Details of a Researcher - Kurihara, Toshihide

Papers - Kurihara, Toshihide

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Hydrogen and N-Acetyl-L-Cysteine Rescue Oxidative Stress-Induced Angiogenesis in a Mouse Corneal Alkali-Burn Model

Kubota, Miyuki, Shimmura, Shigeto, Kubota, Shunsuke, Miyashita, Hideyuki, Kato, Naoko, Noda, Kousuke, Ozawa, Yoko, Usui, Tomohiko, Ishida, Susumu, Umezawa, Kazuo, Kurihara, Toshihide, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 52 ( 1 ) 427 - 433 2011.01

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate the role of reactive oxygen species (ROS) as the prime initiators of the angiogenic response after alkali injury of the cornea and observe the effects of antioxidants in preventing angiogenesis.
METHODS. The corneal epithelia of SOD-1-deficient mice or wild-type (WT) mice were removed after application of 0.15 N NaOH to establish the animal model of alkali burn. ROS production was semiquantitatively measured by dihydroethidium (DHE) fluorescence. Angiogenesis was visualized by CD31 immunohistochemistry. The effects of the specific NF-kappa B inhibitor DHMEQ, the antioxidant N-acetyl-L-cysteine (NAC), and hydrogen (H(2)) solution were observed.
RESULTS. ROS production in the cornea was enhanced immediately after alkali injury, as shown by increased DHE fluorescence (P < 0.01). NF-kappa B activation and the upregulation of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) were significantly enhanced (P < 0.01), leading to a significantly larger area of angiogenesis. Angiogenesis in SOD-1(-/-) mice corneas were significantly higher in WT mice (P < 0.01), confirming the role of ROS. Pretreatment with the specific NF-kappa B inhibitor DHMEQ or the antioxidant NAC significantly reduced corneal angiogenesis by downregulating the NF-kappa B pathway (P < 0.01) in both WT and SOD-1(-/-) mice. Furthermore, we showed that irrigation of the cornea with hydrogen (H 2) solution significantly reduced angiogenesis after alkali-burn injury (P < 0.01).
CONCLUSIONS. Immediate antioxidant therapy with H(2)-enriched irrigation solution is a new potent treatment of angiogenesis in cornea to prevent blindness caused by alkali burn. (Invest Ophthalmol Vis Sci. 2011;52:427-433) DOI:10.1167/iovs.106167

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Regulation of Posttranscriptional Modification as a Possible Therapeutic Approach for Retinal Neuroprotection

Ozawa, Yoko, Kurihara, Toshihide, Tsubota, Kazuo, Okano, Hideyuki

JOURNAL OF OPHTHALMOLOGY (HINDAWI LTD) 2011 663 - 665 2011

Research paper (scientific journal), Accepted, ISSN 2090-004X

Understanding pathogenesis at the molecular level is the first step toward developing new therapeutic approaches. Here, we review the molecular mechanisms of visual dysfunction in two common diseases, innate chorioretinal inflammation and diabetic retinopathy, and the role of the ubiquitin-proteasome system (UPS) in both processes. In innate chorioretinal inflammation, interleukin-6 family ligands induce STAT3 activation in photoreceptors, which causes UPS-mediated excessive degradation of the visual substance, rhodopsin. In diabetic retinopathy, angiotensin II type 1 receptor (AT1R) signaling activates ERK in the inner layers of the retina, causing UPS-mediated excessive degradation of the synaptic vesicle protein, synaptophysin. This latter effect may decrease synaptic activity, in turn adversely affecting neuronal survival. Both mechanisms involve increased UPS activity and the subsequent excessive degradation of a protein required for visual function. Finally, we review the therapeutic potential of regulating the UPS to protect tissue function, citing examples from clinical applications in other medical fields.

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Neural Degeneration in the Retina of the Streptozotocin-Induced Type 1 Diabetes Model

Ozawa, Yoko, Kurihara, Toshihide, Sasaki, Mariko, Ban, Norimitsu, Yuki, Kenya, Kubota, Shunsuke, Tsubota, Kazuo

EXPERIMENTAL DIABETES RESEARCH (HINDAWI PUBLISHING CORPORATION) 2011 1433 - 1439 2011

Research paper (scientific journal), Accepted, ISSN 1687-5214

Diabetic retinopathy, a vision-threatening disease, has been regarded as a vascular disorder. However, impaired oscillatory potentials (OPs) in the electroretinogram (ERG) and visual dysfunction are recorded before severe vascular lesions appear. Here, we review the molecular mechanisms underlying the retinal neural degeneration observed in the streptozotocin-(STZ-) induced type 1 diabetes model. The renin-angiotensin system (RAS) and reactive oxygen species (ROS) both cause OP impairment and reduced levels of synaptophysin, a synaptic vesicle protein for neurotransmitter release, most likely through excessive protein degradation by the ubiquitin-proteasome system. ROS also decrease brain-derived neurotrophic factor (BDNF) and inner retinal neuronal cells. The influence of both RAS and ROS on synaptophysin suggests that RAS-ROS crosstalk occurs in the diabetic retina. Therefore, suppressors of RAS or ROS, such as angiotensin II type 1 receptor blockers or the antioxidant lutein, respectively, are potential candidates for neuroprotective and preventive therapies to improve the visual prognosis.

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Resveratrol Prevents Light-Induced Retinal Degeneration via Suppressing Activator Protein-1 Activation

Kubota, Shunsuke, Kurihara, Toshihide, Ebinuma, Mari, Kubota, Miyuki, Yuki, Kenya, Sasaki, Mariko, Noda, Kousuke, Ozawa, Yoko, Oike, Yuichi, Ishida, Susumu, Tsubota, Kazuo

AMERICAN JOURNAL OF PATHOLOGY (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 177 ( 4 ) 1725 - 1731 2010.10

Research paper (scientific journal), Accepted, ISSN 0002-9440

Light damage to the retina accelerates retinal degeneration in human diseases and rodent models. Recently, the polyphenolic phytoalexin resveratrol has been shown to exert various bioactivities in addition to its classical antioxidant property. In the present study, we investigated the effect of resveratrol on light-induced retinal degeneration together with its underlying molecular mechanisms. BALB/c mice with light exposure (5000-lux white light for 3 hours) were orally pretreated with resveratrol at a dose of 50 mg/kg for 5 days. Retinal damage was evaluated by TdT-mediated dUTP nick-end labeling, outer nuclear layer morphometry, and electroretinography. Administration of resveratrol to mice with light exposure led to a significant suppression of light-induced pathological parameters, including TdT-mediated dUTP nick-end labeling-positive retinal cells, outer nuclear layer thinning, and electroretinography changes. To clarify the underlying molecular mechanisms, the nuclear translocation of activator protein-1 subunit c-fos was evaluated by enzyme-linked immunosorbent assay, and the retinal activity of sirtuin 1 was measured by deacetylase fluorometric assay. Retinal activator protein-1 activation, up-regulated following light exposure, was significantly reduced by application of resveratrol. In parallel, retinal sirtuin 1 activity, reduced in animals with light damage, was significantly augmented by resveratrol treatment. Our data suggest the potential use of resveratrol as a therapeutic agent to prevent retinal degeneration related to light damage. (Am J Pathol 2010, 1 77:1725-1731; DOI: 10.2353/ajpath.2010.100098)

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Astrocyte Hypoxic Response Is Essential for Pathological But Not Developmental Angiogenesis of the Retina

Weidemann, Alexander, Krohne, Tim U., Aguilar, Edith, Kurihara, Toshihide, Takeda, Norihiko, Dorrell, Michael I., Simon, M. Celeste, Haase, Volker H., Friedlander, Martin, Johnson, Randall S.

GLIA (WILEY-LISS) 58 ( 10 ) 1177 - 1185 2010.08

Research paper (scientific journal), Accepted, ISSN 0894-1491

Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type-specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia-regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell-specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia-inducible transcription factors HIF-1 alpha and HIF-2 alpha, and the negative regulator of HIF alpha, von Hippel Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte-derived VEGF is not essential for this process. In contrast, using a model of oxygen-induced ischemic retinopathy, we show that astrocyte-derived VEGF is essential for hypoxia-induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis, and ischemia-driven, pathological neovascularization. (C) 2010 Wiley-Liss, Inc.

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von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina

Kurihara, Toshihide, Kubota, Yoshiaki, Ozawa, Yoko, Takubo, Keiyo, Noda, Kousuke, Simon, M. Celeste, Johnson, Randall S., Suematsu, Makoto, Tsubota, Kazuo, Ishida, Susumu, Goda, Nobuhito, Suda, Toshio, Okano, Hideyuki

DEVELOPMENT (COMPANY OF BIOLOGISTS LTD) 137 ( 9 ) 1563 - 1571 2010.05

Research paper (scientific journal), Accepted, ISSN 0950-1991

In early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. This process is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. In this study, we seek other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the point of birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor alpha s (HIF-alpha s) under normoxic, but not hypoxic, conditions. To examine the role of oxygen-sensing mechanisms in retinal circulatory system transition, we generated retina-specific conditional-knockout mice for VHL (Vhl(alpha-CreKO) mice). These mice exhibit arrested transition from the fetal to the adult circulatory system, persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelial growth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1 alpha gene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the fetal to the adult circulatory system in the retina.

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Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes.

Sasaki M, Ozawa Y†, Kurihara T, Kubota S, Yuki K, Noda K, Kobayashi S, Ishida S, Tsubota K

Diabetologia. (SPRINGER) 53 ( 5 ) 971 - 979 2010.05

Accepted, ISSN 0012-186X

Diabetic retinopathy is a progressive neurodegenerative disease, but the underlying mechanism is still obscure. Here, we focused on oxidative stress in the retina, and analysed its influence on retinal neurodegeneration, using an antioxidant, lutein.
C57BL/6 mice with streptozotocin-induced diabetes were constantly fed either a lutein-supplemented diet or a control diet from the onset of diabetes, and their metabolic data were recorded. In 1-month-diabetic mice, reactive oxygen species (ROS) in the retina were measured using dihydroethidium and visual function was evaluated by electroretinograms. Levels of activated extracellular signal-regulated kinase (ERK), synaptophysin and brain-derived neurotrophic factor (BDNF) were also measured by immunoblotting in the retina of 1-month-diabetic mice. In the retinal sections of 4-month-diabetic mice, histological changes, cleaved caspase-3 and TUNEL staining were analysed.
Lutein did not affect the metabolic status of the diabetic mice, but it prevented ROS generation in the retina and the visual impairment induced by diabetes. ERK activation, the subsequent synaptophysin reduction, and the BDNF depletion in the diabetic retina were all prevented by lutein. Later, in 4-month-diabetic mice, a decrease in the thickness of the inner plexiform and nuclear layers, and ganglion cell number, together with increase in cleaved caspase-3- and TUNEL-positive cells, were avoided in the retina of lutein-fed mice.
The results indicated that local oxidative stress that has a neurodegenerative influence in the diabetic retina is prevented by constant intake of a lutein-supplemented diet. The antioxidant, lutein may be a potential therapeutic approach to protect visual function in diabetes.

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Retinal phototoxicity in a novel murine model of intraocular lens implantation

Kurihara, Toshihide, Omoto, Masahiro, Noda, Kousuke, Ebinuma, Mari, Kubota, Shunsuke, Koizumi, Haruna, Yoshida, Satoru, Ozawa, Yoko, Shimmura, Shigeto, Ishida, Susumu, Tsubota, Kazuo

MOLECULAR VISION (MOLECULAR VISION) 15 ( 290 ) 323 - 329 2009.12

Research paper (scientific journal), Accepted, ISSN 1090-0535

Purpose: To establish a novel murine intraocular lens (IOL) implantation model to study the protective effects of colored-IOLs against retinal phototoxicity.
Methods: Two-millimeter diameter IOL buttons were created from IOLs for clinical use. Extra-capsular crystalline lens extraction and IOL implantation were performed in BALB/c mice using a technique similar to human cataract surgery. For light exposure experiments, mice were exposed to 5,000 LUX of white light for 24 h on the day after surgery. To investigate the protective effects of yellow IOL against light exposure, ERG measurements were conducted in vivo, followed by TdT-mediated dUTP Nick-End Labeling (TUNEL) and outer nuclear layer (ONL) thickness measurement of retinal tissue in yellow or clear IOL-implanted mice and control mice without surgery.
Results: IOLs were successfully implanted in all animals, and IOL buttons without haptics were well stabilized in the capsular bag. Murine eyes developed posterior capsule opacification (PCO) after IOL implantation by postoperative day 5 at the latest. In contrast to the clear IOL-implanted animals stimulated by light exposure, the yellow IOL-implanted animals had significantly reduced numbers of TUNEL-positive cells and retained thickness of the ONL. The ERG showed that yellow IOL implantation prevents a decrease of amplitude in both the a-wave and b-wave compared with clear IOL implantation.
Conclusions: We established a new animal model of IOL implantation and demonstrated the protective effects of colored-IOL against retinal phototoxicity after cataract surgery.

Bilateral acute functional disturbance in the retina following placental abruption

Kurihara, Toshihide, Ohde, Hisao, Inoue, Makoto, Ogata, Masaro, Shinoda, Kei, Tsubota, Kazuo

JAPANESE JOURNAL OF OPHTHALMOLOGY (SPRINGER TOKYO) 53 ( 6 ) 663 - 665 2009.11

Research paper (scientific journal), Accepted, ISSN 0021-5155

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Involvement of Hyaluronan and Its Receptor CD44 with Choroidal Neovascularization

Mochimaru, Hiroshi, Takahashi, Eri, Tsukamoto, Nobuo, Miyazaki, Junichiro, Yaguchi, Tomonori, Koto, Takashi, Kurihara, Toshihide, Noda, Kousuke, Ozawa, Yoko, Ishimoto, Takatsugu, Kawakami, Yutaka, Tanihara, Hidenobu, Saya, Hideyuki, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 50 ( 9 ) 4410 - 4415 2009.09

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. CD44 is a cell-surface adhesion molecule and receptor for hyaluronan (HA), one of the major extracellular matrix components. The purpose of the present study was to clarify a role of HA and CD44 in the development of choroidal neovascularization (CNV).
METHODS. Laser photocoagulation was used to induce CNV in C57BL/6 mice or CD44-deficient mice. The mRNA expression of CD44 and HA synthase (HAS)-2 in the retinal pigment epithelium (RPE)-choroid complex was evaluated by DNA microarray and real-time RT-PCR analyses 3 days after laser treatment. HA synthesis and CD44 expression were examined by immunohistochemistry 1 week after photocoagulation. Mice with laser-induced CNV were systemically administered the HA synthesis inhibitor 4-methylumbelliferone (MU) or an antiCD44-neutralizing antibody. The response of CNV was analyzed by volumetric measurements 1 week after photocoagulation. Macrophage infiltration into CNV lesions was evaluated by real-time RT-PCR for F4/80 3 days after laser-induced injury.
RESULTS. The induction of CNV led to a significant increase in expression of CD44 and HAS2 mRNA. HA and CD44 were immunopositive in the CNV lesions. Compared with vehicle treatment, the systemic application of MU significantly attenuated CNV volume in a dose-dependent fashion, together with macrophage infiltration into the lesions. Consistently, antibody-based blockade of CD44 resulted in a significant reduction of CNV volume, compared with the isotype control. In contrast, genetic ablation of CD44 significantly augmented CNV formation together with HA accumulation and macrophage infiltration, compared with wild-type mice.
CONCLUSIONS. These results indicate a significant role of HA and its receptor CD44 in the development of CNV. (Invest Ophthalmol Vis Sci. 2009;50:4410-4415) DOI: 10.1167/iovs.08-3044

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Prevention of Ocular Inflammation in Endotoxin-Induced Uveitis with Resveratrol by Inhibiting Oxidative Damage and Nuclear Factor-kappa B Activation

Kubota, Shunsuke, Kurihara, Toshihide, Mochimaru, Hiroshi, Satofuka, Shingo, Noda, Kousuke, Ozawa, Yoko, Oike, Yuichi, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 50 ( 7 ) 3512 - 3519 2009.07

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. Resveratrol is known as one of the antioxidant polyphenols contained in red wine and grape skin. The purpose of the present study was to investigate the role of resveratrol in ocular inflammation in endotoxin-induced uveitis (EIU).
METHODS. EIU was induced in male C57/B6 mice at the age of 6 weeks by a single intraperitoneal injection of lipopolysaccharide (LPS). Animals had received oral supplementation of resveratrol at the doses of 5, 50, 100, or 200 mg/kg for 5 days until LPS injection. Twenty-four hours after LPS administration, leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique. Retinal and retinal pigment epithelium (RPE)-choroidal levels of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear translocation of nuclear factor (NF)-kappa B p65 were evaluated by enzyme-linked immunosorbent assay. Retinal and RPE-choroidal activities of silent information regulator two ortholog (SIRT) 1 were measured by deacetylase fluorometric assay.
RESULTS. Resveratrol pretreatment led to significant and dose-dependent suppression of leukocyte adhesion to retinal vessels of EIU mice compared with vehicle application. Protein levels of MCP-1 and ICAM-1 in the retina and the RPE-choroid of EIU animals were significantly reduced by resveratrol administration. Importantly, resveratrol-treated animals showed significant decline of retinal 8-OHdG generation and nuclear NF-kappa B P65 translocation, both of which were upregulated after EIU induction. RPE-choroidal SIRT1 activity, reduced in EIU animals, was significantly augmented by treatment with resveratrol.
CONCLUSIONS. Resveratrol prevented EIU-associated cellular and molecular inflammatory responses by inhibiting oxidative damage and redox-sensitive NF-kappa B activation. (Invest Ophthalmol Vis Sci. 2009; 50: 3512-3519) DOI: 10.1167/iovs.08-2666

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Neuroprotective Effect of an Antioxidant, Lutein, during Retinal Inflammation

Sasaki, Mariko, Ozawa, Yoko, Kurihara, Toshihide, Noda, Kousuke, Imamura, Yutaka, Kobayashi, Saori, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 50 ( 3 ) 1433 - 1439 2009.03

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. Lutein has been the focus of recent study as a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate, with the use of a mouse endotoxin-induced uveitis (EIU) model, the neuroprotective effects of lutein against retinal neural damage caused by inflammation.
METHODS. EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Each animal was given a subcutaneous injection of lutein or vehicle three times: concurrently with and 3 hours before and after the LPS injection. Analysis was carried out 24 hours after EIU induction. Levels of rhodopsin protein and STAT3 activation were analyzed by immunoblotting. Lengths of the outer segments of the photoreceptor cells were measured. Dark-adapted full-field electroretinograms were recorded. Oxidative stress in the retina was analyzed by dihydroethidium and fluorescent probe. Expression of glial fibrillary acidic protein (GFAP) was shown immunohistochemically.
RESULTS. The EIU-induced decrease in rhodopsin expression followed by shortening of the outer segments and reduction in a-wave amplitude were prevented by lutein treatment. Levels of STAT3 activation, downstream of inflammatory cytokine signals, and reactive oxygen species (ROS), which are both upregulated during EIU, were reduced by lutein. Pathologic change of Muller glial cells, represented by GFAP expression, was also prevented by lutein.
CONCLUSIONS. The present data revealed that the antioxidant lutein was neuroprotective during EIU, suggesting a potential approach for suppressing retinal neural damage during inflammation. (Invest Ophthalmol Vis Sci. 2009; 50: 1433-1439) DOI: 10.1167/iovs.08-2493

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NEW RETRACTOR CAPABLE OF ASPIRATING FLUIDS DURING SCLERAL BUCKLING SURGERY

Kurihara, Toshihide, Suzuki, Kotaro, Noda, Kousuke, Kawamura, Ryosuke, Nagai, Norihiro, Shinoda, Hajime, Shinoda, Kei, Tsubota, Kazuo, Ishida, Susumu, Inoue, Makoto

RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES (LIPPINCOTT WILLIAMS & WILKINS) 29 ( 10 ) 1542 - 1544 2009.01

Research paper (scientific journal), Accepted, ISSN 0275-004X

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Roles of STAT3/SOCS3 pathway in regulating the visual function and ubiquitin-proteasome-dependent degradation of rhodopsin during retinal inflammation

Ozawa, Yoko, Nakao, Keiko, Kurihara, Toshihide, Shimazaki, Takuya, Shimmura, Shigeto, Ishida, Susumu, Yoshimura, Akihiko, Tsubota, Kazuo, Okano, Hideyuki

JOURNAL OF BIOLOGICAL CHEMISTRY (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC) 283 ( 36 ) 24561 - 24570 2008.09

Research paper (scientific journal), Accepted, ISSN 0021-9258

Inflammatory cytokines cause tissue dysfunction. We previously reported that retinal inflammation down-regulates rhodopsin expression and impairs visual function by an unknown mechanism. Here, we demonstrate that rhodopsin levels were preserved by suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of STAT3 activation. SOCS3 was expressed mainly in photoreceptor cells in the retina. In the SOCS3-deficient retinas, rhodopsin protein levels dropped sooner, and the reduction was more profound than in the wild type. Visual dysfunction, measured by electroretinogram, was prolonged in retina-specific SOCS3 conditional knockout mice. Visual dysfunction and decreased rhodopsin levels both correlated with increased STAT3 activation enhanced by SOCS3 deficiency. Interleukin 6, one of the inflammatory cytokines found during retinal inflammation, activated STAT3 and decreased rhodopsin protein in adult retinal explants. This was enhanced by inhibiting SOCS3 function in vitro, indicating that rhodopsin reduction was not a secondary effect in the mutant mice. Interestingly, in the inflamed SOCS3-deficient adult retina, rhodopsin decreased post-transcriptionally at least partly through ubiquitin-proteasome-dependent degradation accelerated by STAT3 activation and not transcriptionally as in the developing retina, on which we reported previously. A STAT3-dependent E3 ubiquitin ligase, Ubr1, was responsible for rhodopsin degradation and was up-regulated in the inflamed SOCS3-deficient retinas. These results indicate that in wild-type animals, a decrease in rhodopsin during inflammation is minimized by endogenous SOCS3. However, when STAT3 activation exceeds some threshold beyond the compensatory activity of endogenous SOCS3, rhodopsin levels decrease. These findings suggest SOCS3 as a potential therapeutic target molecule for protecting photoreceptor cell function during inflammation.

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Angiotensin II type 1 receptor signaling contributes to synaptophysin degradation and neuronal dysfunction in the diabetic retina

Kurihara, Toshihide, Ozawa, Yoko, Nagai, Norihiro, Shinoda, Kei, Noda, Kousuke, Imamura, Yutaka, Tsubota, Kazuo, Okano, Hideyuki, Oike, Yuichi, Ishida, Susumu

DIABETES 57 ( 8 ) 2191 - 2198 2008.08

Research paper (scientific journal), ISSN 0012-1797

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Retinal dysfunction and progressive retinal cell death in SOD1-deficient mice

Hashizume, Kouhei, Hirasawa, Manabu, Imamura, Yutaka, Noda, Setsuko, Shimizu, Takahiko, Shinoda, Kei, Kurihara, Toshihide, Noda, Kousuke, Ozawa, Yoko, Ishida, Susumu, Miyake, Yozo, Shirasawa, Takuji, Tsubota, Kazuo

AMERICAN JOURNAL OF PATHOLOGY (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 172 ( 5 ) 1325 - 1331 2008.05

Research paper (scientific journal), Accepted, ISSN 0002-9440

The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase (SOD1) in Mice leads to many different phenotypes that resemble accelerated aging. The purpose of this study was to examine the morphology and physiology of the sensory retina in Sod1(-/-) mice. The amplitudes of the a- and b-waves of electroretinograms elicited by stimuli of different intensity were reduced in senescent Sod1(-/-) mice, and this reduction in amplitude was more pronounced with increasing age. Retinal morphometric analyses showed a reduced number of nuclei in both the inner nuclear cell layer and outer nuclear cell layer. Electron microscopy revealed swollen cells and degenerated mitochondria in the inner nuclear cell and outer nuclear cell layer of senescent Sod1(-/-) mice indicating necrotic cell death. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed no significant differences in the number of apoptotic cells between Sod1(-/-) and wild-type mice, and activated caspase-3 could not be detected in the retina of Sod1(-/-) mice. In addition to the age-related macular degeneration-like phenotypes previously reported, Sod1(-/-) mice also present progressive retinal degeneration. Our results indicate that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated retinal degeneration.

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Angiotensin II type 1 receptor signaling contributes to synaptophysin degradation and neuronal dysfunction in the diabetic retina

Kurihara, Toshihide, Ozawa, Yoko, Tsubota, Kazuo, Okano, Hideyuki, Ishida, Susumu

NEUROSCIENCE RESEARCH 61 S220 - S220 2008

Research paper (scientific journal), ISSN 0168-0102

Suppression of choroidal neovascularization by dendritic cell vaccination targeting VEGFR2

Mochimaru, Hiroshi, Nagai, Norihiro, Hasegawa, Go, Kudo-Saito, Chie, Yaguchi, Tomonori, Usui, Yoshihiko, Kurihara, Toshihide, Koto, Takashi, Satofuka, Shingo, Shinoda, Hajime, Ozawa, Yoko, Tsubota, Kazuo, Kawakami, Yutaka, Ishida, Susumu

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 48 ( 10 ) 4795 - 4801 2007.10

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate whether the induction of cellular immunity against vascular endothelial growth factor receptor ( VEGFR) 2 inhibits the development of choroidal neovascularization (CNV).
METHODS. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGFR2 was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of the epitope peptide-pulsed mature DCs three times at 6-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, mice were killed to harvest the choroid and splenocytes. CNV volume was evaluated by volumetric measurements. To confirm the specific immunogenicity of the epitope peptides in C57BL/6 mice, CD8 T cells isolated from harvested splenocytes were restimulated to measure interferon (IFN)-gamma and tumor necrosis factor (TNF)-gamma production through enzyme-linked immunospot assay and ELISA. To determine the T-cell subset responsible for the immunotherapy, mice were intraperitoneally injected with an anti-CD4 or anti-CD8 depletion antibody.
RESULTS. CNV volume was significantly lower in mice immunized with the VEGFR2 epitope peptide than in those not immunized or immunized with a control peptide gp70. Cytokine assays showed the peptide-specific production of IFN-gamma and TNF-alpha from the CD8 T cells in a dose-dependent manner. In vivo depletion of CD8, but not CD4, T cells significantly reversed the suppressive effect of the VEGFR2 peptide-pulsed DC vaccination on CNV to the level observed in nonimmunized or gp70-immunized animals.
CONCLUSIONS. These results indicate that the VEGFR2 peptide-specific induction of cellular immunity inhibits CNV through the cytotoxicity of CD8 T cells. Results of the present study suggested the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for CNV.

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Eicosapentaenoic acid is anti-inflammatory in preventing choroidal neovascularization in mice

Koto, Takashi, Nagai, Norihiro, Mochimaru, Hiroshi, Kurihara, Toshihide, Izumi-Nagai, Kanako, Satofuka, Shingo, Shinoda, Hajime, Noda, Kousuke, Ozawa, Yoko, Inoue, Makoto, Tsubota, Kazuo, Oike, Yuichi, Ishida, Susumu

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 48 ( 9 ) 4328 - 4334 2007.09

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate the role of eicosapentaenoic acid (EPA), the major omega-3 polyunsaturated fatty acid (PUFA), in the development of choroidal neovascularization (CNV), together with underlying molecular mechanisms.
METHODS. Six-week-old C57BL/ 6 mice were fed with laboratory chow with 5% EPA or the omega-6 PUFA linoleic acid ( LA) for 4 weeks. Laser photocoagulation was performed to induce CNV, and the volume of CNV tissue was evaluated by volumetric measurements. The expression and production of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, vascular endothelial growth factor ( VEGF) and interleukin (IL)-6 in the retinal pigment epithelium (RPE)-choroid in vivo, and stimulated b-End3 endothelial cells and RAW264.7 macrophages in vitro were evaluated by RT-PCR and ELISA. Fatty acid composition in the serum and the RPE-choroid was analyzed by gas chromatography and high-performance liquid chromatography, respectively. Serum levels of C-reactive protein (CRP), IL-6, VEGF, MCP-1, and soluble ICAM-1 were examined by ELISA.
RESULTS. The CNV volume in EPA-fed animals was significantly suppressed compared with that in control mice, whereas the LA-rich diet did not affect CNV. The mRNA expression and protein levels of ICAM- 1, MCP-1, VEGF, and IL- 6 after CNV induction were significantly reduced in EPA-supplemented mice. In vitro, EPA application led to significant inhibition of mRNA and protein levels of ICAM- 1 and MCP-1 in endothelial cells and VEGF and IL- 6 in macrophages. EPA- fed mice exhibited significantly higher levels of EPA and lower levels of the omega-6 PUFA arachidonic acid in the serum and the RPE-choroid than control animals. EPA supplementation also led to significant reduction of serum levels of IL- 6 and CRP after CNV induction.
CONCLUSIONS. The present study demonstrates for the first time that an EPA- rich diet results in significant suppression of CNV and CNV-related inflammatory molecules in vivo and in vitro. These results suggest that frequent consumption of omega-3 PUFAs may prevent CNV and lower the risk of blindness due to age-related macular degeneration.

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Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neovascularization

Izumi-Nagai, Kanako, Nagai, Norihiro, Ozawa, Yoko, Mihara, Masahiko, Ohsugi, Yoshiyuki, Kurihara, Toshihide, Koto, Takashi, Satofuka, Shingo, Inoue, Makoto, Tsubota, Kazuo, Okano, Hideyuki, Oike, Yuichi, Ishida, Susumu

AMERICAN JOURNAL OF PATHOLOGY (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 170 ( 6 ) 2149 - 2158 2007.06

Research paper (scientific journal), Accepted, ISSN 0002-9440

Interleukin (IL)-6, a potent proinflammatory cytokine, is suggested to be a risk factor for choroidal neovascularization (CNV) because of its increased levels in the serum of patients with age-related macular degeneration; however, the role of IL-6 in CNV has not been defined. The present study reveals the critical contribution of IL-6 signaling and its downstream STAT3 pathway to the murine model of laser-induced CNV. CNV induction by laser treatment stimulated IL-6 expression in the retinal pigment epithelium-choroid complex, and antibody-based blockade of M-6 receptor or genetic ablation of M-6 led to significant suppression of CNV. CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages, and M-6 receptor blockade resulted in selectively inhibited phosphorylation of STAT3 but not extracellular signal-regulated kinase 1/2. Consistently, pharmacological blockade of STAT3 pathway also suppressed CNV. in addition, M-6 receptor neutralization led to significant inhibition of the in vivo and in vitro expression of inflammation-related molecules including monocyte chemotactic protein, intercellular adhesion molecule-1, and vascular endothelial growth factor, and of macrophage infiltration into CNV. These results indicate the significant involvement of H,6 receptor-mediated activation of STAT3 inflammatory pathway in CNV generation, suggesting the possibility of M-6 receptor blockade as a therapeutic strategy to suppress CNV associated with age-related macular degeneration.

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