Biomedical subjects
Publications and source records attributed to Satoshi Ogawa.
In Japan, the number of sudden death is estimated to be about 100,000/year and 60% of them are sudden cardiac death(SCD). And the cause of almost SCD is death of arrhythmia. The prevention of SCD by antiarrhythmic drugs is limited. So for the high risk group of SCD, ICD is needed. On the other hand, CPR of the bystander and the use of AED are important to avoid SCD. We must spread the education and training for the citizens.
Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2. Co-transfection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractive stimulation of reporter gene expression. Immunohistochemistry of adult porcine pituitary confirmed the colocalization of the Prop-1 and FSHbeta subunit. This study is the first to report that Prop-1 participates in the regulation of FSHbeta gene. The present finding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD), since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.
Type 2 diabetes is one of the most prevalent and serious metabolic diseases in the world, and insulin resistance and pancreatic beta-cell dysfunction are the hallmarks of the disease. In this study, we have shown that endoplasmic reticulum (ER) stress, which is provoked under diabetic conditions, plays a crucial role in the insulin resistance found in diabetes by modifying the expression of oxygen-regulated protein 150 (ORP150), a molecular chaperone that protects cells from ER stress. Sense ORP overexpression in the liver of obese diabetic mice significantly improved insulin resistance and markedly ameliorated glucose tolerance. Conversely, expression of antisense ORP150 in the liver of normal mice decreased insulin sensitivity. The phosphorylation state of IRS-1 and Akt, which are key molecules for insulin signaling, and the expression levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, key enzymes of gluconeogenesis, were also altered by ORP150 overexpression. This is the first report showing that ER stress plays a crucial role in the insulin resistance found in diabetes and thus could be a potential therapeutic target for diabetes.
OBJECTIVES: We sought to determine the influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) induction on post-myocardial infarction (MI) remodeling, especially in relation to the inflammatory response and myocardial fibrosis. BACKGROUND: Granulocyte-macrophage colony-stimulating factor modifies wound healing by promoting monocytopoiesis and infiltration of monocytes and macrophages into injured tissue; however, the effect of GM-CSF induction on the infarct healing process and myocardial fibrosis is unclear. METHODS: A model of MI was produced in Wistar rats by ligation of the left coronary artery. The MI animals were randomized to receive GM-CSF inducer (romurtide 200 microg/kg/day for 7 consecutive days) (MI/Ro) or saline (MI/C). RESULTS: Echocardiographic and hemodynamic studies on day 14 revealed increased left ventricular (LV) end-diastolic dimension, decreased fractional shortening, elevated LV end-diastolic pressure, and decreased LV maximum rate of isovolumic pressure development in MI/Ro compared with MI/C. Immunoblotting showed that expression of transforming growth factor (TGF)-beta1 in the infarcted site on day 3 after MI was decreased in MI/Ro compared with MI/C. In the infarcted site, TGF-beta1, collagen type I and type III messenger ribonucleic acid (mRNA) expression on day 3, and collagen content on day 7 were reduced in MI/Ro compared with MI/C, in association with marked infarct expansion. In MI/Ro, monocyte chemoattractant protein-1 mRNA level and the degree of infiltration of monocyte-derived macrophages (ED-1-positive)were greater in the infarcted site on day 7 than those in MI/C. CONCLUSIONS: The GM-CSF induction by romurtide facilitated infarct expansion in association with the promotion of monocyte recruitment and inappropriate collagen synthesis in the infarcted region during the early phase of MI.
Mammalian cardiomyocytes lose their capacity to proliferate during terminal differentiation. We have previously reported that the expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and its partner cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. Here we show that the D1NLS/CDK4 cells, after their entry into the cell cycle, accumulated cyclin-dependent kinase inhibitor p27 in the nuclei and decreased the cyclin-dependent kinase 2 (CDK2) activity, leading to early cell cycle arrest. Biochemical analysis demonstrated that Skp2-dependent p27 ubiquitylation was remarkably suppressed in cardiomyocytes, whereas Skp2, a component of Skp1-Cullin-F-box protein ubiquitin ligase, was more actively ubiquitylated compared with proliferating rat fibroblasts. Specific degradation of p27 by co-expressing Skp2 or p27 small interfering RNA caused an increase of CDK2 activity and overrode the limited cell cycle. These data altogether indicate that the impaired Skp2-dependent p27 degradation is causally related to the loss of proliferation in cardiomyocytes. This provides a novel insight in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation.
Bone marrow (BM) cells are reported to contribute to the process of regeneration following myocardial infarction. However, the responsible BM cells have not been fully identified. Here, we used 2 independent clonal studies to determine the origin of bone marrow (BM)-derived cardiomyocytes. First, we transplanted single CD34(-) c-kit(+)Sca-1(+) lineage(-) side population (CD34(-)KSL-SP) cells or whole BM cells from mice ubiquitously expressing enhanced green fluorescent protein (EGFP) into lethally irradiated mice, induced myocardial infarction (MI), and treated the animals with granulocyte colony-stimulating factor (G-CSF) to mobilize stem cells to the damaged myocardium. At 8 weeks after MI, from 100 specimens we counted only 3 EGFP(+) actinin(+) cells in myocardium of CD34(-) KSL-SP cells in mice that received transplants, but more than 5000 EGFP(+) actinin(+) cells in whole BM cell in mice that received transplants, suggesting that most of EGFP(+) actinin(+) cells were derived from nonhematopoietic BM cells. Next, clonally purified nonhematopoietic mesenchymal stem cells (MSCs), cardiomyogenic (CMG) cells, that expressed EGFP in the cardiomyocyte-specific manner were transplanted directly into BM of lethally irradiated mice, MI was induced, and they were treated with G-CSF. EGFP(+) actinin(+) cells were observed in the ischemic myocardium, indicating that CMG cells had been mobilized and differentiated into cardiomyocytes. Together, these results suggest that the origin of the vast majority of BM-derived cardiomyocytes is MSCs.
BACKGROUND: The endoplasmic reticulum (ER) is recognized as an organelle that participates in folding secretory and membrane proteins. The ER responds to stress by upregulating ER chaperones, but prolonged and/or excess ER stress leads to apoptosis. However, the potential role of ER stress in pathophysiological hearts remains unclear. METHODS AND RESULTS: Mice were subjected to transverse aortic constriction (TAC) or sham operation. Echocardiographic analysis demonstrated that mice 1 and 4 weeks after TAC had cardiac hypertrophy and failure, respectively. Cardiac expression of ER chaperones was significantly increased 1 and 4 weeks after TAC, indicating that pressure overload by TAC induced prolonged ER stress. In addition, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells increased, and caspase-3 was cleaved in failing hearts. The antagonism of angiotensin II type 1 receptor prevented upregulation of ER chaperones and apoptosis in failing hearts. On the other hand, angiotensin II upregulated ER chaperones and induced apoptosis in cultured adult rat cardiac myocytes. We also investigated possible signaling pathways for ER-initiated apoptosis. The CHOP- (a transcription factor induced by ER stress), but not JNK- or caspase-12-, dependent pathway was activated in failing hearts by TAC. Pharmacological ER stress inducers upregulated ER chaperones and induced apoptosis in cultured cardiac myocytes. Finally, mRNA levels of ER chaperones were markedly increased in failing hearts of patients with elevated brain natriuretic peptide levels. CONCLUSIONS: These findings suggest that pressure overload by TAC induces prolonged ER stress, which may contribute to cardiac myocyte apoptosis during progression from cardiac hypertrophy to failure.
The 150 kDa oxygen-regulated protein (ORP150) is an inducible endoplasmic reticulum (ER) chaperone with cytoprotective properties in settings of cell stress, such as ischemia/reperfusion (I/R). Renal tissue from patients with acute renal failure displayed strong induction of ORP150 in tubular epithelium. In a rodent model of renal I/R injury, ORP150 was expressed in both the ischemic and contralateral kidney, principally in the thick ascending loop of Henle (TAL) and distal tubules. Cultured renal epithelial cells exposed to hypoxic or hyperosmotic conditions displayed induction of ORP150. Renal tubular epithelial cells stably transfected with ORP150 sense or antisense cDNA displayed a strong correlation between ORP150 expression and vulnerability to hypoxic/osmotic stress; higher levels of ORP150 were protective, whereas lower levels increased susceptibility to cell death. Compared with nontransgenic controls, transgenic mice overexpressing ORP150 subjected to renal I/R displayed a blunted rise of serum creatinine and blood urea nitrogen, and enhanced survival of TAL, consistent with cytoprotection. In contrast, heterozygous ORP150+/- mice, with lower levels of ORP150, showed enhanced renal injury. These data are consistent with the possibility that ORP150 exerts cytoprotective effects in renal tubular epithelia subjected to I/R injury and suggest a key role for ER stress in the renal tubular response to acute renal failure.
The possibility of producing stable oil-in-water (O/W) emulsions containing oil droplets surrounded by multiple layer interfacial membranes from food grade ingredients has been demonstrated. These emulsions were produced using a three stage process that relies on the adsorption of charged biopolymers to oppositely charged surfaces. Emulsions (0.5 wt % corn oil, 0.1 wt % lecithin, 0.0078 wt % chitosan, 0.02 wt % pectin, and 100 mM acetic acid, pH 3.0) containing oil droplets stabilized by lecithin-chitosan-pectin membranes were formed using this interfacial layer-by-layer deposition process. The droplets in these emulsions had good stability to aggregation over a wide range of pH values and salt concentrations (pH 4-8 at 0 mM NaCl and pH 3-8 at 100 mM NaCl). This technology could be extremely useful to the food industry for the creation of O/W emulsions with improved properties or novel applications, e.g., improved stability to environmental stresses, protection of labile substances, controlled release, and triggered release.
GPR54 is a novel G protein-coupled receptor speculated to be essential for sexual development. However, its role in the regulation of GnRH types is unknown. To address this issue, we cloned GPR54 from the brain of a cichlid fish (tilapia Oreochromis niloticus) and determined its expression in immature and mature males using our newly developed technique: laser-captured microdissection of single digoxigenin-labeled GnRH neurons coupled with real-time quantitative PCR. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids and exhibits 56% identity to human GPR54. Absolute copies of GnRH1 and GnRH3, not GnRH2, mRNAs were significantly high in mature compared with immature males. At the single-cell level, only in mature males, GnRH1 mRNA levels were inversely related to GPR54 mRNA (P < 0.002). GPR54 was expressed in a significantly high percentage (45.0-60.0%) of mature GnRH1, GnRH2, and GnRH3 neurons and in immature GnRH3 neurons, which had migrated to the vicinity of their final locations in the brain; on the contrary, only 5.0% of immature GnRH1 and GnRH2 neurons had GPR54 transcripts (P < 0.001). Thus, using a novel innovative single-cell gene profiling technique, we provide evidence of the structure of a nonmammalian GPR54, which is highly conserved during evolution and is expressed in GnRH1, GnRH2, and GnRH3 neurons. Furthermore, we propose that the expression of GPR54 is a "stop signal" for GnRH1, GnRH2, and GnRH3 neuronal migration, leading to suppression of cell growth and modulation of GnRH secretion, which is important for normal sexual development.
OBJECTIVES: This study investigated the possibility of achieving angiogenesis by using gene-modified cells as a vector. BACKGROUND: Although gene therapy for peripheral circulation disorders has been studied intensively, the plasmid or viral vectors have been associated with several disadvantages, including unreliable transfection and uncontrollable gene expression. METHODS: Human hepatocyte growth factor (hHGF) and thymidine kinase (TK) expression plasmids were serially transfected into NIH3T3 cells, and permanent transfectants were selected (NIH3T3 + hHGF + TK). Unilateral hindlimb ischemia was surgically induced in BALB/c nude mice, and cells were transplanted into the thigh muscles. All effects were assessed at four weeks. RESULTS: The messenger ribonucleic acid expression and protein production of hHGF were confirmed. Assay of growth inhibition by ganciclovir revealed that the 50% (median) inhibitory concentration of NIH3T3 + hHGF + TK was 1000 times lower than that of NIH3T3 + hHGF. The NIH3T3 + hHGF + TK group had a higher laser Doppler blood perfusion index, higher microvessel density, wider microvessel diameter, and lower rate of hindlimb necrosis, as compared with the plasmid- and adenovirus-mediated hHGF transfection groups or the NIH3T3 group. The newly developed microvessels were accompanied by smooth muscle cells, as well as endothelial cells, indicating that they were on the arteriolar or venular level. Laser Doppler monitoring showed that the rate of blood perfusion could be controlled by oral administration of ganciclovir. The transplanted cells completely disappeared in response to ganciclovir administration for four weeks. CONCLUSIONS: Gene-modified cell transplantation therapy induced strong angiogenesis and collateral vessel formation that could be controlled externally with ganciclovir.
Hypoxia-inducible factor 1 (HIF-1) is a key regulator of O(2) homeostasis, which regulates the expression of several genes linked to angiogenesis and energy metabolism. Tumor hypoxia has been shown to be associated with poor prognosis in a variety of tumors, and HIF-1 induced by hypoxia plays pivotal roles in tumor progression. The presence of putative HIF-1-binding sites on the promoter of human telomerase reverse transcriptase gene (hTERT) prompted us to examine the involvement of HIF-1 in the regulation of hTERT and telomerase in tumor hypoxia. The telomeric repeat amplification protocol (TRAP) assay revealed that hypoxia activated telomerase in cervical cancer ME180 cells, with peak induction at 24-48 h of hypoxia. Notably, hTERT mRNA expression was upregulated at 6-12 h of hypoxia, concordant with the elevation of HIF-1 protein levels at 6 h. hTERT protein levels were subsequently upregulated at 24 h and later. Luciferase assays using reporter plasmids containing hTERT core promoter revealed that hTERT transcription was significantly activated in hypoxia and by HIF-1 overexpression, and that the two putative binding sites within the core promoter are responsible for this activation. Chromatin immunoprecipitation assay identified the specific binding of HIF-1 to these sites (competing with c-Myc), which was enhanced in hypoxia. The present findings suggest that hypoxia activates telomerase via transcriptional activation of hTERT, and that HIF-1 plays a critical role as a transcription factor. They also suggest the existence of novel mechanisms of telomerase activation in cancers, and have implications for the molecular basis of hypoxia-induced tumor progression and HIF-1-based cancer gene therapy.
We have previously reported that leukemia inhibitory factor (LIF) gradually increased cardiac L-type Ca2+ channel current (I(CaL)), which peaked at 15 minutes in both adult and neonatal rat cardiomyocytes, and this increase was blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. This study investigated the molecular basis of LIF-induced augmentation of I(CaL) in rodent cardiomyocytes. LIF induced phosphorylation of a serine residue in the alpha(1c) subunit (Ca(v)1.2) of L-type Ca2+ channels in cultured rat cardiomyocytes, and this phosphorylation was inhibited by PD98059. When constructs encoding either a wild-type or a carboxyl-terminal-truncated rabbit Ca(v)1.2 subunit were transfected into HEK293 cells, LIF induced phosphorylation of the resultant wild-type protein but not the mutant protein. Cotransfection of constitutively active mitogen-activated protein kinase kinase also resulted in phosphorylation of the Ca(v)1.2 subunit in the absence of LIF stimulation. In in-gel kinase assays, extracellular signal-regulated kinase phosphorylated a glutathione S-transferase fusion protein of the carboxyl-terminal region of Ca(v)1.2 (residues 1700 through 1923), which contains the consensus sequence Pro-Leu-Ser-Pro. A point mutation within this consensus sequence, which results in a substitution of alanine for serine at residue 1829 (S1829A), was sufficient to abolish the LIF-induced phosphorylation. LIF increased I(CaL) in HEK cells transfected with wild-type Ca(v)1.2 but not with the mutated version. These results provide direct evidence that LIF phosphorylates the serine residue at position 1829 of the Ca(v)1.2 subunit via the actions of extracellular signal-regulated kinase and that this phosphorylation increases I(CaL) in cardiomyocytes.
OBJECTIVES: We sought to define the electrophysiologic property of the rabbit heart associated with autoimmunity against the second extracellular loop of the beta(1)-adrenergic receptor. BACKGROUND: Sudden death of patients with cardiomyopathy, probably due to lethal ventricular arrhythmias, can be predicted by the presence of autoantibodies against the second extracellular loop of the beta(1)-adrenergic receptor. METHODS: Rabbits were immunized by repetitive subcutaneous administration of a synthetic peptide corresponding to the second extracellular loop of beta(1)-adrenergic receptors (beta group; n = 30) for a mean of 4.2 months. Control rabbits received only vehicle (control group; n = 30). RESULTS: One of the rabbits in the beta group died suddenly during the observation period, but none of the control animals died. The prevalence of sustained ventricular tachycardia was significantly higher in the beta group (beta: 4 of 27 vs. control: 0 of 30), and a standard microelectrode experiment revealed prolongation of the action potential duration (APD) in the right ventricular papillary muscle (beta: 156 +/- 5 ms vs. control: 131 +/- 4 ms; p < 0.05). Early afterdepolarization (EAD) was observed in one rabbit in the beta group (1 of 26), but not in any animals in the control group (0 of 17). A dose of 100 nmol/l of E-4031 induced EAD in the beta group (10 of 10), but not in the control group, except for one rabbit (1 of 10). The whole-cell, patch-clamp experiment on left ventricular M cells showed significant decreases in transient outward current (I(to1)) (-43%) and slowly activated delayed rectifier current (I(Ks)) densities (-33%), whereas the inward-rectifying K current (I(K1)) and rapidly activated delayed rectifier current (I(Kr)) densities remained unchanged. CONCLUSIONS: Long-term immunization against the second extracellular loop of the beta(1)-adrenergic receptor caused EAD and APD prolongation and decreased the K-channel density, suggesting that an arrhythmic substrate via autoimmune mechanisms is present in cardiomyopathic patients who have autoantibodies directed against the receptors.
The endoplasmic reticulum (ER) stress response contributes to neuronal survival in ischemia and neurodegenerative processes. ORP150 (oxygen-regulated protein 150)/HSP12A (heat shock protein 12A), a novel stress protein located in the ER, was markedly induced in Purkinje cells maximally at 4-8 d after birth, a developmental period corresponding to their vulnerability to cell death. Both terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling analysis and immunostaining using anti-activated caspase-3 antibody revealed that transgenic mice with targeted neuronal overexpression of ORP150 (Tg ORP150) displayed diminished cell death in the Purkinje cell layer and increased numbers of Purkinje cells up to 40 d after birth (p < 0.01), compared with those observed in heterozygous ORP150/HSP12A-deficient (ORP150+/-) mice and wild-type littermates (ORP150+/+). Cultured Purkinje cells from Tg ORP150 mice displayed resistance to both hypoxia- and AMPA-induced stress. Behavioral analysis, using rotor rod tasks, indicated impairment of cerebellar function in Tg ORP150 animals, consistent with the concept that enhanced survival of Purkinje cells results in dysfunction. These data suggest that ER chaperones have a pivotal role in Purkinje cell survival and death and thus may highlight the importance of ER stress in neuronal development.
OBJECTIVES: We tested the hypothesis that angiotensin II type 1 receptor blocker (ARB) may improve beta-adrenergic receptor (AR) coupling in heart failure (HF) after myocardial infarction (MI). BACKGROUND: Beta-AR desensitization is one of the mechanisms underlying the transition from compensated to decompensated HF. Beta-adrenergic receptor kinase-1 (ARK1), which can be induced by protein kinase C (PKC) in vitro, is activated in the failing myocardium, resulting in beta-AR uncoupling. METHODS: Models of MI in rats were produced by ligation of left coronary artery. Four weeks after surgery, they were randomized to vehicle (MI/control [C]) or candesartan (10 mg/kg/day) treatment (MI/ARB). Sham-operated rats, or shams, served as controls. RESULTS: After two weeks of treatment, echocardiography and hemodynamics showed that the left ventricular (LV) dimension increased and that the percent of fractional shortening and maximum rate of rise in left ventricular pressure (dP/dt) decreased in MI rats compared with shams. There were no differences in these indexes between MI/C and MI/ARB. An increase in maximum dP/dt under isoproterenol (ISO) stimulation was attenuated in MI/C but improved in MI/ARB. Reductions in the percentage of high-affinity sites of beta-AR and ISO-stimulated cyclic adenosine monophosphate production in noninfarcted myocardium were also improved by ARB treatment. Up-regulation of beta-ARK1 and PKC-epsilon isoform protein levels and activation of PKC in noninfarcted myocardium from MI/C were both inhibited by ARB treatment. CONCLUSIONS: Treatment with ARB during the chronic phase of MI improved beta-AR coupling in noninfarcted myocardium without affecting basal LV function. Cross-talk between beta-AR and angiotensin signaling through beta-ARK1 and PKC-epsilon may be responsible for the phenomenon.
The purpose of this study was to clarify predictors of vessel remodeling following directional coronary atherectomy (DCA). Negative remodeling after DCA leads to restenosis. However, little is known about the predictors of the vessel remodeling. Serial IVUS was performed in 43 lesions. The vessel remodeling was defined as adaptive if vessel area at follow-up minus postprocedure vessel area was > 0 mm2, or as constrictive if < 0 mm2. Adaptive remodeling occurred in 21 (49%) lesions. Postprocedure percent plaque area was smaller in the adaptive group (32.9% +/- 5.7% vs. 45.5% +/- 8.8%; P < 0.005). At follow-up, vessel area was larger in the adaptive group. However, plaque area was similar between the two groups. As a result, lumen area was larger in the adaptive group. Multivariate analyses showed that postprocedure percent plaque area < 40% was the only predictor of adaptive remodeling (odds ratio, 6.68; P < 0.05).
Ventricular septal rupture is a serious complication of acute myocardial infarction. We experienced a case of septal rupture immediately after primary angioplasty with thrombolysis, whose angiographic findings were similar to those of coronary perforation. The progression of septal rupture was delineated by the serial angiograms.