Masamitsu Shimazawa

Publications of Masamitsu Shimazawa

• Candesartan, an angiotensin II type 1 receptor antagonist, inhibits pathological retinal neovascularization by downregulating VEGF receptor-2 expression.

  Authors: Shinsuke Nakamura, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hideaki Hara

  European journal of pharmacology. 04/2012;

  Several studies have examined the anti-angiogenic effects of angiotensin II type 1 (AT(1)) receptor antagonists; however, the mechanisms underlying these effects are currently unclear. In the presentSeveral studies have examined the anti-angiogenic effects of angiotensin II type 1 (AT(1)) receptor antagonists; however, the mechanisms underlying these effects are currently unclear. In the present study, we examined the efficacy and the mechanism of candesartan, an AT(1) receptor antagonist, in suppressing pathological retinal neovascularization. We used an in vivo murine oxygen-induced retinopathy (OIR) model and also studied the in vitro proliferation and migration of human retinal microvascular endothelial cells (HRMECs) induced by vascular endothelial growth factor (VEGF)-A. The regulation of angiogenesis-associated genes such as hypoxia-inducible factor (HIF-1α), VEGF-A, VEGF receptor-1, and VEGF receptor-2 was evaluated with real-time RT-PCR in the OIR model. In the OIR model, candesartan suppressed the pathological neovascularization in a dose-dependent manner, but did not prevent the physiological angiogenesis. However, candesartan did not inhibit VEGF-A-induced proliferation or migration in HRMECs in the in vitro study. When administered interperitoneally in the OIR model, candesartan reduced the upregulation of VEGF receptor-2 in the retina, but had no effects in the other angiogenesis-related genes, such as HIF-1α, VEGF-A, and VEGF receptor-1. These findings indicate that candesartan inhibited the retinal pathological neovascularization, at least in part, by suppressing the expression of VEGF receptor-2, independent of VEGF signaling cascade. Therefore, candesartan may be a useful therapeutic target for the inhibition of retinal neovascularization that has a low risk of serious side effects.

• CROCETIN, A CAROTENOID DERIVATIVE, INHIBITS VEGF-INDUCED ANGIOGENESIS VIA SUPPRESSION OF P38 PHOSPHORYLATION.

  Authors: Naofumi Umigai, Junji Tanaka, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hideaki Hara

  Current neurovascular research. 04/2012;

  We evaluated the protective effects of crocetin against angiogenesis induced by vascular endothelial growth factor (VEGF). Crocetin, the aglycone of crocin carotenoids, is found in saffron crocusWe evaluated the protective effects of crocetin against angiogenesis induced by vascular endothelial growth factor (VEGF). Crocetin, the aglycone of crocin carotenoids, is found in saffron crocus (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). The effects of crocetin on VEGF-induced angiogenesis were examined by in vitro tube formation assays and following 14-day co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. The anti-angiogenic mechanism of crocetin was evaluated by examining its effects on VEGF-induced proliferation and migration of human retinal microvascular endothelial cells (HRMECs) and phosphorylation of p38. Vascular endothelial (VE)-cadherin, zonula occludens (ZO-1) and occludin, which are adherens and tight junction proteins, respectively, play a major role in the control of vascular permeability. Therefore, we tested effects of crocetin on adhesion molecule dissociation induced by VEGF. Crocetin significantly suppressed VEGF-induced tube formation by HUVECs and migration of HRMECs. It also significantly inhibited phosphorylation of p38 and protected VE-cadherin expression. These findings indicate that crocetin suppresses the VEGF-induced angiogenesis by inhibiting migration and that the inhibition of phosphorylated-p38 and protection of VE-cadherin expression may be involved in its underlying mechanism of action.

• Role of Oxidative Stress in Retinal Photoreceptor Cell Death in N-Methyl-N-nitrosourea-Treated Mice.

  Authors: Kazuhiro Tsuruma, Mika Yamauchi, Yuta Inokuchi, Sou Sugitani, Masamitsu Shimazawa, Hideaki Hara

  Journal of pharmacological sciences. 02/2012; 118(3):351-62.

  This study aimed to investigate whether oxidative stress contributes to retinal cell death in a mouse model of photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU). We measured in vitroThis study aimed to investigate whether oxidative stress contributes to retinal cell death in a mouse model of photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU). We measured in vitro MNU-induced radical production in retinal cell cultures of murine 661W photoreceptor-derived cells; RGC-5, a mouse ganglion cell line; and primary retinal cells. The addition of MNU induced oxidative radical generation in 661W and primary retinal cells, but not in RGC-5 cells. Edaravone, a free radical scavenger, at 1 µM reduced MNU-induced radical production in 661W and primary retinal cells. To induce in vivo retinal photoreceptor degeneration in mice, we administered 60 mg/kg MNU by intraperitoneal injection. We intravenously administered 1 mg/kg edaravone immediately and at 6 h after the MNU injection. Retinal photoreceptor degeneration was evaluated by measuring the thickness of the outer nuclear layer (ONL) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and by oxidative stress markers. MNU caused photoreceptor cell loss at 7 days after administration. Edaravone inhibited ONL thinning and reduced TUNEL-positive cells and the oxidative stress markers. These findings indicate that MNU leads to selective photoreceptor degradation via oxidative stress in vitro and in vivo and may help to understand the pathogenic mechanism of retinitis pigmentosa.

• Diacylglycerol Kinase β Knockout Mice Exhibit Attention-Deficit Behavior and an Abnormal Response on Methylphenidate-Induced Hyperactivity.

  Authors: Mitsue Ishisaka, Kenichi Kakefuda, Atsushi Oyagi, Yoko Ono, Kazuhiro Tsuruma, Masamitsu Shimazawa, Kiyoyuki Kitaichi, Hideaki Hara

  PloS one. 01/2012; 7(5):e37058.

  Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKβ is one of the subtypes of the DGK family and regulates many intracellular signalingDiacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKβ is one of the subtypes of the DGK family and regulates many intracellular signaling pathways in the central nervous system. Previously, we demonstrated that DGKβ knockout (KO) mice showed various dysfunctions of higher brain function, such as cognitive impairment (with lower spine density), hyperactivity, reduced anxiety, and careless behavior. In the present study, we conducted further tests on DGKβ KO mice in order to investigate the function of DGKβ in the central nervous system, especially in the pathophysiology of attention deficit hyperactivity disorder (ADHD). DGKβ KO mice showed attention-deficit behavior in the object-based attention test and it was ameliorated by methylphenidate (MPH, 30 mg/kg, i.p.). In the open field test, DGKβ KO mice displayed a decreased response to the locomotor stimulating effects of MPH (30 mg/kg, i.p.), but showed a similar response to an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.3 mg/kg, i.p.), when compared to WT mice. Examination of the phosphorylation of extracellular signal-regulated kinase (ERK), which is involved in regulation of locomotor activity, indicated that ERK1/2 activation induced by MPH treatment was defective in the striatum of DGKβ KO mice. These findings suggest that DGKβ KO mice showed attention-deficit and hyperactive phenotype, similar to ADHD. Furthermore, the hyporesponsiveness of DGKβ KO mice to MPH was due to dysregulation of ERK phosphorylation, and that DGKβ has a pivotal involvement in ERK regulation in the striatum.

• Morphological and functional changes in the retina after chronic oxygen-induced retinopathy.

  Authors: Shinsuke Nakamura, Shunsuke Imai, Hiromi Ogishima, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hideaki Hara

  PloS one. 01/2012; 7(2):e32167.

  The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tendsThe mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment.

• An alteration in the lateral geniculate nucleus of experimental glaucoma monkeys: in vivo positron emission tomography imaging of glial activation.

  Authors: Masamitsu Shimazawa, Yasushi Ito, Yuta Inokuchi, Hajime Yamanaka, Tomohiro Nakanishi, Takuya Hayashi, Bin Ji, Makoto Higuchi, Tetsuya Suhara, Kazuyuki Imamura, Makoto Araie, Yasuyoshi Watanabe, Hirotaka Onoe, Hideaki Hara

  PloS one. 01/2012; 7(1):e30526.

  We examined lateral geniculate nucleus (LGN) degeneration as an indicator for possible diagnosis of glaucoma in experimental glaucoma monkeys using positron emission tomography (PET). ChronicWe examined lateral geniculate nucleus (LGN) degeneration as an indicator for possible diagnosis of glaucoma in experimental glaucoma monkeys using positron emission tomography (PET). Chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the left eyes of 5 cynomolgus monkeys. Glial cell activation was detected by PET imaging with [(11)C]PK11195, a PET ligand for peripheral-type benzodiazepine receptor (PBR), before and at 4 weeks after laser treatment (moderate glaucoma stage). At mild, moderate, and advanced experimental glaucoma stages (classified by histological changes based on the extent of axonal loss), brains were stained with cresyl violet, or antibodies against PBR, Iba-1 (a microglial marker), and GFAP (an activated astrocyte marker). In laser-treated eyes, IOP was persistently elevated throughout all observation periods. PET imaging showed increased [(11)C]PK11195 binding potential in the bilateral LGN at 4 weeks after laser treatment; the increase in the ipsilateral LGN was statistically significant (P<0.05, n = 4). Immunostaining showed bilateral activations of microglia and astrocytes in LGN layers receiving input from the laser-treated eye. PBR-positive cells were observed in LGN layers receiving input from laser-treated eye at all experimental glaucoma stages including the mild glaucoma stage and their localization coincided with Iba-1 positive microglia and GFAP-positive astrocytes. These data suggest that glial activation occurs in the LGN at a mild glaucoma stage, and that the LGN degeneration could be detected by a PET imaging with [(11)C]PK11195 during the moderate experimental glaucoma stage after unilateral ocular hypertension. Therefore, activated glial markers such as PBR in the LGN may be useful in noninvasive molecular imaging for diagnosis of glaucoma.

• Protective Effects of Astaxanthin from Paracoccus carotinifaciens on Murine Gastric Ulcer Models.

  Authors: Kenta Murata, Atsushi Oyagi, Dai Takahira, Kazuhiro Tsuruma, Masamitsu Shimazawa, Takashi Ishibashi, Hideaki Hara

  Phytotherapy research : PTR. 12/2011;

  The purpose of this study was to investigate the effect of astaxanthin extracted from Paracoccus carotinifaciens on gastric mucosal damage in murine gastric ulcer models. Mice were pretreated withThe purpose of this study was to investigate the effect of astaxanthin extracted from Paracoccus carotinifaciens on gastric mucosal damage in murine gastric ulcer models. Mice were pretreated with astaxanthin for 1 h before ulcer induction. Gastric ulcers were induced in mice by oral administration of hydrochloride (HCl)/ethanol or acidified aspirin. The effect of astaxanthin on lipid peroxidation in murine stomach homogenates was also evaluated by measuring the level of thiobarbituric acid reactive substance (TBARS). The free radical scavenging activities of astaxanthin were also measured by electron spin resonance (ESR) measurements. Astaxanthin significantly decreased the extent of HCl/ethanol- and acidified aspirin-induced gastric ulcers. Astaxanthin also decreased the level of TBARS. The ESR measurement showed that astaxanthin had radical scavenging activities against the 1,1-diphenyl-2-picrylhydrazyl radical and the superoxide anion radical. These results suggest that astaxanthin has antioxidant properties and exerts a protective effect against ulcer formation in murine models. Copyright © 2011 John Wiley & Sons, Ltd.

• Increased expression of tight junctions in ARPE-19 cells under endoplasmic reticulum stress.

  Authors: Tadanobu Yoshikawa, Nahoko Ogata, Hiroshi Izuta, Masamitsu Shimazawa, Hideaki Hara, Kanji Takahashi

  Current eye research. 12/2011; 36(12):1153-63.

  To investigate the effects of endoplasmic reticulum (ER) stress on the tight junctions of the retinal pigment epithelial (RPE) cells in vitro. ER stress was induced in cultured ARPE-19 cells, a humanTo investigate the effects of endoplasmic reticulum (ER) stress on the tight junctions of the retinal pigment epithelial (RPE) cells in vitro. ER stress was induced in cultured ARPE-19 cells, a human RPE cell line, by exposure to tunicamycin (TM) or to thapsigargin (TG). After 6, 12, 24 and 48 hours of exposure, the expressions of GRP78/Bip (Bip), C/EBP-homologous protein (CHOP), vascular endothelial growth factor (VEGF), zonula occludens (ZO)-1, occludin and claudin-1 were determined by real-time RT-PCR. Immunoblot analysis and/or immunohistochemistry for proteins of tight junctions and ER stress markers, viz., Bip, activating transcription factor (ATF) 6, CHOP, and caspase-4, were performed at 48 hours after the exposure. Enzyme-linked immunosorbent assay was used to determine the concentration of VEGF165. Transepithelial electrical resistance (TER) of the ARPE-19 cells was determined to measure the permeability. The expressions of the mRNAs and/or proteins of Bip, CHOP, ATF6 and caspase-4 were significantly increased in ARPE-19 cells under ER stress induced by TM and TG. The mRNAs of VEGF were also increased by both TM and TG. However, the concentration of VEGF165 was not significantly increased after 48 hours exposure to TM and TG compared to that of the control in the apical chamber medium. The proteins and mRNAs of occludin and claudin-1 were significantly increased by TM and TG, and that of ZO-1 was significantly increased by TG. Immunohistochemistry showed that the staining of ZO-1, occludin and claudin-1 under ER stress was stronger than that of the control. A significant increase of TER was observed after exposure to TM and TG. The increased expressions of tight junction molecules by TM- or TG-exposed ARPE-19 cells indicate that ER stress can alter the function of RPE cells and may be involved in the pathogenesis of age-related macular degeneration.

• Ligation of the pterygopalatine and external carotid arteries induces ischemic damage in the murine retina.

  Authors: Hiromi Ogishima, Shinsuke Nakamura, Tomohiro Nakanishi, Shunsuke Imai, Mamoru Kakino, Fumiya Ishizuka, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hideaki Hara

  Investigative ophthalmology & visual science. 11/2011; 52(13):9710-20.

  This study aimed to characterize the functional and morphologic changes in a murine model of ocular ischemic disease caused by vascular occlusion. Retinal ischemia was induced by unilateral ligationThis study aimed to characterize the functional and morphologic changes in a murine model of ocular ischemic disease caused by vascular occlusion. Retinal ischemia was induced by unilateral ligation of the pterygopalatine artery (PPA) and the external carotid artery (ECA) in anesthetized mice. Changes in ocular blood flow and retinal circulation were evaluated by three different methods: laser speckle blood flow imaging, fundus imaging, and fluorescein isothiocyanate angiography. Five days after reperfusion following 3- or 5-hour ischemia, an electroretinogram (ERG) was recorded, and the retinal histology was examined and quantified. The effects of a free radical scavenger, edaravone, using the model were evaluated by ERG and histologic analysis. The ligation of both the PPA and the ECA significantly reduced ocular blood flow and narrowed the blood vessels. Five hours of ischemia reduced the a-wave, b-wave, and oscillatory potential amplitudes of the ERG. The number of cells in the ganglion cell layer and the thickness of both the inner plexiform layer and the inner nuclear layer were reduced in the ischemic group. Retinal ischemia caused an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the inner layer after 21-hour reperfusion following 3-hour ischemia and 19-hour reperfusion following 5-hour ischemia. Edaravone (1 mg/kg, administered intraperitoneally) significantly reduced the retinal ischemic damage. These findings indicate that the murine model in which both the PPA and the ECA are ligated may be useful to clarify the pathologic mechanisms of retinal ischemic diseases and to evaluate neuroprotective drugs that target retinal ischemic injury.

• Apoptosis-inducing factor and cyclophilin a cotranslocate to the motor neuronal nuclei in amyotrophic lateral sclerosis model mice.

  Authors: Hirotaka Tanaka, Hiroki Shimazaki, Masataka Kimura, Hiroshi Izuta, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hideaki Hara

  CNS neuroscience & therapeutics. 10/2011; 17(5):294-304.

  Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease whose mechanism is not understood. Recently, it was reported that apoptosis-inducing factor (AIF) was involved in motor neuronalAmyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease whose mechanism is not understood. Recently, it was reported that apoptosis-inducing factor (AIF) was involved in motor neuronal cell death in ALS model mice, and AIF-induced neuronal cell death by interacting with cyclophilin A (CypA). However, it is unknown whether the CypA and AIF-complex induces chromatinolysis in ALS. Therefore, in the present study, we investigated the process of motor neuron degeneration as the disease progresses and to determine whether the CypA-AIF complex would play a role in inducing motor neuronal cell death in mutant superoxide dismutase 1 (SOD1)(G93A) ALS model mice. We prepared the nuclear fractions of spinal cords and demonstrated the nuclear translocation of CypA with AIF in SOD1(G93A) mice by immunoprecipitation. The localization of CypA and AIF in the spinal cords was assessed by immunohistochemistry. In the spinal cords of SOD1(G93A) mice, the expressions of CypA and AIF were detected in the motor neurons, and CypA and AIF cotranslocated to the motor neuronal nuclei with CypA. Furthermore, the expression of CypA was detected in GFAP-positive astrocytes, but not in CD11b-positive microglial cells. On the other hand, these findings were not detected in the spinal cords of wild-type mice. From these results, we suggest that CypA and AIF may play cooperative and pivotal roles in motor neuronal death in the murine ALS model.

• Heparin-binding EGF-like growth factor is required for synaptic plasticity and memory formation.

  Authors: Atsushi Oyagi, Shigeki Moriguchi, Atsumi Nitta, Kenta Murata, Yasuhisa Oida, Kazuhiro Tsuruma, Masamitsu Shimazawa, Kohji Fukunaga, Hideaki Hara

  Brain research. 09/2011; 1419:97-104.

  Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of epidermal growth factor (EGF) family, is a potent mitogenic peptide for various types of cells. HB-EGF is widelyHeparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of epidermal growth factor (EGF) family, is a potent mitogenic peptide for various types of cells. HB-EGF is widely expressed in central nervous system, including hippocampus and cerebral cortex, and is considered to play pivotal roles in the developing and adult nervous system. In this study, we assessed the role of HB-EGF in learning and memory by testing HB-EGF conditional knock-out mice (KO) in two different learning tasks, and evaluated the long-term potentiation (LTP) in hippocampus slices from these mice. The HB-EGF KO mice were impaired in spatial memory in the Morris water maze and in fear learning in a passive avoidance test. HB-EGF KO mice also showed an impaired LTP, and reduction in activity of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and phosphorylated GluR1. We also found that the levels of neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or glial cell line-derived neurotrophic factor (GDNF), were altered in several brain regions in the HB-EGF KO mice. These results confirm the importance of the HB-EGF in synaptic plasticity and memory formation.

• Central Changes in Glaucoma: Neuroscientific Study Using Animal Models

  Authors: Kazuyuki Imamura, Masamitsu Shimazawa, Hirotaka Onoe, Yasuyoshi Watanabe, Kiyoshi Ishii, Chihiro Mayama, Takafumi Akasaki, Satoshi Shimegi, Hiromichi Sato, Kazuhiko Nakadate, Hideaki Hara, Makoto Araie

  09/2011;

  ISBN: 978-953-307-567-9

• Edaravone-loaded liposome eyedrops protect against light-induced retinal damage in mice.

  Authors: Hiroki Shimazaki, Kohei Hironaka, Takuya Fujisawa, Kazuhiro Tsuruma, Yuichi Tozuka, Masamitsu Shimazawa, Hirofumi Takeuchi, Hideaki Hara

  Investigative ophthalmology & visual science. 08/2011; 52(10):7289-97.

  To investigate the pharmacologic effects of eyedrops containing liposomes loaded with edaravone (3-methyl-1-phenyl-2-pyrazolin-5-1) against light-induced retinal damage in mice. Edaravone wasTo investigate the pharmacologic effects of eyedrops containing liposomes loaded with edaravone (3-methyl-1-phenyl-2-pyrazolin-5-1) against light-induced retinal damage in mice. Edaravone was incorporated into submicron-sized liposomes (ssLips) by the calcium acetate gradient method. Retinal damage in mice was induced in dark-adapted mice by exposure to white light at 8000 lux for 3 hours. Edaravone-loaded ssLips were dropped into the left eye just before and after light exposure and then three times daily for 5 days after light exposure. Retinal damage was evaluated by recording the scotopic electroretinogram (ERG) and measuring the thickness of the outer nuclear layer (ONL) and by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. The scavenging capacity of reactive oxygen species (ROS) of edaravone-loaded ssLips was determined using a murine cone photoreceptor cell line (661W). The human corneal and conjunctival cell lines were exposed to edaravone-loaded ssLips to determine cytotoxicity. Eyedrop administration of edaravone-loaded ssLips significantly prevented both the decrease in a- and b-wave amplitudes of flash ERG and the shrinkage of the ONL compared with the control group (treated with empty ssLips) after 5 days of light exposure. The edaravone-loaded ssLips prevented the increase in the numbers of TUNEL-positive cells after 48 hours of light exposure. This marked protection was not found in the group treated with free edaravone. Edaravone-loaded ssLips showed a stronger inhibition of in vitro light-induced ROS production and cell death than did free edaravone. The ssLips showed little cytotoxicity toward ocular cell lines. Edaravone-loaded ssLips protected against light-induced retinal dysfunction by eyedrop administration. Liposomal eyedrops may become one of the therapeutic candidates for drug delivery to posterior eye segments.

• Involvement of Bid and caspase-2 in endoplasmic reticulum stress- and oxidative stress-induced retinal ganglion cell death.

  Authors: Rumi Uchibayashi, Kazuhiro Tsuruma, Yuta Inokuchi, Masamitsu Shimazawa, Hideaki Hara

  Journal of neuroscience research. 07/2011; 89(11):1783-94.

  Endoplasmic reticulum (ER) stress and oxidative stress are involved in many diseases, including retinal disorders, causing toxicity in various tissues and cells; however, intracellular signaling ofEndoplasmic reticulum (ER) stress and oxidative stress are involved in many diseases, including retinal disorders, causing toxicity in various tissues and cells; however, intracellular signaling of ER stress and cross-talk between ER stress and oxidative stress are unknown in retinal ganglion cells (RGC), whose degeneration is associated with glaucoma. The aim of the study was to clarify the mechanisms of ER stress- and oxidative stress-induced RGC death, using cultured retinal ganglion cells (RGC-5) in vitro and N-methyl-D-aspartate (NMDA)- or ER stress-induced retinal damage in mice in vivo. We focused on both BH3-interacting domain death agonist (Bid) and caspase-2, which work as apoptosis promotion factors. In an in vitro study, both Bid and caspase-2 inhibitors protected against RGC-5 death from ER stress or oxidative stress. A caspase-2 inhibitor did not inhibit Bid cleavage, although a Bid inhibitor reduced the increase of caspase-2 activity in ER stress-induced RGC-5 death. A Bid inhibitor also reduced the increase of caspase-2 activity in oxidative stress-induced RGC-5 death. Moreover, both Bid and caspase-2 inhibitors reduced the increase of caspase-3 activity. In an in vivo study, a Bid inhibitor inhibited NMDA- or ER stress-induced retinal damage. These findings indicate that a common mechanism through Bid and caspase-2 exists in both ER stress- and oxidative stress-induced RGC death and that they are activated in the order of Bid, caspase-2, and caspase-3.

• Fasudil and ozagrel in combination show neuroprotective effects on cerebral infarction after murine middle cerebral artery occlusion.

  Authors: Akihiro Koumura, Junya Hamanaka, Koh Kawasaki, Kazuhiro Tsuruma, Masamitsu Shimazawa, Isao Hozumi, Takashi Inuzuka, Hideaki Hara

  The Journal of pharmacology and experimental therapeutics. 07/2011; 338(1):337-44.

  Rho kinase (ROCK), one of the serine/threonine kinases, is involved in pathologic conditions, and its activation causes neuronal cell death. Fasudil, a selective ROCK inhibitor, has been reported toRho kinase (ROCK), one of the serine/threonine kinases, is involved in pathologic conditions, and its activation causes neuronal cell death. Fasudil, a selective ROCK inhibitor, has been reported to cause increased cerebral blood flow (CBF) in the ischemic brain and protect against neuronal cell death by inhibiting ROCK. Ozagrel, a thromboxane A(2) synthase inhibitor, inhibits platelet aggregation and causes vasodilatation, thereby increasing CBF in cerebral thrombosis. The present study evaluates the combination therapy of fasudil and ozagrel on focal brain ischemia induced by middle cerebral artery occlusion (MCAO) in mice. Each monotherapy of fasudil at 10 mg/kg i.p. and ozagrel at 30 mg/kg i.p. significantly reduced cerebral infarction. The combination therapy of fasudil (3 mg/kg i.p.) and ozagrel (10 mg/kg i.p.), which are noneffective doses, resulted in reduction of cerebral infarction, and the protective effect was observed up to 5 min, but not 3 h, after reperfusion. Regional CBF after MCAO and phosphorylation of endothelial nitric-oxide synthase (NOS) significantly increased in response to the combination therapy, whereas these effects were not observed with monotherapy of either drug. The protective effect of combination treatment was antagonized by the treatment of a NOS inhibitor, nitro-l-arginine methyl ester hydrochloride. These findings indicate that the combination treatment of fasudil and ozagrel exhibits additive effects for neuroprotection after MCAO. These findings indicate that the combination treatment of fasudil and ozagrel may be useful as a potential therapeutic strategy for the treatment of stroke.

• Purple rice (Oryza sativa L.) extract and its constituents inhibit VEGF-induced angiogenesis.

  Authors: Junji Tanaka, Shinsuke Nakamura, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hiroshi Shimoda, Hideaki Hara

  Phytotherapy research : PTR. 06/2011; 26(2):214-22.

  The study evaluated the protective effects of purple rice (Oryza sativa L.) bran extract (PRE) and its constituents, cyanidin and peonidin, against angiogenesis induced by vascular endothelial growthThe study evaluated the protective effects of purple rice (Oryza sativa L.) bran extract (PRE) and its constituents, cyanidin and peonidin, against angiogenesis induced by vascular endothelial growth factor (VEGF). The effects of VEGF and PRE were examined by in vitro tube formation assays and following 14-day co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. The antiangiogenic mechanism of PRE was evaluated by VEGF-induced proliferation and migration of HUVECs and/or human retinal microvascular endothelial cells (HRMECs) and phosphorylation of extracellular signal-regulated kinase (ERK) and p38. The PRE significantly suppressed VEGF-induced tube formation, proliferation and migration in HUVECs and HRMECs as well as phosphorylation of ERK and p38. Cyanidin and peonidin also suppressed the proliferation and migration induced by VEGF. These findings indicate that PRE and anthocyanidins suppress VEGF-induced angiogenesis by inhibiting proliferation and migration and suggest that the inhibition of phosphorylated-ERK and -p38 may be involved in the underlying mechanism.

• Novel situations of endothelial injury in stroke--mechanisms of stroke and strategy of drug development: protective effects of antiplatelet agents against stroke.

  Authors: Masamitsu Shimazawa, Hideaki Hara

  Journal of pharmacological sciences. 05/2011; 116(1):30-5.

  Stroke is the second cause of mortality worldwide, and intravenous administration of tissue plasminogen activator (t-PA) within 3 h of symptom onset is the only treatment proven effective forStroke is the second cause of mortality worldwide, and intravenous administration of tissue plasminogen activator (t-PA) within 3 h of symptom onset is the only treatment proven effective for re-establishment of cerebral blood flow following acute ischemic stroke. However, its widespread application remains limited by its narrow therapeutic time window and the related risks of intracranial hemorrhage. On the other hand, in patients with atherothrombotic risk, antiplatelet agents are widely used to decrease the risk of occlusive arterial events. All of these drugs are used during coronary interventions and in the medical management of acute coronary syndromes. In contrast, only aspirin, cilostazol, and thienopyridine derivatives (ticlopidine and clopidogrel) are used in the long-term prevention of cerebrovascular events in patients with risk of recurrence. In this paper, we introduce recent clinical findings on antiplatelet therapies for secondary prevention after ischemic stroke and describe basic research that has focused on cerebrovascular protection by cilostazol, which has a unique pharmacological profile.

• Blockade of phosphodiesterase-III protects against oxygen-glucose deprivation in endothelial cells by upregulation of VE-cadherin.

  Authors: Mitsunori Ishiguro, Yukiya Suzuki, Keisuke Mishiro, Mamoru Kakino, Kazuhiro Tsuruma, Masamitsu Shimazawa, Shinichi Yoshimura, Toru Iwama, Hideaki Hara

  Current neurovascular research. 03/2011; 8(2):86-94.

  We recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activatorWe recently reported that a phosphodiesterase-III inhibitor, cilostazol, prevented the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tissue plasminogen activator (tPA) and that it reversed tPA-induced cell damage by protecting the neurovascular unit, particularly endothelial cells. However, the mechanisms of cilostazol action are still not clearly defined. The adheren junction (AJ) protein, VE-cadherin, is a known mediator of endothelial barrier sealing and maintenance. Therefore, we tested whether cilostazol might promote expression of adhesion molecules in endothelial cells, thereby preventing deterioration of endothelial barrier functions. Human brain microvascular endothelial cells were exposed to 6-h oxygen-glucose deprivation (OGD). We compared cilostazol with aspirin treatments and examined 2 representative AJ proteins: VE-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1). A protein kinase A (PKA) inhibitor, LY294002 (a PI3-K inhibitor), db-cAMP, and RP-cAMPS were used to assess the roles of cAMP, PKA, and PI3-K signaling, respectively, in cilostazol-induced responses. Cilostazol and db-cAMP prevented OGD-stress injury in endothelial cells by promoting VE-cadherin expression, but not PECAM-1. Aspirin did not prevent cell damage. P13-K inhibition by LY294002 had no influence on the effects of cilostazol, but inhibition of cAMP/PKA with PKA inhibitor and Rp-cAMPS suppressed cilostazol-induced inhibition of cell damage and promotion of VE-cadherin expression. In contrast, OGD stress had no detectable effects on VEGF, VEGF receptor, or angiopoietin-1 levels. Cilostazol promotes VE-cadherin expression through cAMP/PKA-dependent pathways in brain endothelial cells; thus, cilostazol effects on adhesion molecule signaling may provide protection against OGD stress in endothelial cells.

• An arylidene-thiazolidinedione derivative, GPU-4, without PPARγ activation, reduces retinal neovascularization.

  Authors: Shinsuke Nakamura, Kei Hayashi, Haruka Takizawa, Tetsuji Murase, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hiroki Kakuta, Hideko Nagasawa, Hideaki Hara

  Current neurovascular research. 02/2011; 8(1):25-34.

  Retinal angiogenesis is a leading cause of blindness, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Vascular endothelial growth factor (VEGF) isRetinal angiogenesis is a leading cause of blindness, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. Vascular endothelial growth factor (VEGF) is one of the major angiogenesis factors, and induces endothelial cell proliferation and migration. VEGF stimulates NADPH oxidase to produce reactive oxygen species (ROS), and ROS induce the transcription factors and genes involved in angiogenesis. In the present study, we demonstrated that GPU-4, 5-arylidene-2,4-thiazolidinedione derivative, demonstrates anti-angiogenic activity regarding human retinal microvascular endothelial cells (HRMECs) and retinal neovascularization in a mouse model of retinopathy of prematurity. GPU-4 inhibited the VEGF-induced radicals, proliferation, and migration in HRMECs without a PPARγ-mediated effect. Furthermore, systemic administration of GPU-4 inhibited the development of retinal neovascularization in a murine oxygen-induced retinopathy model but did not exert revascularization of the capillary-free area, which shows normal physiological revascularization. These findings indicate that GPU-4 suppressed in vitro and in vivo retinal neovascularization partly by a radical scavenging effect, suggesting that GPU-4 might be a potential therapeutic agent candidate for proliferative diseases of the retinal vasculature.

• Tissue kallikrein inhibits retinal neovascularization via the cleavage of vascular endothelial growth factor-165.

  Authors: Shinsuke Nakamura, Nobutaka Morimoto, Kazuhiro Tsuruma, Hiroshi Izuta, Yoshika Yasuda, Noriaki Kato, Tsunehiko Ikeda, Masamitsu Shimazawa, Hideaki Hara

  Arteriosclerosis, thrombosis, and vascular biology. 02/2011; 31(5):1041-8.

  Tissue kallikrein, a widely used vasodilator for the treatment of hypertension and peripheral circulatory disorder, acts by releasing kinin, a potent vasodilator peptide. To identify the role ofTissue kallikrein, a widely used vasodilator for the treatment of hypertension and peripheral circulatory disorder, acts by releasing kinin, a potent vasodilator peptide. To identify the role of tissue kallikrein in retinal neovascularization, we investigated the antiangiogenic effect by using an in vitro and in vivo angiogenesis model. Tissue kallikrein in vitreous fluid was markedly elevated in proliferative diabetic retinopathy patients compared with that in control patients with macular hole and epiretinal membrane. Tissue kallikrein inhibited vascular endothelial growth factor-165 (VEGF165)-induced tube formation, proliferation, and migration in vitro angiogenesis model via suppression of the VEGF165-induced phosphorylation of VEGF receptor-2. Furthermore, tissue kallikrein cleavage of VEGF165 was on the C-terminal side, which was analyzed by Western blotting and mass spectrometry. When administered subcutaneously, tissue kallikrein reduced the pathological vascular changes in retinal neovascularization induced in neonatal mice by returning the retina to normoxia after exposure to hyperoxia. These findings indicate that tissue kallikrein is partly involved in pathogenesis of proliferative diabetic retinopathy and may be a promising therapeutic agent that could cleave VEGF165 itself when administered by a peripheral route.