Hideaki Okumichi

Platelet-derived growth factor receptor alpha is associated with oxidative stress-induced retinal cell death.

PURPOSE The purpose of this study was to investigate the role played by platelet-derived growth factor-α (PDGFRα) in oxidative stress-induced retinal cell death. A previous proteomic study from our laboratory showed that expression of PDGFRα is elevated considerably in the retinas of an animal model of glaucoma-the excitatory amino acid carrier (EAAC) 1-deficient (EAAC1-/-) mouse. METHODS Retinal sites and expression patterns of PDGFRα were determined by immunohistochemistry in the retinas of EAAC1-/- and control CRL:CD1(ICR) mice. A retinal cell line was exposed to hydrogen peroxide, and expression PDGFRα determined by Western blot analysis. Effects of PDGF-AA and PDGFRα-siRNA on hydrogen peroxide-induced retinal cell death were examined. RESULTS PDGFRα was detected in the retinal ganglion cell layer (RGL) of both EAAC1-/- and ICR mice, and was also localized in the internal nuclear layer (INL) of EAAC1-/- mice. While treatment with excess PDGF-AA had no additional effect on retinal cell death, expression of PDGFRα increased with exposure to hydrogen peroxide. Hydrogen peroxide-induced retinal cell death was inhibited by exposure to PDGF-AA via phosphatidylinositol 3 kinase (PI3K); cell death was promoted by PDGFRα-siRNA. CONCLUSIONS PDGFRα is expressed in mouse retina, where it is essential for retinal cell survival under conditions of oxidative stress.

GABAA receptors are associated with retinal ganglion cell death induced by oxidative stress

A previously proteomic study from our laboratory showed that the expression of the protein of the GABA A receptor beta1 subunit was significantly increased in the retina of EAAC1-deficient (EAAC1-/-) mice, a mouse model of glaucoma. The purpose of this study was to investigate the role played by GABA A receptor beta1 subunit in the death of retinal ganglion cells (RGCs) caused by oxidative stress. The retinal sites and expression pattern of GABA A receptor beta1 subunit were determined by immunohistochemistry in the retina of ICR mice. A RGC line, RGC-5, was exposed to GABA A receptor agonist and antagonist, and the cell viability was determined by the MTS assay. The effect of GABA A receptor antagonist on the death of RGCs, and the activation of oxidative stress signaling induced by hydrogen peroxide was investigated. Our results showed that the GABA A receptor beta1 subunit was expressed on the RGCs of ICR mice. GABA A receptor agonist induced RGCs death, and this death was inhibited by bicuculline, a GABA A receptor antagonist. The hydrogen peroxide-induced death of RGCs was reduced by GABA A receptor antagonist, and the oxidative stress signaling activated by hydrogen peroxide was also inhibited. These results indicate that GABA A receptors are expressed on RGCs and may play a role in the death of RGCs induced by oxidative stress.

Proteomic analyses of retina of excitatory amino acid carrier 1 deficient mice

BackgroundExcitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1-/-) mice.ResultsProteomic analyses and two-dimensional gel electorphoresis were performed on the retina of EAAC1-/- mice, and the results were compared to that of wild type mice. The protein spots showing significant differences were selected for identification by mass spectrometric analyses. Thirteen proteins were differentially expressed; nine proteins were up-regulated and five proteins were down-regulated in EAAC1-/- retina. Functional clustering showed that identified proteins are involved in various cellular process, e.g. cell cycle, cell death, transport and metabolism.ConclusionWe identified thirteen proteins whose expression is changed in EAAC-/- mice retinas. These proteins are known to regulate cell proliferation, death, transport, metabolism, cell organization and extracellular matrix.Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1-/-) mice. Proteomic analyses and two-dimensional gel electorphoresis were performed on the retina of EAAC1-/- mice, and the results were compared to that of wild type mice. The protein spots showing significant differences were selected for identification by mass spectrometric analyses. Thirteen proteins were differentially expressed; nine proteins were up-regulated and five proteins were down-regulated in EAAC1-/- retina. Functional clustering showed that identified proteins are involved in various cellular process, e.g. cell cycle, cell death, transport and metabolism. We identified thirteen proteins whose expression is changed in EAAC-/- mice retinas. These proteins are known to regulate cell proliferation, death, transport, metabolism, cell organization and extracellular matrix.

The signs of ocular-surface disorders after switching from latanoprost to tafluprost/timolol fixed combination: a prospective study

Purpose To evaluate the ocular-surface safety of a 0.001% benzalkonium chloride-containing tafluprost/timolol fixed combination (TTFC) in patients with primary open-angle glaucoma (POAG) or ocular hypertension who have inadequate intraocular pressure (IOP) control with latanoprost monotherapy. Methods This study is a multicenter, prospective, single-arm, open-label clinical study. Patients with POAG or ocular hypertension who have inadequate IOP control with latanoprost monotherapy were considered eligible. After providing informed consent, patients continued latanoprost monotherapy for 12 weeks, followed by a switch to TTFC. We evaluated the extent of ocular-surface damage using superficial punctate keratopathy (SPK) score, tear breakup time (TBUT), hyperemia score, IOP, systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate at 0, 4, and 12 weeks after switching. Results A total of 68 patients were enrolled, of whom, 64 patients were included in the final analysis. No significant changes in SPK score, TBUT, or hyperemia score were observed at 4 and 12 weeks compared with week 0. IOP decreased significantly at 4 (13.9±2.5 mmHg) and 12 (14.1±2.5 mmHg) weeks, relative to week 0 (15.3±2.7 mmHg). No significant changes in either SBP or DBP were observed during the study, although patients’ mean heart rate decreased significantly after switching to TTFC. Adverse drug reactions to TTFC occurred in seven patients including two incidences of asthma and one of arrhythmia, and no events were serious. Conclusion The ocular-surface safety of TTFC is not significantly different to that of latanoprost. Furthermore, switching from latanoprost to TTFC in patients with insufficient IOP control has additive IOP-lowering effects. TTFC is an effective approach for patients receiving latanoprost monotherapy who require more intensive IOP reduction.

Cullin5 Reduces Retinal Cell Death Induced by Glutamate Toxicity

Purpose: To investigate the expression in the mouse retina and the function in a retinal cell of cullin5 that is known as a protein that is associated with ubiquitilation. Methods: The retinal sites of expressing cullin5 were determined by immunohistochemistry with a specific antibody for cullin5. Retinal cells were transfected with HA-tagged cullin5 using an adenovirus system, and retinal cells were exposed to L-glutamate with or without an over-expression of cullin5. Results: Cullin5 was expressed in the retina of C57BL/6N mice and the retinal ganglion cell layer (RGL) of retina was strongly immunostained. Further, cullin5 was localized in the retinal ganglion cell (RGC). An over-expression of cullin5 reduced the percentage of retinal cell deaths induced by L-glutamate. Conclusions: The expression of cullin5 on retinal cells and reduction in the percentage of dead retinal cells induced by L-glutamate suggest that cullin5 has neuroprotective properties in retinal cells.