Clinicopathological findings in refractory diabetic macular edema: A case report.
Takayuki Tanaka, Satoru Kase, Michiyuki Saito, Ikuyo Hirose, Miyuki Murata, Emi Takakuwa, Susumu Ishida
Biomedical reports, 20, 1, 13, 13, 2024年01月, ［国際誌］
英語, The present study describes the case of a patient with refractory diabetic cystoid macular edema who underwent vitrectomy with en bloc removal of the cystoid lesion component. The current study also performed histopathological and immunohistochemical analyses of the cystoid lesion component to assess fibrin/fibrinogen and advanced glycation end-products (AGEs) immunoreactivity. A 69-year-old Japanese man presented with visual loss in the left eye due to residual cystoid macular edema (CME) refractory to anti-vascular endothelial growth factor therapy. Best-corrected visual acuity was 1.2 in the right eye (OD) and 0.5 in the left eye (OS). Fundus examination showed dot hemorrhages and hard exudates in the peri-macular region with pan-retinal photocoagulation scars in both eye. Swept-source optical coherence tomography revealed CME with slight hyperreflectivity in the cyst OS. A total of 3 months after the initial visit, pars plana vitrectomy was performed, and the translucent solidified component within the cystoid lesion was isolated. Histopathologically, the excised component was elliptical in shape, measuring 0.7x0.4 mm and exhibited homogeneous eosinophilic material without cellular components. No membranous structure was observed surrounding the component. Immunohistochemistry demonstrated that the tissue was positive for fibrin/fibrinogen and weakly positive for AGEs, but was negative for glial fibrillary acidic protein, type 1 collagen and receptor for AGEs. To the best of our knowledge, the present case report is the first to histopathologically examine the contents of refractory CME, and to immunohistochemically demonstrate that fibrin in diabetic CME may be post-translationally modified by AGEs. These results suggested that fibrin in CME may escape degradation by plasmin due to post-translational modifications.

Phosphorylation of αB-Crystallin Involves Interleukin-1β-Mediated Intracellular Retention in Retinal Müller Cells: A New Mechanism Underlying Fibrovascular Membrane Formation.
Taku Yamamoto, Satoru Kase, Akihiro Shinkai, Miyuki Murata, Kasumi Kikuchi, Di Wu, Yasushi Kageyama, Masami Shinohara, Tomohiko Sasase, Susumu Ishida
Investigative ophthalmology & visual science, 64, 10, 20, 20, 2023年07月03日, ［国際誌］
英語, 研究論文（学術雑誌）, PURPOSE: Chronic inflammation plays a pivotal role in the pathology of proliferative diabetic retinopathy (PDR), in which biological alterations of retinal glial cells are one of the key elements. The phosphorylation of αB-crystallin/CRYAB modulates its molecular dynamics and chaperone activity, and attenuates αB-crystallin secretion via exosomes. In this study, we investigated the effect of phosphorylated αB-crystallin in retinal Müller cells on diabetic mimicking conditions, including interleukin (IL)-1β stimuli. METHODS: Human retinal Müller cells (MIO-M1) were used to examine gene and protein expressions with real-time quantitative PCR, enzyme linked immunosorbent assay (ELISA), and immunoblot analyses. Cell apoptosis was assessed by Caspase-3/7 assay and TdT-mediated dUTP nick-end labeling staining. Retinal tissues isolated from the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic animal model with obesity, and fibrovascular membranes from patients with PDR were examined by double-staining immunofluorescence. RESULTS: CRYAB mRNA was downregulated in MIO-M1 cells with the addition of 10 ng/mL IL-1β; however, intracellular αB-crystallin protein levels were maintained. The αB-crystallin serine 59 (Ser59) residue was phosphorylated with IL-1β application in MIO-M1 cells. Cell apoptosis in MIO-M1 cells was induced by CRYAB knockdown. Immunoreactivity for Ser59-phosphorylated αB-crystallin and glial fibrillary acidic protein was colocalized in glial cells of SDT fatty rats and fibrovascular membranes. CONCLUSIONS: The Ser59 phosphorylation of αB-crystallin was modulated by IL-1β in Müller cells under diabetic mimicking inflammatory conditions, suggesting that αB-crystallin contributes to the pathogenesis of PDR through an anti-apoptotic effect.

Effects of Mirogabalin on Hyperalgesia and Chronic Ocular Pain in Tear-Deficient Dry-Eye Rats.
Kasumi Kikuchi, Yoshiaki Tagawa, Miyuki Murata, Susumu Ishida
Investigative ophthalmology & visual science, 64, 5, 27, 27, 2023年05月01日, ［国際誌］
英語, 研究論文（学術雑誌）, PURPOSE: Patients with dry eye disease (DED) sometimes complain of ocular pain. DED-related ocular pain has many similarities with neuropathic pain. Mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium channels, is approved for treating neuropathic pain in Japan. This study aimed to investigate the effect of mirogabalin on hyperalgesia and chronic ocular pain in a rat DED model. METHODS: DED was induced in female Sprague Dawley rats by unilaterally excising the external lacrimal gland (ELG) and Harderian gland (HG). After 4 weeks of ELG and HG removal, tear production (pH threads) and corneal epithelial damage (fluorescein staining) were evaluated. Corneal hyperalgesia and chronic pain were analyzed, respectively, by measuring capsaicin-induced eye-wiping behavior and c-Fos expression in the trigeminal nucleus. Mirogabalin (10 or 3 mg/kg) was evaluated for effects on DED-induced hyperalgesia and chronic ocular pain. RESULTS: Tear production was significantly lower in DED-induced eyes than in control eyes. Corneal damage was significantly higher in DED eyes than in control eyes. Hyperalgesia and chronic ocular pain were detected 4 weeks after ELG and HG removal. Five days of mirogabalin administration significantly suppressed capsaicin-induced eye-wiping behavior, which indicated the suppression of ocular hyperalgesia. Administration of 10 mg/kg mirogabalin significantly reduced c-Fos expression in the trigeminal nucleus, which indicated the amelioration of chronic ocular pain. CONCLUSIONS: Mirogabalin suppressed DED-induced hyperalgesia and chronic ocular pain in a rat DED model. Our findings suggested that mirogabalin might effectively alleviate chronic ocular pain in patients with DED.

Involvement of Angiopoietin 2 and vascular endothelial growth factor in uveitis.
Kayo Suzuki, Daiju Iwata, Kenichi Namba, Keitaro Hase, Miki Hiraoka, Miyuki Murata, Nobuyoshi Kitaichi, Richard Foxton, Susumu Ishida
PloS one, 18, 11, e0294745, 2023年, ［国際誌］
英語, 研究論文（学術雑誌）, PURPOSE: Angiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation. METHODS: We measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated. RESULTS: The protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (P<0.05). The retinochoroidal mRNA levels of Ang2 and VEGFA were significantly upregulated in EAU mice compared to controls (n = 6, P<0.05). Although there was no significant difference, treatment with anti-VEGFA antibody reduced the clinical and histopathological scores. However, treatment with anti-Ang2 antibody reduced the clinical and histopathological scores (n = 18-20, P<0.05). Furthermore, these scores were further decreased when treated by inhibiting both Ang2 and VEGFA. CONCLUSIONS: Based on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

Relationship between Brachial-Ankle Pulse Wave Velocity and Fundus Arteriolar Area Calculated Using a Deep-Learning Algorithm.
Kanae Fukutsu, Michiyuki Saito, Kousuke Noda, Miyuki Murata, Satoru Kase, Ryosuke Shiba, Naoki Isogai, Yoshikazu Asano, Nagisa Hanawa, Mitsuru Dohke, Manabu Kase, Susumu Ishida
Current eye research, 47, 11, 1534, 1537, 2022年11月, ［国際誌］
英語, 研究論文（学術雑誌）, PURPOSE: Retinal vessels reflect alterations related to hypertension and arteriosclerosis in the physical status. Previously, we had reported a deep-learning algorithm for automatically detecting retinal vessels and measuring the total retinal vascular area in fundus photographs (VAFP). Herein, we investigated the relationship between VAFP and brachial-ankle pulse wave velocity (baPWV), which is the gold standard for arterial stiffness assessment in clinical practice. METHODS: Retinal photographs (n = 696) obtained from 372 individuals who visited the Keijinkai Maruyama Clinic for regular health checkups were used to analyze VAFP. Additionally, the baPWV was measured for each patient. Automatic retinal-vessel segmentation was performed using our deep-learning algorithm, and the total arteriolar area (AA) and total venular area (VA) were measured. Correlations between baPWV and several parameters, including AA and VA, were assessed. RESULTS: The baPWV was negatively correlated with AA (R = -0.40, n = 696, P < 2.2e-16) and VA (R = -0.36, n = 696, P < 2.2e-16). Independent variables (AA, sex, age, and systolic blood pressure) selected using the stepwise method showed a significant correlation with baPWV. The estimated baPWV, calculated using a regression equation with variables including AA, showed a better correlation with the measured baPWV (R = 0.70, n = 696, P < 2.2e-16) than the estimated value without AA (R = 0.68, n = 696, P < 2.2e-16). CONCLUSIONS: AA and VA were significantly correlated with baPWV. Moreover, baPWV estimated using AA correlated well with the actual baPWV. VAFP may serve as an alternative biomarker for evaluating systemic arterial stiffness.

Placental growth factor stabilizes VEGF receptor-2 protein in retinal pigment epithelial cells by downregulating glycogen synthase kinase 3 activity.
Miyuki Murata, Kousuke Noda, Satoru Kase, Keitaro Hase, Di Wu, Ryo Ando, Susumu Ishida
The Journal of biological chemistry, 298, 9, 102378, 102378, 2022年09月, ［国際誌］
英語, 研究論文（学術雑誌）, Placental growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family of proteins that participate in angiogenesis and vasculogenesis. Anti-VEGF therapy has become the standard treatment for ocular angiogenic disorders in ophthalmological practice. However, there is emerging evidence that anti-VEGF treatment may increase the risk of atrophy of the retinal pigment epithelium (RPE), which is important for the homeostasis of retinal tissue. Whereas the cytoprotective role of VEGF family molecules, particularly that of VEGF A (VEGFA) through its receptor VEGF receptor-2 (VEGFR-2), has been recognized, the physiological role of PlGF in the retina is still unknown. In this study, we explored the role of PlGF in the RPE using PlGF-knockdown RPE cells generated by retrovirus-based PlGF-shRNA transduction. We show that VEGFA reduced apoptosis induced by serum starvation in RPE cells, whereas the antiapoptotic effect of VEGFA was abrogated by VEGFR-2 knockdown. Furthermore, PlGF knockdown increased serum starvation-induced cell apoptosis and unexpectedly reduced the protein level of VEGFR-2 in the RPE. The antiapoptotic effect of VEGFA was also diminished in PlGF-knockdown RPE cells. In addition, we found that glycogen synthase kinase 3 activity was involved in proteasomal degradation of VEGFR-2 in RPE cells and inactivated by PlGF via AKT phosphorylation. Overall, the present data demonstrate that PlGF is crucial for RPE cell viability and that PlGF supports VEGFA/VEGFR-2 signaling by stabilizing the VEGFR-2 protein levels through glycogen synthase kinase 3 inactivation.

Serum advanced glycation end-products and αB-crystallin in diabetic retinopathy patients.
Taku Yamamoto, Satoru Kase, Miyuki Murata, Susumu Ishida
Biomedical reports, 16, 4, 28, 28, 2022年04月, ［国際誌］
英語, 研究論文（学術雑誌）, αB-crystallin, one of the small heat shock proteins, which is also known as HSPB5, has cytoprotective effects under inflammatory conditions. Advanced glycation end-products (AGE) are produced through non-enzymatic glycation under conditions of hyperglycemia and they contribute to angiogenesis and inflammation. The aim of this study was to examine the levels of serum αB-crystallin and AGE concentrations in blood samples collected from proliferative diabetic retinopathy (PDR) patients. Blood samples were collected from seven diabetic patients with PDR and eight patients without diabetes mellitus who underwent vitrectomy due to PDR and idiopathic macular diseases, respectively, in a single center. The levels of serum αB-crystallin and AGE were measured by ELISA and correlations were assessed statistically. The serum levels (mean ± SEM) of AGE were significantly higher in PDR patients (28.41±0.46 µg/ml) than in patients with non-diabetic macular diseases (25.76±0.60 µg/ml; P=0.015), whereas there was no significant difference in serum αB-crystallin levels. There was one patient with an extremely high level of αB-crystallin, who was treated with systemic corticosteroid due to chronic autoimmune inflammatory diseases. The current prospective study showed that serum AGE levels were significantly higher in PDR patients; however, no correlations between serum AGE and αB-crystallin levels were identified.

Downregulation of AlphaB-crystallin in Retinal Pigment Epithelial Cells Exposed to Diabetes-related Stimuli In Vivo and In Vitro.
D I Wu, Satoru Kase, Y E Liu, Atsuhiro Kanda, Miyuki Murata, Susumu Ishida
In vivo (Athens, Greece), 36, 1, 132, 139, 2022年, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND/AIM: AlphaB-crystallin plays a pivotal role in many diseases. However, the involvement of alphaB-crystallin in retinal pigment epithelial (RPE) cells with diabetes stimuli remains unknown. The aim of this study is to examine the alterations of RPE cells and alphaB-crystallin expression in diabetic models in vivo and in vitro. MATERIALS AND METHODS: Diabetic conditions in mice were induced by streptozotocin (STZ). The thickness of the RPE/choroid complex was measured by optical coherence tomography (OCT). Periodic acid-Schiff (PAS) staining was used to investigate the choriocapillaris in histological sections of murine eyeballs and oxidative stress was evaluated using immunofluorescence with anti-4-hydroxynonenal (HNE) antibody. AlphaB-crystallin expression was examined in the RPE/choroid complex using ELISA. Real-Time PCR was performed to evaluate the alphaB-crystallin expression in cultured human RPE cells with high glucose or following advanced glycation end-products (AGE) stimulation. RESULTS: In diabetic mice, OCT-based RPE/choroidal layers were thickened 2 months after STZ stimulation, where PAS-positive dilated choriocapillaris was noted. Immunoreactivity of 4-HNE was strongly observed in the RPE layer, from which a significant number of RPE cells was lost. Meanwhile, alphaB-crystallin expression in 2-month STZ mice was significantly lower compared to controls. In accordance with these results, in vitro data showed that the alphaB-crystallin expression was also significantly lower in RPE cells with high glucose or following AGE stimulation compared to untreated cells. CONCLUSION: In both types of diabetic models the expression of alphaB-crystallin was found to be downregulated in RPE cells and was associated with increased levels of oxidative stress.

A Novel Deep Learning Architecture for Vascular Area Measurement in Fundus Images
Kanae Fukutsu, Michiyuki Saito, Kousuke Noda, Miyuki Murata, Satoru Kase, Ryosuke Shiba, Naoki Isogai, Yoshikazu Asano, Nagisa Hanawa, Mitsuru Dohke, Manabu Kase, Susumu Ishida
Ophthalmology Science, 100004, 100004, Elsevier BV, 2021年02月
研究論文（学術雑誌）

ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein.
Kanae Fukutsu, Miyuki Murata, Kasumi Kikuchi, Shiho Yoshida, Kousuke Noda, Susumu Ishida
Frontiers in medicine, 8, 717602, 717602, 2021年, ［国際誌］
英語, 研究論文（学術雑誌）, Objective: Acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently modulates cellular function. Our previous study demonstrated that acrolein induces glial cell migration, a pathological hallmark of diabetic retinopathy; however, the detailed cellular mechanism remains unclear. The purpose of this study was to investigate the role of acrolein in retinal glial cell migration by focusing on rho-associated coiled-coil-containing protein kinases (ROCKs). Methods: Immunofluorescence staining for ROCK isoforms was performed using sections of fibrovascular tissue obtained from the eyes of patients with proliferative diabetic retinopathy (PDR). Rat retinal Müller glial cell line, TR-MUL5, was stimulated with acrolein and the levels of ROCK1 were evaluated using real-time PCR and western blotting. Phosphorylation of the myosin-binding subunit of myosin light chain phosphatase [myosin phosphatase target subunit 1, (MYPT1)] and myosin light chain 2 (MLC2) was assessed. The cell migration rate of TR-MUL5 cells exposed to acrolein and/or ripasudil, a non-selective ROCK inhibitor, was measured using the Oris cell migration assay. Results: ROCK isoforms, ROCK1 and ROCK2, were positively stained in the cytosol of glial cells in fibrovascular tissues. In TR-MUL5 cells, the mRNA expression level of Rock1, but not Rock2, was increased following acrolein stimulation. In line with the PCR data, western blotting showed increase in ROCK1 and cleaved ROCK1 protein in TR-MUL5 cells stimulated with acrolein. N-acetylcysteine (NAC) suppressed acrolein-associated Rock1 upregulation in TR-MUL5 cells. Acrolein augmented the phosphorylation of MYPT1 and MLC2 and increased the cell migration rate of TR-MUL5 cells, both of which were abrogated by ripasudil. Conclusions: Our study demonstrated that ROCK1 mediates the migration of retinal glial cells promoted by the unsaturated aldehyde acrolein.

Involvement of Müller Glial Autoinduction of TGF-β in Diabetic Fibrovascular Proliferation Via Glial-Mesenchymal Transition.
Di Wu, Atsuhiro Kanda, Ye Liu, Kousuke Noda, Miyuki Murata, Susumu Ishida
Investigative ophthalmology & visual science, 61, 14, 29, 29, 2020年12月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Purpose: Müller glial-mesenchymal transition (GMT) is reported as the fibrogenic mechanism promoted by TGF-β-SNAIL axis in Müller cells transdifferentiated into myofibroblasts. Here we show the multifaceted involvement of TGF-β in diabetic fibrovascular proliferation via Müller GMT and VEGF-A production. Methods: Surgically excised fibrovascular tissues from the eyes of patients with proliferative diabetic retinopathy were processed for immunofluorescence analyses of TGF-β downstream molecules. Human Müller glial cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and ELISA, respectively. Immunoblot analyses were performed to detect TGF-β signal activation. Results: Müller glial cells in patient fibrovascular tissues were immunopositive for GMT-related molecular markers, including SNAIL and smooth muscle protein 22, together with colocalization of VEGF-A and TGF-β receptors. In vitro administration of TGF-β1/2 upregulated TGFB1 and TGFB2, both of which were suppressed by inhibitors for nuclear factor-κB, glycogen synthase kinase-3, and p38 mitogen-activated protein kinase. Of the various profibrotic cytokines, TGF-β1/2 application exclusively induced Müller glial VEGFA mRNA expression, which was decreased by pretreatment with small interfering RNA for SMAD2 and inhibitors for p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Supporting these findings, TGF-β1/2 stimulation to Müller cells increased the phosphorylation of these intracellular signaling molecules, all of which were also activated in Müller glial cells in patient fibrovascular tissues. Conclusions: This study underscored the significance of Müller glial autoinduction of TGF-β as a pathogenic cue to facilitate diabetic fibrovascular proliferation via TGF-β-driven GMT and VEGF-A-driven angiogenesis.

増殖糖尿病網膜症患者の硝子体中アクロレイン結合蛋白と単球走化性因子　　　　　　　　　　　　　　　
村田 美幸, 福津 佳苗, 野田 航介, 吉田 志帆, 神田 敦宏, 石田 晋
糖尿病合併症, 34, Suppl.1, 262, 262, (一社)日本糖尿病合併症学会, 2020年11月
日本語

機械学習によって自動算出された網膜動静脈面積と血圧脈波の関係　　　　　　　　　　　　　　　
福津 佳苗, 齋藤 理幸, 野田 航介, 村田 美幸, 加瀬 諭, 柴 涼介, 磯貝 直己, 道家 充, 石田 晋
眼科臨床紀要, 13, 10, 683, 684, 眼科臨床紀要会, 2020年10月
日本語

Blockade of Platelet-Derived Growth Factor Signaling Inhibits Choroidal Neovascularization and Subretinal Fibrosis in Mice.
Ye Liu, Kousuke Noda, Miyuki Murata, Di Wu, Atsuhiro Kanda, Susumu Ishida
Journal of clinical medicine, 9, 7, 2020年07月15日, ［国際誌］
英語, 研究論文（学術雑誌）, Neovascular age related macular degeneration (nAMD) leads to severe vision loss worldwide and is characterized by the formation of choroidal neovascularization (CNV) and fibrosis. In the current study, we aimed to investigate the effect of blockade for platelet derived growth factor receptor-β (PDGFR-β) on the formation of choroidal neovascularization and fibrosis in the laser-induced CNV model in mice. Firstly, the presence of PDGFR-β in CNV lesions were confirmed. Intravitreal injection of PDGFR-β neutralizing antibody significantly reduced the size of CNV and subretinal fibrosis. Additionally, subretinal hyperreflective material (SHRM), a landmark feature on OCT as a risk factor for subretinal fibrosis formation in nAMD patients was also suppressed by PDGFR-β blockade. Furthermore, pericytes were abundantly recruited to the CNV lesions during CNV formation, however, blockade of PDGFR-β significantly reduced pericyte recruitment. In addition, PDGF-BB stimulation increased the migration of the rat retinal pericyte cell line, R-rPCT1, which was abrogated by the neutralization of PDGFR-β. These results indicate that blockade of PDGFR-β attenuates laser-induced CNV and fibrosis through the inhibition of pericyte migration.

Regulation of Spermine Oxidase through Hypoxia-Inducible Factor-1α Signaling in Retinal Glial Cells under Hypoxic Conditions.
Di Wu, Kousuke Noda, Miyuki Murata, Ye Liu, Atsuhiro Kanda, Susumu Ishida
Investigative ophthalmology & visual science, 61, 6, 52, 52, 2020年06月03日, ［国際誌］
英語, 研究論文（学術雑誌）, Purpose: Acrolein, a highly reactive unsaturated aldehyde, is known to facilitate glial cell migration, one of the pathological hallmarks in diabetic retinopathy. However, cellular mechanisms of acrolein generation in retinal glial cells remains elusive. In the present study, we investigated the role and regulation of spermine oxidase (SMOX), one of the enzymes related to acrolein generation, in retinal glial cells under hypoxic condition. Methods: Immunofluorescence staining for SMOX was performed using sections of fibrovascular tissues obtained from patients with proliferative diabetic retinopathy. Expression levels of polyamine oxidation enzymes including SMOX were analyzed in rat retinal Müller cell line 5 (TR-MUL5) cells under either normoxic or hypoxic conditions. The transcriptional activity of Smox in TR-MUL5 cells was evaluated using the luciferase assay. Levels of acrolein-conjugated protein, Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), and hydrogen peroxide were measured. Results: SMOX was localized in glial cells in fibrovascular tissues. Hypoxia induced SMOX production in TR-MUL5 cells, which was suppressed by silencing of hypoxia-inducible factor-1α (Hif1a), but not Hif2a. Transcriptional activity of Smox was regulated through HIF-1 binding to hypoxia response elements 2, 3, and 4 sites in the promoter region of Smox. Generation of FDP-Lys and hydrogen peroxide increased in TR-MUL5 cells under hypoxic condition, which was abrogated by SMOX inhibitor MDL72527. Conclusions: The current data demonstrated that hypoxia regulates production of SMOX, which plays a role in the generation of oxidative stress inducers, through HIF-1α signaling in Müller glial cells under hypoxic condition.

Glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced Müller glial galectin-1 expression via HIF-1α destabilization.
Atsuhiro Kanda, Ikuyo Hirose, Kousuke Noda, Miyuki Murata, Susumu Ishida
Journal of cellular and molecular medicine, 24, 8, 4589, 4599, 2020年04月, ［国際誌］
英語, 研究論文（学術雑誌）, Galectin-1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin-1β-driven inflammatory pathway for galectin-1 expression in vitro and in vivo. Here, we show glucocorticoid-mediated inhibitory mechanism against hypoxia-inducible factor (HIF)-1α-involved galectin-1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia-induced increases in galectin-1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia-stimulated cells decreased HIF-1α protein, but not mRNA, together with its DNA-binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co-immunoprecipitation revealed that glucocorticoid-transactivated TSC22D3 interacted with HIF-1α, leading to degradation of hypoxia-stabilized HIF-1α via the ubiquitin-proteasome pathway. Silencing TSC22D3 reversed glucocorticoid-mediated ubiquitination of HIF-1α and subsequent down-regulation of HIF-1α and galectin-1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes-induced retinal HIF-1α and galectin-1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co-localization of galectin-1 and HIF-1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid-transactivated TSC22D3 attenuates hypoxia- and diabetes-induced retinal glial galectin-1/LGALS1 expression via HIF-1α destabilization, highlighting therapeutic implications for DR in the era of anti-vascular endothelial growth factor treatment.

SDT fattyラット水晶体におけるポリオール経路と酸化ストレスの継時的解析　　　　　　　　　　　　　　　
菊地 香澄, 村田 美幸, 野田 航介, 神田 敦宏, 加瀬 諭, 影山 靖, 篠原 雅巳, 笹瀬 智彦, 石田 晋
日本眼科学会雑誌, 124, 臨増, 242, 242, (公財)日本眼科学会, 2020年03月
日本語

アクロレインによる網膜ミュラーグリア細胞の遊走作用とROCK1の関与　　　　　　　　　　　　　　　
福津 佳苗, 村田 美幸, 野田 航介, 吉田 志帆, 神田 敦宏, 石田 晋
日本眼科学会雑誌, 124, 臨増, 275, 275, (公財)日本眼科学会, 2020年03月
日本語

Pathological Role of Unsaturated Aldehyde Acrolein in Diabetic Retinopathy.
Miyuki Murata, Kousuke Noda, Susumu Ishida
Frontiers in immunology, 11, 589531, 589531, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, With increasing prevalence of diabetes and a progressively aging society, diabetic retinopathy is emerging as one of the global leading causes of blindness. Recent studies have shown that vascular endothelial growth factor (VEGF) plays a central role in the pathogenesis of diabetic retinopathy and anti-VEGF agents have become the first-line therapy for the vision-threatening disease. However, recent studies have also demonstrated that diabetic retinopathy is a multifactorial disease and that VEGF-independent mechanism(s) also underlie much of the pathological changes in diabetic retinopathy. Acrolein is a highly reactive unsaturated aldehyde and is implicated in protein dysfunction. As acrolein is common in air pollutants, previous studies have focused on it as an exogenous causative factor, for instance, in the development of respiratory diseases. However, it has been discovered that acrolein is also endogenously produced and induces cell toxicity and oxidative stress in the body. In addition, accumulating evidence suggests that acrolein and/or acrolein-conjugated proteins are associated with the molecular mechanisms in diabetic retinopathy. This review summarizes the pathological roles and mechanisms of endogenous acrolein production in the pathogenesis of diabetic retinopathy.

Cytoprotective Effect of Astaxanthin in a Model of Normal Intraocular Pressure Glaucoma.
Kasumi Kikuchi, Zhenyu Dong, Yasuhiro Shinmei, Miyuki Murata, Atsuhiro Kanda, Kosuke Noda, Takayuki Harada, Susumu Ishida
Journal of ophthalmology, 2020, 9539681, 9539681, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies. Lowering intraocular pressure is currently the only way to treat glaucoma, but it is often insufficient to inhibit the progression of the disease. Glaucoma is a multifactorial disease, and the involvement of oxidative stress has recently received much attention. In the present study, we investigated the cytoprotective effect of astaxanthin (AST) on RGC degeneration using a normal-tension glaucoma (NTG) mouse model, which lacks the glutamate/aspartate transporter (Glast) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure. Three-week-old Glast± mice were given intraperitoneal injections of AST at 10, 30, or 60 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. Five weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. We also used a retrograde labeling technique to quantify the number of RGCs. Additionally, the phosphorylated (p) IκB/total IκB ratio and the 4-hydroxynonenal (HNE) were measured in retinal tissues. The number of RGCs in Glast± mice was significantly decreased compared with that in control mice. RGC loss was suppressed by the administration of AST at 60 mg/kg/day, compared with vehicle alone. Following AST administration, the concentration of 4-HNE in the retina was also suppressed, but the pIκB/IκB ratio did not change. Our study revealed that the antioxidative stress effects of AST inhibit RGC degeneration in the retina and may be useful in the treatment of NTG.

Diabetic Cataract in Spontaneously Diabetic Torii Fatty Rats.
Kasumi Kikuchi, Miyuki Murata, Kousuke Noda, Satoru Kase, Yoshiaki Tagawa, Yasushi Kageyama, Masami Shinohara, Tomohiko Sasase, Susumu Ishida
Journal of diabetes research, 2020, 3058547, 3058547, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, Spontaneously Diabetic Torii (SDT) fatty rat is a novel animal model of type 2 diabetes with obesity. SDT fatty rats develop hyperglycemia, dyslipidemia, and other diabetic complications including ocular disorders; however, diabetic cataract formation in SDT fatty rats has not been fully investigated. The aim of the current study was to investigate the characteristics of cataract in the SDT fatty rats. The mean body weight of SDT fatty rats is larger than that of age-matched Sprague-Dawley (SD) rats and control animals until 8 weeks of age, and thereafter the growing speed decreased until the end of observation at 16 weeks of age. Blood glucose levels in SDT fatty rats were significantly higher than those in SD rats throughout the observational period. Slit-lamp examination revealed that no rats showed cataract formation at 5 weeks of age; however, SDT fatty rats gradually developed cortical cataract and posterior subcapsular cataract, both of which are the common types of cataract in patients with type 2 diabetes. The levels of glucose, sorbitol, and fructose were higher in the lens tissues of SDT fatty rats in comparison with that of SD rats. Furthermore, the level of 4-hydroxynonenal (4-HNE) was higher in the lens of SDT fatty rats than in that of SD rats. By contrast, total glutathione (GSH) concentration was lower in the lens of SDT fatty rats than in that of SD rats. The present study demonstrated that the cataractogenesis in SDT fatty rats resembled human diabetic cataract formation, indicating that SDT fatty rats serve as a potential animal model in researches on human cataract associated with type 2 diabetes and obesity.

網膜色素上皮細胞における胎盤成長因子のアポトーシス抑制効果　　　　　　　　　　　　　　　
村田 美幸, 野田 航介, 長谷 敬太郎, 神田 敦宏, 石田 晋
眼科臨床紀要, 12, 11, 850, 850, 眼科臨床紀要会, 2019年11月
日本語

Unsaturated Aldehyde Acrolein Promotes Retinal Glial Cell Migration.
Miyuki Murata, Kousuke Noda, Shiho Yoshida, Michiyuki Saito, Akio Fujiya, Atsuhiro Kanda, Susumu Ishida
Investigative ophthalmology & visual science, 60, 13, 4425, 4435, 2019年10月01日, ［国際誌］
英語, 研究論文（学術雑誌）, Purpose: To investigate the effect of the unsaturated aldehyde acrolein on retinal glial cell migration. Methods: Müller glial cell markers expression in TR-MUL5 were confirmed by RT-PCR and immunostaining. Cell viability and migration rate of TR-MUL5 cells were assessed after the stimulation with acrolein. DNA microarray analysis was performed to analyze changes in the expression levels of migration-related genes in Müller glial cells stimulated with acrolein. Real-time PCR and ELISA were performed to validate DNA microarray analysis results. Inhibitors of C-X-C motif chemokine ligand 1 (CXCL1), one of the genes highly upregulated after the exposure to acrolein, and blockers of its receptor, CXCR2, were used to investigate the role of the CXCL1-CXCR2 axis on glial cell migration. CXCL1 concentration was measured in vitreous fluid samples obtained from proliferative diabetic retinopathy (PDR) and nondiabetic control eyes. CXCL1 and CXCR2 expression in glial cells of fibrovascular tissues obtained from PDR patients was examined by immunostaining. Results: At a high concentration, acrolein (100 μM) significantly decreased cell viability. However, in moderate, sublethal concentrations (25-50 μM), acrolein induced cell migration and substantially increased the production of CXCL1 in TR-MUL5 cells. CXCL1 concentration was significantly elevated in vitreous fluids of PDR patients, and CXCL1 and CXCR2 were present in glial cells in fibrovascular tissues of PDR patients. CXCL1 stimulation increased glial cell migration in a dose-dependent manner, which was abrogated by the neutralization of the CXCL1-CXCR2 axis. Conclusions: Our data demonstrate that acrolein promotes retinal Müller glial cell migration by enhancing CXCL1 production.

Spontaneously Diabetic Torii fattyラット網膜における炎症性サイトカインの発現解析　　　　　　　　　　　　　　　
菊地 香澄, 野田 航介, 村田 美幸, 神田 敦宏, 加瀬 諭, 影山 靖, 篠原 雅巳, 笹瀬 智彦, 石田 晋
糖尿病合併症, 33, Suppl.1, 271, 271, (一社)日本糖尿病合併症学会, 2019年09月
日本語

網膜色素上皮細胞における胎盤成長因子による血管内皮増殖因子受容体 2の安定化機序　　　　　　　　　　　　　　　
村田 美幸, 野田 航介, 長谷 敬太郎, Di Wu, 神田 敦宏, 石田 晋
糖尿病合併症, 33, Suppl.1, 271, 271, (一社)日本糖尿病合併症学会, 2019年09月
日本語

Spontaneously Diabetic Torii fattyラットにおける糖尿病白内障の検討　　　　　　　　　　　　　　　
菊地 香澄, 野田 航介, 村田 美幸, 田川 義晃, 神田 敦宏, 加瀬 諭, 影山 靖, 篠原 雅巳, 笹瀬 智彦, 石田 晋
日本眼科学会雑誌, 123, 臨増, 166, 166, (公財)日本眼科学会, 2019年03月
日本語

A novel pathway of LPS uptake through syndecan-1 leading to pyroptotic cell death.
Shigetoshi Yokoyama, Yan Cai, Miyuki Murata, Takeshi Tomita, Mitsuhiro Yoneda, Lei Xu, Aprile L Pilon, Raul E Cachau, Shioko Kimura
eLife, 7, 2018年12月07日, ［国際誌］
英語, 研究論文（学術雑誌）, Intracellular lipopolysaccharide (LPS) triggers the non-canonical inflammasome pathway, resulting in pyroptosis of innate immune cells. In addition to its well-known proinflammatory role, LPS can directly cause regression of some tumors, although the underlying mechanism has remained unknown. Here we show that secretoglobin(SCGB)3A2, a small protein predominantly secreted in airways, chaperones LPS to the cytosol through the cell surface receptor syndecan-1; this leads to pyroptotic cell death driven by caspase-11. SCGB3A2 and LPS co-treatment significantly induced pyroptosis of macrophage RAW264.7 cells and decreased cancer cell proliferation in vitro, while SCGB3A2 treatment resulted in reduced progression of xenograft tumors in mice. These data suggest a conserved function for SCGB3A2 in the innate immune system and cancer cells. These findings demonstrate a critical role for SCGB3A2 as an LPS delivery vehicle; they reveal one mechanism whereby LPS enters innate immune cells leading to pyroptosis, and they clarify the direct effect of LPS on cancer cells.

血管径自動解析プログラムによる網膜動静脈比と血圧脈波の相関　　　　　　　　　　　　　　　
齋藤 理幸, 野田 航介, 星川 靖裕, 小林 正彦, 道家 充, 村田 美幸, 齋藤 航, 加瀬 学, 石田 晋
眼科臨床紀要, 11, 11, 850, 851, 眼科臨床紀要会, 2018年11月
日本語

糖尿病網膜症におけるポリアミン代謝の病態関与　　　　　　　　　　　　　　　
野田 航介, 村田 美幸, 神田 敦宏, 石田 晋
日本糖尿病眼学会誌, 22, 132, 132, 日本糖尿病眼学会, 2018年09月
日本語

Proteolytic cleavage of vascular adhesion protein-1 induced by vascular endothelial growth factor in retinal capillary endothelial cells
Yoshida S, Murata M, Noda K, Matsuda T, Saito M, Saito W, Kanda A, Ishida S
Jpn J Ophthalmol, 62, 2, 256, 264, 2018年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Soluble Vascular Adhesion Protein-1 Mediates Spermine Oxidation as Semicarbazide-Sensitive Amine Oxidase: Possible Role in Proliferative Diabetic Retinopathy.
Murata M, Noda K, Kawasaki A, Yoshida S, Dong Y, Saito M, Dong Z, Ando R, Mori S, Saito W, Kanda A, Ishida S
Current eye research, 42, 12, 1, 10, 2017年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, UNLABELLED: Purpose/Aim of the study: To explore the possible role of vascular adhesion protein-1 (VAP-1) via its enzymatic function as a semicarbazide-sensitive amine oxidase (SSAO) in the pathogenesis of proliferative diabetic retinopathy (PDR). MATERIALS AND METHODS: The levels of soluble VAP-1/SSAO and the unsaturated aldehyde acrolein (ACR)-conjugated protein, Nε-(3-formyl-3, 4-dehydropiperidino) lysine adduct (FDP-Lys), were measured in vitreous fluid samples of PDR and non-diabetic patients using ELISA. Recombinant human VAP-1/SSAO (rhVAP-1/SSAO) was incubated with spermine, with or without semicarbazide or RTU-1096 (a specific inhibitor for VAP-1/SSAO). Immunofluorescence assays were performed to assess the localization of VAP-1/SSAO and FDP-Lys in fibrovascular tissues from patients with PDR. The impact of ACR on cultured retinal capillary endothelial cells was assessed using a cell viability assay and total glutathione (GSH) measurements. RESULTS: The levels of sVAP-1/SSAO and FDP-Lys were elevated in the vitreous fluid of patients with PDR. Incubation of rhVAP-1 with spermine resulted in the generation of hydrogen peroxide and FDP-Lys and the production was inhibited by semicarbazide and RTU-1096. In fibrovascular tissues, FDP-Lys and VAP-1/SSAO were present in endothelial cells. ACR stimulation reduced GSH levels in the cultured endothelial cells in a dose-dependent manner and caused cellular toxicity. CONCLUSIONS: Our results indicate the pathological role of sVAP-1/SSAO to generate hydrogen peroxide and toxic aldehyde ACR, both of which are associated with oxidative stress, as a consequence of spermine oxidation in eyes with PDR.

Vascular Adhesion Protein-1 Blockade Suppresses Ocular Inflammation After Retinal Laser Photocoagulation in Mice
Takashi Matsuda, Kousuke Noda, Miyuki Murata, Akiko Kawasaki, Atsuhiro Kanda, Yukihiko Mashima, Susumu Ishida
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 58, 7, 3254, 3261, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2017年06月, ［査読有り］
英語, 研究論文（学術雑誌）, PURPOSE. To investigate the effect of the vascular adhesion protein-1 (VAP-1) inhibitor RTU-1096 on retinal morphologic changes and ocular inflammation after retinal laser photocoagulation in mice.
METHODS. C57BL/6JJcl mice were fed a diet containing RTU-1096, a specific inhibitor for VAP-1, or a control diet ad libitum for 7 days. Laser photocoagulation was performed on the peripheral retina of the animals. The semicarbazide sensitive amine oxidase (SSAO) activities in plasma and chorioretinal tissues were measured. Optical coherence tomography (OCT) images were acquired before and at 1, 3, and 7 days after laser photocoagulation, and thickness of the individual retinal layers was measured. Intravitreal leukocyte infiltration was assessed by histologic analysis. The expression level of intercellular adhesion molecule-1 (ICAM-1) in retinal tissues were examined by quantitative real-time PCR.
RESULTS. One day after laser photocoagulation, the thickness of the outer nuclear layer (ONL) increased in the laser group compared with in the control group, and RTU-1096 administration abrogated the ONL thickening. Histologic analysis and OCT observation revealed that laser photocoagulation caused infiltration of inflammatory cells and the appearance of hyperreflective foci at the vitreoretinal surface, both of which were suppressed by RTU-1096 administration. In addition, systemic administration of RTU-1096 reduced upregulation of the leukocyte adhesion molecules ICAM-1 in the retina.
CONCLUSIONS. The current data indicate that VAP-1/SSAO inhibition may be a potential therapeutic strategy for the prevention of macular edema secondary to scatter laser photocoagulation in patients with ischemic retinal diseases such as diabetic retinopathy.

Evaluation of the Safety and Tolerability of Conjunctival Ring for Posterior Segment of the Eye
Satoshi Kinoshita, Takeshi Ohguchi, Kousuke Noda, Miyuki Murata, Shin-ichi Yasueda, Haruka Obata, Toru Matsunaga, Tsutomu Fukushima, Atsuhiro Kanda, Susumu Ishida
CURRENT EYE RESEARCH, 42, 8, 1149, 1154, TAYLOR & FRANCIS INC, 2017年, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: To evaluate the safety and tolerability of conjunctival rings (CRs), a novel device for drug delivery to the posterior segment of the eye.Methods: In animal studies, CRs containing 5% dexamethasone sodium phosphate (DSP) or vehicle solution were placed on the right and left eyes of C57BL/6J mice, respectively. Contact lenses (CLs) containing vehicle solution were used as a control. Twenty-four hours after placement of the CRs, corneal fluorescein staining was graded based on the McDonald-Shadduck scoring system, ranging from 0 to 4. In humans, CRs containing vehicle solution were placed on the right eye of healthy volunteers for 9 hours. The corneal curvature, corneal thickness, intraocular pressure, visual acuity, tear production (Schirmer I test), tear film break-up time and fluorescein staining scores of the cornea (scores ranging from 0 to 3) and conjunctiva (scores ranging from 0 to 6) were assessed before and after wearing the CRs. The release characteristics of DSP from CRs were also evaluated.Results: In animal experiments, corneal fluorescein staining scores were 1 or less in all the groups, and there was no significant difference between the CR group and the CL group. In the preclinical safety evaluation of CR for humans, ophthalmic examination revealed that CR caused no significant changes in all the parameters investigated including corneal curvature (p = 0.77), corneal thickness (p = 0.96), intraocular pressure (p = 0.59), visual acuity (p = 0.14), Schirmer I test results (p = 0.76), tear film break-up time (p = 0.68), corneal fluorescein staining scores (p = 0.64), and conjunctival fluorescein staining scores (p = 0.52). The DSP release from CRs occurs within a few hours, which is similar to the drug-release property of medicated CL, as reported previously.Conclusions: The current data showed the safety and tolerability of CR as a drug delivery device for the treatment of posterior segment diseases.

Localization of Acrolein-Lysine Adduct in Fibrovascular Tissues of Proliferative Diabetic Retinopathy
Yoko Dong, Kousuke Noda, Miyuki Murata, Shiho Yoshida, Wataru Saito, Atsuhiro Kanda, Susumu Ishida
CURRENT EYE RESEARCH, 42, 1, 111, 117, TAYLOR & FRANCIS INC, 2017年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: To determine the presence of N-epsilon-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), unsaturated aldehyde acrolein-derived lipoxidation end-product, in fibrovascular tissues obtained from patients with proliferative diabetic retinopathy (PDR).Methods: Fibrovascular tissues were collected from 11 eyes of 11 patients with PDR and paraffin-embedded tissue sections were prepared. Tissue localization of FDP-Lys was studied by immunohistochemistry. Signal intensity was quantified by two masked evaluators and graded into three discrete categories. The relationship between FDP-Lys staining and vascular density was analyzed. In addition, subcellular localization of FDP-Lys was studied by immunofluorescent microscopy. The impact of acrolein on cell viability and proliferation was assessed and the expression level of heme oxygenase-1 (HO-1) mRNA was quantified by real-time polymerase chain reaction (PCR) in cultured retinal microvascular endothelial cells.Results: In fibrovascular tissues, FDP-Lys staining was found in vascular components containing CD34-positive cells and alpha smooth muscle actin (-SMA)-positive cells, and clusters of rabbit anti-glial fibrillary acid protein (GFAP)-positive cells. Immunofluorescent staining depicted subcellular localization of FDP-Lys in the nucleus and cytoplasm of the cells. Morphological analysis revealed that fibrovascular tissues with FDP-Lys staining in vascular components showed high vascular density. Exposure of cultured endothelial cells to high concentration of acrolein resulted in the decrease of cell viability and proliferation, whereas lower concentration of acrolein increased cell viability and proliferation. Sublethal concentration of acrolein upregulated HO-1 mRNA expression in retinal microvascular endothelial cells.Conclusions: The current data demonstrated the presence of FDP-Lys in fibrovascular tissues and indicate its involvement in fibrovascular proliferation in PDR.

Regulation of vascular endothelial growth factor-C by tumor necrosis factor-α in the conjunctiva and pterygium.
Dong Y, Kase S, Dong Z, Fukuhara J, Tagawa Y, Ishizuka ET, Murata M, Shinmei Y, Ohguchi T, Kanda A, Noda K, Ishida S
International journal of molecular medicine, 38, 2, 545, 550, SPANDIDOS PUBL LTD, 2016年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Vascular endothelial growth factor C (VEGF-C) plays an important role in the development of a pterygium through lymphangiogenesis. We examined the association between VEGF-C and tumor necrosis factor- (TNF-) in the pathogenesis of pterygia. Cultured conjunctival epithelial cells were treated with TNF-, and the gene expression levels of VEGFC were evaluated by quantitative polymerase chain reaction (qPCR) and VEGF-C protein expression levels were measured using an enzyme-linked immunosorbent assay (ELISA). In addition, using ELISA, we evaluated the VEGF-C protein expression in the supernatants of cultured conjunctival epithelial cells, in which we neutralized TNF- using anti-TNF- antibody. The gene expression of tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), known as TNF receptor 1 (TNFR1), was confirmed using reverse transcription PCR in cultured conjunctival epithelial cells. Immunofluorescence microscopy was used to examine the localization of VEGF-C and TNFR1 in pterygium tissues and TNFR1 expression in cultured conjunctival epithelial cells. Immunohistochemistry was used to examine the localization of TNFR1 in pterygia and normal conjunctival tissues. VEGFC gene expression increased in cultured conjunctival epithelial cells 24 h after the addition of TNF-. The secretion of VEGF-C protein was significantly increased 48 h after the stimulation of cultured conjunctival epithelial cells with TNF-. Increased VEGF-C protein secretion stimulated by TNF- was significantly reduced by anti-TNF- neutralizing antibody treatment. In cultured conjunctival epithelial cells, TNFRSF1A and TNFR1 were expressed. TNFR1 was immunolocalized in normal conjunctival tissues and in human pterygium tissues as well as in VEGF-C-positive epithelial cells from human pterygia. Our data demonstrate that TNF- mediates VEGF-C expression, which plays a critical role in the pathogenesis of pterygia.

Phosphorylation of alphaB-crystallin in epiretinal membrane of human proliferative diabetic retinopathy.
Dong Y, Dong Z, Kase S, Ando R, Fukuhara J, Kinoshita S, Inafuku S, Tagawa Y, Ishizuka ET, Saito W, Murata M, Kanda A, Noda K, Ishida S
International journal of ophthalmology, 9, 8, 1100, 1105, 2016年, ［査読有り］

Alteration of N-Glycan Profiles in Diabetic Retinopathy
Saori Inafuku, Kousuke Noda, Maho Amano, Tetsu Ohashi, Chikako Yoshizawa, Wataru Saito, Miyuki Murata, Atsuhiro Kanda, Shin-Ichiro Nishimura, Susumu Ishida
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 56, 9, 5316, 5322, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）, PURPOSE. To investigate the alteration of vitreal N-glycans in patients with proliferative diabetic retinopathy (PDR).
METHODS. Plasma and vitreous samples were collected from 17 patients (10 females and 7 males) with PDR (PDR group) and 17 nondiabetic patients (8 females and 9 males) with epiretinal membrane (ERM) and idiopathic macular hole (MH) (non-diabetes mellitus [DM] group). Profiles of N-glycans were analyzed by a glycoblotting-based high-throughput protocol that we recently developed. Human retinal microvascular endothelial cells (HRMECs) were cultivated with culture media containing either low glucose (5 mM) or high glucose (25 mM), and expression levels of sialyltransferases were analyzed by real-time PCR and ELISA.
RESULTS. Amount of N-glycans in the vitreous fluid of the PDR group was significantly higher than that of the non-DM group (495.5 +/- 37.4 vs. 142.7 +/- 30.8 pmol/100 mu g protein, P < 0.005), whereas there was no significant difference in the plasma samples between the PDR and the non-DM group. In addition, profile analysis showed that N-glycans with sialic acids increased in the vitreous of the PDR group (328.4 +/- 25.8 pmol/100 mu g protein) compared to the non-DM group (92.1 +/- 21.2 pmol/100 mu g protein, P < 0.0005). Expression levels of sialyltransferases ST3GAL1 and ST3GAL4 were upregulated in the HRMECs after high-glucose stimulation. Consistent with the real-time PCR data, high-glucose stimulation elevated the protein levels of ST3GAL1 (117.4 +/- 14.9 pg/mg, P < 0.01) and ST3GAL4 (6.1 +/- 0.9 pg/mg, P < 0.05) in the HRMECs compared with the cells cultured with low-glucose culture media (ST3GAL1, 64.4 +/- 5.8 pg/mg; ST3GAL4, 3.8 +/- 0.3 pg/mg).
CONCLUSIONS. Our data demonstrate distinct changes in the N-glycan profile and an increase in sialylated N-glycans in eyes with PDR.

Specific inhibition of serine/arginine-rich protein kinase attenuates choroidal neovascularization
Zhenyu Dong, Kousuke Noda, Atsuhiro Kanda, Junichi Fukuhara, Ryo Ando, Miyuki Murata, Wataru Saito, Masatoshi Hagiwara, Susumu Ishida
MOLECULAR VISION, 19, 536, 543, MOLECULAR VISION, 2013年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: To investigate the applicability of serine/arginine-rich protein kinase (SRPK)-specific inhibitor, SRPIN340, for attenuation of choroidal neovascularization (CNV) formation using a mouse model.
Methods: Laser photocoagulation was performed to induce CNV in C57BL/'6J mice, followed by intravitreal injection of SRPIN340 or vehicle. Seven days after the treatment, the CNV size was evaluated using a flatmount technique. Protein levels of vascular endothelial growth factor (VEGF) and inflammation-associated molecules, such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, in the retinal pigment epithelium-choroid complex were measured with enzyme-linked immunosorbent assay. Expression levels of total Vegf, exon 8a-containing Vegf isoforms, and F4/80 (a specific marker for macrophage) were assessed using real-time PCR.
Results: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the vehicle, SRPIN340 significantly decreased the protein levels of VEGF, MCP-1, ICAM-1, and consequently inhibited macrophage infiltration. Furthermore, SRPIN340 suppressed the gene expression levels of total Vegf and exon 8a-containing Vegf isoforms.
Conclusions: SRPIN340, a specific inhibitor of SRPK, suppressed Vegf expression and attenuated CNV formation. Our data suggest the possibility that SRPIN340 is applicable for neovascular age-related macular degeneration as a novel chemical therapeutics.

Tissue Kallikrein Attenuates Choroidal Neovascularization via Cleavage of Vascular Endothelial Growth Factor
Junichi Fukuhara, Kousuke Noda, Miyuki Murata, Shiho Namba, Satoshi Kinoshita, Zhenyu Dong, Ryo Ando, Anton Lennikov, Atsuhiro Kanda, Susumu Ishida
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 54, 1, 274, 279, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2013年01月, ［査読有り］
英語, 研究論文（学術雑誌）, PURPOSE. To investigate the antiangiogenic properties of tissue kallikrein in a murine model of laser-induced choroidal neovascularization (CNV).
METHODS. CNV was induced in male C57BL/6J mice by laser photocoagulation. The animals received daily subcutaneous injections of tissue kallikrein (50 lg/kg) or vehicle control for 2 days before the laser photocoagulation, and this treatment continued until sample collection. Seven days after laser injury, the CNV size was quantified. The levels of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 were assessed by enzyme-linked immunosorbent assay 3 days after laser injury. Cleavage of mouse VEGF with tissue kallikrein was assessed in vivo and in vitro. The protein levels of bradykinin were assessed in the RPE-choroid complexes and hearts.
RESULTS. A significant decrease in CNV size was observed in animals treated with tissue kallikrein (27,168.3 +/- 2432.2 mu m(2)) compared with vehicle-treated controls (36,374.6 +/- 3204.1 mu m(2), P < 0.05). Tissue kallikrein treatment significantly reduced MCP-1, ICAM-1, and IL-6 levels in RPE-choroid complexes. Furthermore, immunoblotting showed the bands, presumably corresponding to the fragmented VEGF(164) protein, in the samples of both mouse VEGF preincubated with tissue kallikrein and RPE-choroid complexes obtained from animals treated with tissue kallikrein. In addition, bradykinin was unchanged in the RPE-choroid complexes of animals treated with tissue kallikrein, whereas the level of bradykinin was increased in the heart obtained from these experimental animals.
CONCLUSIONS. The current data indicate that kallikrein exhibits antiangiogenic properties by cleaving VEGF164 in a laser-induced CNV model. (Invest Ophthalmol Vis Sci. 2013; 54: 274279) DOI: 10.1167/iovs.12-10512

ALPHAB-CRYSTALLIN EXPRESSION IN EPIRETINAL MEMBRANE OF HUMAN PROLIFERATIVE DIABETIC RETINOPATHY
Zhenyu Dong, Satoru Kase, Ryo Ando, Junichi Fukuhara, Wataru Saito, Atsuhiro Kanda, Miyuki Murata, Kousuke Noda, Susumu Ishida
RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES, 32, 6, 1190, 1196, LIPPINCOTT WILLIAMS & WILKINS, 2012年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: To examine the expression of alphaB-crystallin and its colocalization with vascular endothelial growth factor in the epiretinal membrane of human proliferative diabetic retinopathy.
Methods: Ten epiretinal membranes of proliferative diabetic retinopathy and three normal retinas surgically excised were included in this study. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with alphaB-crystallin, vascular endothelial growth factor, and CD31 antibodies.
Results: AlphaB-crystallin was expressed in all epiretinal membranes examined. The immunolocalization of alphaB-crystallin was detected in the cytoplasm of CD31-positive endothelial cells, but not in normal retinal blood vessels. Furthermore, alphaB-crystallin immunoreactivity was colocalized in vascular endothelial growth factor-positive endothelial cells in proliferative diabetic retinopathy membranes.
Conclusion: AlphaB-crystallin was expressed in proliferative diabetic retinopathy membranes, and colocalized with vascular endothelial growth factor-positive neovessels. AlphaB-crystallin may play a potential role in the pathogenesis of epiretinal membranes in proliferative diabetic retinopathy, together with vascular endothelial growth factor. RETINA 32:1190-1196, 2012

Soluble Vascular Adhesion Protein-1 Accumulates in Proliferative Diabetic Retinopathy
Miyuki Murata, Kousuke Noda, Junichi Fukuhara, Atsuhiro Kanda, Satoru Kase, Wataru Saito, Yoko Ozawa, Satsuki Mochizuki, Shioko Kimura, Yukihiko Mashima, Yasunori Okada, Susumu Ishida
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 53, 7, 4055, 4062, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2012年06月, ［査読有り］
英語, 研究論文（学術雑誌）, PURPOSE. Vascular adhesion protein (VAP)-1, a multifunctional molecule with adhesive and enzymatic properties, is expressed at the surface of vascular endothelial cells of mammals. It also exists as a soluble form (sVAP-1), which is implicated in oxidative stress via its enzymatic activity. This study explores a link between increased level of sVAP-1 and oxidative stress in proliferative diabetic retinopathy (PDR) with a focus on mechanistic components to form sVAP-1 by shedding from retinal endothelial cells.
METHODS. Protein levels of sVAP-1 and N epsilon-(hexanoyl) lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with PDR or non-PDR were measured by ELISA. The mechanism of VAP-1 shedding under diabetic condition, exposure to high glucose and/or inflammatory cytokines, was explored using cultured retinal capillary endothelial cells.
RESULTS. Protein level of sVAP-1 was increased and correlated with HEL in the vitreous fluid of patients with PDR. Retinal capillary endothelial cells released sVAP-1 when stimulated with high glucose or inflammatory cytokines, such as TNF-alpha and IL-1 beta in vitro. Furthermore, matrix metalloproteinase-2 and -9, type IV collagenases, were the key molecules to mediate the protein cleavage of VAP-1 from retinal capillary endothelial cells.
CONCLUSIONS. Our data for the first time provide evidence on the link between sVAP-1 and type IV collagenases in the pathogenesis of PDR. (Invest Ophthalmol Vis Sci. 2012;53:4055-4062) DOI:10.1167/iovs.12-9857

Amelioration of ultraviolet-induced photokeratitis in mice treated with astaxanthin eye drops
Anton Lennikov, Nobuyoshi Kitaichi, Risa Fukase, Miyuki Murata, Kousuke Noda, Ryo Ando, Takeshi Ohguchi, Tetsuya Kawakita, Shigeaki Ohno, Susumu Ishida
MOLECULAR VISION, 18, 49, 455, 464, MOLECULAR VISION, 2012年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: Ultraviolet (UV) acts as low-dose ionizing radiation. Acute UVB exposure causes photokeratitis and induces apoptosis in corneal cells. Astaxanthin (AST) is a carotenoid, present in seafood, that has potential clinical applications due to its high antioxidant activity. In the present study, we examined whether topical administration of AST has preventive and therapeutic effects on UV-photokeratitis in mice.
Methods: C57BL/6 mice were administered with AST diluted in polyethylene glycol (PEG) in instillation form (15 mu l) to the right eye. Left eyes were given vehicle alone as controls. Immediately after the instillation, the mice, under anesthesia, were irradiated with UVB at a dose of 400 mJ/cm(2). Eyeballs were collected 24 h after irradiation and stained with H&E and TUNEL. In an in vitro study, mouse corneal epithelial (TKE2) cells were cultured with AST before UV exposure to quantify the UV-derived cytotoxicity.
Results: UVB exposure induced cell death and thinning of the corneal epithelium. However, the epithelium was morphologically well preserved after irradiation in AST-treated corneas. Irradiated corneal epithelium was significantly thicker in eyes treated with AST eye drops, compared to those treated with vehicles (p < 0.01), in a doses dependent manner. Significantly fewer apoptotic cells were observed in AST-treated eyes than controls after irradiation (p < 0.01). AST also reduced oxidative stress in irradiated corneas. The in vitro study showed less cytotoxicity of TKE2 cells in AST-treated cultures after UVB-irradiation (p < 0.01). The cytoprotective effect increased with the dose of AST.
Conclusions: Topical AST administration may be a candidate treatment to limit the damages by UV irradiation with wide clinical applications.

Immunolocalization of Vascular Adhesion Protein-1 in Human Conjunctival Tumors
Junichi Fukuhara, Satoru Kase, Kousuke Noda, Miyuki Murata, Mika Noda, Ryo Ando, Zhenyu Dong, Atsuhiro Kanda, Susumu Ishida
OPHTHALMIC RESEARCH, 48, 1, 33, 37, KARGER, 2012年, ［査読有り］
英語, 研究論文（学術雑誌）, Objective: We analyzed the expression and immunolocalization of vascular adhesion protein (VAP)-1 in conjunctival tumors and normal conjunctival tissue of humans. Methods: Nine conjunctival tumors, including pyogenic granuloma and extranodal marginal zone B-cell lymphoma (EMZL), and 2 normal conjunctivas were analyzed by immunohistochemistry for VAP-1 and CD31 expression. Results: Immunoreactivity for VAP-1 was detected in the lumen of microvessels in pyogenic granuloma and in EMZLs. In contrast, normal bulbar conjunctival tissues demonstrated weak cytoplasmic immunoreactivity for VAP-1 in the blood vessels. Conclusions: The immunolocalization of VAP-1 varied in the histopathology of the conjunctiva, involving the pathology of inflammatory conjunctival disorders. Copyright (c) 2012 S. Karger AG, Basel

Tissue factor expression in human pterygium
Ryo Ando, Satoru Kase, Tsutomu Ohashi, Zhenyu Dong, Junichi Fukuhara, Atsuhiro Kanda, Miyuki Murata, Kousuke Noda, Nobuyoshi Kitaichi, Susumu Ishida
MOLECULAR VISION, 17, 8-10, 63, 69, MOLECULAR VISION, 2011年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Purpose: A pterygium shows tumor-like characteristics, such as proliferation, invasion, and epithelial-mesenchymal transition (EMT). Previous reports suggest that tissue factor (TF) expression is closely related to the EMT of tumor cells, and subsequent tumor development. In this study, we analyzed the expression and immunolocalization of TF in pterygial and normal conjunctival tissues of humans.
Methods: Eight pterygia and three normal bulbar conjunctivas, surgically removed, were used in this study. Formalin-fixed, paraffin-embedded tissues were submitted for immunohistochemical analysis with anti-TF antibody. Double staining immunohistochemistry was performed to assess TF and alpha-smooth muscle actin (alpha-SMA) or epidermal growth factor receptor (EGFR) expression in the pterygia.
Results: Immunoreactivity for TF was detected in all pterygial tissues examined. TF immunoreactivity was localized in the cytoplasm of basal, suprabasal, and superficial epithelial cells. The number of TF-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival epithelial cells (p<0.001). TF immunoreactivity was detected in alpha-SMA-positive or -negative pterygial epithelial cells. EGFR immunoreactivity was detected in pterygial epithelium, which was colocalized with TF.
Conclusions: These results suggest that TF plays a potential role in the pathogenesis and development of a pterygium, and that TF expression might be involved through EMT-dependent and -independent pathways.

Requirement of STAT3 activation for maximal collagenase-1 (MMP-1) induction by epidermal growth factor and malignant characteristics in T24 bladder cancer cells
M Itoh, T Murata, T Suzuki, M Shindoh, K Nakajima, K Imai, K Yoshida
ONCOGENE, 25, 8, 1195, 1204, NATURE PUBLISHING GROUP, 2006年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Signal transducers and activators of transcription (STATs) are latent transcription factors that mediate cytokine- and growth factor-induced transcription. Constitutive activation of STAT3 has been shown in human cancers and transformed cell lines. We report that STAT3, but not STAT1 and STAT5, becomes phosphorylated in response to epidermal growth factor (EGF) and achieves maximal induction of collagenase-1 (MMP1) transcription by interacting with c-JUN. Phosphorylation of STAT3 protein is biphasic: the first peak within 30 min and the second peak between 4 and 8 h. Association of STAT3 with c-JUN is detected and its constituting STAT3 is increasingly phosphorylated. The STAT and AP-1 elements are necessary for effective induction of MMP-1 promoter by EGF. Mutation of AP-1 element closely located at the STAT site abolishes the binding not only of c-JUN but also of STAT3 to MMP-1 promoter, resulting in the loss of the responsiveness to EGF. By blocking STAT3 activity with the dominant-negative form, we show the requirement of STAT3 for EGF induction of MMP-1 and MMP-10 (stromelysin-2). Furthermore, expression of the dominant-negative STAT3 is sufficient to inhibit the constitutive and EGF-inducible cell migration and invasion and the tumor formation in nude mice. These results demonstrate that STAT3 phosphorylation and its possible interaction with c-JUN are required for the strong responsiveness of MMP-1 to EGF, and STAT3 activation is crucial for exhibition of malignant characteristics in T24 bladder cancer cells.

E1AF degradation by a ubiquitin-proteasome pathway
A Takahashi, F Higashino, M Aoyagi, K Yoshida, M Itoh, M Kobayashi, Y Totsuka, T Kohgo, M Shindoh
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 327, 2, 575, 580, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2005年02月, ［査読有り］
英語, 研究論文（学術雑誌）, E1AF is a member of the ETS family of transcription factors. In mammary tumors. overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway. which has some effect on E1AF function. (C) 2004 Elsevier Inc. All rights reserved.

EWS/ETS fusions activate telomerase in Ewing's tumors
A Takahashi, F Higashino, M Aoyagi, K Yoshida, M Itoh, S Kyo, T Ohno, T Taira, H Ariga, K Nakajima, M Hatta, M Kobayashi, H Sano, T Kohgo, M Shindoh
CANCER RESEARCH, 63, 23, 8338, 8344, AMER ASSOC CANCER RESEARCH, 2003年12月, ［査読有り］
英語, 研究論文（学術雑誌）, EWS/ETS is a chimeric protein identified in most Ewing's sarcomas. Although EWS/ETS has been shown to activate transcription as a transcription factor, the detailed targets of EWS/ETS in transformed cells have not been clarified. Herein, we demonstrate that telomerase is a new target of EWS/ETS fusions. Both telomerase activity and the expression level of telomerase reverse transcriptase (TERT) mRNA were up-regulated in NIH3T3 cells transformed by EWS/E1AF and EWS/FLI1 as well as in two Ewing's sarcoma cell lines. Luciferase assay using the TERT promoter revealed that EWS/E1AF and EWS/FLI1 function as positive regulators of TERT transcription in an ETS binding site-independent manner. EWS/ETS appeared to be included in the initiation complex of TERT transcription and to cooperate with CREB-binding protein (CBP)/ p300. When EWS/FLI1 was knocked down in Ewing's sarcomas cells by RNA interference, the expression level of TERT mRNA and the telomerase activity were significantly decreased. These findings indicate that EWS/ETS fusion proteins activate human telomerase activity in Ewing's tumors through up-regulation of TERT gene expression, probably as a transcriptional coactivator.

Nuclear export of glucocorticoid receptor is enhanced by c-Jun N-terminal kinase-mediated phosphorylation
M Itoh, M Adachi, H Yasui, M Takekawa, H Tanaka, K Imai
MOLECULAR ENDOCRINOLOGY, 16, 10, 2382, 2392, ENDOCRINE SOC, 2002年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The c-Jun N-terminal kinase (JNK) phosphorylates the glucocorticoid receptor (GR) and inhibits GR-mediated transcription. However, the biological effect of the GR phosphorylation remains unknown. Here we demonstrate that activated JNK phosphorylates human GR at Ser226 and enhances its nuclear export after withdrawal of a ligand for GR, dexamethasone. At I h after dexamethasone withdrawal, green fluorescent protein-GR molecules were mostly retained at the nucleus, whereas UV exposure enhanced its nuclear export, and approximately 30-40% of cells revealed distinct nuclear export. JNK overexpression alone mimics UV exposure and enhanced GR export accompanied by inhibition of GR-mediated transcription. However, mutation of the Ser226 JNK phosphorylation site in GR abrogated UV-mediated enhancement of GR nuclear export. Furthermore, overexpression of a dominant negative SEK1 mutant also abrogated the effects of UV exposure on GR export. Taken together, these findings suggest that JNK-mediated phosphorylation of the GR-Ser226 enhances GR nuclear export and may contribute to termination of GR-mediated transcription.

Involvement of p27(KIP1) degradation by Skp2 in the regulation of proliferation in response to wounding of corneal epithelium
K Yoshida, K Nakayama, H Nagahama, T Harada, C Harada, J Imaki, A Matsuda, K Yamamoto, M Ito, S Ohno, K Nakayama
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 43, 2, 364, 370, ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2002年02月, ［査読有り］
英語, 研究論文（学術雑誌）, PURPOSE. To examine the expression of the p27(KIP1) in the normal and epithelial-scraped cornea and whether degradation of p27(KIP1) by Skp2 is involved in the regulation of cell proliferation in response to wounding of the corneal epithelium.
METHODS. C57B16, p27(KIP1-/-), Skp2(-/-), and Skp2(-/-)/p27(KIP1-/-) double-knockout mice were examined. Normal and epithelial-scraped corneas were analyzed by imniunocyto-chemistry using anti-p27(KIP1) antibody. Cells in the S phase of DNA synthesis were analyzed by immunocytochemistry using anti-bromodeoxyuridine (BrdU) antibody.
RESULTS. The p27(KIP1) was expressed in basal cells of the central and peripheral region of the cornea and limbus. This expression was not detected 24 hours after the epithelial scraping, when there were many cells in the S phase of DNA synthesis in the corneal epithelium. There were no obvious differences in the thickness and anti-BrdU staining in the corneal epithelium of p27(KIP1-/-) mice from that of control animals. Twenty-four hours after epithelial scraping in the Skp2(-/-) mice, the corneal epithelium was thinner than in wild-type mice and had many p27(KIP1)-positive cells and few BrdU-positive cells. In contrast. 24 hours after epithelial scraping in the Skp2(-/-)/p27(KIP1-/-) double-knockout mice, the corneal epithelium was as thick as in wild-type mice and had many BrdU-positive cells.
CONCLUSIONS. These results suggest that degradation of p27(KIP1) by Skp2 is involved in the regulation of cell proliferation in response to wounding of the corneal epithelium.

ニューラルネットワークによる眼底写真の網膜動静脈検出
福津佳苗, 齋藤理幸, 野田航介, 村田美幸, 加瀬諭, 柴涼介, 磯貝直己, 道家充, 石田晋, 日本眼循環学会講演抄録集, 36th, 2019年

眼科のトランスレーショナルリサーチ 多機能蛋白質に着目した糖尿病網膜症に対する創薬研究　　　　　　　　　　　　　　　
野田 航介, 村田 美幸, 稲福 沙織, 松田 剛, 吉田 志帆, 董 陽子, 木下 哲志, 安藤 亮, 藤谷 顕雄, 齋藤 理幸, 董 震宇, 森 祥平, 加瀬 諭, 吉澤 史子, 齋藤 航, 神田 敦宏, 石田 晋, 眞島 行彦, 笹瀬 智彦, 天野 麻穂, 大橋 哲, 西村 伸一郎, 今川 貴仁, 白仁田 明生, 日本眼科学会雑誌, 122, 3, 223, 248, 2018年03月
近年の基礎研究は、血管内皮増殖因子(vascular endothelial growth factor:VEGF)が糖尿病網膜症の病態形成に主要な役割を演じることを明らかとした。そして、同分子群に対する阻害薬の臨床応用は糖尿病網膜症の治療予後を劇的に改善し、現在我々はanti-VEGF eraと呼ばれるこの時代において同疾患の治療体系が刻々と変貌するのを目の当たりにしている。かつては光凝固と硝子体手術のみが進行した糖尿病網膜症に対する治療手段であったことを考えると隔世の感がある。しかしながらその一方で、情報システムの革新と研究技術の進歩を背景に蓄積される基礎および臨床研究の成果は、網膜症病態の複雑さ、VEGF単独阻害による治療の限界、そしてその弊害の可能性なども浮き彫りにした。そして、その必然としてVEGF以外の病態責任分子を標的とした糖尿病網膜症に対する創薬研究が全世界で現在行われ、複数の分子標的製剤が糖尿病網膜症の治療オプションとなるpost anti-VEGF eraが目前に迫ってきている。糖尿病網膜症の発症および進展には、慢性炎症、そして酸化ストレスの関与が知られている。本研究においては、糖鎖など新規標的分子の探索的研究を行うとともに、この二つの病態に関わる分子としてvascular adhesion protein-1(VAP-1)/semicarbazide sensitive amine oxidase(SSAO)の糖尿病網膜症病態における役割についての検討を主に行った。VAP-1/SSAOは血管内皮細胞に発現する白血球接着分子だが、その一方で酵素活性も持つ多機能蛋白質"moonlighting protein"であり、慢性炎症と酸化ストレスの双方に関わる重要な分子の一つである。本研究ではVAP-1/SSAOが糖尿病網膜症の病態形成に白血球接着分子として関与する一方、遊離型蛋白質としてその眼内に蓄積すること、そしてその機序にVEGFや蛋白質分解酵素matrix metalloproteinasesが関与することを明らかにした。また、VAP-1/SSAOは酵素として過酸化水素および不飽和アルデヒドの一種アクロレインを産生し、血管内皮細胞における酸化ストレス亢進に寄与することとその機序を見出した。以上の検討結果に基づいて、本稿では糖尿病網膜症におけるVAP-1/SSAO阻害剤による治療可能性について述べたい。(著者抄録), (公財)日本眼科学会, 日本語

増殖糖尿病網膜症患者の硝子体中アクロレイン結合蛋白FDP-lysine濃度　　　　　　　　　　　　　　　
森 祥平, 村田 美幸, 野田 航介, 鈴木 智浩, 齋藤 理幸, 安藤 亮, 加瀬 諭, 齋藤 航, 神田 敦宏, 石田 晋, 眼科臨床紀要, 10, 11, 920, 921, 2017年11月
眼科臨床紀要会, 日本語

網膜グリア細胞に対するアクロレインの作用　　　　　　　　　　　　　　　
野田 航介, 村田 美幸, 吉田 志帆, 神田 敦宏, 石田 晋, 糖尿病合併症, 31, Suppl.1, 347, 347, 2017年10月
(一社)日本糖尿病合併症学会, 日本語

糖尿病網膜症におけるポリアミン代謝の病態関与　　　　　　　　　　　　　　　
野田 航介, 村田 美幸, 神田 敦宏, 石田 晋, 糖尿病合併症, 30, Suppl.1, 248, 248, 2016年09月
(一社)日本糖尿病合併症学会, 日本語

糖尿病網膜症の硝子体および血漿におけるN型糖鎖プロファイル　　　　　　　　　　　　　　　
稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋, 日本糖尿病眼学会誌, 20, 114, 114, 2016年03月
日本糖尿病眼学会, 日本語

Alteration of N-glycan Profiles in Diabetic Retinopathy
Saori Inafuku, Kousuke Noda, Maho Amano, Miyuki Murata, Wataru Saito, Tetsu Ohashi, Atsuhiro Kanda, Shin-Ichiro Nishimura, Susumu Ishida, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 56, 7, 2015年06月
ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 英語, 研究発表ペーパー・要旨（国際会議）

糖尿病網膜症の硝子体および血漿におけるN型糖鎖の網羅的解析　　　　　　　　　　　　　　　
稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 吉澤 史子, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋, 日本眼科学会雑誌, 119, 臨増, 160, 160, 2015年03月
(公財)日本眼科学会, 日本語

糖負荷刺激によるシアル酸転移酵素の誘導　　　　　　　　　　　　　　　
稲福 沙織, 野田 航介, 天野 麻穂, 大橋 哲, 齋藤 航, 村田 美幸, 神田 敦宏, 西村 紳一郎, 石田 晋, 日本眼科学会雑誌, 119, 臨増, 199, 199, 2015年03月
(公財)日本眼科学会, 日本語

SRPK阻害薬による脈絡膜血管新生の抑制効果　　　　　　　　　　　　　　　
董 震宇, 野田 航介, 斉藤 航, 木下 哲志, 福原 淳一, 安藤 亮, 村田 美幸, 神田 敦宏, 萩原 正敏, 石田 晋, 日本眼科学会雑誌, 117, 臨増, 289, 289, 2013年03月
(公財)日本眼科学会, 日本語

増殖糖尿病網膜症におけるVEGF阻害剤の硝子体内VAP-1濃度への影響　　　　　　　　　　　　　　　
安藤 亮, 野田 航介, 村田 美幸, 難波 志帆, 木下 哲志, 福原 淳一, 董 震宇, アントン・レニコフ, 齋藤 航, 神田 敦宏, 石田 晋, 日本眼科学会雑誌, 116, 臨増, 265, 265, 2012年03月
(公財)日本眼科学会, 日本語

ヒト翼状片におけるTissue Factorの発現　　　　　　　　　　　　　　　
安藤 亮, 加瀬 諭, 大橋 勉, 董 震宇, 福原 淳一, アントン・レニコフ, 神田 敦宏, 村田 美幸, 野田 航介, 石田 晋, 日本眼科学会雑誌, 115, 臨増, 203, 203, 2011年04月
(公財)日本眼科学会, 日本語

アスタキサンチン点眼による紫外線急性角膜炎の軽症化　　　　　　　　　　　　　　　
アントン・レニコフ, 北市 伸義, 深瀬 理沙, 村田 美幸, 野田 航介, 大野 重昭, 石田 晋, 日本眼科学会雑誌, 115, 臨増, 227, 227, 2011年04月
(公財)日本眼科学会, 日本語

増殖糖尿病網膜症の線維血管膜におけるalphaB-crystallinの発現　　　　　　　　　　　　　　　
董 震宇, 加瀬 諭, 安藤 亮, 福原 淳一, アントン・レニコフ, 神田 敦宏, 村田 美幸, 野田 航介, 斎藤 航, 石田 晋, 日本眼科学会雑誌, 115, 臨増, 231, 231, 2011年04月
(公財)日本眼科学会, 日本語

増殖糖尿病網膜症患者の硝子体液におけるVAP-1濃度　　　　　　　　　　　　　　　
福原 淳一, 野田 航介, 村田 美幸, 齋藤 航, 董 震宇, 安藤 亮, アントン・レニコフ, 神田 敦宏, 石田 晋, 日本眼科学会雑誌, 115, 臨増, 231, 231, 2011年04月
(公財)日本眼科学会, 日本語

2022年08月 - 現在
日本眼薬理学会　　　　　　　　　　　　　　　

2019年05月 - 現在
日本糖尿病眼学会　　　　　　　　　　　　　　　

2018年05月 - 現在
日本網膜硝子体学会　　　　　　　　　　　　　　　

2005年04月 - 現在
日本癌学会　　　　　　　　　　　　　　　

1999年06月 - 現在
日本分子生物学会　　　　　　　　　　　　　　　

滲出型加齢黄斑変性の網膜色素上皮障害における毒性アルデヒドアクロレインの関与
科学研究費助成事業
2024年04月01日 - 2027年03月31日
村田 美幸, 野田 航介
日本学術振興会, 基盤研究(C), 北海道大学, 24K12777

網膜ミュラー細胞のグリア間葉移行におけるアルファBクリスタリンの関与
科学研究費助成事業
2024年04月01日 - 2027年03月31日
加瀬 諭, 村田 美幸
日本学術振興会, 基盤研究(C), 北海道大学, 24K12797

加齢黄斑変性におけるセリン/スレオニンキナーゼLRRK2の病態意義解明
科学研究費助成事業
2021年04月01日 - 2025年03月31日
野田 航介, 村田 美幸, 矢部 一郎, 加瀬 諭
加齢黄斑変性(AMD)の病態基盤には網膜色素上皮細胞(RPE)の変性や細胞死が関与している。興味深いことに、AMD患者ではパーキンソン病の発症リスクが有意に高いとする報告が近年相次いでおり、パーキンソン病が神経変性疾患であることから、この2つの疾患には共通病態が存在する可能性があると仮説を立てた。本研究の目的は、「AMDにおけるパーキンソン病との共通病態を探索すること」である。
研究開始初年度は、まずヒト摘出眼球切片を用いてRPEにおけるαシヌクレインおよびリン酸化αシヌクレインの局在を免疫組織染色によって検討した。その結果、ヒト眼球組織のRPEにおいてαシヌクレインとリン酸化αシヌクレインの比較的強い染色シグナルが確認された。次に、RPEにおけるαシヌクレインおよびリン酸化αシヌクレインの産生機序を検討するため、in vitro実験系を用いて検討を行なった。血清除去条件下でヒト培養RPE株hTERT RPE-1を24時間培養した後のαシヌクレインおよびリン酸化αシヌクレインの変化をwestern blottingで検討した。その結果、血清除去によってhTERT RPE-1におけるαシヌクレインおよびリン酸化αシヌクレインのタンパク量は増加していた。また同条件下では、パーキンソン病発症に重要な役割を演じているとされるleucine-rich repeat kinase 2 (LRRK2)のmRNA発現およびタンパク量も増加した。
リン酸化αシヌクレインはオリゴマーを形成することで神経細胞の変性を引き起こすとされる。また、αシヌクレインのリン酸化およびその蓄積には前述のLRRK2が関与するとする報告がある。以上のことは、パーキンソン病と同様に、加齢などの要因によるRPEの変性にもリン酸化αシヌクレインが関与している可能性を示している。
日本学術振興会, 基盤研究(B), 北海道大学, 21H03091

糖尿病網膜症病態におけるアクロレインの炎症誘導機構
科学研究費助成事業
2021年04月01日 - 2024年03月31日
村田 美幸, 野田 航介
糖尿病網膜症は重篤な視機能障害をきたすため、その病態の解明は眼科学における重要な課題である。我々は過去に、増殖糖尿病網膜症患者の 線維血管組織において網膜グリア細胞に不飽和アルデヒドの一種であるアクロレインが結合した蛋白が蓄積していることを報告した。アクロレ インは反応性が高く、様々な蛋白と結合することでその機能異常を惹起するため、神経変性疾患や悪性腫瘍など多様な疾患病態で注目されてい る分子である。本研究では、糖尿病網膜症におけるアクロレインの炎症誘導機構について検討をおこなう。
昨年度までに、アクロレインがラット培養網膜グリア細胞株TR-MUL5において、単球走化性因子(monocyte chemoattractant protein-1、MCP-1)を含む複数の炎症関連分子の発現を誘導することが明らかになった。アクロレインによるMCP-1の発現誘導は酸化ストレス阻害剤であるN-acetyl-cysteine の添加により抑制されたことから、アクロレインによる酸化ストレスの亢進がMCP-1の誘導に関わることが示唆された。トランスウェルの下方にTR-MUL5を播種し、アクロレインを添加して24時間後に上層のウェルにマクロファージ細胞株RAW264.7を播種したところ、アクロレイン無添加群に比べ有意にRAW264.7の遊走の亢進が認められた。MCP-1の受容体であるCCR2の阻害剤をRAW264.7に添加したところ遊走能の亢進が抑制されたことから、アクロレイン刺激によりTR-MUL5からの分泌が増加したMCP-1が、RAW264.7の遊走を促進していることが示された。これらのことから、アクロレインに網膜グリア細胞からのMCP-1の分泌を亢進させ、同細胞へのマクロファージの遊走を促すという作用があることが明らかになった。
日本学術振興会, 基盤研究(C), 北海道大学, 21K09667

Glycer-AGEによる網膜神経病態の機序解明と糖尿病網膜症に対する創薬
科学研究費助成事業
2020年04月01日 - 2024年03月31日
石田 晋, 村田 美幸, 神田 敦宏
糖尿病網膜症は重篤な視機能障害をきたす疾患であり、その病態理解は眼科学における重要課題である。これまでの糖尿病網膜症治療はその血管病態を制御することを目的とされてきたが、近年、糖尿病網膜症における「神経病態」が注目されている。しかし、糖尿病網膜症における神経保護的治療法は現在のところ確立されていない。
グリセルアルデヒド由来の終末糖化産物(Glycer-AGE)は、その細胞障害性の強さからToxic AGE(TAGE)とも呼ばれ、糖尿病網膜症を含む様々な疾患での病態形成への関与が報告されている。申請者グループの検討により、このTAGEが糖尿病モデルマウスの視細胞変性に寄与していることが明らかになった。本研究では、TAGEによる網膜神経病態の機序を解明し、さらにTAGEを標的とした創薬開発を行い、糖尿病網膜症の新規治療法開発を目指す。
本研究におけるこれまでの検討により、ヒト培養網膜色素上皮細胞にグリセルアルデヒドを添加してTAGE化反応を誘導し、抗TAGEモノクローナル抗体で免疫沈降をおこなって質量分析に供し、グリセルアルデヒドを添加しないコントロール群と比較することで、網膜色素上皮細胞内でTAGE化される複数のタンパク質を同定している。また、ヒト培養網膜色素上皮細胞にグリセルアルデヒドと牛血清アルブミン(BSA)を反応させて作製したTAGE化BSAを添加し、TAGE負荷が網膜色素上皮細胞機能に及ぼす影響について現在解析を進めている。
日本学術振興会, 基盤研究(B), 北海道大学, 20H03837

糖尿病網膜症病態における炎症細胞遊走と不飽和アルデヒドの関与について　　　　　　　　　　　　　　　
アカデミックサポート
2021年04月 - 2022年03月
福津佳苗, 村田美幸, 野田航介
バイエル薬品株式会社, 基礎研究, 北海道大学大学院医学研究院眼科学教室, BASJ20210401022

糖尿病網膜症における毒性アルデヒド-アクロレインの産生機構
科学研究費助成事業
2017年04月01日 - 2020年03月31日
村田 美幸, 野田 航介
Vascular adhesion protein (VAP)-1は血管内皮細胞に発現するタンパクであり、白血球接着分子として炎症に関与する一方、アミン酸化酵素としての活性も有する。アクロレインは高反応性の不飽和アルデヒドであり、外因性の毒性物質として煙草などに含まれる他、ポリアミンと呼ばれる生体内分子の酸化などにより内因性にも産生されることが近年報告されている。我々は糖尿病網膜症患者の硝子体においてアクロレイン結合蛋白とVAP-1が共に増加し、正の相関を示すことを見出した。本研究により、VAP-1がポリアミンの一種であるスペルミンを酸化してアクロレインを生成することが明らかになった。
日本学術振興会, 基盤研究(C), 北海道大学, 17K11442

網膜の恒常性維持における胎盤増殖因子(PIGF)の役割の解析
科学研究費助成事業
2015年 - 2015年
村田 美幸
研究目的
血管新生や血管形成に関与する増殖因子には、血管内皮増殖因子(vascular endothelial growth factor, VEGF)-A、VEGF-B、VEGF-C、VEGF-D、VEGF-EおよびPIGF-1、PIGF-2の7種類が知られ、VEGFファミリーと呼ばれている。この中で、VEGF-Aは糖尿病網膜症や加齢黄斑変性などにおいて眼内血管新生を促し病態を悪化させる重要な因子であることが明らかとなり、VEGF-Aを標的とする阻害剤がすでに良好な治療結果をもたらしている。近年開発されたVEGF阻害薬はその標的分子も多様化し、VEGF-AのみならずPIGFを含む複数のVEGFファミリーを阻害する薬剤である。PIGFは血管新生や炎症などに関与する事が報告されているが、その生理的作用は未だ明らかになっていない。本研究では、網膜の恒常性維持に重要な働きをする網膜色素上皮細胞(retinal pigment epithelium, RPE)におけるPIGFの役割を解析した。
研究方法
培養ヒトRPE細胞にPIGF shRNAを導入して遺伝子発現を抑制し、non target shRNAを導入したコントロールと比較をおこなった。
研究結果
PIGF発現を抑制したヒトRPE株では、VEGF受容体(VEGFR)2のタンパク発現が低下していること、VEGFR2下流シグナルである細胞外シグナル調節キナーゼ(extracellular signal-regulated kinase, ERK)1/2やv-akt murine thymoma viral oncogene homolog(AKT)のリン酸化が抑制されることが明らかになった。VEGFR2のmRNA発現に変化は見られなかったことから、PIGFはVEGFR2タンパクの安定性に関与している可能性が示された。
日本学術振興会, 奨励研究, 北海道大学, 15H00579

網膜色素上皮細胞における上皮間葉移行関連転写因子の探索
科学研究費助成事業
2010年 - 2011年
村田 美幸
加齢黄斑変性(AMD)の病型の一つ、滲出型AMDの病態を特徴づける脈絡膜新生血管(CNV)はその終末期病態として線維化をきたし、視力障害の原因となる。近年,組織の線維化においてEpithelial-Mesenchymal Transition(上皮間葉移行:EMT)が着目されているが、ヒトCNV組織検体を用いた我々の検討結果ではEMTを制御する転写因子の一つSnailがヒトCNV中の網膜色素上皮細胞(RPE)に高発現しており,線維化をきたしている組織ほどその陽性率が高いことが明らかとなった。この結果はCNV中に含まれるRPEがEMTを生じていること、EMTの転写因子SnailがCNVの線維化に関与する可能性を示唆していた。
本研究では、培養ヒトRPE細胞株ARPE19を用いたin vitro実験系により、EMT関連転写因子snailの制御機構を解析した。CNV組織において発現が確認されている各種サイトカイン(transforming growth factor (TGF)-b, tumor-necrosis factor (TNF)-a, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF)など)を用いてARPE19細胞を刺激したところ、TGF-b刺激による転写因子Snailの発現増加がRT-PCRおよび免疫組織染色によって確認された。本結果は、ヒトCNV中に存在するTGF-bがEMT転写因子Snailの発現を誘導し、CNV組織においてRPEにEMT変化を惹起するということを示唆していた。
日本学術振興会, 若手研究(B), 北海道大学, 22791644