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ABSTRACT: Rett syndrome (RTT) is a neurodevelopmetal disorder associated with mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. MeCP2-deficient mice recapitulate the neurological degeneration observed in RTT patients. Recent studies indicated a role of not only neurons but also glial cells in neuronal dysfunction in RTT. We cultured astrocytes from MeCP2-null mouse brain and examined astroglial gene expression, growth rate, cytotoxic effects, and glutamate (Glu) clearance. Semi-quantitative RT-PCR analysis revealed that expression of astroglial marker genes, including GFAP and S100β, was significantly higher in MeCP2-null astrocytes than in control astrocytes. Loss of MeCP2 did not affect astroglial cell morphology, growth, or cytotoxic effects, but did alter Glu clearance in astrocytes. When high extracellular Glu was added to the astrocyte cultures and incubated, a time-dependent decrease of extracellular Glu concentration occurred due to Glu clearance by astrocytes. Although the shapes of the profiles of Glu concentration versus time for each strain of astrocytes were grossly similar, Glu concentration in the medium of MeCP2-null astrocytes were lower than those of control astrocytes at 12 and 18 h. In addition, MeCP2 deficiency impaired downregulation of excitatory amino acid transporter 1 and 2 (EAAT1/2) transcripts, but not induction of glutamine synthetase (GS) transcripts, upon high Glu exposure. In contrast, GS protein was significantly higher in MeCP2-null astrocytes than in control astrocytes. These findings suggest that MeCP2 affects astroglial genes expression in cultured astrocytes, and that abnormal Glu clearance in MeCP2-deficient astrocytes may influence the onset and progression of RTT.
PLoS ONE 01/2012; 7(4):e35354. · 4.09 Impact Factor
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ABSTRACT: The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.
Biochemical and Biophysical Research Communications 08/2011; 413(1):128-35. · 2.48 Impact Factor
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Hepatology Research 06/2011; 41(6):594-6. · 2.20 Impact Factor
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ABSTRACT: The aim of this study is to investigate the role of hepatic stellate cells (HSCs) and the effect of vitamin A administration on liver damage induced by bile duct ligation (BDL) and administration of CCl(4).
Two types of animal model were used; one was BDL as a model of biliary atresia, the other was CCl(4)-induced hepatic fibrosis. Pathological changes of the liver with or without administration of vitamin A were compared by light and electron microscopy with focusing on HSCs in each experimental group. Immunohistochemical examination was performed with anti-keratinocyte growth factor (KGF), anti-alpha-smooth muscle actin (α-SMA), and anti-glial fibrillary acidic protein (GFAP) antibodies, as markers of fibrosis.
On light microscopic findings, periportal inflammation with bile ductular proliferation was obvious in BDL group and pericentral necrosis with fatty degeneration was observed in CCl(4) group, both of which were ameliorated by subcutaneous injection of vitamin A. Electron microscopy showed lipid droplets were almost depleted in the HSCs treated with BDL or CCl(4), which improved with vitamin A administration. Immunohistochemistry demonstrated that enhanced expression of all three fibrotic markers in the BDL group was diminished by vitamin A administration.
Although most of our data are qualitative observation, vitamin A may ameliorate hepatic fibrosis in the BDL model by restoring vitamin A in the HSCs.
Pediatric Surgery International 02/2011; 27(8):863-70. · 1.25 Impact Factor
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ABSTRACT: Mutations in methyl-CpG-binding protein 2 (MeCP2) gene cause the neurodevelopmental disorder Rett syndrome (RTT). Here, we describe a new experimental system that efficiently elucidates the role of MeCP2 in neural development. MeCP2-null and control ES cells were generated by adenoviral conditional targeting and examined for maintenance of the undifferentiated ES cell state, neurogenesis, and gliogenesis during in vitro differentiation. In addition, dopamine release and electrophysiological features of neurons differentiated from these ES cells were examined. Loss of MeCP2 did not affect undifferentiated ES cell colony morphology and growth, or the timing or efficiency of neural stem cell differentiation into Nestin-, TuJ- or TH-positive neurons. In contrast, gliogenesis was drastically accelerated by MeCP2 deficiency. Dopamine production and release in response to a depolarizing stimulus in MeCP2-null ES-derived dopaminergic neurons was intact. However, MeCP2-null differentiated neurons showed significantly smaller voltage-dependent Na(+) currents and A-type K(+) currents, suggesting incomplete maturation. Thus, MeCP2 is not essential for maintenance of the undifferentiated ES cell state, neurogenesis, or dopaminergic function; rather, it is principally involved in inhibiting gliogenesis. Altered neuronal maturity may indirectly result from abnormal glial development and may underlie the pathogenesis of RTT. These data contribute to a better understanding of the developmental roles of MeCP2 and the pathogenesis of RTT.
Brain research 11/2010; 1360:17-27. · 2.46 Impact Factor
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ABSTRACT: The incidence of diabetes is increasing worldwide, yet current treatments are not always effective for all patient or disease types.
Here, we summarize the biologic and clinical roles of leptin in diabetes, and discuss candidate viral vectors that may be employed in the clinical use of central leptin gene therapy for diabetes.
We discuss how studies on leptin, a regulator of the insulin-glucose axis, have significantly advanced our understanding of the roles of energy homeostasis and insulin resistance in the pathogeneses of metabolic syndrome and diabetes. Recent studies have demonstrated the long-term therapeutic effects of central leptin gene therapy in obesity and diabetes via decreased insulin resistance and increased glucose metabolism. Many of these studies have employed viral vectors, which afford high in vivo gene transduction efficiencies compared with non-viral vectors.
Adeno-associated viral vectors are particularly well suited for central leptin gene therapy owing to their low toxicity and ability to drive transgene expression for extended periods.
Expert opinion on biological therapy 10/2010; 10(10):1405-14. · 3.22 Impact Factor
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Shusaku Miyata,
Genzou Takemura, Ken-Ichiro Kosai,
Tomoyuki Takahashi,
Masayasu Esaki,
Longhu Li,
Hiromitsu Kanamori,
Rumi Maruyama,
Kazuko Goto,
Akiko Tsujimoto,
Toshiaki Takeyama,
Tomonori Kawaguchi,
Takamasa Ohno,
Kazuhiko Nishigaki,
Takako Fujiwara,
Hisayoshi Fujiwara,
Shinya Minatoguchi
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ABSTRACT: Activation of Fas signaling is a key mediator of doxorubicin cardiotoxicity, which involves both cardiomyocyte apoptosis and myocardial inflammation. In this study, acute cardiotoxicity was induced in mice by doxorubicin, and some mice simultaneously received an intramuscular injection of adenoviral vector encoding mouse soluble Fas (sFas) gene (Ad.CAG-sFas), an inhibitor of Fas/Fas ligand interaction. Two weeks later, left ventricular dilatation and dysfunction were apparent in the LacZ-treated control group, but both were significantly mitigated in the sFas-treated group. The in situ nick-end labeling-positive rate were similar in the two groups, and although electron microscopy revealed cardiomyocyte degeneration, no apoptotic structural features and no activation of caspases were detected, suggesting an insignificant role of apoptosis in this model. Instead, sFas treatment reversed doxorubicin-induced down-regulation of GATA-4 and attenuated ubiquitination of myosin heavy chain and troponin I to preserve these sarcomeric proteins. In addition, doxorubicin-induced significant leukocyte infiltration, fibrosis, and oxidative damage to the myocardium, all of which were largely reversed by sFas treatment. sFas treatment also suppressed doxorubicin-induced p53 overexpression, phosphorylation of c-Jun N-terminal kinase, c-Jun, and inhibitor of nuclear factor-kappaB, as well as production of cyclooxygenase-2 and monocyte chemoattractant protein-1, and it restored extracellular signal-regulated kinase activation. Therefore, sFas gene therapy prevents the progression of doxorubicin-induced acute cardiotoxicity, with accompanying attenuation of the cardiomyocyte degeneration, inflammation, fibrosis, and oxidative damage caused by Fas signaling.
American Journal Of Pathology 02/2010; 176(2):687-98. · 4.89 Impact Factor
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Longhu Li,
Hideshi Okada,
Genzou Takemura, Ken-ichiro Kosai,
Hiromitsu Kanamori,
Masayasu Esaki,
Tomoyuki Takahashi,
Kazuko Goto,
Akiko Tsujimoto,
Rumi Maruyama,
Itta Kawamura,
Tomonori Kawaguchi,
Toshiaki Takeyama,
Takako Fujiwara,
Hisayoshi Fujiwara,
Shinya Minatoguchi
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ABSTRACT: The small leucine-rich proteoglycan decorin is a natural inhibitor of transforming growth factor-beta (TGF-beta) and exerts antifibrotic effects in heart and to stimulate skeletal muscle regeneration. We investigated decorin's chronic effects on postinfarction cardiac remodeling and dysfunction. Myocardial infarction (MI) was induced in mice by left coronary artery ligation. An adenoviral vector encoding human decorin (Ad. CAG-decorin) was then injected into the hindlimbs on day 3 post-MI (control, Ad.CAG-LacZ). Four weeks post-MI, the decorin-treated mice showed significant mitigation of the left ventricular dilatation and dysfunction seen in control mice. Although infarct size did not differ between the two groups, the infarcted wall thickness was greater and the segmental length of the infarct was smaller in decorin-treated mice. In addition, cellular components, including myofibroblasts and blood vessels, were more abundant within the infarcted area in decorin-treated mice, and fibrosis was significantly reduced in both the infarcted and noninfarcted areas of the left ventricular wall. Ten days post-MI, there was greater cell proliferation and less apoptosis among granulation tissue cells in the infarcted areas of decorin-treated mice. The treatment, however, did not affect proliferation and apoptosis of salvaged cardiomyocytes. Although decorin gene therapy did not affect TGF-beta1 expression in the infarcted heart, it inhibited Smad2/3 activation (downstream mediators of TGF-beta signaling). In summary, postinfarction decorin gene therapy mitigated cardiac remodeling and dysfunction by altering infarct tissue noncardiomyocyte dynamics and preventing cardiac fibrosis, accompanying inhibition of Smad2/3 activation.
AJP Heart and Circulatory Physiology 09/2009; 297(4):H1504-13. · 3.71 Impact Factor
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Hideshi Okada,
Genzou Takemura, Ken-Ichiro Kosai,
Akiko Tsujimoto,
Masayasu Esaki,
Tomoyuki Takahashi,
Satoshi Nagano,
Hiromitsu Kanamori,
Shusaku Miyata,
Yiwen Li,
Takamasa Ohno,
Rumi Maruyama,
Atsushi Ogino,
Longhu Li,
Munehiro Nakagawa,
Kenshi Nagashima,
Takako Fujiwara,
Hisayoshi Fujiwara,
Shinya Minatoguchi
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ABSTRACT: We hypothesized that therapy, composed of antiapoptotic soluble Fas (sFas) gene transfer, combined with administration of the cardioprotective cytokine granulocyte colony-stimulating factor (G-CSF), would markedly mitigate cardiac remodeling and dysfunction following myocardial infarction (MI). On the 3rd day after MI induced by ligating the left coronary artery in mice, four different treatments were initiated: saline injection (Group C, n = 26); G-CSF administration (Group G, n = 27); adenoviral transfer of sFas gene (Group F, n = 26); and the latter two together (Group G+F, n = 26). Four weeks post-MI, Group G+F showed better survival than Group C (96 vs. 65%, P < 0.05) and the best cardiac function among the four groups. In Group G, the infarct scar was smaller and less fibrotic, whereas in Group F the scar was thicker, without a reduction in area, and contained abundant myofibroblasts and vascular cells; Group G+F showed both phenotypes. G-CSF exerted a beneficial effect on infarct tissue dynamics through antifibrotic and proliferative effects on granulation tissue; however, it also exerts an adverse proapoptotic effect that leads to thinning of the infarct scar. sFas appeared to offset the latter drawback. In vitro study using cultured myofibroblasts derived from the infarct tissue revealed that G-CSF increased proliferating activity of those cells accompanying activation of Akt and signal transducer and activator of transcription 3, while accelerating Fas-mediated apoptosis with increasing Bax-to-Bcl-2 ratio. The results suggest that combined use of G-CSF administration and sFas gene therapy is a potentially powerful tool against post-MI heart failure.
AJP Heart and Circulatory Physiology 01/2009; 296(3):H616-26. · 3.71 Impact Factor
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Takehito Kondo,
Genzou Takemura, Ken-Ichiro Kosai,
Takamasa Ohno,
Tomoyuki Takahashi,
Masayasu Esaki,
Kazuko Goto,
Rumi Maruyama,
Ichijiro Murata,
Shinya Minatoguchi,
Takako Fujiwara,
Hisayoshi Fujiwara
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ABSTRACT: 1. In the present study, we examined the effects of inhibiting transforming growth factor (TGF)-beta in a mouse model of diabetic nephropathy. 2. An adenovirus harbouring the gene encoding soluble TGF-beta type II receptor (Ad.CAG-sTbetaRII), a competitive inhibitor of TGF-beta, was injected into hindlimb muscles (systemic delivery) of mice 5 weeks after the induction of diabetes with streptozotocin. The control group was injected with an adenovirus encoding the LacZ gene (Ad-LacZ). 3. Five weeks after administration, anti-TGF-beta gene therapy was found to have had no effect on renal function, albuminuria or glucose metabolism in mice with diabetic nephropathy. Nonetheless, this gene therapy did significantly reduce fibrosis in both glomeruli and renal tubules. These effects were accompanied by attenuation of the increased expression of alpha-smooth muscle actin normally seen in kidneys of diabetic mice and better preservation of glomerular cell numbers, although the thickness of the glomerular capillary basement membrane was unchanged. The plasma concentration of soluble TGF-beta type II receptor peaked on Day 7 after treatment, but was undetectable by Day 14. Moreover, a second treatment with Ad.CAG-sTbetaRII failed to prolong the interval of gene product expression in the blood. 4. The present anti-TGF-beta gene therapy showed a significant antifibrotic effect in a model of diabetic nephropathy, but failed to improve renal function. The inadequacy of the observed effect is likely due to the relatively short interval of gene product expression. This problem will have to be overcome if gene therapies for slowly progressing diseases, like diabetic nephropathy, are to be realised.
Clinical and Experimental Pharmacology and Physiology 11/2008; 35(11):1288-93. · 1.85 Impact Factor
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Masayasu Esaki,
Genzou Takemura, Ken-ichiro Kosai,
Tomoyuki Takahashi,
Shusaku Miyata,
Longhu Li,
Kazuko Goto,
Rumi Maruyama,
Hideshi Okada,
Hiromitsu Kanamori,
Atsushi Ogino,
Hiroaki Ushikoshi,
Shinya Minatoguchi,
Takako Fujiwara,
Hisayoshi Fujiwara
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ABSTRACT: Hepatocyte growth factor (HGF) reportedly exerts beneficial effects on the heart following myocardial infarction and during nonischemic cardiomyopathy, but the precise mechanisms underlying the latter have not been well elucidated. We generated nonischemic cardiomyopathy in mice by injecting them with doxorubicin (15 mg/kg ip). Two weeks later, when cardiac dysfunction was apparent, an adenoviral vector encoding human HGF gene (Ad.CAG-HGF, 1x10(11) particles/mouse) was injected into the hindlimb muscles; LacZ gene served as the control. Left ventricular dilatation and dysfunction normally seen 4 wk after doxorubicin administration were significantly mitigated in HGF-treated mice, as were the associated cardiomyocyte atrophy/degeneration and myocardial fibrosis. Myocardial expression of GATA-4 and a sarcomeric protein, myosin heavy chain, was downregulated by doxorubicin, but the expression of both was restored by HGF treatment. The protective effect of HGF against doxorubicin-induced cardiomyocyte atrophy was confirmed in an in vitro experiment, which also showed that neither cardiomyocyte apoptosis nor proliferation plays significant roles in the present model. Upregulation of c-Met/HGF receptor was noted in HGF-treated hearts. Among the mediators downstream of c-Met, the activation of extracellular signal-regulated kinase (ERK) was reduced by doxorubicin, but the activity was restored by HGF. Levels of transforming growth factor-beta1 and cyclooxygenase-2 did not differ between the groups. Our findings suggest the HGF gene delivery exerts therapeutic antiatrophic/degenerative and antifibrotic effects on myocardium in cases of established cardiac dysfunction caused by doxorubicin. These beneficial effects appear to be related to HGF-induced ERK activation and upregulation of c-Met, GATA-4, and sarcomeric proteins.
AJP Heart and Circulatory Physiology 03/2008; 294(2):H1048-57. · 3.71 Impact Factor
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ABSTRACT: The technical limitations of isolating target cells have restricted the utility of pluripotent embryonic stem (ES) cells. For example, early cardiac (i.e., precontractile) cells have not been isolated from ES cells. Here, we find that direct expression of reporter genes under cell-specific promoters-the currently available strategy for isolating cells lacking cell-specific surface markers-is ineffective for isolating progenitor cells. This was due to the weak activity of cell-specific promoters, particularly in ES cells at early stages. We show that adenoviral conditional targeting efficiently isolates viable ES cell-derived target cells without harmful effects. In this strategy, we employ the alpha-myosin heavy chain and Nkx2.5 promoter to visualize and purify efficiently differentiated and primitive cells of the cardiac lineage, respectively. While the former cells predominantly expressed sarcomeric proteins and maintained contractile function, the latter demonstrated neither of these features, but rather exhibited expression patterns characteristic of a mixture of primitive cells and cardiomyocytes. Interestingly, smooth muscle actin was predominantly expressed in the latter cells, and both functionally known and unknown genes were systematically identified, demonstrating the benefits of this system. Thus, our method facilitates molecular and cellular studies of development and ES cell-derived cell therapy.
Molecular Therapy 12/2006; 14(5):673-83. · 6.87 Impact Factor
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ABSTRACT: Based on the finding that telomerase is reactivated solely in cancer cells, the human telomerase reverse transcriptase (hTERT) promoter has recently been used to target cancer cells by gene therapy. The recent, surprising observation that telomerase is physiologically activated even in normal somatic cells during S-phase has raised concerns as to the safety of this methodology. To clarify this issue, the present study carefully examined the changes in endogenous telomerase activities, hTERT mRNA expression, and hTERT promoter-based transgene expression in normal and cancer cells at synchronized phases of the cell cycle. Telomerase activity and hTERT expression were detected at variable, but relatively high, levels in all 12 cancer cell lines, while both were undetectable in the 11 normal cell lines. In HepG2 cancer cells, the highest levels of hTERT expression and telomerase activity, seen in the G(1)/S- and S-phases, were 2-3-fold higher than the lowest levels of both, observed in G(0)-phase and during asynchronization. No hTERT expression or telomerase activitiy could be detected in normal WI-38 fibroblasts at any phase of the cell cycle, including S-phase. Consequently, activity of the shorter hTERT promoter, which was transferred into HepG2 cancer cells via adenovirus transduction, was stronger than that of the longer hTERT promoter at all phases and that of two representatives of ubiquitously strong promoters, at both S-phase and asynchronization, but not at G(0)-phase. In contrast, neither of hTERT promoters induced detectable transgene expressions in normal WI-38 cells at any cell cycle phase, including S-phase. These results, particularly the lack of problematic levels of S-phase-specific activation of hTERT promoters in normal cells, have promising implications for hTERT promoter-based targeted gene therapy of cancer.
International Journal of Oncology 10/2006; 29(3):681-8. · 2.40 Impact Factor
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Ngin Cin Khai,
Tomoyuki Takahashi,
Hiroaki Ushikoshi,
Satoshi Nagano,
Kentaro Yuge,
Masayasu Esaki,
Takao Kawai,
Kazuko Goto,
Yoshiteru Murofushi,
Takako Fujiwara,
Hisayoshi Fujiwara, Ken-ichiro Kosai
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ABSTRACT: It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF.
Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration.
Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice.
HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.
Journal of Hepatology 07/2006; 44(6):1046-54. · 9.26 Impact Factor
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ABSTRACT: Whether bone marrow cells injected following acute myocardial infarction (MI) transdifferentiate into cardiomyocytes remains controversial, and how these cells affect repair-related cytokines is not known.
Autologous bone marrow-derived mononuclear cells (BM-MNCs) labeled with DiI, 1,1'-dioctadecyl-1 to 3,3,3',3'-tetramethylindocarbocyanine perchlorate, or saline were intravenously injected into rabbits 5 h following a 30-min ischemia and reperfusion protocol, and cardiac function and the general pathology of the infarcted heart were followed up 1 and 3 months post-MI. To search for regenerated myocardium, electron microscopy as well as confocal microscopy were performed in the infarcted myocardium 7 days post-MI. Expression levels of repair-related cytokines were evaluated by immunohistochemistry and Western blotting.
Improvements in cardiac function and reductions in infarct size were observed in the BM-MNC group 1 month and 3 months post-MI. Using electron microscopy 7 days after infarction, clusters of very immature (fetal) and relatively mature cardiomyocytes undergoing differentiation were identified in the infarcted anterior LV wall in the BM-MNC group, though their numbers were small. These cells contained many small and dense DiI particles (a BM-MNC marker), indicating that cardiomyocytes had regenerated from the injected BM-MNCs. The expression of both transforming growth factor-beta, which stimulates collagen synthesis and matrix metalloproteinase-1, a collagenase, were both down-regulated 7 days and 1 month post-MI in the BM-MNC group. Stromal cell-derived factor-1, which is known to recruit BM-MNCs into target tissues, was overexpressed in the infarcted areas of BM-MNC hearts 7 days post-MI.
Intravenous transplantation of BM-MNCs leads to the development of BM-MNC-derived myocyte-like cells and regulates the expression of repair-related cytokines that facilitate repair following myocardial infarction.
Cardiovascular Research 03/2006; 69(2):476-90. · 6.06 Impact Factor
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Tomoyo Kagawa,
Genzou Takemura, Ken-ichiro Kosai,
Ichijiro Murata,
Takamasa Ohno,
Tomoyuki Takahashi,
Masayasu Esaki,
Rumi Maruyama,
Takako Fujiwara,
Hiroshige Ohashi,
Hisayoshi Fujiwara
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ABSTRACT: Effect of hepatocyte growth factor (HGF) has scarcely been determined on diabetic nephropathy.
Adenovirus encoding human HGF gene or LacZ gene (as the control) was injected into the hindlimb muscles of the C57BL/KsJ-db/db (db/db) mice at the age of 12 weeks, a model of genetic diabetes. Diabetic nephropathy was then evaluated at the age of 24 weeks.
The urine volume and albumin excretion progressively decreased in the control, whereas they remained unchanged in the HGF-treated group during the 12-week follow-up. The HGF gene therapy did not affect glucose metabolism. However, it resulted in a better renal function as evaluated by creatinine clearance (Ccr) than the control; Ccr was progressively worsened in controls (0.14 +/- 0.02 liters/day) whereas unchanged in the HGF gene-treated group (0.38 +/- 0.09 liters/day, p < 0.05). Kidneys of the HGF gene-treated mice showed glomeruli with greater area and cell population, smaller glomerular sclerotic index, and less fibrosis in both glomeruli and renal tubules, where apoptotic rate of glomerular endothelial cells and that of tubular epithelial cells were significantly decreased. TGF-beta1 expression was significantly decreased in kidneys of the HGF gene-treated group. Finally, the HGF treatment significantly improved the long-term survival of db/db mice.
The HGF gene delivery thus appeared to slow down the aggravation of diabetic nephropathy in db/db mice by attenuating progression from the hyperfiltration phase into the sclerotic phase through antiapoptotic and antifibrotic actions. The present findings suggest that the HGF gene delivery can be a novel therapeutic approach against diabetic nephropathy.
Nephron Physiology 01/2006; 102(3-4):p92-102. · 2.55 Impact Factor
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Kentaro Yuge,
Tomoyuki Takahashi,
Satoshi Nagano,
Yasuhiro Terazaki,
Yoshiteru Murofushi,
Hiroaki Ushikoshi,
Takao Kawai,
Ngin Cin Khai,
Toshikazu Nakamura,
Hisayoshi Fujiwara, Ken-ichiro Kosai
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ABSTRACT: Hepatocyte growth factor (HGF) gene therapy may have potential for treating chronic hepatitis (CH) and liver cirrhosis (LC). However, the lack of an HGF gene therapy study on hepatomas that are often associated with CH or LC, together with the stimulatory effects of HGF on many types of cancer, may hamper its application. This study explored the effects of adenoviral HGF gene transduction and their mechanisms on two types of hepatoma cells (hepatoblastoma and hepatocellular carcinoma) in in vitro experiments. Both types of hepatomas were revealed to have higher adenoviral gene transduction efficiencies and more efficient expressions of the HGF transgene, which successfully activated the HGF receptor/c-Met in an autocrine fashion, than those of other types of cancer. Notably, not only HGF, but also adenoviral infection, inhibited DNA synthesis, whereas only HGF but not adenoviral infection exerted a potent apoptotic effect. Moreover, adenoviral HGF gene transduction additively exerted inhibitory effects on cisplatin-treated hepatomas. In conclusion, inhibitory and apoptotic effects of adenoviral HGF gene transduction in hepatomas in contrast to potent mitogenic and antiapoptotic effects of HGF for hepatocytes are not only of biological interest, but also pose clinical benefits for adenoviral HGF gene therapy for CH and LC.
International Journal of Oncology 08/2005; 27(1):77-85. · 2.40 Impact Factor
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Hiroaki Ushikoshi,
Tomoyuki Takahashi,
Xuehai Chen,
Ngin Cin Khai,
Masayasu Esaki,
Kazuko Goto,
Genzou Takemura,
Rumi Maruyama,
Shinya Minatoguchi,
Takako Fujiwara,
Satoshi Nagano,
Kentaro Yuge,
Takao Kawai,
Yoshiteru Murofushi,
Hisayoshi Fujiwara, Ken-ichiro Kosai
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ABSTRACT: Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are cardiogenic and cardiohypertrophic growth factors. Although the therapeutic effects of IGF and HGF have been well demonstrated in injured hearts, it is uncertain whether natural upregulation of HB-EGF after myocardial infarction (MI) plays a beneficial or pathological role in the process of remodeling. To answer this question, we conducted adenoviral HB-EGF gene transduction in in vitro and in vivo injured heart models, allowing us to highlight and explore the HB-EGF-induced phenotypes. Overexpressed HB-EGF had no cytoprotective or additive death-inducible effect on Fas-induced apoptosis or oxidative stress injury in primary cultured mouse cardiomyocytes, although it significantly induced hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. Locally overexpressed HB-EGF in the MI border area in rabbit hearts did not improve cardiac function or exhibit an angiogenic effect, and instead exacerbated remodeling at the subacute and chronic stages post-MI. Namely, it elevated the levels of apoptosis, fibrosis, and the accumulation of myofibroblasts and macrophages in the MI area, in addition to inducing left ventricular hypertrophy. Thus, upregulated HB-EGF plays a pathophysiological role in injured hearts in contrast to the therapeutic roles of IGF and HGF. These results imply that regulation of HB-EGF may be a therapeutic target for treating cardiac hypertrophy and fibrosis.
Laboratory Investigation 08/2005; 85(7):862-73. · 3.64 Impact Factor
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ABSTRACT: Although a conditionally replicating adenovirus (CRA) exhibiting cancer-selective replication and induction of cell death is an innovative potential anticancer agent, current imperfections in cancer specificity and efficient viral replication limit the usefulness of this technique. Here, we constructed survivin-responsive CRAs (Surv.CRAs), in which expression of the wild-type or mutant adenoviral early region 1A (E1A) gene is regulated by the promoter of survivin, a new member of the inhibitor of apoptosis gene family. We explored the cancer specificity and effectiveness of viral replication of Surv.CRAs, evaluating their potential as a treatment for cancer. The survivin promoter was strongly activated in all cancers examined at levels similar to or even higher than those seen for representative strong promoters; in contrast, low activity was observed in normal cells. Surv.CRAs efficiently replicated and potently induced cell death in most types of cancer. In contrast, minimal viral replication in normal cells did not induce any detectable cytotoxicity. A single injection of Surv.CRAs into a preestablished tumor expressing survivin, even at relatively low levels, induced significant tumor death and inhibition of tumor growth. Furthermore, Surv.CRAs were superior to telomerase-dependent CRAs, one of the most effective CRAs that have been examined to date, both in terms of cancer specificity and efficiency. Thus, Surv.CRAs are an attractive potential anticancer agent that could effectively and specifically treat a variety of cancers.
Cancer Research 07/2005; 65(12):5284-91. · 7.86 Impact Factor
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ABSTRACT: The use of recombinant adenovirus as a vehicle for gene transfer into ependymal cells is a potential therapeutic tool for the treatment of various neural disorders. However, gene transfer into the ependymal cells of the ventricular wall is associated with high-level expression of the transferred gene, which declines rapidly. The purpose of this study is to understand the cause of this early decline in gene expression.
Different doses of adenovirus-expressing beta-galactosidase (Ad-beta-gal) were injected into the lateral brain ventricle of C57BL/6 mice, and the brains were observed histologically and with magnetic resonance (MR) imaging for a month.
Inoculation of the lateral ventricle with more than 1 x 10(8) viral particles (2.6 x 10(6) pfu) resulted in a rapid decline of beta -gal expression. MR imaging indicated gradual ventriculomegaly and histological analysis showed the loss of the ependymal cells from the ventricular wall, lymphocytes infiltration near the wall, degeneration of myelinated fibers and apoptosis in the external capsule. Reactive astrocytes proliferated in the external capsule 17 days following inoculation. To avoid this irreversible brain atrophy, the inoculated adenovirus should be reduced to less than 1 x 10(7) particles (2.6 x 10(5) pfu) in mice.
Our results indicate the presence of a unique and diffuse immune response of the brain; therefore, the clinical use of recombinant virus for intraventricular gene transfer must be carefully evaluated.
Neurological Research 07/2005; 27(4):378-86. · 1.52 Impact Factor
