weeks after the end of DOX-administration. At 16 weeks of age, endothelin-1 mRNA and atrial natriuretic peptide mRNA were also significantly increased, likewise, and TUNEL positive cells were significantly increased in the ventricles of DOX-treated rats. The activity of caspase-3 in the ventricles was also significantly increased compared to that of untreated rats at 16 weeks. However, the activity of caspase-8 and the expression level of Fas-ligand mRNA were comparable with those of the untreated rats. In conclusion, DOX induces apoptosis through the activation of caspase-3, suggesting that apoptosis has an important role in the progression of cardiomyopathy due to DOX.

DOI： 10.1254/jphs.FP0050980

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHYSIOLOGICAL SOCIETY JAPAN  

Background: Hypoxia-inducible factor (HIF)-1 alpha regulates the transcription of lines of genes, including vascular endothelial growth factor (VEGF), a major gene responsible for angiogenesis. Several recent studies have demonstrated that a nonhypoxic pathway via nitric oxide (NO) is involved in the activation of HIF-1 alpha. However, there is no direct evidence demonstrating the release of angiogenic factors by cardiomyocytes through the nonhypoxic induction pathway of HIF-1 alpha in the heart. Therefore we assessed the effects of an NO donor, S-Nitroso-N-acetylpenicillamine (SNAP) on the induction of VEGF via HIF-1 alpha under normoxia, using primary cultured rat cardiomyocytes (PRCMs). Methods and Results: PRCMs treated with acetylcholine (ACh) or SNAP exhibited a significant production of NO. SNAP activated the induction of HIF-1 alpha protein expression in PRCMs during normoxia. Phosphatidlylinositol 3-kinase (PI3K)-dependent Akt phosphorylation was induced by SNAP and was completely blocked by wortmannin, a PI3K inhibitor, and N-G-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor. The SNAP treatment also increased VEGF protein expression in PRCMs. Furthermore, conditioned medium derived from SNAP-treated cardiomyocytes phosphorylated the VEGF type-2 receptor (Flk-1) of human umbilical vein endothelial cells (a fourfold increase compared to the control group, p < 0.001, n = 5) and accelerated angiogenesis. Conclusion: Our results suggest that cardiomyocytes produce VEGF through a nonhypoxic HIF-1 alpha induction pathway activated by NO, resulting in angiogenesis.

DOI： 10.2170/physiolsci.RP002305

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Background - Myocardial ischemia (MI) leads to derangements in cellular electrical stability and the generation of lethal arrhythmias. Vagal nerve stimulation has been postulated to contribute to the antifibrillatory effect. Here, we suggest a novel mechanism for the antiarrhythmogenic properties of vagal stimulation during acute MI.
Methods and Results - Under anesthesia, Wistar rats underwent 30 minutes of left coronary artery (LCA) ligation with vagal stimulation (MI-VS group, n = 11) and with sham stimulation (MI-SS group, n = 12). Eight of the 12 rats in the MI-SS group had ventricular tachyarrhythmia (VT) during 30-minute LCA ligation; on the other hand, VT occurred in only 1 of the 11 rats in the MI-VS group (67% versus 9%, respectively). Atropine administration abolished the antiarrhythmogenic effect of vagal stimulation. Immunoblotting revealed that the MI-SS group showed a marked reduction in the amount of phosphorylated connexin43 (Cx43), whereas the MI-VS group showed only a slight reduction compared with the sham operation and sham stimulation group (37 +/- 20% versus 79 +/- 18%). Immunohistochemistry confirmed that the MI-induced loss of Cx43 from intercellular junctions was prevented by vagal stimulation. In addition, studies with rat primary-cultured cardiomyocytes demonstrated that acetylcholine effectively prevented the hypoxia-induced loss of phosphorylated Cx43 and ameliorated the loss of cell-to-cell communication as determined by Lucifer Yellow dye transfer assay, which supports the in vivo results.
Conclusions - Vagal nerve stimulation exerts antiarrhythmogenic effects accompanied by prevention of the loss of phosphorylated Cx43 during acute MI and thus plays a critical role in improving ischemia-induced electrical instability.

DOI： 10.1161/CIRCULATIONAHA.104.525493

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Electrical stimulation of the vagal efferent nerve improves the survival of myocardial infarcted rats. However, the mechanism for this beneficial effect is unclear. We investigated the effect of acetylcholine (ACh) on hypoxia-inducible factor (HIF)-1 alpha using rat cardiomyocytes under normoxia and hypoxia. ACh posttranslationally regulated HIF-1 alpha and increased its protein level under normoxia. ACh increased Akt phosphorylation, and wortmannin or atropine blocked this effect. Hypoxia-induced caspase-3 activation and mitochondrial membrane potential collapse were prevented by ACh. Dominant-negative HIF-1 alpha inhibited the cell protective effect of ACh. In acute myocardial ischemia, vagal nerve stimulation increased HIF-1 alpha expression and reduced the infarct size. These results suggest that ACh and vagal stimulation protect cardiomyocytes; through the PI3K/Akt/HIF-1 alpha pathway. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.02.065

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In demyelinating diseases, the mechanisms of how oligodendrocyte (OLG) progenitor cells are affected in the demyelinated area remain to be elucidated. To investigate one aspect of the mechanisms, we focused on the role of tenascin C in regulating the migratory mobility of the progenitor cells via beta-catenin. By cDNA subtraction screening, we found tenascin C expression to be increased in OLG progenitors (rat primary O2A cells). Tenascin C inhibited the migration of OLG progenitors and CG-4 cells, and beta-catenin accumulated at focal adhesions in these cells. These changes were associated with the inactivation of canonical wnt signaling. Overexpression of the wnt-signaling antagonist Dapper prevented the migration of CG-4 cells. This suggests that inactivation of the wnt signal is responsible for impaired migration of OLG caused by tenascin C. Our results suggest that tenascin C is involved in the impaired mobility of OLG progenitor cells through increased amounts of adhesion complex as well as the prevention of wnt signaling.

DOI： 10.1016/j.febslet.2004.05.022

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Background-Two subtypes of endothelin (ET) receptors, ETA and ETB, are distributed in vascular smooth muscle cells to cause contraction and proliferation. Vascular endothelial cells express only ETB receptors, which cause NO release. Although ETA receptor blockade is reported to be effective in ameliorating vascular remodeling, there is no report on the long-term effect of ETB receptor blockade on vascular remodeling after injury.
Methods and Results-ETB receptor-knockout (KO) mice, which were genetically rescued from lethal intestinal aganglionosis, and wild-type (WT) mice underwent complete ligation of the right common carotid artery, ie, a blood flow cessation model of vascular remodeling. Fourteen days after ligation, the intimal area, the ratio of intimal to medial areas, and the stenotic ratio in the ligated artery of KO mice were significantly increased compared with those of WT mice. The expression level of ET-1 mRNA in the ligated artery of KO mice was increased similarly to that of WT mice, whereas tissue NOx levels in lesions of KO mice were significantly lower than those of WT mice. Long-term treatment with the ETA receptor antagonist TA-0201 (0.5 mg . kg(-1) . d(-1)) significantly ameliorated vascular stenosis in both groups. Long-term treatment with the ETB receptor antagonist A-192.621 (30 mg . kg(-1) . d(-1)) worsened vascular remodeling in WT mice.
Conclusions-We demonstrated that-inhibition of the ETB receptor system is harmful for vascular remodeling after injury, the mechanism of which is partly attributed to decreased NO release, in KO mice. These results suggest that the overall effect of vascular ETB receptors is antiproliferative in the injured artery.

DOI： 10.1161/01.CIR.0000032004.56585.2A

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Background-Hypoxia-inducible factor (HIF)-1 alpha is an important transcriptional factor that activates the gene expression of glycolytic enzymes, which are activated as compensation for impaired beta -oxidation of fatty acid in the failing heart. We reported that cardiac endothelin (ET)-1 expression is markedly increased in heart failure. The mechanism, however, is unknown. Because we found an HIF-1 alpha binding site in the 5 ' -promoter region of the ET-1 gene, we hypothesized that HIF-1 alpha is involved in this mechanism.
Methods and Results-In rat cardiomyocytes, luciferase assay and electrophoretic mobility shift assay showed that HIF-1 alpha transcriptionally activates ET-1 gene expression by direct interaction with the predicted DNA binding site in the 5 ' -promoter region. HIF-1 alpha mRNA and ET-1 mRNA in the failing heart increased during the aggravation of heart failure in vivo in animal models, ie, rats with myocardial infarction and hamsters with cardiomyopathy. In cultured cardiomyocytes treated with a mitochondrial inhibitor, HIF-1 alpha mRNA and ET-1 mRNA were markedly increased with activated glycolysis, and antisense oligonucleotide for HIF-1 alpha largely inhibited the increased gene expression of ET-1.
Conclusions-The present study revealed a novel molecular mechanism of upregulation of myocardial ET-1 in heart failure, indicating that induction of HIF-1 alpha to stimulate glycolysis as an adaptation in heart failure against impaired energy metabolism alternatively causes an elevation of cardiac ET-1 gene expression as a maladaptation.

DOI： 10.1161/01.cir.103.19.2387

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Renin plays a key role in controlling blood pressure through its specific cleavage of angiotensinogen to generate angiotensin I (AI), Although possible existence of the other angiotensin forming enzymes has been discussed to date, its in vivo function remains to be elucidated. To address the contribution of renin, we generated renin knockout mice. Homozygous mutant mice show neither detectable levels of plasma renin activity nor plasma AI, lowered blood pressure 20-30 mm Hg less than normal, increased urine and drinking volume, and altered renal morphology as those observed in angiotensinogen-deficient mice. We recently found the decreased density in granular layer cells of hippocampus and the impaired blood-brain barrier function in angiotensinogen-deficient mice. Surprisingly, however, such brain phenotypes were not observed in renin-deficient mice. Our results demonstrate an indispensable role for renin in the circulating angiotensin generation and in the maintenance of blood pressure, but suggest a dispensable role for renin in the blood-brain barrier function.

DOI： 10.1074/jbc.275.1.5

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Astrocytes in the central nervous system have physiologically important roles in the response to brain injury(1,2). Brain damage results in disruption of the blood-brain barrier (BBB), producing detachment of astrocyte endfeet from endothelial cells(3). The resultant leakage of serum proteins from loosened tight junctions between endothelial cells produces brain edema. At the same time, reactive astrocytes migrate to the injured area, where they proliferate and produce extracellular matrix(4-6), thereby reconstituting the BBB. As astrocytes are known to express angiotensinogen(7,8), which is the precursor of angiotensins (Al to AIV), we have investigated a possible functional contribution of angiotensinogen or one of its metabolites to BBB reconstitution. The astrocytes of angiotensinogen knockout mice had very attenuated expression of glial fibrially acidic protein and decreased laminin production in response to cold injury, and ultimately incomplete reconstitution of impaired BBB function. Although these abnormalities were rescued by administration of All or AIV, the restoration of BBB function was not inhibited by All type 1 and 2 receptor antagonists. These findings provide evidence that astrocytes with angiotensins are required for functional maintenance of the BBB.

DOI： 10.1038/2070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

We examined intrarenal localization of angiotensin II type 1 receptor (AT1) mRNA in kidneys of normal adult male Munich Wistar rats using the methods of reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. For RT-PCR, we used a rat AT1 subtype A (AT1A)-specific oligonucleotide primer pair. To semiquantitatively assess the expression level of AT1 mRNA among several regions of kidney, AT1 cDNA was coamplified with beta-actin cDNA. When compared to the level in the adrenal gland (expressed as 100%), the level of AT1 mRNA was markedly higher in glomeruli (273 +/- 69%), followed in intensity by the renal papilla (151 +/- 57%), renal cortex (139 +/- 19%), and renal medulla (114 +/- 35%). In situ hybridization studies, using a 479 bp nucleotide fragment from AT1A-coding exon as a probe, also revealed a glomerular preponderant pattern of AT1 mRNA localization. Thus, within the glomerulus, AT1 mRNA localized in mesangial areas, predominantly at the vascular pole. In the vascular components of the juxtaglomerular apparatus (JGA), namely the terminal portion of the afferent arteriole (that is, immunohistochemically renin-positive site) and extraglomerular mesangial cells, the latter showed AT1 mRNA localization in the non-manipulated kidney, while AT1 mRNA was undetectable in the arteriole outside the JGA. The kidneys of rats treated with an angiotensin I converting enzyme inhibitor (ACEI) showed extension of the AT1 mRNA localization on the afferent arteriole toward the interlobular artery. We speculate that the observed pattern of AT1 mRNA localizing in the glomerular vascular pole and JGA may account for the unique physiological and pathophysiological actions of angiotensin II (Ang II), that is: (1) the renal hemodynamic effect of Ang II involves simultaneous increases in afferent and efferent arteriolar resistances and a reduction in ultrafiltration coefficient; (2) experimental glomerular sclerosis, sensitive to pharmacological inhibition of angiotensin II, is commonly prominent at the vascular pole; and (3) a negative feedback relationship exists between angiotensin II and renin, presumably via the AT1 on the JGA.

DOI： 10.1038/ki.1993.174

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

Previous studies in experimental models of progressive renal failure have shown that the capacity of antihypertensive drugs to protect glomeruli from sclerosis is often unpredictable from their effect on systemic blood pressure. The present study was undertaken to ascertain whether this systemic blood pressure-independent structure-preserving effect of antihypertensives, particularly angiotensin II converting enzyme inhibitors (ACEI), is confined to the glomerulus or not, as well as whether this effect is mediated via angiotensin II (Ang II). The following experimental drug regimens were used in the rat model of subtotal nephrectomy (sNPX): so-called triple therapy [TRX: a combination of reserpine 5 mg/liter drinking water (DW), hydralazine 80 mg/liter DW and hydrochlorothiazide 25 mg/liter DW], or ACEI (either captopril, CPL, 600 mg/liter DW, enalapril, ENL, 400 mg/liter DW or lisinopril, LSL, 200 mg/liter DW). or a novel Ang II receptor antagonist (Ang IIR, L-158.809, 20 mg/liter DW). These dosages were identified in pilot studies to be the minimum required to control systemic blood pressure in the early phase up to 12 weeks. In addition, a separate group was treated with a higher dose of L-158,809 (80 mg/liter DW) with equipotent systemic pressor effect. Treatment was initiated eight weeks after subtotal nephrectomy following renal biopsy, and animals were sacrificed at 16 weeks. In ACEI treated rats, carotid arterial wall thickening (WT), defined as ratio of media thickening to radius of outer vessel wall, was similar to normal age-matched control (0.073 in all ACEI treated rats, vs. 0.074 in normal control) and significantly less than with TRX (ratio 0.118) or untreated sNPX (0.130). Even more remarkably, coronary arteriole WT in ACEI-treated rats averaged 0.139, a value less than one half and one third of TRX (0.298) and untreated sNPX control (0.388), respectively. Similar results were obtained for mesenteric artery WT. These findings were closely paralleled by changes of glomerular sclerosis. In untreated sNPX control rats, glomerular sclerosis increased from biopsy to autopsy specimens by an average of 458%. Although TRX dampened the degree of increase in sclerosis to on average 212%, this protective effect was far less than that achieved by ACEI. In the latter, sclerosis increased on average only 65% from biopsy to autopsy. Although all ACEIs were more effective than TRX, captopril and lisinopril groups showed greatest benefit at these doses. Ang IIR also protected renal and extrarenal structures with 34% increase of sclerosis index in low dose and WT 0.088, 0.117 and 0.112, respectively in carotid, mesenteric and coronary arteries. Higher dose had greater protective effects, although systemic pressures were comparable to the low dose. The findings indicate that in minimum dosages required for controlling systemic blood pressures ACEI and Ang IIR are more potent than other vasodilators in protecting glomerular structure. This greater structure-preserving potency of ACEI and Ang IIR is not limited to the glomerulus, but extends to other systemic vascular beds, where variable lesions also develop in chronic renal failure. The similar results with Ang IIR and ACEI further indicate that the progressive morphological damage of glomerular and extraglomerular vasculatures in chronic renal failure involves local action of angiotensin II.

DOI： 10.1038/ki.1992.259

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

We tested the effect of angiotensin I converting enzyme inhibitor (ACEI) on established glomerular sclerosis. Starting eight weeks after subtotal nephrectomy (sNPX), rats were given enalapril for four weeks in a dose of 50 (Group II, N = 5) or 200 mg/liter drinking water (Group III, N = 5). A third group of sNPX rats not given ACEI served as control (Group I, N = 10). Glomerular sclerosis index (SI, 0 to 4 scale) was assessed three-dimensionally on serial thin sections for individual glomeruli at biopsy (Bx, 8 weeks), and divided into four different ranks of severity and compared to autopsy (Ax, 12 weeks). In Group I control rats, 48% of the glomeruli at Bx had SI between 0 and 1 (rank 1, average: 0.49 +/- 0.06), 36% between 1 and 2 (rank 2, average: 1.53 +/- 0.06), 9% between 2 and 3 (rank 3, average: 2.45 +/- 0.12) and 7% between 3 and 4 (rank 4, average: 3.54 +/- 0.10). Glomeruli of the same rats at Ax were ranked according to severity of sclerosis, and then divided into percentile groups, corresponding to the percent of distribution at Bx. The 48% least sclerotic glomeruli at Ax had average SI of 0.69 +/- 0.08, the next 36% 2.58 +/- 0.11, and next 9% 3.97 +/- 0.02 and the most sclerotic 7% 4.00 +/- 0.00. Thus, sclerosis advanced during the last four weeks after biopsy in all glomeruli, with more accelerated progression occurring toward later stages of sclerosis. The Bx and Ax specimens of Group II rats were analyzed in an identical fashion, showing virtually no appreciable increase in SI from Bx to Ax in glomeruli with SI < 2.0 at Bx. In contrast, SI increased in the most sclerotic glomeruli as much as that found in Group I control rats. SI at the whole kidney level, assessed on a single thin section, in Group I increased from Bx to Ax, on average by 176 +/- 58% (P < 0.005). SI also increased in Group II, but to a significantly lesser degree, on average by 49 +/- 17%. More remarkably, whole kidney SI decreased markedly and significantly in two Group III rats by 57 to 71% (on average 12 +/- 24% reduction for all). From 8 to 12 weeks both systemic and glomerular pressures were normalized (approximately 112 mm Hg and approximately 52 mm Hg, respectively) in Group II and Group III, contrasting the typically elevated values in Group I (approximately 180 mm Hg and approximately 63 mm Hg). These results indicate that ACEI exerts variable effect on established glomerular sclerosis even within the same kidney. Thus, whereas ACEI has little or no effect on glomeruli with advanced sclerosis, it effectively preserves the structure of glomeruli with early or no sclerotic lesions. Moreover, ACEI in dosages in excess of required antihypertensive dose imparts additional benefit to glomerular structure, having a potential to reverse early glomerular lesions.

DOI： 10.1038/ki.1991.200

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1017/S2040174423000363

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Publisher：Cold Spring Harbor Laboratory  

Adult heart mostly contains long-lived postmitotic cardiomyocytes and non-cardiomyocytes that have proliferative potential. Here, we isolated cardiomyocytes and non-cardiomyocytes from young and aged mouse heart, and performed transcriptome analyses by RNA sequencing to understand the differences of gene expression in postmitotic and proliferative cells. Gene ontology analyses revealed that genes associated with inflammatory response were upregulated in aged cardiac myocytes, whereas genes including two ATP synthases in mitochondrial respiratory complex V (Atp5landAtp5J2) and two NADH dehydrogenases in complex I (Ndufa11andNdufv3) were significantly downregulated. In aged non-cardiomyocytes, genes related to inflammatory responses were also upregulated, while genes involved in cell cycle and DNA replication process were downregulated. We also found that the expression levels of some small nucleolar RNAs (snoRNAs) are decreased cardiomyocytes with aging. snoRNAs are deeply involved in RNA modification such as pseudouridylation stabilizing ribosomal RNA (rRNA) and mRNA splicing. Therefore, the age-related reduction in snoRNA expression may lead to the destabilization of rRNA, splicing dysfunction, and ultimately a decrease in protein synthesis capacity. A comparison with transcriptome results obtained for non-cardiomyocytes suggests that the decline in the expression of mitochondria-related genes and snoRNAs accompanying aging is specific to cardiomyocytes, implying their potential utility as one of novel aging markers in postmitotic cells.

DOI： 10.1101/2023.08.20.554007

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Publisher：Cold Spring Harbor Laboratory  

Abstract

We have previously identified the novel splicing variant of mousePparγ(Pparγ1sv) and proposed the synergistic regulation of the early stage of adipocyte differentiation byPparγ1svandPparγ2. Here, we report the finding of PPARγ-binding sites within thePparγgene locus and its importance in adipogenesis and propose the positive feedback regulation ofPparγ1svandPparγ2expression during the adipocyte differentiation of 3T3-L1 cells.

DOI： 10.1101/2023.08.03.551916

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Language：English   Publishing type：Research paper (scientific journal)  

We previously developed a stress-induced premature senescence (SIPS) model in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1). To clarify the involvement of mitochondrial function in our SIPS model, MRC-5 cells were treated with MG132 or BAFA1 along with an inhibitor targeting either the electron transport chain complex I or complex III, or with a mitochondrial uncoupler. SIPS induced by MG132 or BAFA1 was significantly attenuated by short-term co-treatment with the complex III inhibitor, antimycin A (AA), but not the complex I inhibitor, rotenone or the mitochondrial uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. By co-treatment with AA, mitochondrial and intracellular reactive oxygen species levels, accumulation of protein aggregates and mitochondrial unfolded protein responses (UPRmt ) were remarkably suppressed. Furthermore, AA co-treatment suppressed the hyperpolarization of the mitochondrial membrane and the induction of mitophagy observed in MG132-treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis.

DOI： 10.1111/febs.16785

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Publisher：Cold Spring Harbor Laboratory  

Abstract

We previously reported a novel compound called S-nitroso-N-pivaloyl-D-penicillamine (SNPiP), which was screened from a group of nitric oxide (NO) donor compounds with a basic chemical structure of S-nitroso-N-acetylpenicillamine (SNAP), to activate the non-neuronal acetylcholine (NNA) system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The NNA-activating effects included increased resilience to ischemia, modulation of energy metabolism preference, and activation of angiogenesis. Here, we performed transcriptome analysis of SNPiP-treated mice ventricles to elucidate how SNPiP exerts beneficial effects on cardiac function. A time-course study (24 and 48 h after SNPiP administration) revealed that SNPiP initially induced Wnt and cGMP-protein kinase G (PKG) signaling pathways, along with upregulation of genes involved in cardiac muscle tissue development and oxytocin signaling pathway. We also observed enrichment of glycolysis-related genes in SNPiP-treated samples, indicating a metabolic shift from oxidative phosphorylation to glycolysis following SNPiP administration in the hearts. Additionally, SNPiP significantly upregulated atrial natriuretic peptide (ANP) and sarcolipin (SLN), which play crucial roles in calcium handling and cardiac performance. These findings suggest that SNPiP may have therapeutic potential based on the pleiotropic mechanisms elucidated in this study.

DOI： 10.1101/2023.06.20.545771

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AIMS: We previously showed that hindlimb ischemia-reperfusion (IR) enhanced glucose uptake in the liver through the activation of the parasympathetic nervous system. Although we suggested that the key glucose transporter (GLUT) in this hepatic glucose uptake was GLUT4 by western blotting, the molecular weight of GLUT4 was nearly the same as that of GLUT2, which is predominantly expressed in the liver. We primarily conducted a histological evaluation to determine whether IR specifically accelerates the overexpression of GLUT4, rather than GLUT2, in the hepatocytes in vitro and in vivo. MAIN METHODS: A total of 54 male C57BL/6J mice were used and subjected to 3 min hindlimb ischemia repeated three times with 3 min interval. Focusing on the area connecting portal and central veins, the GLUT4 and GLUT2 expression in the hepatocytes were examined by real-time PCR and immunohistochemically. Moreover, the alteration of GLUT4 and GLUT2 expression by acetylcholine in the primary hepatocytes were examined by immunofluorescence. KEY FINDINGS: IR significantly upregulated the GLUT4, rather than GLUT2, expression in both mRNA and protein in the liver. Histological examination revealed marked glycogen storage in zone1, the periportal area, coincident with the enhanced GLUT4 immunoreactivity, in the IR-treated liver. Incubation of primary hepatocytes with acetylcholine induced the appearance of GLUT4 on the membrane peripheries. SIGNIFICANCE: The overexpression of GLUT4 on the membrane peripheries contributed to increasing glucose uptake found in IR-treated livers. This acceleration of glucose uptake via GLUT4 may induce marked glycogen storage in zone1 through energy production linked with increased glucose preference.

DOI： 10.1016/j.lfs.2021.120261

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DOI： 10.1016/j.autneu.2022.102957

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Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title><sec>
<title>Background</title>
Acetylcholine (ACh) plays a crucial role in the function of the heart. Recent evidence suggests that cardiomyocytes possess a non-neuronal cholinergic system (NNCS) that comprises of choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AChE) and type-2 muscarinic ACh receptors (M₂AChR) to synthesize, release, degrade ACh as well as for ACh to transduce a signal. NNCS is linked to cardiac cell survival, angiogenesis and glucose metabolism. Impairment of these functions are hallmarks of diabetic heart disease (DHD). The role of the NNCS in DHD is unknown. The aim of this study was to examine the effect of diabetes on cardiac NNCS and determine if activation of cardiac NNCS is beneficial to the diabetic heart.


</sec><sec>
<title>Methods</title>
Ventricular samples from type-2 diabetic humans and db/db mice were used to measure the expression pattern of NNCS components (ChAT, CHT1, VAChT, AChE and M₂AChR) and glucose transporter-4 (GLUT-4) by western blot analysis. To determine the function of the cardiac NNCS in the diabetic heart, a db/db mouse model with cardiac-specific overexpression of <italic>ChAT</italic> gene was generated (db/db-ChAT-tg). Animals were followed up serially and samples collected at different time points for molecular and histological analysis of cardiac NNCS components and prosurvival and proangiogenic signaling pathways.


</sec><sec>
<title>Results</title>
Immunoblot analysis revealed alterations in the components of cardiac NNCS and GLUT-4 in the type-2 diabetic human and db/db mouse hearts. Interestingly, the dysregulation of cardiac NNCS was followed by the downregulation of GLUT-4 in the db/db mouse heart. Db/db-ChAT-tg mice exhibited preserved cardiac and vascular function in comparison to db/db mice. The improved function was associated with increased cardiac ACh and glucose content, sustained angiogenesis and reduced fibrosis. These beneficial effects were associated with upregulation of the PI3K/Akt/HIF1α signaling pathway, and increased expression of its downstream targets—GLUT-4 and VEGF-A.


</sec><sec>
<title>Conclusion</title>
We provide the first evidence for dysregulation of the cardiac NNCS in DHD. Increased cardiac ACh is beneficial and a potential new therapeutic strategy to prevent or delay the development of DHD.


</sec>

DOI： 10.1186/s12933-021-01231-8

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Other Link： http://link.springer.com/article/10.1186/s12933-021-01231-8/fulltext.html

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/2211-5463.13332

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Proteolytic activity declines with age, resulting in the accumulation of aggregated proteins in aged organisms. To investigate how disturbance in proteostasis causes cellular senescence, we developed a stress-induced premature senescence (SIPS) model, in which normal human fibroblast MRC-5 cells were treated with the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1) for 5 days. Time-course studies revealed a significant increase in intracellular reactive oxygen species (ROS) and mitochondrial superoxide during and after drug treatment. Mitochondrial membrane potential initially decreased, suggesting temporal mitochondrial dysfunction during drug treatment, but was restored along with mitochondrial accumulation after drug treatment. AMP-activated protein kinase alpha was notably activated during treatment; thereafter, intracellular ATP levels significantly increased. SIPS induction by MG132 or BAFA1 was partially attenuated by co-treatment with vitamin E or rapamycin, in which the levels of ROS, mitochondrial accumulation, and protein aggregates were suppressed, implying the critical involvement of oxidative stress and mitochondrial function in SIPS progression. Rapamycin co-treatment also augmented the expression of HSP70 and activation of AKT, which could recover proteostasis and promote cell survival, respectively. Our study proposes a possible pathway from the disturbed proteostasis to cellular senescence via excess ROS production as well as functional and quantitative changes in mitochondria.

DOI： 10.1111/febs.16249

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/febs.16249

Language：Japanese   Publisher：日本医科大学医学会  

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Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Low body weight at birth has been shown to be a risk factor for future metabolic disorders, as well as stress response abnormalities and depression. We showed that low-birthweight rats had prolonged high blood corticosterone levels after stress exposure, and that an increase in Gas5 lncRNA, a decoy receptor for glucocorticoid receptors (GRs), reduces glucocorticoid responsiveness. Thus, we concluded that dampened pituitary glucocorticoid responsiveness disturbed the glucocorticoid feedback loop in low-birthweight rats. However, it remains unclear whether such glucocorticoid responsiveness is suppressed solely in the pituitary or systemically. The expression of Gas5 lncRNA increased only in the pituitary, and the intact induction of expression of the GR co-chaperone factor Fkbp5 against dexamethasone was seen in the liver, muscle, and adipose tissue. Intervention with a methyl-modulator diet (folate, VB12, choline, betaine, and zinc) immediately before or one week after delivery reversed the expression level of Gas5 lncRNA in the pituitary of the offspring. Consequently, it partially normalized the blood corticosterone levels after restraint stress exposure. In conclusion, the mode of glucocorticoid response in low-birthweight rats is impaired solely in the pituitary, and intervention with methyl-modulators ameliorates the impairment, but with a narrow therapeutic time window.

DOI： 10.3390/ijms22189767

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Smoking during pregnancy is one of the causes of low birthweight. Ingestion of nicotine during pregnancy has various metabolic impacts on the fetus and offspring. According to the developmental origins of health and disease theory, low birthweight is a risk factor for developing various non-communicable diseases, including diabetes. We hypothesized that when nicotine-induced low-birthweight rats, when exposed to a high-fat diet (HFD) after growth, are predisposed to glucose intolerance as a result of a mismatch between the eutrophic environment and small body size. Therefore, we investigated whether hyperinsulinemia was caused by exposure of nicotine-induced low-birthweight rats to HFD, including whether this phenomenon exhibited possible sex differences. The average birthweight and body weight at weaning day of offspring from nicotine-administered dams was lower than those of controls. The offspring from nicotine-administered dams did not show rapid fat accumulation after exposure to HFD, and weight and body fat ratio of these animals did not differ from those of the controls. Blood glucose levels did not differ between the groups, but insulin levels increased only in male HFD-exposed offspring from nicotine-administered dams. Similarly, only in HFD-exposed male from nicotine-administered dams showed decreases in the insulin receptor expression in the liver. We conclude that male rats subjected to prenatal nicotine exposure develop hyperinsulinemia when exposed to HFD after growth. Our results suggest that decreased expression of insulin receptors in the liver may be involved in the mechanism underlying hyperinsulinemia in low-birthweight offspring, a phenomenon that appeared to exhibit a sex-specific bias.

DOI： 10.3389/fendo.2021.694336

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.neuint.2020.104934

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Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.neuint.2020.104943

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: We previously established that the non-neuronal cardiac cholinergic system (NNCCS) is equipped with cardiomyocytes synthesizes acetylcholine (ACh), which is an indispensable endogenous system, sustaining cardiac homeostasis and regulating an inflammatory status, by transgenic mice overexpressing choline acetyltransferase (ChAT) gene in the heart. However, whole body biological significances of NNCCS remain to be fully elucidated. METHODS AND RESULTS: To consolidate the features, we developed heart-specific ChAT knockdown (ChATKD) mice using 3 ChAT-specific siRNAs. The mice developed cardiac dysfunction. Factors causing it included the downregulation of cardiac glucose metabolism along with decreased signal transduction of Akt/HIF-1alpha/GLUT4, leading to poor glucose utilization, impairment of glycolytic metabolites entering the tricarboxylic (TCA) cycle, the upregulation of reactive oxygen species (ROS) production with an attenuated scavenging potency, and the downregulated nitric oxide (NO) production via NOS1. ChATKD mice revealed a decreased vagus nerve activity, accelerated aggression, more accentuated blood basal corticosterone levels with depression-like phenotypes, several features of which were accompanied by cardiac dysfunction. CONCLUSION: The NNCCS plays a crucial role in cardiac homeostasis by regulating the glucose metabolism, ROS synthesis, NO levels, and the cardiac vagus nerve activity. Thus, the NNCCS is suggested a fundamentally crucial system of the heart.

DOI： 10.1186/s12576-020-00787-6

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Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pone.0238223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Medical Association of Nippon Medical School  

Background: The antidiabetic drug teneligliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor with a thiazolidine-specific structure. This study aimed to investigate whether teneligliptin can activate PPARγ directly and/or indirectly in cell-based assays. Methods: Promoter assays using the reporter construct driven under the control of the SV40 promoter and the PPAR response element (PPRE) were performed. Luciferase activity was measured after a 3-day incubation of vector-transduced cells with various concentrations of teneligliptin. Results: Treatment of the cells with 50 μM teneligliptin significantly transactivated a reporter gene. The presence of the PPARγ antagonist, GW9662, did not affect the activation of PPRE-reporter expression by teneligliptin. Conclusion: We found that teneligliptin could increase PPARγ activity in cell-based assays irrespective of the PPARγ ligand-binding domain.

DOI： 10.1272/jnms.2018_85-15

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/978-981-10-5526-3_6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X(2) purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X(2) purinoceptors acquire permeability to large cations, such as N-methyl-D-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, alpha,beta-methylene ATP did not produce a cation current, whereas ATP gamma S and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl- 2', 4'-sulfonic acid. Accordingly, P2X(2) purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X(2) purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.
NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X(2) purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X(2) purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

DOI： 10.1152/jn.00506.2016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Cardiomyocytes possess a non-neuronal cardiac cholinergic system (NNCCS) regulated by a positive feedback system; however, its other regulatory mechanisms remain to be elucidated, which include the epigenetic control or regulation by the female sex steroid, estrogen. Here, the NNCCS was shown to possess a circadian rhythm; its activity was upregulated in the light-off phase via histone acetyltransferase (HAT) activity and downregulated in the light-on phase. Disrupting the circadian rhythm altered the physiological choline acetyltransferase (ChAT) expression pattern. The NNCCS circadian rhythm may be regulated by miR-345, independently of HAT, causing decreased cardiac ChAT expression. Murine cardiac ChAT expression and ACh contents were increased more in female hearts than in male hearts. This upregulation was downregulated by treatment with the estrogen receptor antagonist tamoxifen, and in contrast, estrogen reciprocally regulated cardiac miR-345 expression. These results suggest that the NNCCS is regulated by the circadian rhythm and is affected by sexual dimorphism.

DOI： 10.1007/s12265-017-9750-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: The majority of Parkinson's disease (PD) cases are sporadic and idiopathic suggesting that this neurodegenerative disorder is the result of both environmental and genetic factors. Stress and neuroinflammation are among the factors being investigated for their possible contributions to PD. Experiments in rodents showed that severe chronic stress can reduce the number of dopaminergic neurons in the substantia nigra pars compacta (SNc); the same cells that are lost in PD. These actions are at least in part mediated by increased oxidative stress. Here, we tested the hypothesis that the interleukin-13 receptor alpha 1 (IL-13Ra1), a cytokine receptor whose activation increases the vulnerability of dopaminergic neurons to oxidative damage, participates in the stress-dependent damage of these neurons.
Methods: Mice were subject to daily sessions of 8 h (acute) stress for 16 weeks (5 days a week), a procedure previously showed to induce loss of dopaminergic neurons in the SNc. The source and the kinetics of interleukin-13 (IL-13), the endogenous ligand of IL-13Ra1, were evaluated 0, 1, 3, 6, and 8 h and at 16 weeks of stress. Identification of IL-13 producing cell-type was performed by immunofluorescent and by in situ hybridization experiments. Markers of oxidative stress, microglia activation, and the number of dopaminergic neurons in IL-13Ra1 knock-out animals (II13ra1(Y/-)) and their wild-type littermates (II13ra1(Y/+)) were evaluated at 16 weeks of stress and at 20 weeks, following a 4 week nonstressed period and compared to non-stressed mice.
Results: IL-13 was expressed in microglial cells within the SN and in a fraction of the tyrosine hydroxylase-positive neurons in the SNc. IL-13 levels were elevated during daily stress and peaked at 6 h. 16 weeks of chronic restraint stress significantly reduced the number of SNc dopaminergic neurons in II13ra1(Y/+) mice. Neuronal loss at 16 weeks was significantly lower in II13ra1(Y/-) mice. However, the loss of dopaminergic neurons measured at 20 weeks, after 4 weeks of non-stress following the 16 weeks of stress, was similar in II13ra1(Y/+) and Il13ra1Y/-mice.
Conclusions: IL-13, a cytokine previously demonstrated to increase the susceptibility of SNc dopaminergic neurons to oxidative stress, is elevated in the SN by restraint stress. Lack of IL-13Ra1 did not prevent nor halted but delayed neuronal loss in the mouse model of chronic restraint stress. IL-13/IL-13Ra1 may represent a target to reduce the rate of DA neuronal loss that can occur during severe chronic restraint stress.

DOI： 10.1186/s12974-017-0862-1

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.bbi.2016.11.028

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Corticotrophin-releasing factor (CRF) regulates the hypothalamic-pituitary-adrenal axis response to stress through its type 1 receptor (CRF1) in the corticotrophs of the anterior pituitary. Although CRF1 mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF1 protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177-188 sequence of the first extracellular loop of the rat CRF1. Using the antiserum, CRF1-like immunoreactivity (CRF1-LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF1-LI also appeared in the intermediate lobe cells and on the fibre-like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF1 (male: 27.1 +/- 3.0%, female: 18.0 +/- 3.0%), followed by lactotrophs (male: 6.7 +/- 3.0%, female: 12.1 +/- 1.3%), gonadotrophs (male: 2.6 +/- 1.0%, female: 7.5 +/- 0.5%), thyrotrophs (male: 2.9 +/- 0.1%, female: 5.3 +/- 1.2%) and somatotrophs (male: 1.1 +/- 0.3%, female: 1.2 V 0.5%). The percentage of CRF1-LI-positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF1-LI-positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF1 in the intermediate lobe (98.9 +/- 0.2%). CRF1-LI and the percentage of CRF1-LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 mu g/kg bw). These results suggest that CRF1 is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF1 are regulated by glucocorticoids in the rat anterior pituitary.

DOI： 10.1111/jne.12440

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have previously demonstrated that the pharmacotherapy with donepezil, an acetylcholinesterase inhibitor, suppresses cardiac remodeling in a mouse model of ischemic heart failure after myocardial infarction (MI). However, the precise mechanisms of the cardioprotective effect of donepezil have not been completely delineated. Because post-ischemic inflammation is a pathological key event in the cardiac remodeling process following MI, we investigated the hypothesis that donepezil acts as an inhibitor of inflammatory mediators. RAW 264.7 murine macrophage cells were pretreated with donepezil (100 mu M) prior to a pro-inflammatory stimulation by administration of lipopolysaccharide (LPS, 10 ng/ml). Donepezil significantly reduced intra- and extracellular levels of various kinds of inflammatory mediators such as TNF-alpha, IL-1 beta, IL-2, IL-6 and IL-18 after the LPS stimulation, and attenuated LPS-induced nuclear translocation of nuclear factor -kappa B (NF-kappa B). These results indicate that donepezil possesses an anti-inflammatory property. However, the inhibitory effect of donepezil on the macrophage inflammatory responses was never reproduced by ACh, nor was disrupted by ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to inhibit the inflammatory responses in LPS-stimulated macrophage cells. These results suggest that a cholinergic anti-inflammatory pathway would not be involved in the anti-inflammatory effect of donepezil and that the specific characteristics of donepezil in suppressing the LPS-induced cytokine release and the NF-kappa B activation would be independent of its acetylcholinesterase inhibition. The present study showed that donepezil exerts an anti-inflammatory effect independently of acetylcholinesterase inhibitory action, thereby donepezil may contribute to cardioprotection during cardiac remodeling process in an ischemic heart failure after MI. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2016.06.053

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of dopaminergic (DA) neurons in the nigrostriatal and mesolimbic pathways including ventral tegmental area (VTA). Although several factors for the neuronal loss have been suggested, most of the PD cases are sporadic and idiopathic. In our previous study, we demonstrated the first evidence that solely chronic restraint stress (RS) induced the DA neuronal loss in the substantia nigra (SN). In this study, we further investigated whether chronic stress could affect other major DA systems, VTA and tuberoinfundibular system (TIDA), by using immunohistochemical and in situ hybridization techniques. The present study showed that, in the VTA, tyrosine hydroxylase (TH) immunoreactive neurons decreased by 9.8% at 2nd week, 19.2% at 4th week, 39.5% at 8th week, and 40.6% at 16th week during chronic RS as compared to control. Similarly, in the TIDA, the TH neurons decreased by 10.9% at 2nd week, 38.2% at 4th week, 56.3% at 8th week, and 57.1% at 16th week. The in situ hybridization results consistently demonstrated decreases in Th mRNA expressing cells in the VTA and TIDA in a comparable time dependent manner. Thus, exposure to chronic stress may simultaneously induce multiple neuronal loss of DA systems. (C) 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2016.04.008

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We previously reported that satellite cells possess the ability to produce angiogenic factors, including fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in vivo. However, whether C2C12 cells possess a non-neuronal cholinergic system (NNCS) or non-neuronal ACh (NNA) remains to be studied; therefore, we investigated the system using C2C12 cells and its regulatory mechanisms. C2C12 cells synthesized ACh, the level of which was comparable with that of cardiomyocytes, and the synthesis was augmented by the acetylcholinesterase inhibitor galantamine. The ChAT promoter activity was upregulated by nicotine or galantamine, partly through nicotinic receptors for both agents as well as through a non-nicotinic receptor pathway for galantamine. Further, VEGF secretion by C2C12 cells was also increased by nicotine or galantamine through nicotinic receptors as well as partly through non-nicotinic pathways in the case of galantamine. These results suggest that C2C12 cells are equipped with NNCS or NNA, which is positively regulated through nicotinic or non-nicotinic pathways, particularly in the case of galantamine. These results provide a novel concept that myogenic cells expressing NNA can be a therapeutic target for regulating angiogenic factor synthesis. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.intimp.2015.04.057

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Ischemic preconditioning (IPC) renders the targeted organ resistant to prolonged ischemic insults, leading to organoprotection. Among several means to achieve IPC, we reported that remote ischemic preconditioning (RIPC) activates the non-neuronal cardiac cholinergic system (NNCCS) to accelerate de novo ACh synthesis in cardiomyocytes. In the current study, we aimed to optimize a specific protocol to most efficiently activate NNCCS using RIPC. In this study, we elucidated that the protocol with 3 min of ischemia repeated three times increased cardiac ChAT expression (139.2 +/- 0.4%; P &lt; 0.05) as well as ACh (142 +/- 2.0 x 10(-8) M; P &lt; 0.05) and ATP content (2.13 +/- 0.19 mu mol/g tissue; P &lt; 0.05) in the heart. Moreover, in the specific protocol, several characteristic responses against energy starvation and for obtaining adequate energy were observed; therefore, it is suggested that RIPC evokes a robust response by the heart to activate NNCCS through the modification of energy metabolism. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.intimp.2015.06.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

Low birth weight (LBW) is related to increased incidence of common cardiovascular and metabolic disorders, and psychopathologies later in life. Recent studies have suggested that maternal malnutrition affects fetal hypothalamic-pituitary-adrenal (HPA) axis programing although the mechanism is unknown. We demonstrated that LBW offspring delivered from malnourished dams showed prolonged elevated plasma corticosterone concentrations when compared with those of normal-birth-weight (NBW) offspring and impaired downregulation of corticotropin-releasing factor receptor type 1 (CRF-R1, Crhr1) in the anterior pituitary in restraint. Restraint increased expression of miR449a, which we had previously demonstrated to be involved in Crhr1 downregulation, in the anterior pituitary and serum exosomal miR449a contents through glucocorticoids in NBW offspring, but not in LBW offspring. Although plasma corticosterone concentrations were higher at 2000 h than at 0800 h in both LBW and NBW offspring, they were significantly higher in LBW offspring than in NBW offspring at 2000 and 0200 h. There were no significant diurnal changes in miR449a expression levels in the anterior pituitary of either NBW or LBW offspring, but the expression was significantly lower in LBW offspring than in NBW offspring at 1400, 2000, and 0200 h. The expression levels of GAS5, which inhibits glucocorticoid receptor (GR) binding to glucocorticoid-responsive element, in the anterior pituitary of LBW offspring were elevated when compared with those of NBW offspring. The downregulation of GR found in NBW offspring did not occur in restrained LBW offspring. These results indicate that impaired miR449a expression, probably induced by increased GAS5 expression, causes dysregulation of Crhr1 expression in the anterior pituitary, resulting in prolonged HPA axis activation in restrained LBW offspring.

DOI： 10.1530/JOE-14-0537

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Language：English   Publisher：MEDICAL ASSOC NIPPON MEDICAL SCH  

DOI： 10.1272/jnms.81.296

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

We have recently identified that donepezil, an anti-Alzheimer drug, accelerates angiogenesis in a murine hindlimb ischemia (HLI) model. However, the precise mechanisms are yet to be fully elucidated, particularly whether the effects are derived from endothelial cells alone or from other nonvascular cells. Further investigation of the HLI model revealed that nicotine accelerated angiogenesis by activation of vascular endothelial cell growth factor (VEGF) synthesis through nicotinic receptors in myogenic cells, that is, satellite cells, in vivo and upregulated the expression of angiogenic factors, for example, VEGF and fibroblast growth factor 2, in vitro. As a result, nicotine prevented skeletal muscle from ischemia-induced muscle atrophy and upregulated myosin heavy chain expression in vitro. The in vivo anti-atrophy effect of nicotine on muscle was also observed in galantamine, another anti-Alzheimer drug, playing as an allosteric potentiating ligand. Such effects of nicotine were attenuated in alpha 7 nicotinic receptor knockout mice. In contrast, PNU282987, an alpha 7 nicotinic receptor agonist, comparably salvaged skeletal muscle, which was affected by HLI. These results suggest that cholinergic signals also target myogenic cells and have inhibiting roles in muscle loss by ischemia-induced muscle atrophy.

DOI： 10.1016/j.trsl.2014.02.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Background/Aims: We previously suggested that a non-neuronal cholinergic system modulates energy metabolism through the mitochondria. However, the mechanisms responsible for making this system crucial remained undetermined. Methods: In this study, we developed a fusion protein expression vector containing a luciferase gene fused to the folic acid receptor-alpha. gene. Results: This protein of the vector was confirmed to target the plasma membrane of transfected HEK293 cells, and vector-derived luciferase activities and ATP levels in viable cells were positively correlated (r = 0.599). Using this luciferase vector, choline acetyltransferase (ChAT)-expressing cells (i.e., cells with an activated non-neuronal cholinergic system) had increased cellular ATP levels. ChAT-expressing cells also had upregulated IGF-1R and Glut-1 protein expressions as well as increased glucose uptake. This activated non-neuronal cholinergic system with efficient glucose metabolism rendered cells resistant to serum depletion induced cell death. Conclusion: Our results indicate that a non-neuronal cholinergic system is involved in sustaining ATP levels to render cells resistant to a nutrient-deficient environment. Copyright (C) 2014 S. Karger AG, Basel.

DOI： 10.1159/000363042

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Background: Oestrogen is known to play a cardioprotective role in cardiovascular diseases, as demonstrated in a number of animal studies. However, few human studies have investigated sex-based differences with regard to cardiac glucose uptake using F-18-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT).
Methods: Therefore, we evaluated healthy male and female subjects who underwent FDG-PET/CT examination to determine whether there was a sex-related difference in cardiac glucose uptake with age.
Results: In females, the prevalence of maximal FDG uptake (PET score 2) demonstrated a convex pattern with ageing, and it peaked at age 51-60 years in the females, gradually decreasing to a minimum at age &gt; 70 years. In contrast, the prevalence of maximal FDG uptake by age in males was a mirror image of that in females, i.e. it formed a concave pattern with a nadir at 61-70 years, followed by an increase in the prevalence. These findings suggest that female hearts depend more on glucose as an energy substrate as they age, however, efficient glucose uptake is attenuated with increasing age. In contrast, the male heart sustains its glucose uptake capacity at age &gt; 70 years.
Conclusion: This characteristic sex-based difference in cardiac glucose uptake might be related to the female predominance of Takotsubo cardiomyopathy. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.ijcard.2013.03.016

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Our conditional VHL knockout (VHL-KO) mice, having VHL gene deletion induced by tamoxifen, developed severe hypoglycemia associated with disproportionately increased storage of PAS-positive substances in the liver and resulted in the death of these mice. This hypoglycemic state was neither due to impaired insulin secretion nor insulin receptor hypersensitivity. By focusing on insulin-like growth factor I (IGF-I), which has a similar effect on glucose metabolism as the insulin receptor, we demonstrated that IGF-I receptor (IGF-IR) protein expression in the liver was upregulated in VHL-KO mice compared to that in the mice without VHL deletion, as was the expression of glucose transporter (GLUT) 1. The interaction of the receptor for activated C kinase (RACK) 1, which predominantly binds to VHL, was enhanced in VHL-KO livers with IGF-IR, because VHL deletion increased free RACK1 and facilitated the IGF-IR-RACKI interaction. An IGF-IR antagonist retarded hypoglycemic progression and sustained an euglycemic state. These IGF-IR antagonist effects on restoring blood glucose levels also attenuated PAS-positive substance storage in the liver. Because the effect of IGF-I on HIF-1α protein synthesis is mediated by IGF-IR, our results indicated that VHL inactivation accelerated hepatic glucose storage through the upregulation of IGF-IR and GLUT1 and that IGF-IR was a key regulator in VHL-deficient hepatocytes. © 2013 Kurabayashi et al.

DOI： 10.1371/journal.pone.0069139

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We previously demonstrated that inflammatory stress impaired cardiac glucose uptake, using fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) by showing that a proportion of patients with subtle FDG signals was remarkably increased compared with that of normal subjects. The current study assessed the inhibitory effects of cancer-associated stress on cardiac glucose uptake in female cancer patients, and compared the results with those obtained for healthy female subjects. Cardiac glucose uptake was decreased in female cancer, as indicated by the significantly higher number of patients with poor FDG uptake and the lower number of patients with high FDG uptake, compared with the number of healthy subjects with poor and high FDG uptake, respectively. These results suggest that cancer-associated stress inhibited cardiac glucose utilization. From the 78 female cancer patients, 13 oophorectomized patients, who underwent repeated postoperative follow-up examinations by FDG-PET/CT before and after receiving hormone replacement therapy (HRT) with estrogen derivatives, were analyzed to determine the effects of estrogen on cardiac glucose uptake. HRT increased a proportion of patients with high FDG signals to comparable in healthy subjects with high FDG uptake, whereas that of patients with poor FDG uptake decreased. These results suggest that estrogen can improve cardiac glucose uptake in cancer-resected and oophorectomized patients.&lt
. Learning objective: This study presents us a new point of view, which should be taken into account on the pathophysiology in some cardiovascular diseases, the sex-differences of energy substrates for the heart, i.e., estrogen or its derivatives accelerates the preference of glucose by the heart. Unfortunately, a very few studies have studied on this issue, e.g., effects of sex-steroid on cardiac uptake of energy substrate, and therefore, this suggests that cardiologists do not focus on this issue.&gt
. © 2012 Japanese College of Cardiology.

DOI： 10.1016/j.jccase.2012.09.009

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Language：English   Publisher：SPRINGER JAPAN KK  

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Language：English   Publisher：SPRINGER JAPAN KK  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

To study prospectively influences of donepezil, an acetylcholinesterase inhibitor against Alzheimer disease, on cardiovascular system, we evaluated cardiovascular changes occurring during new initialized treatment with donepezil in 49 dementia patients over 6 months. No patient suffered from cardiovascular events. In clinical changes between baseline and the first evaluation after donepezil treatment, heart rate and plasma brain natriuretic peptide (BNP) levels as a marker for heart failure did not change (BNP: 59.62 + 62.71 pg/mL at baseline to 53.18 + 42.34 pg/mL at first evaluation; P = 0.262). We further examined plasma BNP levels in 2 groups into which the patients were divided at baseline according to the cut-off plasma BNP level of 60 pg/mL. In patients with high level of BNP, the BNP levels decreased after administration of donepezil (116.39 +/- 76.58 pg/mL at baseline to 82.24 +/- 46.64 pg/mL at first evaluation; P = 0.011) with the tendency to be reduced in the follow-up period. BNP did not change in patients with low level of BNP. Donepezil seemed to be safe in patients with dementia without symptomatic heart disease and significantly decreased plasma BNP levels in patients with subclinical chronic heart failure.

DOI： 10.1097/FJC.0b013e3182609a74

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

While NF90 has been known to participate in transcription, translation and microRNA biogenesis, physiological functions of this protein still remain unclear. To uncover this, we generated transgenic (Tg) mice using NF90 cDNA under the control of beta-actin promoter. The NF90 Tg mice exhibited a reduction in body weight compared with wild-type mice, and a robust expression of NF90 was detected in skeletal muscle, heart and eye of the Tg mice. To evaluate the NF90 overexpression-induced physiological changes in the tissues, we performed a number of analyses including CT-analysis and hemodynamic test, revealing that the NF90 Tg mice developed skeletal muscular atrophy and heart failure. To explore causes of the abnormalities in the NF90 Tg mice, we performed histological and biochemical analyses for the skeletal and cardiac muscles of the Tg mice. Surprisingly, these analyses demonstrated that mitochondria in those muscular tissues of the Tg mice were degenerated by autophagy. To gain further insight into the cause for the mitochondrial degeneration, we identified NF90-associated factors by peptide mass fingerprinting. Of note, approximately half of the NF90-associated complexes were ribosome-related proteins. Interestingly, protein synthesis rate was significantly suppressed by high-expression of NF90. These observations suggest that NF90 would negatively regulate the function of ribosome via its interaction with the factors involved in the ribosome function. Furthermore, we found that the translations or protein stabilities of PGC-1 and NRF-1, which are critical transcription factors for expression of mitochondrial genes, were significantly depressed in the skeletal muscles of the NF90 Tg mice. Taken together, these findings suggest that the mitochondrial degeneration engaged in the skeletal muscle atrophy and the heart failure in the NF90 Tg mice may be caused by NF90-induced posttranscriptional repression of transcription factors such as PGC-1 and NRF-1 for regulating nuclear-encoded genes relevant to mitochondrial function.

DOI： 10.1371/journal.pone.0043340

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Background/aims. We have reported that tubular epithelial cell injury caused by renal ischemia-reperfusion is attenuated in conditional VHL knockout (VHL-KO) mice and also that induction of hypoxia-inducible factor (HIF) suppresses angiotensin II-accelerated Habu snake venom (HV) glomerulonephropathy in rats. However, it remains unknown whether VHL knockdown protects glomerular endothelial cells from endothelium-targeted glomerulonephritis.
Methods and results. VHL-KO mice with HV glomerulonephropathy (HV GN) had fewer injured glomeruli, a lower mesangiolysis score and reduced blood urea nitrogen levels. Immunoreactivity of vascular endothelial growth factor (VEGF) in the glomerular capillaries was enhanced by VHL knockdown and was conserved even in VHL-KO mice with HV GN, despite HV-attenuating endothelial VEGF expression in vitro. VHL-KO mice showed enhanced nitric oxide (NO) production in glomerular endothelial cells and tubular cells, associated with activated VEGF expression in the kidney (i.e. an activated NO-VEGF axis). The levels of NO in glomeruli and tubules were conserved even in mice with HV GN. In contrast, suppressing NO production in glomerular endothelial cells by an NO synthase inhibitor, N(pi)-nitro-L-arginase, completely blunted the protection of VHL-KO from HV GN. The activated NO-VEGF axis in the kidney of VHL-KO mice was also associated with an elevation in Flk-1 phosphorylation and increased levels of IL-10 and IP-10.
Conclusion. Conditional VHL knockdown may enhance the NO-VEGF axis and protect glomerular endothelial cells from HV GN, thereby providing resistance to injury of tubular epithelial cells and glomerular endothelial cells.

DOI： 10.1093/ndt/gfr176

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background: We have previously demonstrated that the chronic intervention in the cholinergic system by donepezil, an acetylcholinesterase inhibitor, plays a beneficial role in suppressing long-term cardiac remodeling after myocardial infarction (MI). In comparison with such a chronic effect, however, the acute effect of donepezil during an acute phase of MI remains unclear. Noticing recent findings of a cholinergic mechanism for anti-inflammatory actions, we tested the hypothesis that donepezil attenuates an acute inflammatory tissue injury following MI.
Methods and Results: In isolated and activated macrophages, donepezil significantly reduced intra-and extracellular matrix metalloproteinase-9 (MMP-9). In mice with MI, despite the comparable values of heart rate and blood pressure, the donepezil-treated group showed a significantly lower incidence of cardiac rupture than the untreated group during the acute phase of MI. Immunohistochemistry revealed that MMP-9 was localized at the infarct area where a large number of inflammatory cells including macrophages infiltrated, and the expression and the enzymatic activity of MMP-9 at the left ventricular infarct area was significantly reduced in the donepezil-treated group.
Conclusion: The present study suggests that donepezil inhibits the MMP-9-related acute inflammatory tissue injury in the infarcted myocardium, thereby reduces the risk of left ventricular free wall rupture during the acute phase of MI.

DOI： 10.1371/journal.pone.0020629

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Language：English   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Vagal nerve stimulation (VS) has been reported to improve the survival after both acute and chronic myocardial infarction through the release of neurotransmitter ACh. However, the precise mechanism behind its beneficial effect is still unknown. In this study, we demonstrate the upregulation of tumor necrosis factor-alpha (TNF-alpha) and its cell survival TNF receptor-2 (TNFR2) as the mechanism behind VS induced myocardial protection. We investigated the effects of efferent VS on myocardial ischemic injury with in vivo and in vitro mouse models. In in vivo hearts VS significantly increased the expression of TNF-alpha both at the messenger and protein level after 3-hours of myocardial ischemia. In the in vitro studies ACh treatment before hypoxia, induced a significant upregulation of TNF-alpha compared to the untreated cardiomyocytes. Immunofluorescence analysis confirmed the synthesis of TNF-alpha by cardiomyocytes both in vivo and in vitro. VS also significantly reduced the myocardial infarct size (23.9 +/- 5.7% vs. 56 +/- 1.9%) and activated the cell survival Akt cascade system. Further, ACh upregulated the cell survival TNFR2 expression, while downregulating the cell destructive TNF receptor 1 (TNFR1) expression. These results were confirmed using the TNF receptors deficient mice, where the VS mediated protection was lost both in vivo and in vitro in TNFR2 (TNFR2(-/-)) and TNF receptors double knock out (TNFR1(-/-)2(-/-)) mice. VS and ACh protects the heart against acute ischemia or hypoxic injury by differentially regulating the TNF receptor subtypes. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.yjmcc.2010.03.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background: Understanding the basic mechanisms and prevention of any disease pattern lies mainly on development of a successful experimental model. Recently, engineered heart tissue (EHT) has been demonstrated to be a useful tool in experimental transplantation. Here, we demonstrate a novel function for the spontaneously contracting EHT as an experimental model in studying the acute ischemia-induced changes in vitro.
Methodology/Principal Findings: EHT was constructed by mixing cardiomyocytes isolated from the neonatal rats and cultured in a ring-shaped scaffold for five days. This was followed by mechanical stretching of the EHT for another one week under incubation. Fully developed EHT was subjected to hypoxia with 1% O(2) for 6 hours after treating them with cell protective agents such as cyclosporine A (CsA) and acetylcholine (ACh). During culture, EHT started to show spontaneous contractions that became more synchronous following mechanical stretching. This was confirmed by the increased expression of gap junctional protein connexin 43 and improved action potential recordings using an optical mapping system after mechanical stretching. When subjected to hypoxia, EHT demonstrated conduction defects, dephosphorylation of connexin-43, and down-regulation of cell survival proteins identical to the adult heart. These effects were inhibited by treating the EHT with cell protective agents.
Conclusions/Significance: Under hypoxic conditions, the EHT responds similarly to the adult myocardium, thus making EHT a promising material for the study of cardiac functions in vitro.

DOI： 10.1371/journal.pone.0009275

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE INC MEDICAL PUBLISHERS  

Background: We previously reported that chronic vagal nerve Stimulation markedly improved long-term survival after chronic heart failure (CHF) in rats through cardioprotective effects of acetylcholine, independent of the heart rate-slowing mechanism. However, such ail approach is invasive and its safety is unknown in clinical settings. To develop an alternative therapy with a clinically available drug, we examined the chronic effect of oral donepezil, in acetylcholinesterase inhibitor against Alzheimer&apos;s disease, on cardiac remodeling and survival with a murine model of volume-overloaded CHF.
Methods and Results: Four weeks after surgery of aortocaval shunt, CHF mice were randomized into untreated and donepezil-treated groups. Donepezil was orally given at a dosage of 5 mg.kg(-1).day(-1). After 4 weeks of treatment, we evaluated in situ left ventricular (LV) pressure, ex vivo LV pressure-volume relationships, and LV expression of brain natriuretic peptides (BNP). We also observed Survival for 50 days. When compared with the untreated group, the donepezil-treated group had significantly low LV end-diastolic pressure, high LV contractility, and low LV expression of BNP. Donepezil significantly reduced the heart weight and markedly improved the Survival rate during the 50-day treatment period (54% versus 81%, P &lt; .05).
Conclusions: Oral donepezil improves Survival of CHF mice through prevention of pumping failure and cardiac remodeling. (J Cardiac Fail 2009;15:805-811)

DOI： 10.1016/j.cardfail.2009.05.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Chronic heart failure (CHF) is the major cause of death in the developed countries. Calorie restriction is known to improve the recovery in these patients; however, the exact mechanism behind this protective effect is unknown. Here we demonstrate the activation of cell survival PI3kinase/Akt and VEGF pathway as the mechanism behind the protection induced by intermittent fasting in a rat model of established chronic myocardial ischemia (MI). Chronic MI was induced in rats by occlusion of the left coronary artery. Two weeks later, the rats were randomly assigned to a normal feeding group (MI-NF) and an alternate-day feeding group (MI-IF). After 6 weeks of observation, we evaluated the effect of intermittent fasting on cellular and ventricular remodeling and long-term survival after CHF. Compared with the normally fed group, intermittent fasting markedly improved the survival of rats with CHIF (88.5% versus 23% survival, P&lt;0.05). The heart weight body weight ratio was significantly less in the MI-IF group compared to the MI-NF group (3.4 +/- 0.17 versus 3.9 +/- 0.18, P&lt;0.05). Isolated heart perfusion studies exhibited well preserved cardiac functions in the MI-IF group compared to the MI-NF group (P&lt;0.05). Molecular studies revealed the upregulation of angiogenic factors such asHIF-1-alpha (3010 +/- 350% versus 650 +/- 151%), BDNF (523 +/- 32% versus 110 +/- 12%), and VEGF (450 +/- 21% versus 170 +/- 30%) in the fasted hearts. Immunohistochemical studies confirmed increased capillary density (P&lt;0.001) in the border area of the ischemic myrocardium and synthesis VEGF by cardiomyocytes. Moreover fasting also upregulated the expression of other anti-apoptotic factors such as Akt and Bcl-2 and reduced the TUNEL positive apoptotic nuclei in the border zone. Chronic intermittent fasting markedly improves the long-term survival after CHIF by activation through its pro-angiogenic, anti-apoptotic and anti-remodeling effects. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yjmcc.2008.10.027

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Language：English   Publisher：ELSEVIER SCIENCE INC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOSBY-ELSEVIER  

Background: In spite of recent advances in coronary interventional therapy, reperfusion injury is still considered to be a major problem in patients undergoing surgical procedures, such as bypass grafting. Here we demonstrate a novel therapeutic strategy against ischemia-reperfusion injury: vagally mediated prevention of reperfusion-induced opening of mitochondrial permeability transition pore.
Methods: We investigated the effects of efferent vagal stimulation on myocardial reperfusion injury with ex vivo and in vitro rat models. In the ex vivo model the hearts were perfused with intact vagal innervation, which allowed us to study the effects of the vagal nerve on the heart without other systemic effects.
Results: Compared with sham stimulation, vagal stimulation exerted a marked anti-infarct effect irrespective of the heart rate (34% +/- 6% vs 85% +/- 9% at a heart rate of 300 beats/min, 37% +/- 4% vs 43% +/- 5% at a heart rate of 250 beats/min, and 39% +/- 4% vs 88% +/- 7% at a heart rate of 350 beats/min) after a 30-minute period of global ischemia, activated cell-survival Akt cascade, prevented downregulation of the antiapoptotic protein Bcl-2, and suppressed cytochrome-c release and caspase-3 activation. Furthermore, vagal stimulation-treated hearts exhibited a significant improvement in left ventricular developed pressure (78 +/- 5 vs 45 +/- 8 mm Hg) and a significant attenuation in an incremental change in left ventricular end-diastolic pressure during reperfusion. These beneficial effects of vagal stimulation were abolished by a permeability transition pore opener, atractyloside. In the in vitro study with primary-cultured cardiomyocytes, acetylcholine prevented a reoxygenation-induced collapse in mitochondrial transmembrane potential through inhibition of permeability transition pore opening.
Conclusion: Vagal stimulation would be a potential adjuvant therapy for the rescue of ischemic myocardium from reperfusion injury, and the protective effects are independent of its bradycardiac effects.

DOI： 10.1016/j.jtcvs.2008.08.020

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Language：Japanese   Publisher：Japan Heart Foundation  

DOI： 10.11281/shinzo.41.115

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Other Link： http://search.jamas.or.jp/link/ui/2009128118

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Recently, we reported that acetylcholine-induced hypoxia-inducible factor-la protects cardiomyocytes from hypoxia; however, the downstream factors reducing hypoxic stress are unknown. We identified apoptosis inhibitor (AI) gene as being differentially expressed between von Hippel Lindau (VHL) protein-positive cells with high levels of GRP78 expression and VHL-negative cells with lower GRP levels, using cDNA subtraction. AI decreased GRP78 level, suppressed mitochondrial function, reduced oxygen consumption and, ultimately, suppressed hypoxia-induced apoptosis. By contrast, knockdown of the AI gene increased mitochondrial function. Hypoxic cardiomyocytes and ischemic myocardium showed increased AI mRNA expression. These findings suggest that AI is involved in suppressing mitochondrial function, thereby leading to cellular stress eradication and consequently to protection during hypoxia. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2007.12.026

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Background/Aims: The von Hippel-Lindau (VHL) protein functions as an E3 ubiquitin ligase, controlling the stability of hypoxia-inducible factor (HIF). Preinduction of HIF-1 alpha before pathological insult activates a self-defense mechanism and suppresses further aggravation of organ or cellular injury by ischemia. We investigated whether acute inactivation of the VHL gene might play a role in the response of mice to ischemic renal injury.
Methods: We generated tamoxifen-inducible conditional VHL knockout (VHL-KO) mice to inactivate the VHL gene in an acute manner during renal ischemia-reperfusion injury (IRI) induced by bilateral clamping of kidney arteries. Renal IRI is characterized by renal dysfunction and tubular damage.
Results: After the procedure of IRI, blood urea nitrogen (BUN) and creatinine (CRN) levels in control mice were significantly higher (BUN, 138.10 +/- 13.03 mg/dl; CRN, 0.72 +/- 0.16 mg/dl) than in VHL-KO mice (BUN, 52.12 +/- 6.61 mg/dl; CRN, 0.24 +/- 0.04 mg/dl; BUN: p &lt; 0.05; CRN: p &lt; 0.05). Histologically, tubular injury scores were higher in control mice than in VHL-KO mice (p &lt; 0.05).
Conclusion: We suggest that the acute inactivation of the VHL gene contributes to protective effects of ischemic preconditioning in renal tubules of the mouse. Copyright (C) 2008 S. Karger AG, Basel

DOI： 10.1159/000166994

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DOI： 10.1159/000166994

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Language：Japanese  

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Our previous study reveals that connexin (Cx) 43 is targeted by ACh to prevent lethal arrhythmia. Granulocyte colony-stimulating factor (G-CSF), used against ischemic heart failure, may be another candidate, however, with unknown mechanisms. Therefore, we investigated the cellular effects of G-CSF. G-CSF activated the Wnt and Jak2 signals in cardiomyocytes, and up-regulated Cx43 protein and phosphorylation levels. In addition, G-CSF enhanced the localization of Cx43, beta-catenin and cadherin on the plasma membrane. G-CSF inhibited the reduction of Cx43 by enhancing Cx43 anchoring and sustained the cell-cell communication during hypoxia. Consequently, GCSF suppressed ventricular arrhythmia induced by myocardial infarction. As a result, G-CSF could be used as a therapeutic tool for arrhythmia. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2007.09.007

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Background/Aims: We previously demonstrated that angiotensin II ( AII) combined with Habu snake venom ( HV) induces glomerulonephritis ( GN) in rats, with lesions being restricted to the glomeruli 2 days after the administration of both reagents, but the mechanisms inducing GN are unclear. We also indicated a role for hypoxia-inducible factor ( HIF)-1 alpha in attenuating the progression of GN. However, a role of the von Hippel-Lindau ( VHL) protein in GN and mechanisms by which HV regulates the pathogenesis of GN remains unclear. Methods and Results: Immunohistochemical analysis revealed that VHL is weakly expressed in the renal tubules alone; however, HV caused elevated VHL expression in the injured glomeruli including endothelial cells and partially podocytes. Western blot analysis revealed that VHL expression was increased in HV-treated kidney compared with AII-treated or normal kidney. An in vitro study also showed HVinduced elevation in VHL expression. To investigate whether VHL pre-induction causes GN aggravation, we utilized thrombin, an inducer of VHL. Thrombin alone did not cause renal injuries; however, thrombin pre-treatment accelerated the development of GN even 1 day after treatment. Conclusion: We suggest that VHL pre-induction by thrombin aggravates GN, and that the increase in VHL expression due to HV might be involved in accelerating onset of GN. Copyright (C) 2007 S. Karger AG, Basel.

DOI： 10.1159/000103022

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DOI： 10.1159/000103022

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WESTMINSTER PUBL INC  

Endothelin-1 (ET-1) is known to be a principal factor in the pathogenesis of primary pulmonary hypertension (PPH). Recently intravenous administration of epoprosterol improved the survival rate in PPH. However, the effect of epoprosterol on ET-1 remains to be investigated. Therefore, we studied a patient with PPH who was treated with a low dose of epoprosterol and examined the serum concentration of ET-1 during the treatment. Epoprosterol greatly decreased the serum concentration of ET-1 in parallel with improvement of the clinical course, suggesting that ET-1 level may be a marker for treatment of PPH.

DOI： 10.1177/000331970505600519

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The basic helix loop helix-PAS (bHLH-PAS) transcription factors have diverse roles in physiologic responses to the environment and in early development. One bHLH-PAS protein, neuronal PAS domain protein 1 (NPAS1), is reported to be expressed only in the central nervous system beginning at the late embryonic stage, but its function is unknown. Using an anti-NPAS1 antibody, we have shown that NPAS1 expression in cerebral cortex was observed first around embryonic Day 16.5 (E16.5) and was then dispersed throughout the region as cortical development progressed. From the similarity with hypoxia-inducible factor-1alpha (HIF-1alpha), we investigated whether NPAS1 regulates transcription of erythropoietin (EPO), which is the target of HIF-1alpha and is expressed in the brain during the early developmental stage. In the present study, we have shown that NPAS1 binds to the enhancer region of Epo in vivo. A luciferase reporter assay revealed a repressive effect of NPAS1 on hypoxia-responsive element-regulated gene expression. These results raise the possibility that NPAS1 plays a role in late central nervous system development by modulating EPO expression in response to cellular oxygen level. (C) 2005 Wiley-Liss, Inc.

DOI： 10.1002/jnr.20365

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Background/Aim: Among various kidney disease models, there are few rat glomerulonephritis (GN) models that develop in a short time, and with mainly glomerular lesions. Hypoxia-inducible factor (HIF)-1&alpha; is a transcriptional factor that induces genes supporting cell survival, but the involvement of HIF-1&alpha; in attenuating the progression of GN remains to be elucidated. We developed a new model of rat GN by coadministration of angiotensin II (AII) with Habu snake venom (HV) and investigated whether HIF-1&alpha; is involved in renal protection. Methods: Male Wistar rats were unilaterally nephrectomized on day 1, and divided into 4 groups on day 0; N group ( no treatment), HV group, A group ( AII), and H+A group ( HV and AII). To preinduce HIF-1&alpha;, cobalt chloride (CoCl2) was injected twice before injections of HV and AII in 11 rats. Results: GN was detected only in the H+A group; observed first on day 2 and aggravated thereafter. HIF-1&alpha; was expressed in the glomeruli and renal tubules in the A and H+A groups. In the H+A group, GN was remarkably reduced by CoCl2 pretreatment (44.9 to 12.2%, p &lt; 0.01). Conclusion: Both HV and AII were critical for the development of GN, and HIF-1&alpha; remarkably attenuated the progression of GN. Copyright (C) 2005 S. Karger AG, Basel.

DOI： 10.1159/000084575

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DOI： 10.1159/000084575

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Endothelin-1 (ET-1) is known as an aggravating factor of the failing cardiomyocytes and, therefore, a therapeutic method is indispensable to decrease cardiac ET-1 expression. To study the mechanisms of how cardiac ET-1 gene expression can be modified, we investigated the effect of electrical stimulation against cardiomyocytes. Considering the physiology of cardiomyocytes, in vitro cultured cardiomyocytes demonstrate distinctive features from in vivo cardiomyocytes (i.e. the absence of a stretch along with electrical stimulation). In this study, we especially focused on the effect of electrical stimulation. The electrical stimulation reduced the gene expression of ET-1 mRNA in rat primary cultured cardiomyocytes. Furthermore, this effect on the transcriptional modification of ET-1 was also identified in H9c2 cells. Luciferase activity using H9c2 cells was decreased by electrical stimulation in the early phase, suggesting that the attenuation of the ET-1 gene transcription by electrical stimulation should be due to a transcriptional repression. To further investigate a trigger signal involved in the transcriptional repression, phosphorylation of 5'-AMP-activated protein kinase (AMPK) was evaluated. It was revealed that AMPK was phosphorylated in the early phase of electrical stimulation of H9c2 cells as well as in rat primary cultured cardiomyocytes, and that AMPK phosphorylation was followed by ET-1 transcriptional repression, suggesting that electrical stimulation directly regulates AMPK. This study suggests that AMPK activation in cardiomyocytes plays a crucial role in the transcriptional repression of ET-1.

DOI： 10.1097/01.fjc.0000166318.91623.f9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Cardiomyocytes produce endothelin-1. Upregulation of endothelin-1 is induced under hypoxic conditions. It has been reported that tissue hypoxia induces transcriptional factor hypoxia inducible factor-la (HIF-1alpha) expression. It has also been reported that cellular hypoxia leads to HIF-1alpha activation. Previously, we reported that HIF-1alpha a bound to the site in the ET-1 gene promoter region. HIF-1alpha is a master transcriptional factor that controls transcriptional activation of a number of genes responsive to cellular hypoxia, including endothelin-1, vascular endothelial growth factor, erythropoietin and glycolytic enzymes. We aimed to establish the HIF-1alpha-overexpressing cardiomyocytes. Because it has been reported that HIF-1alpha is destroyed by the ubiquitin proteasome pathway under normoxic conditions, we constructed the point-mutated HIF-1alpha a that was exchanged at 564 proline with alanine. The point-mutated HIF-1alpha is not destroyed by ubiquitin proteasome, because ubiquitin ligase cannot bind to the point-mutated area. This mutated HIF-1alpha was transfected to rat cardiomyocytes. Using the protein extraction from the mutated HIF-1alpha transfected cells under normoxic conditions, western blot methods with anti-HIF-1alpha antibody showed that the band was detected at 120 kDa, which is of identical size to HIF-1alpha. In the normoxic conditions of the control cardiomyocyte cells, the band was undetectable. The endothelia-1 mRNA was significantly altered in HIF-1alpha-overexpressing cells. These results indicate that we have established the cells overexpressing-HIF-1alpha, and that these cells produce endothelia-1 mRNA.

DOI： 10.1097/01.fjc.0000166282.01034.8f

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By using cDNA subtraction, we identified an extracellular sulfatase (RsulfFP1) from rat oligodendrocyte progenitor cells (OPCs) whose mRNA expression is down-regulated by tumor necrosis factor-alpha. RsulfFP1 mRNA was expressed specifically in the floor plate and the ventral portion of the rat spinal cord at E15. The expression pattern of RsulfFP1 overlapped with the OPCs, which are also located at the ventral region of the ventricular zone. After this stage, RsulfFP1 expression was attenuated, and the OPCs efficiently migrated throughout the spinal cord. The modification of CG-4 cells, a cell line established from rat O2A cells, by RsulfFP1 activated canonical Wnt signaling. Furthermore, the deletion of RsulfFP1 expression by an antisense oligonucleotide caused impairment of OPC migration in rat spinal cord slice culture. Modification of cells by RsulfFP1 resulted in the increased tyrosine phosphorylation of immunoprecipitated beta-catenin, suggesting that sulfation of the extracellular matrix induced by this sulfatase might be responsible for an increase in Wnt signaling that is involved in the migration of OPCs. Thus, the present study revealed that a sulfatase is responsible for the migration of OPCs and activates intracellular mechanisms that regulate migration.

DOI： 10.1002/jnr.20197

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Endothelin (ET)-1 and ET-2 are potent vasoconstrictor peptides with mitogenic activity. In this study, we investigated roles of ET system in renin-angiotensin system (RAS)-mediated hypertension, using transgenic hypertensive mice (THM) with over-expression of both human renin and angiotensinogen genes. In the first step, it was revealed that expression of ET system was locally enhanced, i.e. increases in cardiac preproET-1 mRNA and renal preproET-2 mRNA in THM, compared with the control (wild type) mice. In the next step, we studied the chronic effects of an ET antagonist (SB209670) on THM. Blood pressure (BP) in THM was significantly higher than that in the normal mice during the investigation. However, in the later phase of the study, from 12 to 20 weeks of treatment, THM receiving SB 209670 showed significantly lower BP than that in THM receiving saline. SB 209670 treatment for 20 weeks significantly attenuated phenotypes of cardiac hypertrophy, vascular wall thickening and hypertensive nephropathy observed in THM, suggesting that the ETA/B receptor antagonist is also effective even in the extraordinarily activated RAS condition. These findings suggest that, organ specifically activated ET system in THM develops the phenotypes, hypertension, cardiac hypertrophy, and hypertensive nephropathy. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2003.07.034

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC HYPERTENSION CENT ACADEMIC SOC, PUBL OFFICE  

Hypertension-induced pathological cardiac hypertrophy (hypertensive heart) and exercise training-induced physiological cardiac hypertrophy (athletic heart) have differences in cardiac properties. We hypothesized that gene expression of energy metabolic enzymes differs between these two types of cardiac hypertrophy. To investigate whether mRNA expression of key enzymes in the long-chain fatty acid (FA), glucose, and lactic acid metabolic pathways differs between these two types of cardiac hypertrophy, we used the hearts of spontaneously hypertensive rats (SHR; 19 weeks old) as a model of the hypertensive heart, swim-trained rats (Trained; 19 weeks old, swimming training for 15 weeks) as a model of the athletic heart, and sedentary Wistar-Kyoto rats (Control; 19 weeks old). SHR developed hypertensive cardiac hypertrophy, of which cardiac function was deteriorated, whereas Trained rats developed an athletic heart, of which cardiac function was enhanced. The mRNA expression of CD36, which involved in uptake of long-chain FA, in the heart was almost never detected in the SHR group. Furthermore, the mRNA expression of key enzymes in the long-chain FA metabolic pathway (acyl CoA synthase [ACoAS], carnitine palmitoyl transferase [CPT]-I, CPT-II, and isocitrate dehydrogenase [ISCD]) in the heart was significantly higher in the SHR group compared with the Control group. The mRNA expression of ACoAS, CPT-I, ISCD, and CD36 in the heart did not differ between Trained group and Control group, whereas that of CPT-II in the Trained group was significantly higher compared with the Control group. The mRNA expression of key enzymes (phosphofructokinase and lactate dehydrogenase) in glycolytic metabolic pathway in the heart was markedly higher in the SHR group compared with the Control group, whereas these mRNA expressions did not differ between Trained group and Control group. These findings suggest that the molecular phenotypes in the energy metabolic system differ in hypertension-induced pathological and exercise training-induced physiological cardiac hypertrophy, and these differences may participate in the differences in cardiac function.

DOI： 10.1291/hypres.26.829

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS  

We investigated whether the type of energy metabolism directly affects cardiac gene expression. During development, the heart switches from glycolysis to fatty acid beta-oxidation in vivo, as demonstrated by the developmental switching of the major isoform of myosin heavy chain (MHC) from beta to alpha. However, the beta-MHC isoform predominates in monocrotaline-induced pulmonary hypertension, a model of right ventricular hypertrophy in vivo. Cultured cardiomyocytes showed a predominance of beta-MHC expression over that of alpha-MHC, the same pattern as in the hypertrophied heart, suggesting that the in vitro condition itself causes the energy metabolism of cardiomyocytes to be switched to glycolysis. Electrical stimulation of cultured cardiomyocytes decreased the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxia-inducible factor-1alpha (HIF-1alpha), but not that of peroxisome-proliferator-activated receptor-gamma co-activator, suggesting that electrical stimulation suppresses the glycolytic system. Furthermore, a higher oxygen content (50%) decreased drastically the expression of GAPDH, HIF-1alpha and endothelin-1 (ET-1), and increased [H-3]palmitate uptake. These findings indicate that the intrinsic energy metabolic system in cultured cardiomyocytes in vitro is predominantly glycolysis, and that the gene expression of cardiac ET-1 parallels the state of the glycolytic system. An antisense oligonucleotide against HIF-1alpha greatly decreased the gene expression of ET-1 and GAPDH, suggesting that cardiac ET-1 gene expression is regulated by cardiac energy metabolism through HIF-1alpha. In conclusion, it is suggested that the pattern of gene expression of ET-1 reflects the level of the glycolytic system in cardiomyocytes, and that enhanced glycolysis regulates the cardiac gene expression of ET-1 via HIF-1alpha.

DOI： 10.1042/CS103S210S

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Language：Japanese   Publishing type：Research paper (other academic)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

In chronic heart failure and acute myocardial infarction, the tissue level of endothelin (ET)-1 in the heart, as well as its plasma level, has been reported to increase markedly. There is, however, little information about what in these pathologic conditions leads to increased production of ET-1, and which type of cell in the heart produces ET-1. We examined the mRNA and peptide expression of ET-1 using cultured rat neonatal cardiomyocytes, in which mitochondrial dysfunction was induced by rotenone, a mitochondrial respiratory chain complex I inhibitor. because one of the common features in failing or ischemic hearts is an alteration in energy metabolism due to mitochondrial dysfunction. Rotenone increased glucose use by the culture cells within 12 h of addition without affecting cell viability, and depressed the mitochondrial membrane potential after 72 h, indicating the induction of mitochondrial dysfunction in cardiomyocytes. Rotenone induced significant increase in the expression level of mRNA for ET-1 within 1 h of addition. In accordance with this finding, immunoreactive ET-1 in culture medium increased 3 times after 24 h of incubation, suggesting active secretion of ET-1 from cultured cells treated with rotenone. Immunocytochemical analysis verified significant increase of ET-1 peptide in cardiomyocytes, confirming the production of ET-1 by cardiomyocytes. These results suggest that derangement of mitochondrial function in cardiomyocytes itself could lead to the increased production of ET-1 in cardiomyocytes, and that this mechanism may contribute to the increased production of ET-1 in failing and ischemic hearts.

DOI： 10.1097/00005344-200112000-00006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To understand the pathophysiological role of endothelin-1 in the failing heart, we constructed a cellular mitochondrial impairment model and demonstrated the effect of endothelin-1. Primary cultured cardiomyocytes from neonatal rats were pretreated with rotenone, a mitochondrial complex I inhibitor, and the cytotoxic effect of endothelin-1 on the cardiomyocytes was demonstrated. Rotenone gradually decreased the pH of the culture medium with incubation time and caused slight cell injury. Endothelin-1 markedly enhanced the effect of rotenone that decreased the pH of the medium and enhanced cellular injury: The enhancement of the decrease in pH and cell injury induced by endothelin-1 was counteracted by the endothelin ETA receptor antagonist BQ123 or by :maintaining the pH of the medium by the addition of 50 mM HEPES. Endothelin-1 markedly increased the uptake of 2-deoxyglucose and lactic acid production when the cardiomyocytes were pretreated with rotenone. These findings suggest that the stimulation of glucose uptake and anaerobic glycolysis followed by the increase in lactic acid accumulation in cardiomyocytes under the condition of mitochondrial impairment may be involved, at least in part, in the cellular injury by endothelin-1. Moreover, these findings suggest the possibility that the effect of endothelin-1 on myocardium is reversed by the condition of the mitochondria; and endogenous endothelin-1 may deteriorate cardiac failure with mitochondrial dysfunction. This may contribute to clarify the beneficial effect of endothelin receptor blockade in improving heart failures. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0014-2999(01)01434-0

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Language：Japanese   Publishing type：Research paper (other academic)  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Circulation Society  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

We investigated whether impairment of myocardial energy metabolism attenuates cardiac function and increases cardiac endothelin-1 (ET-1) gene expression in rats. Three weeks after commencing administration of cobalt chloride (CoCl2), an inhibitor of mitochondrial function, the peak positive first derivative of left ventricular (LV) pressure, an indicator of myocardial contractility, was significantly decreased in the CoCl2-treated rats. LV end-diastolic pressure and right ventricular systolic pressure were increased in the CoCl2-treated rats. Echocardiography showed that fractional shortening was significantly decreased in the CoCl2-treated rats. Myocardial expressions of acyl-CoA synthase mRNA, an enzyme involved in fatty acid utilization, was markedly decreased in the CoCl2-treated rats. Under such conditions, myocardial expression of preproendothelin-1 mRNA and atrial natriuretic peptide (ANP) mRNA, molecular markers of heart failure, was markedly increased in the CoCl2 rats. In conclusion, the data suggest that impairment of myocardial energy metabolism causes hemodynamic abnormality and increases molecular markers of heart failure (ET-1, ANP mRNA). These data suggest that myocardial energy metabolism is one of the factors involved in the upregulation of ET-1 gene expression in the failing heart.

DOI： 10.1097/00005344-200036051-00041

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

It has been reported that at the end stage, apoptosis is involved in the progression of heart failure. It is suggested that cardiac energy metabolism is impaired during the progression of heart failure. Although the mechanism of induction of apoptosis in the failing heart varies according to the model of heart failure, it is not known whether an impairment of energy metabolism in cardiomyocytes is a primary cause of apoptosis. In this study, we applied mitochondrial inhibitors, such as rotenone, cobalt chloride and antimycin A, which inhibit mitochondrial function at different sites of the mitochondrial respiratory chain, to cardiomyocytes. All these reagents markedly decreased 3-(4,5)dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT) reduction activity, an indicator of mitochondrial function, of cardiomyocytes and greatly increased glucose consumption, suggesting that cardiac energy metabolism is switched from beta -oxidation of fatty acid to glycolysis. It was shown that after 48-72 h of treatment with each reagent, apoptosis was shown to occur by DNA laddering and increase in caspase activity. Interestingly, each reagent with a different action site greatly activated caspase-3, but not caspase-8 activity, suggesting that mitochondria are involved in induction of apoptosis. On the other hand, within 24 h of the treatment, when apoptosis of cardiomyocytes was not observed, the treated cardiomyocytes showed a marked increase in preproendothelin-1 and atrial natriuretic peptide (ANP) gene expressions. In conclusion, the present study suggests that mitochondrial dysfunction with impaired energy metabolism elevates gene expression of cardiac ET-1, an aggravating factor in heart failure, and then finally induces apoptosis in cardiomyocytes. The finding of marked increases in expression of molecular markers (ET-1 mRNA and ANP mRNA) in the failing heart, followed by apoptosis in the treated cardiomyocytes suggests that the inhibition of mitochondrial function of cultured cardiomyocytes provides a possible new in vitro model of heart failure.

DOI： 10.1097/00005344-200036051-00061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

We have reported that the expression of endothelin-1 (ET-1) increases in the failing heart. With the progress of heart failure, it has been reported that energy metabolism switches from mitochondrial b-oxidation to glycolysis. Furthermore, it has been reported that apoptosis is induced in the failing heart. However, it is not known how the gene expression of preproendothelin-1 and cellular apoptosis are affected by the mitochondrial dysfunction. Therefore, in order to elucidate this problem, we developed an in vitro model of mitochondrial dysfunction using rotenone, a mitochondrial respiratory chain complex I inhibitor, and studied preproendothelin-1 gene expression and apoptosis. Rotenone greatly increased the gene expression of preproendothelin-1 in cardiomyocytes. This result suggests that the gene expression of preproendothelin-1 is induced by the mitochondrial dysfunction. Furthermore, treatment of cardiomyocytes with rotenone induced an elevation of caspase-3 activity, and caused a marked increase in DNA laddering, an indication of apoptosis. In conclusion, it is suggested that mitochondrial impairment in primary cultured cardiomyocytes induced by rotenone in vitro, mimics some of the pathophysiological features of heart failure in vivo, and that ET-1 may have a role in myocardial dysfunction with impairment of mitochondria in the failing heart.

DOI： 10.1097/00005344-200036051-00062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Angiotensinogen-deleted mice (Agt-KO) show phenotypes of hypotension and renal atrophy. To investigate whether an alternative pathway other than angiotensin II (AII), i.e., processed angiotensin fragments, may play a biological role in nephrogenesis, we analyzed a congenic line of Agt-KO fetuses and neonates derived from two sources: one (Agt-KO/He) from mating with heterozygous angiotensinogen-deleted mice and the other (Agt-KO/Ho) from mating homozygous angiotensinogen-deleted mice. Although Agt-KO/He did not show a typical phenotype at birth, these mice showed papillary atrophy 2 weeks later and thereafter, a marked increase in renal size, i.e., pelvic dilatation. In contrast, Agt-KO/Ho showed renal abnormalities at birth and subsequently died. TUNEL staining and electron microscopy revealed that accelerated papillary apoptosis was present at birth in Agt-KO/Ho and caused abnormal papillary development; however, apoptosis was not detected in Agt-KO/He, suggesting that different mechanisms for the abnormal renal development exist in Agt-KO/He and Agt-KO/Ho. Two-week administration of an angiotensin fragment (3-8), angiotensin IV (AIV), to Agt-KO/He markedly attenuated the renal atrophy, decreasing the incidence from 81% to 14% However, administration of AIV to fetal Agt-KO/Ho through the mother did not decrease the incidence. This is marked contrast to AII, which prevented renal atrophy in both fetal and neonatal periods. It is therefore suggested that AIV is involved in nephrogenesis in a developmental stage-specific manner.

DOI： 10.1007/s004670050703

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We and other groups have reported that endothelin (ET) -1 expression in the heart is altered in the setting of heart diseases. We have also reported that myocardial ET-I is involved in the progression of heart failure, and that an ET receptor antagonist improves long-term survival in heart failure (Nature 384: 353-355, 1996). However, the role of myocardial ET-2 in disease states are not known. To characterize the role of ET-2, we used a) the failing hearts of rats with heart failure caused by myocardial infarction, and b) primary cultured cardiomyocytes subjected to hypoxia. In the failing heart in vivo, ET-1 mRNA increased by 390% compared with that in the non-failing heart, while ET-2 mRNA drastically decreased by 88%. Thus, gene expression of ET1 and ET-2 was reciprocally altered in the failing heart in vivo. In in vitro studies, reciprocal alterations in ET-1 and ET-2 gene expression were also observed in isolated primary cultured cardiomyocytes, subjected to hypoxia. Specifically, acute hypoxic stress induced a significant increase (360% of the basal level) in ET-2 mRNA expression compared with that in normoxic cells, whereas it decreased ET-1 mRNA expression by 62% in primary cultured cardiomyocytes. Although these two crucial conditions, i.e., heart failure in vivo and acute hypoxic stress in vitro, are pathophysiologically distinct from each other, reciprocal alteration of ET-1 and ET-2 gene expression was observed in both cases. To further investigate the regulatory mechanism of the altered gene expression, luciferase analysis was performed using primary cultured cardiomyocytes. ET-2 promoter, which is the 5'-flanking region of preproET-2 gene (5'ET-2), showed a marked increase in luciferase activity during acute hypoxia. In contrast, the luciferase activity of 5'ET-1 (ET-1 promoter) did not change in response to hypoxic stress. The present study suggests that there are transcriptionally distinct regulatory mechanisms for ET-1 and ET-2 expression in cardiomyocytes, and therefore this study may provide a new aspect of cardiac ET system that not only ET-I but also ET-2 can be participated in the pathophysiological conditions.

DOI： 10.1016/S0024-3205(99)00416-6

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：現代医療社  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Circulation Society  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Circulation Society  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PROFESSOR D A SPANDIDOS  

We previously produced angiotensinogen-deficient mice, i.e. mice with deleted renin-angiotensin system (RAS), with a genetic background on C57BL/6J - C57BL/6J-agt (-/-) -, but no C57BL/6J-agt (-/-) which survived long enough to be weaned. In the present study, we attempted to prevent neonatal death and analyzed pathological development in C57BL/6J-agt (-/-). We indicate that mortality in C57BL/6J-agt (-/-) derived from C57BL/6J-agt (+/-) can be reduced by hypodermic saline injection in the 7 days following birth, that hydronephrosis developed by day 14 in association with polydiplasia and polyuria by day 30, and that chronic hypotension occurs. Hydronephrosis is less damaging to electrolyte resorption in younger mice, but not in adults. We also observed that C57BL/6J-agt (-/-) derived from C57BL/6J-agt (-/-) frequently develop fetal hydronephrosis and die of respiratory failure at birth. These results suggest that maternal RAS is associated with structural maturation of kidney and lung in late fetus and that postnatal RAS plays important roles in structural and functional maintenance of the kidneys.

DOI： 10.3892/ijmm.1.3.583

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Circulation Society  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Circulation Society  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

The morphological analysis in a congenic line of angiotensinogen knock-out mice (AgKO) revealed the decreased density in granular layer cells of hippocampus and cerebellum, suggesting neuronal cells of AgKO susceptible to apoptotic cell death. This phenomenon was further studied by culture of the hippocampal neurons with decreased concentration of serum. AgKO neuronal cells, which showed apoptosis by lower concentration of the serum within several hours, however, survived much longer in the presence of angiotensin Pi (AII) and TV (AIV). This anti-apoptotic action was not interfered by AII receptor antagonists, CV11874 and PD123319. These results suggest that the renin-angiotensin system could play a critical role in central nervous system, preventing neuronal cells from apoptosis not only by AII but also AIV. (C) 1997 Elsevier Science Ireland Ltd.

DOI： 10.1016/S0304-3940(97)00605-8

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J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(一社)日本生理学会  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：HUMANA PRESS INC  

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Language：Japanese   Publisher：日本循環制御医学会  

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

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Language：Japanese   Publisher：(一社)日本生理学会  

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Language：Japanese   Publisher：日本循環制御医学会  

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Language：Japanese   Publisher：(一社)日本生理学会  

J-GLOBAL

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Language：Japanese   Publisher：日本循環制御医学会  

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Language：Japanese   Publisher：(一社)日本生理学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本生理学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publisher：Japanese Circulation Society  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publisher：Japanese Circulation Society  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publisher：Japanese Circulation Society  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：English   Publisher：Japanese Circulation Society  

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Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

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Language：English   Publisher：Japanese Circulation Society  

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Language：Japanese   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：English   Publisher：Japan Society of Exercise and Sports Physiology  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=192697

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publisher：社団法人日本循環器学会  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publisher：医歯薬出版  

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Other Link： http://search.jamas.or.jp/link/ui/1997084650

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：The Japanese Society of Hematology  

We report a case of a 2 years and 2 months old male, who showed a normochromic normocytic anemia, Hb 3.7g/dl. The bone marrow aspiration revealed a remarkable decrease of erythroblasts alone. Because of none of other underlying diseases identified, it leads to a diagnosis of aquired idiopathic pure red cell aplasia (PRCA). Pulse therapy of methylprednisolone, followed by one month adminisatration of prednisone did not lead to a remission. However, high dose of &gamma;-globulin pulse therapy restored erythroposiesis in several days, with a rate of hemoglobin increment of 0.5 g/dl/day. 2.5 years after the onset of PRCA, the patient has neither suffered from complications of hematological diseases, nor undersired consequences of the therapy. Although recent advence of therapy regarding PRCA has focussed on cyclosporine A, high dose of &gamma;-globulin pulse therapy with a remarkable effect is worth as an alternative.

DOI： 10.11406/rinketsu.37.352

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Country：Japan

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