Tomokazu Fujimoto

Publications (3)10.29 Total impact

• Article: Involvement of RhoA/Rho-Associated Kinase Signal Transduction Pathway in Dexamethasone-Induced Alterations in Aqueous Outflow.

  Tomokazu Fujimoto, Toshihiro Inoue, Takanori Kameda, Nanako Kasaoka, Miyuki Inoue-Mochita, Naoko Tsuboi, Hidenobu Tanihara
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  ABSTRACT: Purpose. We investigated the involvement of the RhoA/Rho kinase (ROCK) signal transduction pathway in dexamethasone (DEX)-induced changes in aqueous outflow. Methods. Using trabecular meshwork (TM) and Schlemm's canal endothelial (SCE) cells, RhoA activation was evaluated with a pull-down assay and myosin light chain phosphorylation was evaluated by Western blot analysis. Outflow facility was measured in perfused porcine anterior segment organ cultures treated with DEX and/or Y-27632, a selective ROCK inhibitor. The barrier function of the cultured cells on a micropore filter was evaluated by measuring the transendothelial electrical resistance. Collagen, fibronectin, and integrin mRNA expression levels were evaluated by quantitative real-time RT-PCR. Results. Relative RhoA activities increased following stimulation with 100 nM DEX in TM and SCE cells. Perfusion with DEX decreased outflow facility by 31.9 ± 14.3% compared to controls at 24 hours, but not by 50 μM Y-27632 in addition to DEX. The transendothelial electrical resistance of the SCE cell monolayer was increased by 48.6 ± 6.4% and 5.3 ± 5.0% following DEX treatments without and with 10 μM Y-27632, respectively, compared to controls. In TM cells, the mRNA expressions of COL4A1 and fibronectin were increased significantly by DEX treatment, but combined treatment with Y-27632 and DEX significantly inhibited the increase in COL4A1and fibronectin expression. Conclusions. Activation of the Rho/ROCK pathway in SCE cells contributes to the mechanism of DEX-induced changes in aqueous outflow.
  Investigative ophthalmology & visual science 09/2012; 53(11):7097-108. · 3.43 Impact Factor
• Article: The Effect of Monocyte Chemoattractant Protein-1/CC Chemokine Ligand 2 on Aqueous Humor Outflow Facility.

  Naoko Tsuboi, Toshihiro Inoue, Motofumi Kawai, Miyuki Inoue-Mochita, Tomokazu Fujimoto, Nanako Awai-Kasaoka, Akitoshi Yoshida, Hidenobu Tanihara
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  ABSTRACT: Purpose. To investigate the effect of monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 on aqueous humor outflow facility. Methods. Aqueous humor outflow facility was measured in enucleated porcine eyes in a constant pressure perfusion system with or without MCP-1 (1600 ng/mL). Expression of CCR2, an MCP-1 receptor, in Schlemm's canal endothelial (SCE) cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) assay. The effect of MCP-1 (0-1600 ng/mL) on SCE cell viability was evaluated using a WST-8 assay. The effect of MCP-1 (0-800 ng/mL) on SCE-cell monolayer permeability was evaluated with or without a CCR2 antagonist (10 nM) by measuring transendothelial electrical resistance (TEER). The intracellular localization of the gap junction protein ZO-1 was analyzed by immunofluorescence staining of SCE cells. Results. The aqueous humor outflow facility increased significantly from basal levels at 80 minutes after perfusion with MCP-1 compared with control eyes (21.2% ± 6.6% [MCP-1] vs. 5.7 ± 2.5% [control]; P = 0.048). CCR2 was detected by RT-PCR. Cell viability was not affected by MCP-1 treatment. TEER of SCE-cell monolayer at 3 hours after treatment with 800 ng/mL MCP-1 decreased by 21.6 ± 1.7% compared with controls (P = 0.014), and the TEER-decreasing effects of MCP-1 were attenuated by a CCR2 antagonist. Immunocytochemical staining revealed a modest disruption of ZO-1 in MCP-1-treated SCE cells. Conclusions. The present results revealed that MCP-1 increased aqueous humor outflow facility and decreased TEER via CCR2. These findings suggest that MCP-1 modulates aqueous humor outflow through the conventional pathway.
  Investigative ophthalmology & visual science 09/2012; 53(10):6702-7. · 3.43 Impact Factor
• Article: The effect of Rho-associated protein kinase inhibitor on monkey Schlemm's canal endothelial cells.

  Takanori Kameda, Toshihiro Inoue, Masaru Inatani, Tomokazu Fujimoto, Megumi Honjo, Nanako Kasaoka, Miyuki Inoue-Mochita, Nagahisa Yoshimura, Hidenobu Tanihara
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  ABSTRACT: To investigate the effect of a specific inhibitor of Rho-associated protein kinase, Y-27632, on monkey Schlemm's canal endothelial (SCE) cells. SCE cells were isolated from cynomolgus monkey eyes. The effects of Y-27632 on aqueous outflow facility were evaluated using enucleated monkey eyes and a constant-pressure perfusion system. The effect of Y-27632 on the barrier function of the confluent SCE-cell monolayer was evaluated by measuring transendothelial electrical resistance (TEER) and fluorescein permeability. Y-27632-induced changes in the intracellular localization of ZO-1, claudin-5, β-catenin, pan-cadherin, and filamentous actin (F-actin) were examined by immunofluorescence. Gene-expression changes induced by Y-27632 were analyzed with microarray, and the functional categories of changed genes were identified by gene ontology analysis. The concentrations of intracellular calcium ions were estimated using Fluo-4/AM and a fluorescence microscope system. Y-27632 significantly increased the outflow facility and the number of associated giant vacuoles, decreased TEER of the SCE-cell monolayer, and increased the transendothelial flux of fluorescein. Y-27632 disrupted ZO-1 and claudin-5 expression in a confluent SCE-cell monolayer. Among 12,544 genes, Y-27632 treatment increased the expression of 57 genes and decreased the expression of 15 genes. Gene ontology analysis revealed that changed genes were related to various cellular functions, including regulation of calcium ion transport into the cytosol. Y-27632 partially diminished the A23187-induced increase in intracellular calcium ions. Y-27632 increased the permeability of the SCE-cell monolayer in association with disruption of the tight junction, F-actin depolymerization, and changes in various cell functions, including calcium transfer.
  Investigative ophthalmology & visual science 04/2012; 53(6):3092-103. · 3.43 Impact Factor