Papers - Kurihara, Toshihide

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

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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

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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

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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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SOCS3 is required to temporally fine-tune photoreceptor cell differentiation

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

DEVELOPMENTAL BIOLOGY (ACADEMIC PRESS INC ELSEVIER SCIENCE)  303 ( 2 ) 591 - 600 2007.03

Research paper (scientific journal), Accepted,  ISSN  0012-1606

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Suppressor of cytokine signaling 3 (SOCS3) is an intracellular, ligand-induced negative feedback modulator of STAT3 activation that acts during inflammation. Here, we dernonstrate that SOCS3 expression is important for normal retinal development in the perinatal period. STAT3 is highly activated in the late-embryonic retina, then downregulated at postnatal day 0 (PO), presumably by the depletion of upstream ligands. We found that SOCS3 was required after PO to shut down the residual STAT3 activation; this loss of activated STAT3 leads to Rhodopsin expression and rod photoreceptor cell differentiation. SOCS3 deficiency failed to terminate STAT3 activation, thereby delaying expression of Rhodopsin and its upstream transcription factor, crx. Development subsequently continued, but its course was temporally erratic, probably because of faulty compensation. Interestingly, SOCS3 protein expression was first detected postnatally, after STAT3 activation was mostly downregulated. It initially appeared in some of the presumptive photoreceptor cells and gradually spread. SOCS3 mRNA level was constant from the late-embryonic to early-postnatal period. Post-transcriptional inhibition of SOCS3 protein expression maintains a high STAT3 activation during late embryogenesis, and after PO, releasing from the inhibition promptly terminates STAT3 activation. Thus, SOCS3 can act as a temporal fine-tuner of STAT3 activation during photoreceptor cell differentiation. (c) 2006 Elsevier Inc. All rights reserved.

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Neuroprotective effects of angiotensin II type 1 receptor (AT1R) blocker, telmisartan, via modulating AT1R and AT2R signaling in retinal inflammation

Kurihara, Toshihide, Ozawa, Yoko, Shinoda, Kei, Nagai, Norihiro, Inoue, Makoto, Oike, Yuichi, Tsubota, Kazuo, Ishida, Susumu, Okano, Hideyuki

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC)  47 ( 12 ) 5545 - 5552 2006.12

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

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PURPOSE. To investigate the retinal neural damage that occurs during inflammation and the therapeutic effects of the angiotensin II type 1 receptor (AT1R) blocker, telmisartan, using a model of endotoxin- induced uveitis (EIU).
METHODS. The localization of AT1R and AT2R was shown by immunohistochemistry. EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Animals were treated with telmisartan for 2 days and were evaluated 24 hours later. Expression levels of angiotensin II, STAT3 activation induced by inflammatory cytokines, and retinal proteins essential for neural activities (e. g., synaptophysin, rhodopsin) were analyzed by immunoblot. An AT2R antagonist was administered to evaluate the contribution of AT2R signaling in this therapy. Dark-adapted full-field electroretinography ( ERG) was also performed.
RESULTS. AT1R and AT2R were expressed in presynaptic terminals in most of the retinal neurons. AT1R was also expressed in Muller glial cells. During inflammation, angiotensin II expression was elevated, STAT3 was activated, and synaptophysin and rhodopsin expression were reduced. The expression of glial fibrillary acidic protein (GFAP), downstream of STAT3 activation, was induced in Muller glial cells. However, treatment with telmisartan successfully avoided all these changes. An AT2R antagonist lowered synaptophysin expression despite the treatment. STAT3 activity was negatively correlated with rhodopsin expression. Furthermore, ERG responses, which were mostly prevented by telmisartan, were disturbed during inflammation.
CONCLUSIONS. Retinal protein expression and visual function are both disturbed by inflammation. Treatment with the AT1R blocker telmisartan efficiently prevented these signs of retinal neural damage through the reduction of local angiotensin II expression, the blockade of AT1R, and the relative upregulation of AT2R function.

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Pars plana vitrectomy with internal limiting membrane removal for macular hole associated with proliferative diabetic retinopathy.

Kurihara T, Noda K, Ishida S, Inoue M†

Graefes Arch Clin Exp Ophthalmol. (SPRINGER)  243 ( 7 ) 724 - 726 2005.01

Lead author, Accepted,  ISSN  0721-832X

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Background: We describe the outcome of vitreous surgery in three eyes of three patients with macular hole associated with proliferative diabetic retinopathy (PDR) in the absence of fibrovascular proliferation, a combination of conditions where efficacy is incompletely known. Methods: The patients, all male were 62, 65, and 66 years old. Panretinal photocoagulation had been performed preoperatively in all, and one eye had undergone vitreous surgery. No fibrovascular tissue causing macular traction was observed in any case. Fluorescein angiography and optical coherence tomography (OCT) demonstrated persistent diabetic macular edema surrounding the hole. Affected eyes were treated with vitrectomy including internal limiting membrane (ILM) peeling; 20% sulfur hexafluoride gas (SF6) was introduced for tamponade. Results: Anatomical closure of the macular hole as well as resolution of macular edema was achieved in all cases, and vision improved considerably by more than two Snellen lines. Conclusions: Vitreous surgery with ILM peeling was effective for macular hole associated with PDR, attaining not only macular hole closure but also resolution of persistent diabetic macular edema.

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A case of retinal detachment associated with acquired retinoschisis and macular pucker

Toshihide Kurihara, Hajime Shinoda, Tadahiko Eshita, Shizuaki Kitamura, Kei Shinoda, Makoto Inoue, Masaro Ogata

Japanese Review of Clinical Ophthalmology 98 ( 3 ) 205-208 - 208 2004.03

Research paper (scientific journal), Joint Work