Yoshiteru Murofushi

Gifu University Hospital, Gihu, Gifu, Japan

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Publications (8)41.87 Total impact

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    ABSTRACT: The effectiveness of autologous fat injection laryngoplasty may be reduced by resorption of injected fat tissue. The aim of the present study was to clarify the efficacy of fat injection laryngoplasty using autologous fat plus a replication-defective adenoviral vector expressing hepatocyte growth factor, regarding reduction of injected fat tissue resorption. Four female beagle dogs were used in this study. After sedation, a direct laryngoscope was introduced to enable visualisation of the larynx. In each dog, harvested autologous fat plus an adenoviral vector expressing hepatocyte growth factor was injected into the right true vocal fold, and harvested fat plus an adenoviral vector expressing no gene was injected into the left true vocal fold. A total laryngectomy was performed one year after the intracordal fat injection. Coronal sections of the resected whole larynges were made and the following parameters assessed using light and electron microscopy: size of fat area; number of vasculoendothelial cells surrounding adipocytes; and shape of injected adipocytes in the vocal fold. The fat area was significantly larger and the number of vasculoendothelial cells surrounding adipocytes significantly greater in the intracordal fat injection containing adenoviral vector expressing hepatocyte growth factor, compared with the control intracordal fat injection containing adenoviral vector expressing no gene. When viewed under electron microscopy, the injected adipocytes were observed to have grafted better in the intracordal fat injection with hepatocyte growth factor adenoviral vector, compared with the control intracordal fat injection with adenoviral vector expressing no gene. Injection into the vocal fold of autologous fat containing an adenoviral vector expressing hepatocyte growth factor can reduce subsequent resorption of injected fat.
    The Journal of laryngology and otology. Supplement 05/2009;
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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. · 2.66 Impact Factor
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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. · 9.86 Impact Factor
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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. · 2.66 Impact Factor
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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. · 3.96 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. · 8.65 Impact Factor
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    ABSTRACT: Introduction and aims: Mutations in the methyl-CpG-binding protein 2 (MeCP2), which binds methylated DNA and regulates gene expression, cause Rett syndrome (RTT). RTT is a neurodevelopmental disorder characterized by normal early psychomotor development, followed by the loss of psychomotor skills, and by the onset of stereotyped hand movement and of gait disturbances; no effective therapy has been developed for the RTT. MeCP2 gene therapy may be a potential candidate for effective therapy for the RTT, however, due to no experimental trial, the potential effectiveness of conducting MeCP2 gene therapy is unknown from the following biological viewpoints. The expression of MeCP2 begins during embryonic development and the expression levels of MeCP2 are high in all neurons in normal humans and mice after birth, whereas the neurological functions that MeCP2 regulates remain largely unknown. Thus, it is not possible to hypothesize whether the expression of an intact MeCP2 gene can, following the onset, modulate pre-established neurological symptoms. An additional obstacle is the fact that all of the current vectors allow only local gene delivery, although MeCP2 is expressed in all neurons in normal humans. Here we show that local delivery of the MeCP2 gene into the striatum of MeCP2-deleted mice remarkably improved the pre-established symptoms of impaired locomotion and self-injury.Results: The deletion of MeCP2 in hemizygous male (MeCP2−/y) and heterozygous female (MeCP2−/+) mice led to RTT-like neurological symptoms at an onset of around 40 days and 6–10 weeks of ages, respectively. To explore whether the RTT is reversible, we first delivered the MeCP2 gene into the MeCP2-null male mice at 39 or 40 days old, and several behavioral studies were performed 4 and 7 days later The progressive deterioration of the locomotor activity seen in the control Ad.LacZ-treated MeCP2−/y mice, who, in an open field test, visited fewer areas of the field, spent more time being immobile, and reared less than the wild-type controls, was significantly attenuated in the Ad.MeCP2-treated MeCP2−/y mice. Because the classic form of human RTT is exclusively found in females, we performed behavior studies before and after the MeCP2 gene therapy using adult female MeCP2−/+ mice that had revealed severe neurological symptoms. The representative mice, which had been almost immobile before the treatment, acquired voluntary movement at almost normal levels in the open field test several days after the treatment. The improvement of the impaired motor coordination was further proven by the wire suspension test and the dowel test. Furthermore, all of the MeCP2−/+ mice were completely cured of self-injuries by the MeCP2 gene therapy.Conclusion: These experimental findings for the first time suggest that RTT is reversible and treatable, and that MeCP2 plays crucial roles in controlling locomotion and emotion in the striatum.
    Molecular Therapy 01/2005; 11. · 7.04 Impact Factor
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    ABSTRACT: Background, aims and methods: Although conditionally replicating adenovirus (CRA), which shows cancer-selective viral replication and death, is potentially an innovative anti-cancer agent, a current limitation results from the incompleteness of cancer-specific and efficient viral replication. Most importantly, previously reported tissue-specific promoters have the disadvantage of targeting only limited types of cancer, as well as showing less cancer specificity and weak activity even in cancer cells. Here we constructed survivin-responsive CRAs (Surv.CRAs), in which the expression of wild or mutant adenoviral early region 1A gene is regulated by the promoter of survivin, a new member of the inhibitor of apoptosis gene family (Surv.CRAwt and Surv.CRAmt, respectively). Both types of Surv.CRAs have the deletion of E1B55kD, which are driven by the CMV promoter, and contain EGFP marker genes. We carefully explored the cancer-specificity and the effectiveness of viral replication of Surv.CRAs and their therapeutic potential against cancer.Results: Survivin mRNA was expressed in all of the 11 human cancer cells that were derived from various tissue origins, whereas survivin mRNA was very low in normal cells. The survivin promoter was revealed to be strongly active in all these cancer cells studied at levels similar to or even higher than those of representative strong promoters (the RSV and the CMV promoters); in contrast, no detectable activity was observed in normal cells. In careful in vitro experiments (e.g., the speed of the viral replication assessed by the spread of EGFP-positive cells on flowcytometric analysis and by the spread of the cells showing the features of cytopathic effects), both types of Surv.CRAs (i.e., Surv.CRAwt and Surv.CRAmt) efficiently replicated and potently induced death in most types of cancer, in contrast to minimal viral replication resulting in no detectable cytotoxicity in normal cells. A single injection of either of Surv.CRAs into a pre-established tumor expressing survivin, even at a relatively low level, significantly induced tumor death and inhibited tumor growth. There are no different phenotypes in any cells between Sruv.CRAwt and Surv.CRAmt. Furthermore, Surv.CRAs were superior to telomerase (TERT)-dependent CRAs (Tert.CRAs), currently among the best CRAs, both in terms of cancer-specificity and efficiency. Namely, the activity of the survivin promoter was higher than that of the TERT promoter in cancer cells, however, the former is lower than the latter in normal cells. Moreover, Surv.CRAwt is more efficient in replicating in cancer cells than Tert.CRAwt, although the former is more quiescent in normal cells than the latter.Conclusion: Surv.CRAs may be an attractive anti-cancer agent that effectively and specifically treats a variety of cancers.
    Molecular Therapy 01/2005; 11. · 7.04 Impact Factor