Tomokazu Fujimoto

The effect of Rho-associated protein kinase inhibitor on monkey Schlemm's canal endothelial cells.

PURPOSE To investigate the effect of a specific inhibitor of Rho-associated protein kinase, Y-27632, on monkey Schlemms canal endothelial (SCE) cells. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Involvement of RhoA/Rho-associated kinase signal transduction pathway in dexamethasone-induced alterations in aqueous outflow.

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 Schlemms 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 COL4A1 and fibronectin expression. CONCLUSIONS Activation of the Rho/ROCK pathway in SCE cells contributes to the mechanism of DEX-induced changes in aqueous outflow.

Epithelial mesenchymal transition-like phenomenon in trabecular meshwork cells

The trabecular meshwork (TM) is a major component of the conventional outflow pathway and the excess extracellular matrix (ECM), and fibrosis in the TM causes increased outflow resistance. In this study, we first investigated the effects of several ECM components in the induction of an epithelial mesenchymal transition (EMT)-like phenomenon in TM cells. TM cells were isolated from cynomolgus monkeys (Macaca fascicularis). The cells were cultured in ECM-coated dishes and then subjected to both western blot analysis and immunocytochemistry to measure the levels of EMT-associated markers. Cell motility was assessed using wound healing and chemotaxis assays. We found that type I collagen, fibronectin and laminin induced the dissociation of cell-cell contact and elongation of actin stress fibers in the cultured monkey TM cells. In addition, following the same stimulation of the ECM, the expression of mesenchymal markers, such as fibronectin and α-smooth muscle actin, and the phosphorylation of Smad2 increased in the TM cells. Our results showed the significant acceleration of TM cellular motility following stimulation with type I collagen, fibronectin and laminin. These phenomena were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. In addition, siRNA against paxillin was transfected to evaluate the association between paxillin and the EMT-like phenomenon. The knockdown of paxillin expression by transfection with siRNA blocked the EMT-like alteration of the cellular characteristics and chemotaxis toward transforming growth factor-β2 in the cultured TM cells. Our results showed that the ECM-JNK-paxillin pathway induced an EMT-like phenomenon in TM cells, resulting in the abundant expression of fibronectin and activation of motility in TM cells. This EMT-like phenomenon could result in aberrant conditions in the aqueous outflow pathway in glaucomatous eyes.

p38 MAP kinase inhibitor suppresses transforming growth factor-β2-induced type 1 collagen production in trabecular meshwork cells.

Glaucoma is an age-related neurodegenerative disease of retinal ganglion cells, and appropriate turnover of the extracellular matrix in the trabecular meshwork is important in its pathology. Here, we report the effects of Rho-associated kinase (ROCK) and p38 MAP kinase on transforming growth factor (TGF)-β2–induced type I collagen production in human trabecular meshwork cells. TGF-β2 increased RhoA activity, actin polymerization, and myosin light chain 2 phosphorylation. These effects were significantly inhibited by Y-27632, but not SB203580. TGF-β2 also increased promoter activity, mRNA synthesis, and protein expression of COL1A2. These effects were significantly inhibited by SB203580, but not Y-27632. Additionally, Y-27632 did not significantly inhibit TGF-β2–induced promoter activation, or phosphorylation or nuclear translocation of Smad2/3, whereas SB203580 partially suppressed these processes. Collectively, TGF-β2–induced production of type 1 collagen is suppressed by p38 inhibition and accompanied by partial inactivation of Smad2/3, in human trabecular meshwork cells.

Vascular endothelial growth factor - A increases the aqueous humor outflow facility

Purpose Anti-vascular endothelial growth factor (VEGF) antibody therapy is an effective treatment for ocular angiogenesis. Although the intraocular pressure of some patients increases after anti-VEGF therapy, the effects of VEGF-A on the aqueous humor outflow pathway remain unknown. This study investigated the effects of VEGF-A on the aqueous humor outflow pathway. Methods We used human recombinant VEGF121 and VEGF165. Trabecular meshwork (TM) and Schlemm’s canal endothelial (SCE) cells were isolated from the eyes of cynomolgus monkeys. Expression of mRNA coding four VEGF receptors, VEGFR1 (FLT1), VEGFR2 (KDR), neuropilin-1, and neuropilin-2, was examined by RT-PCR. To evaluate the permeability of cell monolayers, we measured transendothelial electrical resistance (TEER). The outflow facility was measured in perfused porcine anterior segment organ cultures treated with 30 ng/mL VEGF121 for 48 h. Results Four VEGF-A-related receptor mRNAs were expressed in TM and SCE cells. The TEER of TM cells was not significantly affected by VEGF121 or VEGF165 treatment. In contrast, the TEER of SCE cells was significantly lower 48 h after treatment with 30 ng/mL VEGF121 to 69.4 ± 12.2% of baseline (n = 10), which was a significant difference compared with the control (P = 0.0001). VEGF165 (30 ng/mL) decreased the TEER of SCE cells at 48 h after treatment to 72.3 ± 14.1% compared with the baseline (n = 10), which was not a significant difference compared with the control (P = 0.0935). Ki8751, a selective VEGFR2 inhibitor, completely suppressed the effect of VEGF121 on SCE cell permeability, although ZM306416, a selective VEGFR1 inhibitor, did not affect the VEGF121-induced decrease in TEER. Perfusion with 30 ng/mL of VEGF121 for 48 h significantly increased the outflow facility compared with the control (47.8 ± 28.5%, n = 5, P = 0.013). Conclusions These results suggest that VEGF-A may regulate the conventional aqueous outflow of SCE cells through VEGFR2.

Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy.

PURPOSE To visualize intravital immune cell dynamics in the subconjunctival tissue during the wound-healing process using multiphoton microscopy. METHODS Gene-targeted mice expressing enhanced green fluorescent protein under the control of the endogenous lysozyme M promoter (LysM-eGFP mice) were anesthetized with isoflurane, and injured by a 10-0 nylon conjunctival suture. Vessels were visualized by intravenous injection of 70 kDa rhodamine-conjugated dextran. Using a multiphoton microscope, the three-dimensional images of the subconjunctival tissue were acquired every minute for 20 minutes before and 0.5, 3, 6, and 72 hours after injury. Raw imaging data were processed for four-dimensional images and analyzed for the number and the velocity of the LysM-eGFP-positive cells using Imaris software. RESULTS The intravital LysM-eGFP-positive cells and the red-labeled vessels were successfully visualized using a multiphoton microscope. The conjunctival and scleral collagen fibers were detected as secondary harmonic generation signals, which were colored blue. Compared with mice without injury, the number of LysM-eGFP-positive cells in the subconjunctival tissue after conjunctival surgery increased in a time-dependent manner. The cell velocities significantly increased until 3 hours after surgery (5.9 ± 3.2 μm/min; P < 0.0001) and the elevated level was sustained until 72 hours after injury (5.9 ± 3.3 μm/min). CONCLUSION This is the first report to visualize and evaluate intravital cellular dynamics during inflammation in the subconjunctival tissue using multiphoton microscopy. This technique may be a useful tool to characterize the molecular mechanisms of the wound-healing process after various ocular injuries, such as glaucoma surgery.

YAP/TAZ Are Essential for TGF-β2–Mediated Conjunctival Fibrosis

Purpose To investigate the roles of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ), the major effector molecules of the Hippo pathway, in TGF-β2-mediated conjunctival fibrosis. Methods Primary human conjunctival fibroblasts were treated with TGF-β2. The expression of YAP/TAZ was examined by Western blot analyses and immunocytochemistry. The expression of fibrotic proteins and genes were evaluated by Western blot analyses and quantitative real-time PCR, respectively. The effects of YAP/TAZ on fibrotic changes were examined by knockdown experiments and the YAP/TAZ inhibitor, verteporfin. Results TGF-β2 stabilized YAP/TAZ and subsequently activated Smad2/3, which led to the transcription of fibrotic genes in human primary conjunctival fibroblasts. These fibrotic genes were differently regulated by YAP/TAZ. Notably, α-smooth muscle actin, fibronectin, collagen I, and collagen IV were primarily regulated by YAP. In contrast, CCN family proteins (CTGF and CYR61) depended on both YAP and TAZ. Mechanistically, YAP/TAZ were located in close proximity to Smad2/3, and in particular, YAP was required for TGF-β2-mediated phosphorylation and the nuclear translocation of Smad2/3. Furthermore, a YAP/TAZ inhibitor markedly suppressed TGF-β2-mediated fibrotic changes in conjunctival fibroblasts. Conclusions YAP/TAZ acted as a molecular hub of TGF-β2 signaling in a cellular model of conjunctival fibrosis. Moreover, verteporfin, a YAP/TAZ inhibitor exerted potent antifibrosis effects by suppressing TGF-β2-YAP/TAZ-Smad signaling. Our study highlights YAP/TAZ as essential regulators of conjunctival fibrosis and shows that inhibition of YAP/TAZ might potentially improve the outcomes of glaucoma filtration surgery.

Interaction Between Pilocarpine and Ripasudil on Intraocular Pressure, Pupil Diameter, and the Aqueous-Outflow Pathway

Purpose To explore interactions between pilocarpine and the ROCK inhibitor, ripasudil, on IOP and pupil diameter in human eyes, and morphological and functional changes in outflow tissues in vitro. Methods IOP and pupil diameter were measured after pilocarpine and/or ripasudil, which were topically applied in healthy subjects. Human trabecular meshwork (HTM) cells were used in a gel contraction assay, for the evaluation of phosphorylation of myosin light chain and cofilin, and immunostaining for cytoskeletal proteins. Porcine ciliary muscle (CM) was used in a CM contraction assay. The permeability of human Schlemms canal endothelial (SCE) cells was evaluated by measuring transendothelial electrical resistance and fluorescein permeability. Results Both pilocarpine and ripasudil significantly reduced IOP in human eyes, but pilocarpine interfered with ripasudil-induced IOP reduction when concomitantly introduced. Ripasudil significantly inhibited gel contraction, TGFβ2-induced stress fiber formation, α-smooth muscle actin expression, and phosphorylation of both myosin light chain and cofilin in HTM cells. Pilocarpine reduced these effects, significantly inhibited the ripasudil-induced HTM cell responses to TGFβ2 stimulation, and increased the permeability in SCE cells. In CM, ripasudil inhibited pilocarpine-stimulated contraction, but ripasudil did not have significant effects on pilocarpine-induced miosis. Conclusions Pilocarpine interfered with the direct effects of ROCK inhibitor on the conventional outflow pathway leading to IOP reduction and cytoskeletal changes in trabecular meshwork cells, but did not affect the relaxation effect of the ROCK inhibitor. It is therefore necessary to consider possible interference between these two drugs, which both affect the conventional outflow.

Decreased MCP-1/CCR2 axis-mediated chemotactic effect of conjunctival fibroblasts after transdifferentiation into myofibroblasts

ABSTRACT The purpose of this study is to investigate the change in chemotactic effects of human conjunctival fibroblasts (HConFs) after transdifferentiation into myofibroblasts, and to explore related molecular mechanisms. HConFs were treated with 5 ng/mL transforming growth factor (TGF)‐&bgr;2 for 48 h to induce transdifferentiation into myofibroblasts. The cytokine concentrations in the conditioned media of HConFs were measured by multiplex bead‐based immunoassays. The Boyden chamber assay was used to assess the chemotactic effects using the monocyte cell line, THP‐1 cells. The concentration of monocyte chemoattractant protein (MCP)‐1 in the conditioned media was decreased after transdifferentiation into myofibroblasts (P < 0.001). The conditioned media of HConFs exerted a chemotactic effect on THP‐1 cells, but this effect decreased after transdifferentiation into myofibroblasts (P = 0.032). The number of migrated THP‐1 cells decreased significantly upon treatment with neutralizing anti‐MCP‐1 antibodies (P = 0.006) and tended to decrease upon treatment with C‐C chemokine receptor (CCR) 2 antagonist. The chemotactic effect of HConFs mediated by the MCP‐1/CCR2 axis was decreased after transdifferentiation into myofibroblasts. HighlightsConjunctival fibroblasts secreted various bioactive factors.Conjunctival fibroblasts possessed chemotactic effect on monocytes assessed by Boyden chamber assay.The chemotactic effect was mediated by MCP‐1/CCR2 axis, and reduced after transdifferentiation into myofibroblast.

Stimulation of the adenosine A3 receptor, not the A1 or A2 receptors, promote neurite outgrowth of retinal ganglion cells

ABSTRACT Among candidate neuroprotective agents, adenosine is thought to be a possible treatment for central nervous system disorders. Adenosine elicits biological effects through four G protein‐coupled receptors (A1, A2A, A2B, and A3). The A2A and A2B receptors stimulate adenylyl cyclase (AC) and increase cyclic adenosine monophosphate (cAMP) levels, whereas A1 and A3 receptors inhibit AC and decrease cAMP levels. Several studies have investigated the effects of adenosine receptors (AdoRs) in glaucoma, because modulation of A1, A2A, or A3 receptor regulates intraocular pressure. In addition, AdoR‐related phenomena may induce neuroprotective effects in retinal neurons. Notably, A1, A2A, and A3 receptor agonists reportedly inhibit retinal ganglion cell (RGC) death in in vitro and in vivo glaucoma models. However, there is limited knowledge of the effects of AdoR activation on neurite outgrowth or the regeneration of RGCs. In this report, we described the role of an AdoR subtype in neurite outgrowth and RGC axonal regeneration. The distribution of AdoRs in the retina was evaluated by immunohistochemical analysis. Using primary cultured rat RGCs in vitro and an optic nerve crush model in vivo, neurite elongation was evaluated after stimulation by the following AdoR agonists: CHA, an A1 receptor agonist; CGS21680, an A2A receptor agonist; BAY60‐6583, an A2B receptor agonist; and 2‐Cl‐IB‐MECA, an A3 receptor agonist. To determine the mechanism of neurite promotion, the candidate molecules of signal transduction associated with the neurite elongation of AdoRs were evaluated by enzyme‐linked immunosorbent assay (ELISA) and Western blot analysis, respectively. All four AdoRs (A1, A2A, A2B, and A3) were present in the inner retinal layers. Among the agonists for AdoR, only 2‐Cl‐IB‐MECA significantly promoted neurite outgrowth in primary cultured RGCs. Signaling pathway analyses showed that 2‐Cl‐IB‐MECA caused upregulated phosphorylation of Akt in cultured RGCs. Additionally, LY294002, an inhibitor of Akt, suppressed the neurite‐promoting effects of the A3 receptor agonist in RGCs. Moreover, 2‐Cl‐IB‐MECA increased the number of regenerating axons in the optic nerve crush model. Taken together, these data indicate that activation of the A3 receptor, not the A1 or A2 receptors, promotes in vitro and in vivo neurite outgrowth during the regeneration of rat RGCs, which is caused by the activation of an Akt‐dependent signaling pathway. Therefore, AdoR activation may be a promising candidate for the development of novel regenerative modalities for glaucoma and other optic neuropathies. HighlightsThe activation of A3 receptor enhances RGC neurite outgrowth in vitro and in vivo.A3 receptor stimulation promotes the neurite length via PI3K/Akt pathway.A3 receptor may be hopeful therapeutic target for neurodegenerative diseases.