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Biomedical subjects

Hiroshi Funakoshi

Publications and source records attributed to Hiroshi Funakoshi.

18 recordsLinked to original sources
PubMed2006

Hepatocyte growth factor attenuates cerebral ischemia-induced increase in permeability of the blood-brain barrier and decreases in expression of tight junctional proteins in cerebral vessels.

Hepatocyte growth factor (HGF) exerts its physiological activities as that of an organotropic factor for regeneration and can prevent ischemia-induced injuries; however, its effect and mechanism of action under in vivo pathophysiological conditions remains to be determined. Recently, we demonstrated that treatment with human recombinant HGF (hrHGF) attenuated the disruption of the blood-brain barrier (BBB) observed after microsphere embolism-induced sustained cerebral ischemia. To see if tight junctional proteins were involved in this attenuation, in the present study, we investigated the effects of HGF on the levels of occludin and zonula occludens (ZO)-1 in cerebrovascular endothelial cells after microsphere embolism. Sustained cerebral ischemia was induced by the injection of 700 microspheres (48 microm diameter) into the right internal carotid artery of rats. hrHGF was injected into the right ventricle of the brain by using an osmotic pump at a dose of 30 microg/7 days per animal. The levels of tight junctional proteins in the endothelial cells were examined by immunohistochemical analysis. Treatment with hrHGF attenuated the decrease in the expression of occludin and ZO-1 proteins in the endothelial cells that occurred after sustained cerebral ischemia. Furthermore, treatment with hrHGF resulted in retention of these tight junctional proteins in fluorescein isothiocyanate (FITC)-albumin-perfused cerebral vessels, which did not leak FITC-albumin in the ipsilateral cortex. These results suggest that HGF-mediated maintenance of the tight junctional proteins in the endothelial cells may be a possible mechanism for the protective effect of HGF against the disruption of the BBB after cerebral ischemia.

Animals↗
PubMed2006

The protective effect of hepatocyte growth factor against cell death in the hippocampus after transient forebrain ischemia is related to the improvement of apurinic/apyrimidinic endonuclease/redox factor-1 level and inhibition of NADPH oxidase activity.

Early oxidative DNA damage is regarded to be an initiator of neuronal apoptotic cell death after cerebral ischemia. Although evidence suggests that HGF has the ability to protect cells from oxidative stress, it remains unclear as to how HGF suppresses oxidative DNA damage after cerebral ischemia. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) is a multifunctional protein in the DNA base repair pathway that is responsible for repairing apurinic/apyrimidinic sites in DNA after oxidation. We demonstrated that both the immunoreactivity and the number of APE/Ref-1-positive cells in the hippocampal CA1 region were decreased after transient forebrain ischemia and that treatment with HGF suppressed this reduction. The expression of Cu/ZnSOD and MnSOD in the hippocampal CA1 region did not change after ischemia, regardless of treatment with or not with HGF. The activity of NADPH oxidase was increased mainly in glia-like cells in the hippocampal CA1 region after ischemia, and this increase was attenuated by HGF treatment. These results suggest that the protective effects of HGF against cerebral ischemia-induced cell death in the hippocampal CA1 region are related to the improvement of neuronal APE/Ref-1 expression and the inhibition of NADPH oxidase activity in glia-like cells.

Apoptosis↗
PubMed2006

Adenoviral gene transfer of hepatocyte growth factor prevents death of injured adult motoneurons after peripheral nerve avulsion.

Hepatocyte growth factor (HGF) exhibits strong neurotrophic activities on motoneurons both in vitro and in vivo. We examined survival-promoting effects of an adenoviral vector encoding human HGF (AxCAhHGF) on injured adult rat motoneurons after peripheral nerve avulsion. The production of HGF in COS1 cells infected with AxCAhHGF and its bioactivity were confirmed by ELISA, Western blot and Madin-Darby canine kidney (MDCK) cell scatter assay. The facial nerve or the seventh cervical segment (C7) ventral and dorsal roots of 3-month-old Fischer 344 male rats were then avulsed and removed from the stylomastoid or vertebral foramen, respectively, and AxCAhHGF, AxCALacZ (adenovirus encoding beta-galactosidase gene) or phosphate-buffered saline (PBS) was inoculated in the lesioned foramen. Treatment with AxCAhHGF after avulsion significantly prevented the loss of injured facial and C7 ventral motoneurons as compared to AxCALacZ or PBS treatment and ameliorated choline acetyltransferase immunoreactivity in these neurons. These results indicate that HGF may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Adenoviridae↗
PubMed2006

Effects of hepatocyte growth factor on phosphorylation of extracellular signal-regulated kinase and hippocampal cell death in rats with transient forebrain ischemia.

Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischemia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IkappaB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischemia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischemia could be related to an ERK-dependent pathway.

Animals↗
PubMed2006

Prevention of apoptosis-inducing factor translocation is a possible mechanism for protective effects of hepatocyte growth factor against neuronal cell death in the hippocampus after transient forebrain ischemia.

Hepatocyte growth factor (HGF) is one of the prospective agents for therapy against a variety of neurologic and neurodegenerative disorders, although the precise mechanisms for the effect of HGF remain to be elucidated. We showed that treatment with HGF protected hippocampal cornu ammonis (CA) subregion 1 neurons from apoptotic cell death after transient forebrain ischemia. Accumulating evidence indicates that ischemia-induced neuronal damage occurs via caspase-independent pathways. In the present study, we focused on the localization of apoptosis-inducing factor (AIF), which is an important protein in the signal-transduction system through caspase-independent pathways, to investigate the possible mechanism for the protective effect of HGF after transient forebrain ischemia. Hepatocyte growth factor attenuated the increase in the expression of AIF protein in the nucleus after transient forebrain ischemia. We further explored the upstream components of AIF translocation. Primary DNA damage induced by Ca(2+) influx and subsequent NO formation are thought to be the initial events for AIF translocation, which results in the subsequent DNA damage by AIF. Hepatocyte growth factor prevented the primary oxidative DNA damage, as was estimated by using anti-8-OHdG (8-hydroxy-2'-deoxyguanosine) antibody. Oxidative DNA damage after ischemia is known to lead to the activation of poly(ADP-ribose) polymerase (PARP) and p53, resulting in AIF translocation. Marked increases in the PAR polymer formation and the expression of p53 protein after ischemia were effectively prevented by HGF treatment. In the present study, we first showed that HGF was capable of preventing neuronal cell death by inhibiting the primary oxidative DNA damage and then preventing the activation of the PARP/p53/AIF pathway.

Animals↗
PubMed2006

Novel therapeutic strategy for stroke in rats by bone marrow stromal cells and ex vivo HGF gene transfer with HSV-1 vector.

Occlusive cerebrovascular disease leads to brain ischemia that causes neurological deficits. Here we introduce a new strategy combining mesenchymal stromal cells (MSCs) and ex vivo hepatocyte growth factor (HGF) gene transferring with a multimutated herpes simplex virus type-1 vector in a rat transient middle cerebral artery occlusion (MCAO) model. Gene-transferred MSCs were intracerebrally transplanted into the rats' ischemic brains at 2 h (superacute) or 24 h (acute) after MCAO. Behavioral tests showed significant improvement of neurological deficits in the HGF-transferred MSCs (MSC-HGF)-treated group compared with the phosphate-buffered saline (PBS)-treated and MSCs-only-treated group. The significant difference of infarction areas on day 3 was detected only between the MSC-HGF group and the PBS group with the superacute treatment, but was detected among each group on day 14 with both transplantations. After the superacute transplantation, we detected abundant expression of HGF protein in the ischemic brain of the MSC-HGF group compared with others on day 1 after treatment, and it was maintained for at least 2 weeks. Furthermore, we determined that the increased expression of HGF was derived from the transferred HGF gene in gene-modified MSCs. The percentage of apoptosis-positive cells in the ischemic boundary zone (IBZ) was significantly decreased, while that of remaining neurons in the cortex of the IBZ was significantly increased in the MSC-HGF group compared with others. The present study shows that combined therapy is more therapeutically efficient than MSC cell therapy alone, and it may extend the therapeutic time window from superacute to acute phase.

Animals↗
PubMed2006

Possible role of scavenger receptor SRCL in the clearance of amyloid-beta in Alzheimer's disease.

Accumulation of beta-amyloid protein (Abeta) in the brain is a hallmark of Alzheimer's disease (AD), and Abeta-mediated pathogenesis could result from increased production of Abeta or insufficient Abeta clearance by microglia, astrocytes, or the vascular system. Cell-surface receptors, such as scavenger receptors, might play a critical role in the binding and clearing of Abeta; however, the responsible receptors have yet to be identified. We show that scavenger receptor with C-type lectin (SRCL), a member of the scavenger receptor family containing coiled-coil, collagen-like, and C-type lectin/carbohydrate recognition domains, is expressed in cultured astrocytes and microglia. In contrast to the low expression of SRCL in the wild-type mouse brain, in a double transgenic mouse model of AD (Tg-APP/PS1), immunohistochemistry showed that SRCL was markedly induced in Abeta-positive astrocytes and Abeta-positive vascular/perivascular cells, which are associated closely with cerebral amyloid angiopathy. In patients with AD, the distribution of SRCL was similar to that seen in the Tg-APP/PS1 temporal cortex. The presence of a large number of SRCL/Abeta double-positive particles in the intracellular compartments of reactive astrocytes and vascular/perivascular cells in Tg-APP/PS1 mice and AD patients suggests a role for SRCL in Abeta clearance. Moreover, CHO-K1 cells transfected with SRCL isoforms were found to bind fibrillar Abeta(1-42). These findings suggest that SRCL could be the receptor involved in the binding or clearing of Abeta by glial and vascular/perivascular cells in AD.

Adult↗
PubMed2005

Inhibition of apoptosis-inducing factor translocation is involved in protective effects of hepatocyte growth factor against excitotoxic cell death in cultured hippocampal neurons.

Although hepatocyte growth factor (HGF) and its receptor are expressed in various regions of the brain, their effects and mechanism of action under pathological conditions remain to be determined. Over-activation of the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor, has been implicated in a variety of neurological and neurodegenerative disorders. We investigated the effects of HGF on the NMDA-induced cell death in cultured hippocampal neurons and sought to explore their mechanisms. NMDA-induced cell death and increase in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were prevented by HGF treatment. Although neither the total amounts nor the mitochondrial localization of Bax, Bcl-2 and Bcl-xL were affected, caspase 3 activity was increased after NMDA exposure. Treatment with HGF partially prevented this NMDA-induced activation of caspase 3. Although the amount of apoptosis-inducing factor (AIF) was not altered, translocation of AIF into the nucleus was detected after NMDA exposure. This NMDA-induced AIF translocation was reduced by treatment with HGF. In addition, increased poly(ADP-ribose) polymer formation after NMDA exposure was attenuated by treatment with HGF. These results suggest that the protective effects of HGF against NMDA-induced neurotoxicity are mediated via the partial prevention of caspase 3 activity and the inhibition of AIF translocation to the nucleus.

Animals↗
PubMed2005

Effects of fukutin deficiency in the developing mouse brain.

The major pathological change in Fukuyama-type congenital muscular dystrophy brain is polymicrogyria. Pathological studies of Fukuyama-type congenital muscular dystrophy brain indicated that protrusion of neurons into the subarachnoid space through breaches in the glia limitans-basal lamina complex is a cardinal pathogenic process in this condition. It remains undetermined, however, whether the defect causing this abnormal migration resides in the migrating neurons or in the glia limitans-basal lamina complex. To elucidate the pathogenesis of brain abnormalities in Fukuyama-type congenital muscular dystrophy, we analyzed histologically and immunohistochemically the developing forebrain in fukutin-deficient chimeric mice and compared it with that in controls (n=4 in each group). In chimeric embryos, ectopia became apparent as early as embryonic day 14, and laminar organization became progressively distorted. The basal lamina of the cortical surface in chimeras showed defects at E14, coinciding with the earliest time point at which ectopia were detected. Immunohistochemical analysis of glycosylated alpha-dystroglycan showed progressive defects coincidental with the disruption of the basal lamina. Neuronal migration was not affected in chimeras, as determined by detection of bromodeoxyuridine-labeled neurons. Extension of radial glial fibers was intact in chimeras. Taken together, disruption of the basal lamina, caused by the loss of interaction between hypoglycosylated alpha-dystroglycan and its ligands, plays a key role in the pathogenesis of cortical dysplasia in Fukuyama-type congenital muscular dystrophy.

Animals↗
PubMed2004

Anxiolytic effect of hepatocyte growth factor infused into rat brain.

BACKGROUND: Hepatocyte growth factor (HGF) has the capacity to selectively direct thalamocortical projections into an intermediate target, the pallidum, and eventually to their final cortical destination. HGF may have a role in the mediation of anxiety. Very little is known about other central behavioral effects of HGF. OBJECTIVE: Our aim was to determine what effect HGF has on anxiety in rats. METHODS: HGF was infused at a constant rate into cerebral lateral ventricles and its effect on anxiety in rats was monitored. RESULTS: In the elevated plus maze test and the black and white box test, HGF administration caused all indicators of anxiety to increase. No significant effect on general locomotor activity was seen. CONCLUSION: HGF infusion into the brain produces an anxiolytic effect.

Animals↗
PubMed2003

Expression of hepatocyte growth factor and c-Met in the anterior horn cells of the spinal cord in the patients with amyotrophic lateral sclerosis (ALS): immunohistochemical studies on sporadic ALS and familial ALS with superoxide dismutase 1 gene mutation.

To clarify the trophic mechanism of residual anterior horn cells affected by sporadic amyotrophic lateral sclerosis (SALS) and familial ALS (FALS) with superoxide dismutase 1 (SOD1) mutations, we investigated the immunohistochemical expression of hepatocyte growth factor (HGF), a novel neurotrophic factor, and its receptor, c-Met. In normal subjects, immunoreactivity to both anti-HGF and anti-c-Met antibodies was observed in almost all anterior horn cells, whereas no significant immunoreactivity was observed in astrocytes and oligodendrocytes. Histologically, the number of spinal anterior horn cells in ALS patients decreased along with disease progression. Immunohistochemically, the number of neurons negative for HGF and c-Met increased with ALS disease progression. However, throughout the course of the disease, certain residual anterior horn cells co-expressed both HGF and c-Met with the same, or even stronger intensity in comparison with those of normal subjects, irrespective of the reduction in the number of immunopositive cells. Western blot analysis revealed that c-Met was induced in the spinal cord of a patient with SALS after a clinical course of 2.5 years, whereas the level decreased in a SALS patient after a clinical course of 11 years 5 months. These results suggest that the autocrine and/or paracrine trophic support of the HGF-c-Met system contributes to the attenuation of the degeneration of residual anterior horn cells in ALS, while disruption of the neuronal HGF-c-Met system at an advanced disease stage accelerates cellular degeneration and/or the process of cell death. In SOD1-mutated FALS patients, Lewy body-like hyaline inclusions (LBHIs) in some residual anterior horn cells exhibited co-aggregation of both HGF and c-Met, although the cytoplasmic staining intensity for HGF and c-Met in the LBHI-bearing neurons was either weak or negative. Such sequestration of HGF and c-Met in LBHIs may suggest partial disruption of the HGF-c-Met system, thereby contributing to the acceleration of neuronal degeneration in FALS patients.

Adult↗
PubMed2003

Hepatocyte growth factor: from diagnosis to clinical applications.

Hepatocyte growth factor (HGF), initially identified and molecularly cloned as a potent mitogen of primary cultured hepatocytes, has multiple activities in a variety of tissues during the course of development and also in various disease states. HGF plays key roles in the attenuation of disease progression as an intrinsic repair factor. It is also evident that HGF levels are regulated under different conditions, for example, during the course of pregnancy, aging, and disease. This review focuses on the levels of HGF in normal and pathophysiological situations and examines the relationships between HGF levels and disease, disease stage, and disease prognosis. The clinical potential of HGF as a treatment for subjects with various diseases is also given attention.

Animals↗
PubMed2002

Overexpression of HGF retards disease progression and prolongs life span in a transgenic mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motoneurons and degeneration of motor axons. We show that overexpression of hepatocyte growth factor (HGF) in the nervous system attenuates motoneuron death and axonal degeneration and prolongs the life span of transgenic mice overexpressing mutated Cu2+/Zn2+ superoxide dismutase 1. HGF prevented induction of caspase-1 and inducible nitric oxide synthase (iNOS) in motoneurons and retained the levels of the glial-specific glutamate transporter (excitatory amino acid transporter 2/glutamate transporter 1) in reactive astrocytes. We propose that HGF may be the first example of an endogenous growth factor that can alleviate the symptoms of ALS by direct neurotrophic activities on motoneurons and indirect activities on glial cells, presumably favoring a reduction in glutamatergic neurotoxicity.

Age of Onset↗
PubMed2002

Localization and functional role of hepatocyte growth factor (HGF) and its receptor c-met in the rat developing cerebral cortex.

Development of the cerebral cortex is a series of precisely timed proliferative, migratory, and maturational processes. Hepatocyte growth factor (HGF) is a pleiotrophic cytokine, which plays important roles in the organogenesis and regeneration of various tissues, both during development and in the adult, due to its mitogenic, motogenic and morphogenic activities. In the present study, we examined expression and functional roles of HGF and c-Met during development of the rat cerebral cortex. Quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed that expression levels of c-met and HGF mRNAs were increased in the cerebral cortex during late embryonic development and peaked at E18. Immunohistochemical analyses revealed that c-Met-immunoreactivity (IR) was localized to the preplate (PP), with weaker-IR in neuroepithelial layer (NE) at embryonic day 14 (E14). At E16, c-Met-IR was present in the cortical plate (CP) and the intermediate zone (IZ), with a weak presence in the ventricular zone (VZ). On the other hand, HGF-IR was present in NE and VZ at E14 and E16, respectively. HGF-IR appeared in cortical plate tissue from E16 onward. Double labeling immunofluorescent cytochemical studies revealed that c-Met-IR was localized both in TuJ-1-IR- and non-TuJ-1-IR-cells, purified from E18 cerebral cortex in vitro, suggesting the presence of c-Met-IR in postmitotic neurons as well as in neuroepithelial cells. c-Met-IR was strong in cell bodies and neurites shortly after in vitro culture, while at 7DIV c-Met-IR decreased in neurites and was evident in growth cones. HGF dose dependently supported neuronal survival in vitro under serum-deprived conditions. In a transwell culture chamber, HGF increased neuronal migration, and co-incubation with functional blocking antibody against HGF abrogated this motogenic effect of HGF. These lines of evidence suggest that HGF is involved in the development and maintenance of cortical neurons during differentiation, motogenesis, neuritogenesis and neuronal survival.

Animals↗
PubMed2002

Molecular cloning, expression and partial characterization of Xksy, Xenopus member of the Sky family of receptor tyrosine kinases.

We isolated a cDNA encoding the Xenopus member of Sky/Axl/Mer receptor tyrosine kinase family (referred as Sky family), termed Xksy. The predicted Xksy protein has conserved structural characteristics of the Sky family: an unique extracellular domain of two immunoglobulin (Ig)-like repeats, two fibronectin type III (FNIII)-like repeats and an intracellular tyrosine kinase. Homology analysis of Xksy showed the highest identity to mammalian Sky protein. In contrast to the predominant expression of sky mRNA in the adult mammalian nervous system, Northern blot analysis showed ubiquitous expression of a single 5.2-kb Xksy mRNA in tissues of the adult Xenopus. RNase protection assays revealed that, during development, Xksy mRNA is expressed from mid neurulation stage. Levels increase through the tadpole stage and become restricted to the head region in embryos by stage 40. Whole-mount in situ hybridization analyses revealed that expression of Xksy is localized to the nervous system of the tadpole stage, including origins of sensory organs and branchial arches. When a chimeric receptor (EGFR-Xksy), composed of the extracellular region of epidermal growth factor (EGF) receptor and the transmembrane/intracellular regions of Xksy, was expressed in a doxycycline repressive manner in HEK 293 cells, EGF-stimulus without doxycycline induced tyrosine phosphorylation of the chimeric receptor and evoke morphological changes. EGF treatment also induced growth modifications of EGFR-Xksy cells. And doxycycline pre-treatment eliminated these activities. These findings suggest that Xksy may play an important role in growth, differentiation and the accurate migration of cells during embryogenesis and early neural development.

Amino Acid Sequence↗
PubMed2002

Identification of Gas6, a putative ligand for Sky and Axl receptor tyrosine kinases, as a novel neurotrophic factor for hippocampal neurons.

Sky (also known as "Tyro3" and "Rse") is a member of the Axl/Sky/Mer receptor tyrosine kinase family and has two immunoglobulin-like repeats and two fibronectin type III-like repeats in the extracellular domain. Gas6 is a ligand for all members of the Axl family, each of which (Axl, Sky, and c-Mer) has different affinities to Gas6. Physiological functions of Sky and Gas6 in the nervous system are not well understood, despite their importance, which is suggested by Sky structural features and its predominant expression in the brain. We found in the RNase protection assays that gas6 and sky mRNAs are expressed in the adult rat hippocampus and are similarly regulated during development. Expression levels were low during embryonic stages but gradually increased during development and reached the highest level in adulthood. Sky, but not Axl, immunoreactivity was observed in the adult hippocampus. Recombinant rat Gas6 attenuated hippocampal neuronal cell death that was caused by serum starvation in vitro, indicating that Gas6 is a novel neurotrophic factor for hippocampal neurons. Gas6 showed regulated expression in the sciatic nerve after nerve transection. Therefore, Sky and Gas6 have neurotrophic roles in the nervous system.

Amino Acid Sequence↗