Masamitsu Shimazawa

Gifu Pharmaceutical University, Gihu, Gifu, Japan

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Publications (207)548.38 Total impact

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    ABSTRACT: Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane protein reported to have neuroprotective effects in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We investigated whether GPNMB is also neuroprotective against brain ischemia reperfusion injury. Focal ischemia/reperfusion injury was induced via filament middle cerebral artery occlusion for 2 h, followed by reperfusion upon withdrawal of the filament. We assessed the neuroprotective effects of GPNMB using transgenic (Tg) mice which over expressing GPNMB or recombinant GPNMB which has the sequence of human extracellular GPNMB. The results showed that GPNMB was up-regulated after ischemia reperfusion injury, and that genomic over-expression of GPNMB significantly ameliorated infarct volume. Next, we investigated the protective mechanisms of GPNMB via western blotting and immunohistochemistry. Phosphorylation of Extracellular Signal-regulated Kinase 1 and 2 (ERK1/2), and protein kinase B (Akt), were increased in the GPNMB Tg group according to western blotting data. Immunohistochemistry analysis showed that GPNMB was expressed not only in neurons, but also in astrocytes, produced labelling patterns similar to that in human brain ischemia. Furthermore, recombinant GPNMB also decreased the infarction volume. These results indicate that GPNMB protected neurons against ischemia reperfusion injury, and phosphor-Akt and phosphor-ERK might be a part of the protective mechanisms, and that the neuroprotective effect of GPNMB was seemingly induced by the extracellular sequence of GPNMB. In conclusion, these findings indicate that GPNMB has neuroprotective effects against ischemia reperfusion injury, via phosphorylation of ERK1/2 and Akt, suggesting that GPNMB may be a therapeutic target for ischemia reperfusion injuries.
    Neuroscience 07/2014; · 3.12 Impact Factor
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    ABSTRACT: Ocular ischemic syndrome is likely stem from retinal ischemia, and which causes visual disorder. The pathological mechanism of ocular ischemic syndrome is still unknown, therefore the optimal treatment for ocular ischemic syndrome remains to be established. Then, this study aimed to evaluate the effects of tissue-derived kallidinogenase in retinal ischemia protection in mice. In the present study, the effects of tissue-derived kallidinogenase (1 or 10μg/kg, i.v.) on ischemia/reperfusion-induced retinal damage in mice were examined by histological, electrophysiological, and permeability analyses. In addition, we assessed phosphorylation of endothelial nitric oxide synthase (eNOS) and nuclear factor-kappa B (NF-κB), which is closely-involved in ischemic injury and permeability. Moreover, the neuroprotective effect of kallidinogenase in an in vitro model of ischemia induced by oxygen-glucose deprivation or hypoxia was examined. The results indicated that kallidinogenase significantly prevented the decrease in ganglion cell number induced by ischemia/reperfusion. Electroretinogram measurements showed that kallidinogenase significantly prevented the ischemia/reperfusion-induced reductions in a- and b-wave amplitudes seen 5 days after ischemia/reperfusion. Moreover, kallidinogenase significantly inhibited the permeability increase induced by ischemia/reperfusion. Similar to the results in vivo, kallidinogenase significantly inhibited the retinal ganglion cell death induced by oxygen-glucose deprivation. Also, kallidinogenase significantly suppressed the hypoxia-induced increase in permeability. However, these effects observed in vitro disappeared when an eNOS inhibitor was used concurrently. These findings suggest that kallidinogenase may prevent ischemia/reperfusion-induced retinal damage, might be through eNOS activation.
    European journal of pharmacology. 05/2014;
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    ABSTRACT: Oxidative stress plays a critical role in mediating tissue injury and neuron death during ischemia-reperfusion injury (IRI). The Keap1-Nrf2 defense pathway serves as a master regulator of endogenous antioxidant defense, and Nrf2 has been attracting attention as a target for the treatment of IRI. In the present study, we evaluated Nrf2 expression in IRI using OKD (Keap1-dependent Oxidative stress Detector) mice and investigated the neuroprotective ability of an Nrf2 activator. We demonstrated temporal changes of Nrf2 expression in the same mice with luciferase assays and the Nrf2 activity time course using western blotting. We also visualized Nrf2 expression in the ischemic penumbra and investigated Nrf2 expression in mice and humans using immunohistochemistry. Endogenous Nrf2 up-regulation was not detected early in IRI, but expression peaked 24h after ischemia. Nrf2 expression was mainly detected in the penumbra, and it was found in neurons and astrocytes in both mice and humans. Intravenous administration of the Nrf2 activator bardoxolone methyl (BARD) resulted in earlier up-regulation of Nrf2 and heme oxygenase-1. Furthermore, BARD decreased infarction volume and improved neurological symptoms after IRI. These findings indicate that earlier Nrf2 activation protects neurons, possibly via effects on astrocytes.
    Free Radical Biology & Medicine 04/2014; · 5.27 Impact Factor
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    ABSTRACT: Blue light is a high-energy or short-wavelength visible light, which induces retinal diseases such as age-related macular degeneration and retinitis pigmentosa. Bilberry (Vaccinium myrtillus L.) and lingonberry (Vaccinium vitis-idaea) contain high amounts of polyphenols (anthocyanins, resveratrol, and proanthocyanidins) and thus confer health benefits. This study aimed to determine the protective effects and mechanism of action of bilberry extract (B-ext) and lingonberry extract (L-ext) and their active components against blue light-emitting diode (LED) light-induced retinal photoreceptor cell damage. Cultured murine photoreceptor (661 W) cells were exposed to blue LED light following treatment with B-ext, L-ext, or their constituents (cyanidin, delphinidin, malvidin, trans-resveratrol, and procyanidin B2). 661 W cell viability was assessed using a tetrazolium salt (WST-8) assay and Hoechst 33342 nuclear staining, and intracellular reactive oxygen species (ROS) production was determined using CM-H2DCFDA after blue LED light exposure. Activation of p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor-kappa B (NF-kappaB), and LC3, an ubiquitin-like protein that is necessary for the formation of autophagosomes, were analyzed using Western blotting. Caspase-3/7 activation caused by blue LED light exposure in 661 W cells was determined using a caspase-3/7 assay kit. B-ext, L-ext, NAC, and their active components improved the viability of 661 W cells and inhibited the generation of intracellular ROS induced by blue LED light irradiation. Furthermore, B-ext and L-ext inhibited the activation of p38 MAPK and NF-kappaB induced by blue LED light exposure. Finally, B-ext, L-ext, and NAC inhibited caspase-3/7 activation and autophagy. These findings suggest that B-ext and L-ext containing high amounts of polyphenols exert protective effects against blue LED light-induced retinal photoreceptor cell damage mainly through inhibition of ROS production and activation of pro-apoptotic proteins.
    BMC Complementary and Alternative Medicine 04/2014; 14(1):120. · 2.08 Impact Factor
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    ABSTRACT: Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm(2)). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light.
    Scientific reports. 01/2014; 4:5223.
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    ABSTRACT: Diabetes is a crucial risk factor for stroke and is associated with increased frequency and poor prognosis. Although endothelial dysfunction is a known contributor of stroke, the underlying mechanisms have not been elucidated. The aim of this study was to elucidate the mechanism by which chronic hyperglycemia may contribute to the worsened prognosis following stroke, especially focusing on mitochondrial alterations. We examined the effect of hyperglycemia on hemorrhagic transformation at 24 hours after middle cerebral artery occlusion (MCAO) in streptozotocin (STZ) -induced diabetic mice. We also examined the effects of high-glucose exposure for 6 days on cell death, mitochondrial functions and morphology in human brain microvascular endothelial cells (HBMVECs) or human endothelial cells derived from induced pluripotent stem cells (iCell endothelial cells). Hyperglycemia aggravated hemorrhagic transformation, but not infarction following stroke. High-glucose exposure increased apoptosis, capase-3 activity, and release of apoptosis inducing factor (AIF) and cytochrome c in HBMVECs as well as affected mitochondrial functions (decreased cell proliferation, ATP contents, mitochondrial membrane potential, and increased matrix metalloproteinase (MMP)-9 activity, but not reactive oxygen species production). Furthermore, morphological aberration of mitochondria was observed in diabetic cells (a great deal of fragmentation, vacuolation, and cristae disruption). A similar phenomena were seen also in iCell endothelial cells. In conclusion, chronic hyperglycemia aggravated hemorrhagic transformation after stroke through mitochondrial dysfunction and morphological alteration, partially via MMP-9 activation, leading to caspase-dependent apoptosis of endothelial cells of diabetic mice. Mitochondria-targeting therapy may be a clinically innovative therapeutic strategy for diabetic complications in the future.
    PLoS ONE 01/2014; 9(8):e103818. · 3.73 Impact Factor
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    ABSTRACT: Although dry age-related macular degeneration (AMD) is one of the major causes of blindness, no effective therapies are developed. In this study, we investigated the effects of SUN N8075, a radical scavenger with neuroprotective properties, against light-induced retinal damage used as the model of dry AMD in mice. After dark adaption for 24 h, we exposed the mice at 8000 lx for 3 h. We evaluated the retinal damage by recording the electroretinagram (ERG) and measuring the thickness of outer nuclear layer (ONL) at 5 d after the light exposure. Retinal apoptotic cells were also detected by terminal deoxynucleotidyl transeferase mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining, and the expression of 8-hydroxy-2-deoxyguanosine (8-OHdG) as an index for oxidative stress at 48 h after exposure to light. In ERG measurement, the intraperitoneal administration of SUN N8075 at 30 mg/kg improved the retinal dysfunction induced by the excess light exposure. In the histological evaluation, SUN N8075 inhibited the reduction of ONL thickness. In addition, SUN N8075 decreased in both numbers of TUNEL- and 8-OHdG-positive cells in ONL. These findings suggest that the systemic administration of SUN N8075 has protective effects on excess light-induced photoreceptor degeneration, via inhibition of oxidative stress.
    Biological & Pharmaceutical Bulletin 01/2014; 37(3):424-30. · 1.85 Impact Factor
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    ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Recently, it has been reported that a mutation in the sigma-1 receptor causes juvenile ALS. Therefore, the function of the sigma-1 receptor may be important in the pathology of ALS. In the present study, we investigated the effect of SA4503, a sigma-1 receptor agonist, against in in vitro and in vivo ALS models. We first investigated whether SA4503, a sigma-1 receptor agonist, prevented superoxide dismutase 1 (SOD1G93A) - and serum free-induced cell death of mice motor neuron cells (NSC34) in in vitro model of an ALS. At concentrations of 1 to 10μM, SA4503 reduced SOD1G93A-induced cell death in a concentration-dependent manner, and BD1047, a sigma-1 receptor antagonist, inhibited the protective effect of SA4503. Next, we investigated whether SA4503 affected the phosphorylation levels of Akt (Ser 473) and extracellular signal-regulated kinase (ERK1/2) and the expression of the sigma-1 receptor. SA4503 promoted the phosphorylation of Akt (Ser 473) and ERK1/2 in a time-dependent manner, but SA4503 did not affect the expression of the sigma-1 receptor. These results suggest that the protective effect of SA4503 might be involved in promoting the phosphorylation of Akt and ERK1/2. We then investigated whether SA4503 suppressed the progression of ALS in an SOD1G93A ALS mouse model. SA4503 did not affect the onset time of ALS. However, it significantly extended the survival time in the SOD1G93A mice compared with a vehicle-treated group. These findings indicate that SA4503 is effective in suppressing motor neuron degeneration and symptom progression in ALS.
    Neuroscience Letters 12/2013; · 2.03 Impact Factor
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    ABSTRACT: Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family. Although it has been reported that PlGF protects against neuronal damage in the brain, little is known about the effects of PlGF in the retina. Therefore, we investigated the effects of PlGF on retinal neuronal cells. To evaluate the effects of PlGF against L-buthionine-(S,R)-sulfoximine (BSO)/glutamate cell death, oxygen-glucose deprivation (OGD)-induced cell death, and light-induced cell death, RGC-5 and 661W cells were used. We evaluated the mechanism responsible for the protective effects of PlGF against retinal neuronal cell death by performing the examinations with U1026, which is a mitogen-activated protein kinase (MEK) inhibitor, and LY294002, which is a phosphoinositide 3-kinase (PI3K) inhibitor. In addition, we measured caspase-3/7 activity in RGC-5 cells and 661W cells. PlGF protected against RGC-5 cell death induced by BSO/glutamate and OGD and against 661W cell death induced by light irradiation. Moreover, an anti-PlGF antibody negated these protective effects. The protective effects of PlGF against OGD-induced RGC-5 cell death and light-induced 661W cell death were suppressed by using an anti-PlGF antibody, U1026, and LY294002. Treatment with PlGF suppressed caspase-3/7 activity in both cell lines. We demonstrated for the first time that PlGF exerts a protective effect by inhibiting the activation of caspase-3/7 through the MEK and PI3K pathway in retinal neuronal cells. These data suggest that PlGF may be an important protective factor in the retina. © 2013 Wiley Periodicals, Inc.
    Journal of Neuroscience Research 11/2013; · 2.97 Impact Factor
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    ABSTRACT: Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose-derived stem cells (ASCs) and ASC-conditioned medium (ASC-CM) against retinal damage and identified the neuroprotective factors in ASC-CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light-induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC-CM and mature adipocyte-conditioned medium were collected after 72 hours of culture. In vitro, H2O2- and light-induced cell death was reduced in a photoreceptor cell line with ASC-CM but not with mature adipocyte-conditioned medium. In vivo, light-induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC-CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal-regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC-CM and progranulin have neuroprotective effects in the light-induced retinal-damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.
    Stem cells translational medicine. 11/2013;
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    ABSTRACT: Dietary carotenoids exhibit various biological activities, including antioxidative activity. In particular, astaxanthin, a type of carotenoid, is well known as a powerful antioxidant. We investigated whether astaxanthin would protect against light-induced retinal damage. In an in vivo study, ddY male mice were exposed to white light at 8,000 lux for 3 h to induce retinal damage. Five days after light exposure, retinal damage was evaluated by measuring electroretinogram (ERG) amplitude and outer nuclear layer (ONL) thickness. Furthermore, expression of apoptotic cells, 8-hydroxy-deoxyguanosine (8-OHdG), was measured. In an in vitro study, retinal damage was induced by white light exposure at 2,500 lux for 24 h, and propidium iodide (PI)-positive cells was measured and intracellular reactive oxygen species (ROS) activity was examined. Astaxanthin at 100 mg/kg inhibited the retinal dysfunction in terms of ERG and ONL loss and reduced the expression of apoptotic and 8-OHdG-positive cells induced by light exposure. Furthermore, astaxanthin protected against increases of PI-positive cells and intracellular reactive oxygen species (ROS) activity in 661W cells. These findings suggest that astaxanthin has protective effects against light-induced retinal damage via the mechanism of its antioxidative effect.
    Journal of Pharmacological Sciences 10/2013; · 2.15 Impact Factor
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    ABSTRACT: Cilostazol is a specific inhibitor of phosphodiesterase III and is widely used to treat ischemic symptoms of peripheral vascular disease. We evaluated the protective effects of cilostazol in a murine model of ocular ischemic syndrome in which retinal ischemia was induced by 5-h unilateral ligation of both the pterygopalatine artery (PPA) and the external carotid artery (ECA) in anesthetized mice. The effects of cilostazol (30 mg/kg, p.o.) on ischemia/reperfusion (I/R)-induced retinal damage were examined by histological, retinal vascular permeability, and electrophysiological analyses. Using immunoblotting, the protective mechanism for cilostazol was evaluated by examining antiinflammatory effects of cilostazol on the expression of tumor necrosis factors-α (TNF-α) and tight junction proteins (ZO-1 and claudin-5), and the phosphorylations of nuclear factor-kappa B (NF-κB) and c-Jun. The histological analysis revealed that I/R decreased the cell number in the ganglion cell layer (GCL) and the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL), and that cilostazol attenuated these decreases. Additionally, cilostazol prevented the hyperpermeability of blood vessels. Electroretinogram (ERG) measurements revealed that cilostazol prevented the I/R-induced reductions in a-, b-, and oscillatory potential (OP) wave amplitudes seen at 5 days after I/R. Cilostazol inhibited the increased expression of TNF-α and the phosphorylation levels of NF-κB and c-Jun in the retina after I/R. In addition, cilostazol prevented TNF-α-induced reduction of ZO-1 and claudin-5 expression in human retinal microvascular endothelial cells (HRMECs). These findings indicate that cilostazol may prevent I/R-induced retinal damage partly through inhibition of TNF-α-induced NF-κB/AP-1 signaling pathway.
    Pharmacology Research & Perspectives. 10/2013; 1(1).
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    ABSTRACT: Human adipose-derived stem cells (hASCs) are present in adult adipose tissue and have been reported to secrete various factors that have neuroprotective effects. In the present study, we examined whether hASC-conditioned medium (hASC-CM) was effective against experimental degenerative retinal disease. Mature adipocytes (MAs) and hASCs were isolated from human subcutaneous adipose tissue. The isolated hASCs were identified based on their capacity for bone and neural differentiation. The effects of hASC-CM against tunicamycin-, H2O2-, and light-induced retinal photoreceptor damage were evaluated in vitro by measuring cell death. Moreover, we identified various factors present in hASC-CM using antibody arrays. Retinal damage induced in mice by exposure to white light was studied in vivo, and photoreceptor damage was evaluated according to the thickness of the outer nuclear layer and electroretinography results. In addition, the effect of hASC-CM on Akt phosphorylation at Ser473was confirmed by western blotting. Finally, the effects of the secreted proteins identified in the hASC-CM on light-induced damage were evaluated in vivo. Isolated hASCs differentiated to osteocytes and neurons. hASC-CM protected against tunicamycin-, H2O2-, and light-induced cell death. In addition, hASC-CM inhibited photoreceptor degeneration and retinal dysfunction after exposure to light. Several proteins secreted by hASCs, such as the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the secreted protein acidic and rich in cysteine (SPARC), protected against light-induced damage in vitro and in vivo. The results of the present study showed that hASC-CM has neuroprotective effects against light-induced retinal damage and suggest that hASCs have a therapeutic potential in retinal degenerative diseases via their secreted proteins, without requiring transplantation.
    Experimental Eye Research 09/2013; · 3.03 Impact Factor
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    ABSTRACT: To improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice. In vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-alpha, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-kappaB (NF-kappaB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells. We found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-kappaB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-alpha in endothelial cells. PGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.
    Journal of Neuroinflammation 08/2013; 10(1):105. · 4.35 Impact Factor
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    ABSTRACT: Inflammatory response plays a critical role in neuronal damage after retinal ischemia-reperfusion (I/R) injury, and is tightly regulated by the toll-like receptor (TLR) 4. This study aimed to determine whether TLR4 is involved with injury in ocular ischemic syndrome mice model and to clarify the downstream pathway of TLR4. To cause retinal ischemia, we ligated the unilateral external carotid artery and the pterygopalatine artery of mice for 5 h. Five days after reperfusion, retinal histological analysis was performed. To examine the downstream pathway of TLR4, we analyzed the changes in phosphorylation of nuclear factor-kappaB (NF-κB) by western blotting. In addition, we evaluated the expression of phosphorylated spleen tyrosine kinase (Syk), which is an adaptor protein of TLR4, and the effects of a Syk inhibitor (piceatannol) against the retinal ischemic damage and TLR4 signaling. TLR4 knock-out (KO) mice significantly inhibited the histological damage induced by I/R compared with wild-type mice. The expression of TLR4 was upregulated after I/R in wild-type mice. The phosphorylation level of NF-κB after I/R in TLR4 KO mice was decreased compared with that in wild-type mice. The phosphorylated Syk expression was upregulated after I/R, and the upregulation was inhibited in TLR4 KO mice. Piceatannol inhibited the histological and functional retinal damage and reduced the phosphorylation level of NF-κB induced by I/R. These data indicate that TLR4 plays a pivotal role in the pathogenesis of ocular ischemic syndrome, and Syk and NF-κB are key molecules in TLR4 signaling in retinal ischemia.
    Investigative ophthalmology & visual science 08/2013; · 3.43 Impact Factor
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    ABSTRACT: Although long-term treatment with the oral anticoagulant warfarin is widely used to prevent cardioembolic ischemic stroke, it has been reported that warfarin can exacerbate hemorrhagic transformation (HT) after cerebral ischemia. We investigated whether cilostazol, a phosphodiesterase-III inhibitor, suppressed the warfarin-induced HT after cerebral ischemia in mice. Male ddY mice were treated with oral warfarin before 3-hour middle cerebral artery occlusion followed by 21-hour reperfusion to induce HT. The duration of warfarin pretreatment was determined by measurement of prothrombin time-international normalized ratio value. Cilostazol or vehicle was administered by intraperitoneal injection immediately after reperfusion. The infarct volume, brain swelling, and brain hemoglobin content were evaluated at 24 hours after middle cerebral artery occlusion. We also evaluated the survival rate of each treated group for 7 days after surgery. To investigate the mechanism underlying cilostazol's effects, the proteins involved in vascular endothelial integrity were investigated using Western blotting. HT volume was exacerbated by warfarin treatment, and cilostazol (3 mg/kg, IP) suppressed this exacerbation (sham, mean±SD, 29.2±13.4 mg/dL; vehicle, 33.3±11.9 mg/dL; warfarin, 379.4±428.9 mg/dL; warfarin+cilostazol 1 mg/kg, 167.5±114.2 mg/dL; warfarin+cilostazol 3 mg/kg, 116.9±152.3 mg/dL). Furthermore, cilostazol improved survival rate and upregulated the expression of tight junction proteins and vascular endothelial cadherin. Cilostazol reduced the warfarin-related risk of HT after ischemia by protecting the vascular endothelial cells. This result suggested that cilostazol administration in patients with acute ischemic stroke might reduce HT.
    Stroke 07/2013; · 6.16 Impact Factor
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    ABSTRACT: BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no effective treatments. Fasudil hydrochloride (fasudil), a potent rho kinase (ROCK) inhibitor, is useful for the treatment of ischemic diseases. In previous report, fasudil improved pathology in mouse models of Alzheimer's disease and spinal muscular atrophy, but there is no evidence in ALS. We therefore investigated its effects on experimental ALS models. EXPERIMENTAL APPROACH: In NSC34 cells, the neuroprotective effect of hydroxyfasudil (M3), an active metabolite of fasudil, and its mechanism were evaluated. Moreover, the effect of fasudil 30 or 100 mg/kg administration via drinking water to mutant superoxide dismutase 1 (SOD1(G93A) ) mice were tested by motor performance, survival time, histological analysis, and its mechanism. KEY RESULTS: M3 prevented motor neuron cell death induced by SOD1(G93A) . Furthermore, M3 suppressed both the increase of ROCK activity and phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and the reduction of phosphorylated protein kinase B (Akt) induced by SOD1(G93A) . These effects of M3 were attenuated by treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002). Moreover, fasudil slowed disease progression, increased survival time, and reduced motor neuron loss, in SOD1(G93A) mice. Moreover, fasudil attenuated the increase of ROCK activity and PTEN, and the reduction of Akt in SOD1G93A mice. CONCLUSIONS AND IMPLICATIONS: These findings indicate that fasudil may be effective at suppressing motor neuron degeneration and symptom progression in ALS. Hence, fasudil may have potential as a therapeutic agent for ALS treatment.
    British Journal of Pharmacology 06/2013; · 5.07 Impact Factor
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    ABSTRACT: Spectral-domain optical coherence tomography (SD-OCT) is an interferometric optical tomography technique and provides high resolution and noninvasive visualization of retinal morphology. The purpose of this study was to assess the utility of thickness maps and quantitative thickness measurements of the ganglion cell complex (GCC: retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer) obtained by SD-OCT of a mouse model of N-methyl-d-aspartate (NMDA)-induced retinal damage. SD-OCT imaging was performed in ddY mice at 1, 3, and 7 days and 1 month after intravitreal injection of NMDA. GCC thickness maps and circle cross-sectional OCT images were made from volumetric OCT images. The GCC thickness was measured on a cross-sectional OCT image on a circle with a radius 300 μm from the center of the optic nerve disc. Histological analysis was conducted by measuring the GCC thickness at the same time intervals. The thickness maps and the quantitative thickness values of GCC showed thickness changes at each time point in the NMDA-treated mice when compared with normal and vehicle-treated mice. Both the OCT sectional images and the histological images revealed increases in GCC thickness at 1 day, followed by decreases from 3 days to 1 month after NMDA injection. The GCC thickness measured using OCT sectional images correlated with the thickness measured using histological images. In conclusion, GCC thickness mapping is a useful method for evaluating NMDA-induced retinal degeneration in mice.
    Experimental Eye Research 05/2013; · 3.03 Impact Factor
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    ABSTRACT: PURPOSE: Although heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been reported to have protective effects against various neuronal cell damage, its role in the retina has not been elucidated. Here, we investigated its role in light-induced photoreceptor degeneration using retinas and ventral forebrain specific Hb-egf knockout (KO) mice. METHODS: Disruption of Hb-egf was confirmed by LacZ staining and RT-PCR. Time-dependent changes in retinal HB-EGF were measured using quantitative RT-PCR and Western blotting. Retinal damage was induced by exposure to light. Recombinant human HB-EGF was injected intravitreally. Electroretinogram (ERG) and histological analyses were performed. To evaluate the effect of HB-EGF against light irradiation-induced cell-death, 661W cells, a transformed mouse cone-cell line, were used. RESULTS: LacZ-positive cells were observed, and Hb-egf deletion was confirmed in the retinas of Hb-egf KO mice. Hb-egf and pro-HB-EGF levels were increased after light exposure in wild-type (WT) mice. Exposure to light reduced the a- and b-wave amplitudes of the dark-adapted ERG, and also ONL thickness, in Hb-egf KO mice versus WT mice. Treatment with HB-EGF improved both the a- and b-wave amplitudes, and the thickness of the ONL. The 661W cell-death induced by light irradiation was exacerbated by Hb-egf knockdown. HB-EGF also protected against light-induced cell-death and reduced ROS production in 661W cells. HB-EGF treatment improved the a-wave amplitudes, and the thickness of the ONL in Hb-egf KO mice. CONCLUSIONS: These data suggest that HB-EGF plays a pivotal role in light-induced photoreceptor degeneration. It therefore warrants investigation as a potential therapeutic target for such light-induced retinal diseases as age-related macular degeneration.
    Investigative ophthalmology & visual science 05/2013; · 3.43 Impact Factor
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    ABSTRACT: Endoplasmic reticulum (ER) stress is involved in various diseases such as ischemia, Alzheimer's disease, and Parkinson's disease. The widely used selective sigma-1 receptor antagonist, N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)-phenyl]-ethylamine monohydrochloride (NE-100), has been shown to suppress ischemia-induced neuronal cell death in the murine hippocampus. In the present study, we investigated whether NE-100 might suppress neuronal cell death that is induced by ER stress in ischemic injury. These studies show that NE-100 protected the ER stress-induced cell death of murine hippocampal HT22 cells, but not the oxidative stress-induced cell death. This suggests that NE-100 may have a protective effect on the ER. However, another sigma-1 receptor antagonist (BD1047) did not suppress ER stress-induced cell death. In addition, NE-100 attenuated the upregulation of C/EBP homologous protein (CHOP) induced by ER stress and upregulated the expression of both the 50-kDa activating transcription factor 6 (p50ATF6) and the 78-kDa glucose-regulated protein (GRP78). However, NE-100 did not impact the expression of phosphorylated eukaryotic initiation factor 2α (p-eIF2α) nor splicing of X-box-binding protein 1 (XBP-1). These findings suggest that NE-100 suppresses ER stress-induced cell death via CHOP expression by the upregulation of GRP78 through ATF6 pathway, independent sigma-1 receptor antagonist effect.
    Biochemical and Biophysical Research Communications 04/2013; · 2.41 Impact Factor

Publication Stats

2k Citations
548.38 Total Impact Points


  • 2004–2014
    • Gifu Pharmaceutical University
      • • Department of Biofunctional Evaluation
      • • Department of Biofunctional Molecules
      Gihu, Gifu, Japan
  • 2010
    • Kobe University
      • Laboratory of Molecular Pharmacology
      Kōbe, Hyōgo, Japan
    • Gifu University Hospital
      Gihu, Gifu, Japan
  • 2000–2008
    • Kyoto University
      • • Primate Research Institute
      • • Department of Pharmacology
      Kyoto, Kyoto-fu, Japan
  • 2004–2006
    • Santen Pharmaceutical Co., Ltd.
      Ōsaka, Ōsaka, Japan
  • 1997–2005
    • Hamamatsu University School of Medicine
      • Division of Pharmacology
      Hamamatu, Shizuoka, Japan
  • 1999
    • Osaka Medical College
      • Department of Ophthalmology
      Takatuki, Ōsaka, Japan