Papers - IKEDA Yasuhiro
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Murakami Y., Matsumoto H., Roh M., Suzuki J., Hisatomi T., Ikeda Y., Miller J., Vavvas D.
Proceedings of the National Academy of Sciences of the United States of America 109 ( 36 ) 14598 - 14603 2012.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Proceedings of the National Academy of Sciences of the United States of America
Retinitis pigmentosa comprises a group of inherited retinal photoreceptor degenerations that lead to progressive loss of vision. Although in most cases rods, but not cones, harbor the deleterious gene mutations, cones do die in this disease, usually after the main phase of rod cell loss. Rod photoreceptor death is characterized by apoptotic features. In contrast, the mechanisms and features of subsequent nonautonomous cone cell death remain largely unknown. In this study, we show that receptor-interacting protein (RIP) kinase mediates necrotic cone cell death in rd10 mice, a mousemodel of retinitis pigmentosa caused by a mutation in a rod-specific gene. The expression of RIP3, a key regulator of programmed necrosis, was elevated in rd10 mouse retinas in the phase of cone but not rod degeneration. Although rd10 mice lacking Rip3 developed comparable rod degeneration to control rd10 mice, they displayed a significant preservation of cone cells. Ultrastructural analysis of rd10 mouse retinas revealed that a substantial fraction of dying cones exhibited necrotic morphology, which was rescued by Rip3 deficiency. Additionally, pharmacologic treatment with a RIP kinase inhibitor attenuated histological and functional deficits of cones in rd10 mice. Thus, necrotic mechanisms involving RIP kinase are crucial in cone cell death in inherited retinal degeneration, suggesting the RIP kinase pathway as a potential target to protect cone-mediated central and peripheral vision loss in patients with retinitis pigementosa.
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Ikeda Y., Hisatomi T., Yoshida N., Notomi S., Murakami Y., Enaida H., Ishibashi T.
Graefe's Archive for Clinical and Experimental Ophthalmology 250 ( 6 ) 809 - 814 2012.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Graefe's Archive for Clinical and Experimental Ophthalmology
Background: Cystoid macular edema (CME) is one of the common complications of retinitis pigmentosa (RP), and is responsible for patient complications such as blurred and reduced visual acuity and for subsequent atrophic changes in the fovea. The objective of this work was to evaluate the clinical efficacy of a topical dorzolamide (a carbonic anhydrase inhibitor) in CME associated with RP. Methods: Sixteen eyes of nine patients with CME secondary to typical forms of RP were included in the study. Baseline visual acuity, visual field, and optical coherence tomography (OCT) measurements were obtained for all patients. All patients used 1% dorzolamide three times daily in each eye. Patients underwent follow-up exams at 1, 3, and 6 months after treatment. The response to treatment was monitored by visual acuity and visual field measurement testing using the Humphrey Field Analyzer (HFA: the central 10-2 Program); in addition, foveal thickness was measured by OCT. Evaluation of macular sensitivity calculated by HFA as the average of 12 central points. Results: Thirteen (81.3%) of 16 eyes showed a clear decrease in retinal thickness after treatment. Evaluation of macular sensitivity, calculated by HFA as the average of 12 central points (with the exception of foveal point data, showed an improvement of more than 1.0 dB in nine (56.3%) of 16 eyes. Moreover, both the mean deviation value and macular sensitivity were significantly improved. No severe side-effects were seen in any of the patients examined. Conclusions: The results demonstrated that a topical dorzolamide is effective for the treatment of CME in patients with RP, and that the positive treatment effects last for up to 6 months. © Springer-Verlag 2011.
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The role of mislocalized phototransduction in photoreceptor cell death of retinitis pigmentosa Reviewed
Nakao T., Tsujikawa M., Notomi S., Ikeda Y., Nishida K.
PLoS ONE 7 ( 4 ) 2012.4
Language：English Publishing type：Research paper (scientific journal) Publisher：PLoS ONE
Most of inherited retinal diseases such as retinitis pigmentosa (RP) cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy. © 2012 Nakao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Notomi S., Hisatomi T., Kanemaru T., Takeda A., Ikeda Y., Enaida H., Kroemer G., Ishibashi T.
American Journal of Pathology 179 ( 6 ) 2798 - 2809 2011.12
Language：English Publishing type：Research paper (scientific journal) Publisher：American Journal of Pathology
Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2′- and 3′-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7 -/- mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
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Yoshida N., Hisatomi T., Ikeda Y., Kohno R., Murakami Y., Imaki H., Ueno A., Fujisawa K., Ishibashi T.
Graefe's Archive for Clinical and Experimental Ophthalmology 249 ( 10 ) 1547 - 1552 2011.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Graefe's Archive for Clinical and Experimental Ophthalmology
Background: Neovascular glaucoma (NVG) is a serious complication for patients with proliferative diabetic retinopathy (PDR). Bevacizumab is a full-length humanized monoclonal antibody that binds all isoforms of vascular endothelial growth factor (VEGF). Recently, encouraging results regarding the off-label use of intravitreal bevacizumab (IVB) for the treatment of NVG have been reported. We evaluated the histology of bevacizumab-treated trabeculectomy specimens to clarify IVB's biological effects on angle neovascularization. Methods: We retrospectively reviewed the charts of a consecutive series of 15 eyes of 13 patients who underwent trabeculectomy to treat NVG caused by PDR. In ten eyes of eight patients, 1.25 mg bevacizumab was injected intravitreally via the pars plana. Using light or electron microscopy, the surgically excised trabecular tissue was compared to that without IVB. Results: Light microscopy revealed decreased edema, fibrin deposition, inflammation and vascular congestion in the trabecular meshwork in specimens with IVB compared to those without IVB. Electron microscopy revealed endothelial cell degeneration in the bevacizumab-treated specimens. Conclusions: The biological effects on angle neovascularization after IVB may involve reduced vascular permeability, decreased inflammatory reaction, loss of vascular function, and endothelial cell degeneration. © 2011 Springer-Verlag.
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Miyazaki M., Ikeda Y., Yonemitsu Y., Goto Y., Murakami Y., Yoshida N., Tabata T., Hasegawa M., Tobimatsu S., Sueishi K., Ishibashi T.
Human Gene Therapy 22 ( 5 ) 559 - 565 2011.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Human Gene Therapy
Lentiviral vectors are promising tools for the treatment of chronic retinal diseases including glaucoma, as they enable stable transgene expression. We examined whether simian immunodeficiency virus (SIV)-based lentiviral vector-mediated retinal gene transfer of human pigment epithelium-derived factor (hPEDF) can rescue rat retinal ganglion cell injury. Gene transfer was achieved through subretinal injection of an SIV vector expressing human PEDF (SIV-hPEDF) into the eyes of 4-week-old Wistar rats. Two weeks after gene transfer, retinal ganglion cells were damaged by transient ocular hypertension stress (110mmHg, 60min) and N-methyl-d-aspartic acid (NMDA) intravitreal injection. One week after damage, retrograde labeling with 4′,6-diamidino-2-phenylindole (DAPI) was done to count the retinal ganglion cells that survived, and eyes were enucleated and processed for morphometric analysis. Electroretinographic (ERG) assessment was also done. The density of DAPI-positive retinal ganglion cells in retinal flat-mounts was significantly higher in SIV-hPEDF-treated rats compared with control groups, in both transient ocular hypertension and NMDA-induced models. Pattern ERG examination demonstrated higher amplitude in SIV-hPEDF-treated rats, indicating the functional rescue of retinal ganglion cells. These findings show that neuroprotective gene therapy using hPEDF can protect against retinal ganglion cell death, and support the potential feasibility of neuroprotective therapy for intractable glaucoma. © 2011 Mary Ann Liebert, Inc.
DOI： 10.1089/hum.2010.132
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Retinitis pigmentosa associated with asteroid hyalosis Reviewed
Ikeda Y., Hisatomi T., Murakami Y., Miyazaki M., Kohno R., Takahashi H., Hata Y., Ishibashi T.
Retina 30 ( 8 ) 1278 - 1281 2010.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Retina
Background: Asteroid hyalosis (AH) is a condition in which cream-colored or white spherical particles are suspended in the vitreous body. Asteroid hyalosis is considered not to cause decreased vision or any other visual symptoms except in rare cases. There have been a few reports of AH in patients with retinitis pigmentosa (RP). Methods: To assess the prevalence of AH in patients with RP, 320 patients with typical forms of RP were studied. One patient was offered a standard three-port vitrectomy, and the spherical particles obtained from her vitrectomy sample were analyzed using an energy-dispersive x-ray spectrometer. Results: Ten patients (two men and eight women) developed AH. Among them, four had bilateral AH and two had rapidly increasing vitreous opacity that led to decreased vision. One patient was a 48-year-old woman with progressive AH in the left eye. After treatment with a vitrectomy, her vision improved from 0.4 to 0.8. The spherical particles were composed of mainly calcium and phosphorus. Conclusion: The prevalence of AH in RP was higher than in previous reports, and we encountered two rare cases of progressive AH with decreased vision. We conclude that AH might lead to decreased vision in patients with RP. Copyright © by Ophthalmic Communications Society, Inc.
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Kohno R., Hata Y., Mochizuki Y., Arita R., Kawahara S., Kita T., Miyazaki M., Hisatomi T., Ikeda Y., Aiello L., Ishibashi T.
American Journal of Ophthalmology 150 ( 2 ) 223 - 229.e1 2010.8
Language：English Publishing type：Research paper (scientific journal) Publisher：American Journal of Ophthalmology
Purpose: To examine the histopathologic effect of a single intravitreal injection of bevacizumab on newly formed vessels in eyes with proliferative diabetic retinopathy (PDR). Design: Interventional case series and laboratory investigation. Methods: Two days after intravitreal injection of bevacizumab (1.25 mg/eye), pars plana vitrectomy or trabeculectomy was performed for the treatment of PDR or neovascular glaucoma (NVG) associated with PDR. Ten surgically removed preretinal proliferative tissues and 6 deep scleral flaps containing trabecular meshwork were fixed in 2% glutaraldehyde or 4% paraformaldehyde and were subjected to transmission electron microscopic analysis, immunohistochemical analysis, and terminal deoxyuridiine triphosphate (dUTP) nick-end labeling staining. Two surgically removed preretinal proliferative tissues and 2 deep scleral flaps from patients with PDR and NVG, but without preoperative intravitreal injection of bevacizumab (IVB), served as controls. Results: In control tissues, vascular endothelial cells possessed many fenestrations and were accompanied by pericytes. Apoptotic vascular endothelial cells frequently were observed in tissue after intravitreal injection of bevacizumab, whereas they were not observed in control tissues. Additionally, no apparent fenestration was observed in newly formed vessels from either proliferative tissue or trabecular meshwork after intravitreal injection of bevacizumab. In both PDR and NVG tissues after intravitreal injection of bevacizumab, overexpression of smooth muscle actin was observed in newly formed vessels, suggesting that the treatment may have increased pericytes on the vasculature as compared with control tissue. Conclusions: Intravitreal injection of bevacizumab may induce changes in immature, newly formed vessels of PDR or NVG tissue, leading to endothelial apoptosis with vascular regression, while inducing normalization of premature vessels by increasing pericyte coverage and reducing vessel fenestration. © 2010 Elsevier Inc. All Rights Reserved.
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Murakami Y., Ikeda Y., Yonemitsu Y., Miyazaki M., Inoue M., Hasegawa M., Sueishi K., Ishibashi T.
Human Gene Therapy 21 ( 2 ) 199 - 209 2010.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Human Gene Therapy
Lentiviral vectors are promising tools for the treatment of chronic retinal diseases, including age-related macular degeneration (AMD), as they enable stable transgene expression. On the other hand, Sendai virus (SeV) vectors provide the unique advantage of rapid gene transfer. Here we show that novel simian immunodeficiency viral vectors pseudotyped with SeV envelope proteins (SeV-F/HN-SIV) achieved rapid, efficient, and long-lasting gene transfer in the mouse retina. Subretinal exposure to SeV-F/HN-SIV vectors for only a few minutes resulted in high-level gene transfer to the retinal pigment epithelium, whereas several hours were required for gene transfer by standard vesicular stomatitis virus G-pseudotyped SIV vectors. Transgene expression continued over a 1-year period. SeV-F/HN-SIV vector-mediated retinal overexpression of soluble Fms-like tyrosine kinase-1 (sFlt-1) or pigment epithelium-derived factor (PEDF) significantly suppressed laser-induced choroidal neovascularization (CNV). Histologically, 6-month-long sustained overexpression of PEDF did not adversely affect the retina; however, that with sFlt-1 resulted in photoreceptor degeneration associated with choroidal circulation defects. These data demonstrate that brief subretinal administration of SeV-F/HN-SIV vectors may facilitate safe and efficient retinal gene transfer, and suggest the therapeutic potential of PEDF with a higher safety profile for treating CNV in AMD patients. © Copyright 2010, Mary Ann Liebert, Inc.
DOI： 10.1089/hum.2009.102
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Ikeda Y., Yonemitsu Y., Miyazaki M., Kohno R., Murakami Y., Murata T., Goto Y., Tabata T., Ueda Y., Ono F., Suzuki T., Ageyama N., Terao K., Hasegawa M., Sueishi K., Ishibashi T.
Human Gene Therapy 20 ( 9 ) 943 - 954 2009.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Human Gene Therapy
A phase 1 clinical trial evaluating the safety of gene therapy for patients with wet age-related macular degeneration (AMD) or retinoblastoma has been completed without problems. The efficacy of gene therapy for Leber's congenital amaurosis (LCA) was reported by three groups. Gene therapy may thus hold promise as a therapeutic method for the treatment of intractable ocular diseases. However, it will first be important to precisely evaluate the efficiency and safety of alternative gene transfer vectors in a preclinical study using large animals. In the present study, we evaluated the acute local (ophthalmic) and systemic toxicity of our simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vectors carrying human pigment epithelium-derived factor (SIV-hPEDF) for transferring genes into nonhuman primate retinas. Transient inflammation and elevation of intraocular pressure were observed in some animals, but these effects were not dose dependent. Electroretinograms (ERGs), including multifocal ERGs, revealed no remarkable change in retinal function. Histopathologically, SIV-hPEDF administration resulted in a certain degree of inflammatory reaction and no apparent structural destruction in retinal tissue. Regarding systemic toxicity, none of the animals died, and none showed any serious side effects during the experimental course. No vector leakage was detected in serum or urine samples. We thus propose that SIVagm-mediated stable gene transfer might be useful and safe for ocular gene transfer in a clinical setting. © Mary Ann Liebert, Inc. 2009.
DOI： 10.1089/hum.2009.048
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Ikeda Y., Yonemitsu Y., Miyazaki M., Kohno R., Murakami Y., Murata T., Tabata T., Ueda Y., Ono F., Suzuki T., Ageyama N., Terao K., Hasegawa M., Sueishi K., Ishibashi T.
Human Gene Therapy 20 ( 6 ) 573 - 579 2009.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Human Gene Therapy
Gene therapy may hold promise as a therapeutic approach for the treatment of intractable ocular diseases, including retinitis pigmentosa (RP). Gene transfer vectors that are able to show long-lasting transgene expression in vivo are highly desirable to treat RP; however, there is a dearth of information regarding long-term transgene expression in the eyes of large animals. We previously reported that the simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vector showed efficient, stable, and safe retinal gene transfer, resulting in significant prevention of retinal degeneration by gene transfer of a neurotrophic factor, human pigment epithelium-derived factor (hPEDF), in rodents. Before applying this strategy in a clinical setting, we here assessed the long-lasting transgene expression of our third-generation SIVagm-based lentiviral vectors in the retinal tissue of nonhuman primates. Approximately 20-50μl of SIV-EGFP (enhanced green fluorescent protein) or SIV-hPEDF was injected into the subretinal space via a glass capillary tube. To detect EGFP expression in the retina, we used a fluorescence fundus camera at various time points after gene transfer. Human PEDF expression was assessed by immunohistochemical analysis, Western blot assay, and enzyme-linked immunosorbent assay. The retinas demonstrated frequent EGFP expression that was preserved for at least 4 years without significant decline. The expression of hPEDF was stable, and occurred mainly in the retinal pigment epithelium. The secreted protein was detected in vitreous and aqueous humor. We thus propose that SIVagm-mediated stable gene transfer might be significantly useful for ocular gene transfer in a clinical setting. © Copyright 2009, Mary Ann Liebert, Inc.
DOI： 10.1089/hum.2009.009
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Ex Vivo Transfer of Nuclear Factor-κB Decoy Ameliorates Hepatic Cold Ischemia/Reperfusion Injury Reviewed
Yoshizumi T., Ikeda Y., Kaneda Y., Sueishi K.
Transplantation Proceedings 41 ( 5 ) 1504 - 1507 2009.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Transplantation Proceedings
Cold ischemia/reperfusion injury of the hepatic graft has been attributed to the release of various inflammatory cytokines. Specific inhibition of these cytokines may improve viability of the hepatic graft upon reperfusion. Herein we have assessed the efficacy of cis element decoy against nuclear factor-κB binding site delivery to the hepatic tissue in a rodent liver transplantation model. At 8 hours after reperfusion of the liver, significant reduction was noted in the livers treated with decoy in the release of cytosolic enzymes from the hepatocytes and in serum tumor necrosis factor α (P < .05). The neutrophilic infiltration into the hepatic grafts was significantly suppressed in the livers treated with decoy oligodeoxynucleotides (ODNs). Decoy ODNs against nuclear factor-κB binding site delivery improved the viability of the hepatic graft against cold ischemia/reperfusion injury in the rodent liver transplantation model. © 2009 Elsevier Inc. All rights reserved.
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Miyazaki M., Ikeda Y., Yonemitsu Y., Goto Y., Kohno R., Murakami Y., Inoue M., Ueda Y., Hasegawa M., Tobimatsu S., Sueishi K., Ishibashi T.
Journal of Gene Medicine 10 ( 12 ) 1273 - 1281 2008.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Gene Medicine
Background: We previously demonstrated that a new lentiviral vector derived from nonpathogenic simian immunodeficiency virus (SIVagm) was efficient and safe for long-lasting retinal gene transfer, and that it provided the significant therapeutic effect of expressing human pigment epithelium-derived factor (hPEDF) in Royal College of Surgeons (RCS) rats. In the present study, to obtain a more pronounced outcome, we assessed the potential synergistic effect of the simultaneous gene transfer of hPEDF and human fibroblast growth factor-2 (hFGF-2) by improved third-generation SIV on RCS rats and retinal degeneration slow (rds) mice, because the former targets the primary neurons, including photoreceptor cells (PCs), whereas the latter is effective for targeting secondary neural cells, including Muller cells. Methods: Vector solution (SIV-hPEDF, SIV-hFGF-2, a 1:1 mixture of SIV-hPEDF and SIV-hFGF-2, or SIV-enhanced green fluorescent protein) was injected into the peripheral subretinal space of 3-week-old RCS rats or rds mice. Histopathological and electroretinographic assessments were made at several points after gene transfer. Results: Administration of SIV-hPEDF or SIV-hFGF-2 significantly delayed the histological PC degeneration and electrical deficit in RCS rats, and these delays were synergistically and significantly pronounced by SIV-hPEDF + SIV-hFGF-2 (1:1 mixture). In rds mice, functional therapeutic effects were observed even by SIV-PEDF, or SIV-FGF-2 alone and, moreover, both SIV-PEDF and SIV-FGF-2 showed higher therapeutic effects. Conclusions: These synergistic rescues of retinitis pigmentosa (RP) model animals are the 'proof concept' that the 'dual' expression of hPEDF and hFGF-2 dramatically improved therapeutic efficacy by keeping lower titers. This strategy may contribute to safer and more effective gene therapy for RP. Copyright © 2008 John Wiley & Sons, Ltd.
DOI： 10.1002/jgm.1257
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Murakami Y., Ikeda Y., Yonemitsu Y., Onimaru M., Nakagawa K., Kohno R., Miyazaki M., Hisatomi T., Nakamura M., Yabe T., Hasegawa M., Ishibashi T., Sueishi K.
American Journal of Pathology 173 ( 5 ) 1326 - 1338 2008.11
Language：English Publishing type：Research paper (scientific journal) Publisher：American Journal of Pathology
Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP. Copyright © American Society for Investigative Pathology.
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Murakami Y., Ikeda Y., Yonemitsu Y., Tanaka S., Kondo H., Okano S., Kohno R., Miyazaki M., Inoue M., Hasegawa M., Ishibashi T., Sueishi K.
Journal of Gene Medicine 10 ( 2 ) 165 - 176 2008.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Gene Medicine
Background: Recombinant Sendai virus vectors (rSeV) constitute a new class of cytoplasmic RNA vectors that have shown efficient gene transfer in various organs, including retinal tissue; however, the related immune responses remain to be overcome in view of clinical applications. We recently developed a novel rSeV from which all envelope-related genes were deleted (rSeV/dFdMdHN) and, in the present study, assess host immune responses following retinal gene transfer. Methods: rSeV/dFdMdHN or conventional F-gene deleted rSeV (rSeV/dF) was injected into subretinal space of adult Wistar rats or C57BL/6 mice. The transgene expression and histopathological findings were assessed at various time points. Immunological assessments, including the expression of proinflammatory cytokines, natural killer (NK)-cell activity, as well as SeV-specific cytotoxic T lymphocytes (CTLs) and antibodies, were performed following vector injection. Results: rSeV/dFdMdHN showed high gene transfer efficiency into the retinal pigment epithelium at an equivalent level to that seen with rSeV/ dF. In the early phase, the upregulation of proinflammatory cytokines, local inflammatory cell infiltration and tissue damage that were all prominently seen in rSeV/dF injection were dramatically diminished using rSeV/dFdMdHN. NK cell activity was also decreased, indicating a reduction of the innate immune response. In the later phase, on the other hand, CTL activity and anti-SeV antibodies were similarly induced, even using rSeV/dFdMdHN, and resulted in transient transgene expression in both vector types. Conclusions: Deletion of envelope-related genes of rSeV dramatically reduces the vector-induced retinal damage and may extend the utility for ocular gene transfer; however, further studies regulating the acquired immune response are required to achieve long-term transgene expression of rSeV. Copyright © 2007 John Wiley & Sons, Ltd.
DOI： 10.1002/jgm.1142
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Interleukin-18 regulates pathological intraocular neovascularization Reviewed
Qiao H., Sonoda K., Ikeda Y., Yoshimura T., Hijioka K., Jo Y., Sassa Y., Tsutsumi-Miyahara C., Hata Y., Akira S., Ishibashi T.
Journal of Leukocyte Biology 81 ( 4 ) 1012 - 1021 2007.4
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Leukocyte Biology
Recently, the proinflammatory cytokine IL-18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen-induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL-18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL-18 reduction in oxygen-treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL-18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL-18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL-18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL-18-binding protein was administered during the OIR recovery phase to neutralize endogenous IL-18, OIR was still apparent on Day 24. We therefore concluded that IL-18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans. © Society for Leukocyte Biology.
DOI： 10.1189/jlb.0506342
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Rapid detection of SAG 926delA mutation using real-time polymerase chain reaction Reviewed
Yoshida S., Yamaji Y., Yoshida A., Ikeda Y., Yamamoto K., Ishibashi T.
Molecular Vision 12 1552 - 1557 2006.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Molecular Vision
Purpose: Mutation 926delA of the arrestin/S-antigen SAG gene is the main cause of Oguchi disease in the Japanese. The purpose of this study was to develop a rapid diagnostic assay to detect mutations in the SAG gene. Methods: Two sequence-specific primers and fluorophore-labeled probes for exon 11 of the SAG gene were designed, and the region spanning the mutations was amplified by polymerase chain reaction (PCR) using the LightCycler detection system (Roche Diagnostics, Mannheim, Germany). The mutations were then identified by melting curve analyses of the hybrid formed between the PCR product and a specific fluorescent probe. Results: We clearly distinguished each SAG genotype (homozygous and heterozygous 926delA and wild type) by the distinct melting peaks at different temperatures. One thermal cycling required approximately 54 min to process, and the results were 100% in concordance with the genotypes determined by DNA sequencing. Conclusions: We have succeeded in developing a rapid method to detect the most frequent mutation in the SAG gene. This method will help in identifying gene mutations associated with Oguchi disease with a rapid and reliable identification or the exclusion of the frequent mutations in the SAG gene. © 2006 Molecular Vision.
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Ikeda Y., Yonemitsu Y., Onimaru M., Nakano T., Miyazaki M., Kohno R., Nakagawa K., Ueno A., Sueishi K., Ishibashi T.
Experimental Eye Research 83 ( 5 ) 1031 - 1040 2006.11
Language：English Publishing type：Research paper (scientific journal) Publisher：Experimental Eye Research
The vascular endothelial growth factor (VEGF) family plays an essential role in vascular development, angiogenesis and lymphangiogenesis. VEGF-A is a key regulator of endothelial cell functions and VEGF-C and VEGF-D are known to stimulate both angiogenesis and lymphangiogenesis. In a surgically removed subretinal vascular membrane of an age-related macular degeneration (AMD) patient, both VEGF-C and VEGF-D were confirmed, in addition to VEGF-A, to be markedly positive in the retinal pigment epithelium (RPE). There is no lymph vessel in ocular tissue, so it is possible that VEGF-C and VEGF-D expression in the RPE play some role in ocular angiogenesis, as well as VEGF-A. Next, we assessed the transition of VEGF-A, -C, and -D expression on several conditions, in human RPE. Hypoxia proverbially induced VEGF-A mRNA expression, meanwhile VEGF-C and VEGF-D mRNA expression was down-regulated. The Ca2+ deprivation from culture medium strongly up-regulated VEGF-A and VEGF-D mRNA expression. Culture on plastic flasks precoated with poly-2-hydroxyethyl methacrylate up-regulated VEGF-D expression. Meanwhile, no significant change of VEGF-C mRNA expression was found in the blockade of cell-cell and/or cell-matrix adhesion. These findings suggest the possibility that VEGF-C and VEGF-D expression in RPE modify the ocular angiogenesis as angiogenic stimulators. © 2006 Elsevier Ltd. All rights reserved.
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Kohno R., Ikeda Y., Yonemitsu Y., Hisatomi T., Yamaguchi M., Miyazaki M., Takeshita H., Ishibashi T., Sueishi K.
Brain Research 1093 ( 1 ) 54 - 70 2006.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Brain Research
It is well known that neural stem/progenitor cells of the central nervous system (CNS) can proliferate to form neurospheres (CNS-neurospheres) that are positive for nestin, an intermediate filament for neural progenitors. Retinal stem/progenitor properties were also isolated from the ciliary body (CB) of the eye where, as in the CNS, such stem/progenitors also form spheres and have been considered to expand only via expansion by their proliferation even from the single-cell level (called spheres of pigment cells from the ciliary margin: PCM-spheres). We here found a new and distinct process underlying the growth of CB cell-derived spheres (CB-spheres) that is unlike the mechanism of CNS- and PCM-sphere expansion; this new process is a cell proliferation-independent incorporation of neighbor spheres and cells cultured at high density (200 cells/μl). The majority of cells in CB-spheres consisted of nestin-negative epithelia-like cells and started to express nestin during the course of their expansion by high-density cultivation. The growth of CNS-neurospheres was sensitive to a cell-cycle inhibitor, whereas the growth of CB-spheres was not seriously affected by cell proliferation; rather, the spheres grew by incorporating other CB-spheres and nestin-negative adherent cells, the latter of which started to express nestin and lost the expression of epithelial markers after being incorporated. These results indicate that CB-spheres do not form by the accumulation of neural progenitors but rather by a reprogramming system from epithelia-like cells for neural differentiation, a clearly distinct mechanism from sphere formation by single-cell expansion of retinal stem/progenitor populations. © 2006 Elsevier B.V. All rights reserved.
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Tumor necrosis factor-α antisense transfer remarkably improves hepatic graft viability Reviewed
Yoshizumi T., Yonemitsu Y., Ikeda Y., Kaneda Y., Yanaga K., Sugimachi K., Sueishi K.
Liver International 26 ( 4 ) 451 - 456 2006.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Liver International
Background: Cold ischemia/reperfusion injury of the hepatic graft, an unsolved problem in liver transplantations, is attributed to the release of inflammatory cytokines, especially the tumor necrosis factor- (TNF) α, from activated Kupffer cells (KC). Therefore, the specific inhibition of TNF-α could improve the viability of the hepatic graft upon reperfusion.: We assessed the efficacy of TNF-α antisense (TNF-AS) oligodeoxynucleotides (ODNs) delivery to KC in a rodent liver transplantation model. Results: Seventy-one percent of the animals that received 6 hours preserved grafts in baths of lactated Ringer's solution (4°C) and were treated with TNF-AS survived for over 14 days. Eighty percent of the animals treated with vehicle, sense ODNs, or balanced salt saline (BSS) died. Four hours after reperfusion of the liver, a significant reduction was noted in livers treated with TNF-AS in the release of cytosolic enzymes from the hepatocytes and the serum TNF-α (P<0.05). The expressions of TNF-α on KC and of intercellular adhesion molecule-1 on sinusoidal endothelial cells were completely suppressed in TNF-AS-treated livers. Conclusions: TNF-AS delivery improves the viability of the hepatic graft, and this technique may solve hepatic graft nonfunction in a clinical setting. © 2006 Blackwell Munksgaard.