A ROCK inhibitor suppresses the transforming growth factor-beta-2-induced endothelial-mesenchymal transition in Schlemm's canal endothelial cells.

In the normal eye, most of the aqueous humor drains through the trabecular meshwork (TM) and Schlemm's canal (SC). The concentration of transforming growth factor beta 2 (TGF-ß2) is increased in the aqueous humor of primary open angle glaucoma patients. TGF-ß2 increases outflow resistance by affecting the TM and SC, and endothelial-mesenchymal transition (EndMT) of SC cells is involved in these changes. Here, we investigated the effect of a ROCK inhibitor on TGF-ß2-induced EndMT in SC cells. The ROCK inhibitor Y-27632 suppressed the TGF-ß2-induced increase in the trans-endothelial electrical resistance (TER) and proliferation of SC cells. Y-27632 suppressed the expression of α-SMA, N-cadherin, and Snail, which are upregulated by TGF-ß2. Moreover, TGF-ß2 decreased mRNA levels of bone morphogenetic protein (BMP) 4 and increased those of the BMP antagonist gremlin (GREM1), but Y-27632 significantly suppressed these changes. Y-27632 also inhibited TGF-ß2-induced phosphorylation of p-38 mitogen-activated protein kinase (MAPK). BMP4 and the p-38 MAPK inhibitor SB203580 suppressed the TGF-ß2-induced TER elevation in SC cells. Moreover, SB203580 suppressed TGF-ß2-induced upregulation of fibronectin, Snail, and GREM1. These results indicate that a ROCK inhibitor inhibited the TGF-ß2-induced EndMT in SC cells, implying the involvement of p38 MAPK and BMP4 signaling.

Elevated soluble vascular endothelial growth factor receptor levels in aqueous humor from patients with different types of glaucoma.

We investigated the aqueous humor levels of vascular endothelial growth factor (VEGF), soluble VEGF receptor (sVEGFR)1, and sVEGFR2 in glaucoma patients and the correlations among them. Aqueous humor was collected from the anterior chamber at the start of glaucoma or cataract surgery. The levels of VEGF and its receptors, sVEGFR1 and sVEGFR2, were measured using multiplex bead-based immunoassays. Aqueous humor samples were obtained from 79 participants: 21 with primary open angle glaucoma (POAG), 22 with uveitic glaucoma (UG), 19 with neovascular glaucoma (NVG), and 17 with cataracts as controls. sVEGFR1 levels were significantly higher in NVG than in the other cases (NVG, 2839.8 pg/mL, P < 0.001). The sVEGFR2 levels of glaucoma patients were significantly higher than those of the controls (POAG, 699.0 pg/mL; UG, 866.2 pg/mL; NVG, 1198.1 pg/mL; P < 0.001). In the aqueous humor of glaucoma patients, sVEGFR1 and sVEGFR2 levels were positively correlated (POAG, P = 0.0196; UG, P = 0.0047; NVG, P = 0.0050). VEGF levels were negatively correlated with both sVEGFR1 (P = 0.0197) and sVEGFR2 (P = 0.0015) in POAG patients. In UG patients, the correlation between VEGF and sVEGFR1 levels was negative (P = 0.0144). sVEGFR2 levels were increased in various glaucomatous eyes. sVEGFR levels were negatively correlated with VEGF levels in some glaucoma types, implying that sVEGFRs may modulate the effects of aqueous VEGF in glaucoma pathogenesis.

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance.

Transforming growth factor-beta 2 (TGF-ß2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-ß2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-ß2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-ß2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-ß2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm's canal (MSC) cell monolayers was decreased by TGF-ß2 treatment. SAHA inhibited the effects of TGF-ß2 on the permeability of these cells. TGF-ß2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-ß2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-ß2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-ß signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-ß. These results imply that SAHA prevents TGF-ß2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-ß signaling in TM and MSC cells.

Potential roles of the IL-6 family in conjunctival fibrosis.

Elevated intraocular pressure (IOP) is a significant risk factor for vision loss due to glaucoma, which is a major cause of blindness worldwide. Glaucoma filtration surgery (GFS) is an important method to reduce IOP by guidance of aqueous humor into a newly built filtration bleb in the conjunctiva; management of the wound healing mechanism is essential for the success of GFS. Here, we investigated the roles of interleukin (IL)-6 family members during the wound healing process after GFS. At the surgical site, the expression levels of genes encoding IL-6, oncostatin M (OSM), their receptors, and collagen I were elevated at 3 h after GFS, whereas the levels of genes encoding transforming growth factor (TGF)-ß, α-smooth muscle actin (SMA), type IV collagen, and fibronectin were elevated at 3 days after GFS. IL-6 trans-signaling and OSM signaling suppressed TGF-ß-induced expression of α-SMA and collagen IV, as well as activation of the non-canonical TGF-ß pathway, suggesting that IL-6 and OSM may aid in controlling the phase transition from inflammation to proliferation and remodeling. The suppressive effects of OSM were accompanied by STAT3 activation, such that STAT1 function was complementary to STAT3. Taken together, these observations indicated that IL-6 family members constitute early response genes after GFS, which can suppress TGF-ß-induced expression of late response genes at the surgical site after GFS.

Correction: TGF-ß-induced activation of conjunctival fibroblasts is modulated by FGF-2 and substratum stiffness.

[This corrects the article DOI: 10.1371/journal.pone.0242626.].

TGF-ß-induced activation of conjunctival fibroblasts is modulated by FGF-2 and substratum stiffness.

PURPOSE: This study aimed to investigate the effects of substratum stiffness on the sensitivity of human conjunctival fibroblasts to transforming growth factor (TGF)-ß, and to explore the molecular mechanism of action. METHODS: Human conjunctival fibroblasts were cultured on collagen-coated plastic or silicone plates. The stiffness of the silicone plates was 0.2 or 64 kPa. Cells were treated by 2.5 ng/mL TGF-ß2 with or without fibroblast growth factor (FGF)-2 (0-100 ng/mL) for 24 h or 48 h. The protein expression levels were determined by Western blot analysis. Cell proliferation was assessed using the WST-8 assay. RESULTS: FGF-2 suppressed the TGF-ß-induced expression of α-smooth muscle actin (SMA) and collagen type I (Col I), but not fibronectin (FN). Both FGF-2 and TGF-ß2 increased cell proliferation without an additive effect. The induction of α-SMA by TGF-ß2 was decreased on the soft substratum, without any change in the expression level or subcellular location of Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). FGF-2 suppressed TGF-ß-induced α-SMA expression even on the soft substratum. CONCLUSIONS: FGF-2 treatment and a soft substratum suppressed TGF-ß-induced transdifferentiation of conjunctival fibroblasts into myofibroblasts. FGF-2 attenuated the TGF-ß-induced expression of α-SMA, even on a soft substratum.

DNA methyltransferase inhibitor suppresses fibrogenetic changes in human conjunctival fibroblasts.

Purpose: This study aimed to clarify the effects of a DNA methyltransferase inhibitor on fibrogenetic changes in human conjunctival fibroblasts (HConF). Methods: HConF were pretreated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) for 48 h. After one passage, the cells were treated with 5 ng/ml of transforming growth factor (TGF)-ß2 for 48 h, and the expression levels of α-smooth muscle actin (α-SMA), extracellular matrix proteins, and phosphorylated Smad3 were evaluated with western blotting. A fusion construct between the COL1A2 promoter and the luciferase gene was introduced into the HConF after the first passage, and the construct's activity was detected via a luciferase reporter gene assay. Results: TGF-ß2-induced upregulation of α-SMA was suppressed by pretreatment with 5-Aza-dC (0.1, 1.0, and 10 µM) in a dose-dependent manner. Upregulation of type I collagen was also suppressed by 10 µM 5-Aza-dC pretreatment. In contrast, 5-Aza-dC had no inhibitory effect on the expression of fibronectin or phosphorylated Smad3. However, COL1A2 promoter activity was suppressed with 5-Aza-dC pretreatment. Conclusions: In HConF, fibrogenetic changes were partly suppressed with a DNA methyltransferase inhibitor, suggesting an indirect inhibitory effect of the inhibitor on the COL1A2 promoter in HConF.

YAP/TAZ Are Essential for TGF-ß2-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-ß2-mediated conjunctival fibrosis. Methods: Primary human conjunctival fibroblasts were treated with TGF-ß2. 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-ß2 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-ß2-mediated phosphorylation and the nuclear translocation of Smad2/3. Furthermore, a YAP/TAZ inhibitor markedly suppressed TGF-ß2-mediated fibrotic changes in conjunctival fibroblasts. Conclusions: YAP/TAZ acted as a molecular hub of TGF-ß2 signaling in a cellular model of conjunctival fibrosis. Moreover, verteporfin, a YAP/TAZ inhibitor exerted potent antifibrosis effects by suppressing TGF-ß2-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.

Interleukin-6-mediated trans-signaling inhibits transforming growth factor-ß signaling in trabecular meshwork cells.

Glaucoma is one of the major causes of blindness, and transforming growth factor-ß2 (TGF-ß2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-ß2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-ß in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-ß signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-ß2 stimulation. To elucidate the relationship between TGF-ß2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-ß-induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-ß-induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-ß-mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-ß were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-ß-induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6-mediated trans-signaling potently represses TGF-ß signaling in HTM cells.

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

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.

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

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)-ß2 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.

Inhibition of Rho Kinase Induces Antioxidative Molecules and Suppresses Reactive Oxidative Species in Trabecular Meshwork Cells.

PURPOSE: To investigate the effect of rho kinase inhibitors on oxidative stress in trabecular meshwork (TM) cells. METHODS: TM cells were isolated from the eyes of cynomolgus monkeys. Y-27632 and menadione were used to inhibit rho kinase and induce production of reactive oxygen species (ROS), respectively. The cynomolgus monkey array and 12,613 probes were used in DNA microarray analysis, and the affected genes were categorized using gene ontology analysis. The mRNA levels of the target genes were confirmed by real-time RT-PCR. Intracellular oxidative stress was detected using a fluorescent reagent sensitive to ROS. Cell viability was assessed by the WST-8 assay. RESULTS: Gene ontology analysis revealed upregulation of genes involved in antioxidant activity, and upregulation of catalase was confirmed by real-time RT-PCR after 30 min treatment with Y-27632. Production of ROS was increased by menadione, and the effect was partly suppressed by pretreatment with Y-27632. At a lower dose of menadione, Y-27632 stimulated TM cells and significantly increased their viability following menadione treatment compared to control cells. CONCLUSION: Using microarray analysis, Y-27632 was shown to upregulate antioxidative genes including catalase and partially reduce ROS production and cell death by oxidative stress caused by menadione.

Molecular Mechanisms Underlying the Filtration Bleb-Maintaining Effects of Suberoylanilide Hydroxamic Acid (SAHA).

Purpose: Suberoylanilide hydroxamic acid (SAHA) has been shown to support the maintenance of experimental filtration blebs in animal models. This study was performed to investigate the molecular mechanisms underlying the bleb-maintaining effects of SAHA in modulating wound healing activities of conjunctival fibroblasts. Methods: Human conjunctival fibroblasts (HConFs) were pretreated with SAHA before treatment with TGF-ß2. Microarray-based screening was used to investigate the gene expression profiles. Gene ontology (GO) analysis was conducted to categorize the gene functions. The expression of TGF-ß-induced signaling molecules, α-smooth muscle actin, and extracellular matrix (ECM) proteins were evaluated by Western blot analyses. Multiplex immunoassay was performed to evaluate supernatant cytokine concentrations. Tube formation assay was used to evaluate angiogenesis using human umbilical vein endothelial cells. Results: GO analysis showed that SAHA, in the presence of TGF-ß2, induced changes in expression of genes involved in the TGF-ß receptor signaling pathway, cell proliferation, extracellular matrix organization, inflammatory responses, and angiogenesis. Subsequent in vitro experiments showed that SAHA partly inhibited the phosphorylation of Smad2, Smad3, and Akt. SAHA pretreatment potently suppressed TGF-ß2-driven cell proliferation, myofibroblast differentiation, contraction, ECM production, and angiogenic cytokine expression. The supernatant of HConFs treated with SAHA inhibited tube formation. Conclusions: SAHA has been shown to suppress angiogenesis and activation of conjunctival fibroblasts partly via inhibition of Smad and non-Smad TGF-ß signaling. This in vitro study provides new evidence for the molecular basis of the potential bleb-maintaining effects of SAHA, a novel candidate drug in modulating scar formation after glaucoma filtration surgery.

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.

The effects of ripasudil (K-115), a Rho kinase inhibitor, on activation of human conjunctival fibroblasts.

The most common cause of glaucoma surgery failure is scar formation induced by activation of wound-healing responses and resultant fibrosis at the surgical site. We investigated the effects of ripasudil, a Rho kinase inhibitor, on activation of human conjunctival fibroblasts (HConF). HConF were pretreated with different concentrations of ripasudil for 1 h before addition of transforming growth factor (TGF)-ß2, followed by incubation for 48 h. TGF-ß2-treated fibroblasts exhibited a significant increase in expression of α-smooth muscle actin (α-SMA), a marker of fibroblast-to-myofibroblast differentiation, and this increase was significantly suppressed, in a dose-dependent manner, by pretreatment with ripasudil. Ripasudil pretreatment also significantly attenuated TGF-ß2-induced fibronectin production and collagen gel contraction. TGF-ß2 increased both the number of viable cells and the number of cells in the G2/M phase of the cell cycle; these effects were attenuated by pretreatment with ripasudil. In addition, we explored the effects of ripasudil on stimulation of HConF by activated macrophages. Human monocytic cell line THP-1 cells were differentiated into M1 or M2 macrophage-like cells, and HConF were treated with conditioned media derived from these macrophages in the presence or absence of ripasudil. Conditioned medium from M2 macrophage-like cells induced a significant increase in α-SMA expression, viable cell numbers, and gel contraction, all of which were significantly suppressed by ripasudil. Thus, overall, ripasudil attenuated activation of human conjunctival fibroblasts. Ripasudil may be of therapeutic utility, preventing excessive scarring after glaucoma filtration surgery.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 µM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM cells.

p38 MAP kinase inhibitor suppresses transforming growth factor-ß2-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)-ß2-induced type I collagen production in human trabecular meshwork cells. TGF-ß2 increased RhoA activity, actin polymerization, and myosin light chain 2 phosphorylation. These effects were significantly inhibited by Y-27632, but not SB203580. TGF-ß2 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-ß2-induced promoter activation, or phosphorylation or nuclear translocation of Smad2/3, whereas SB203580 partially suppressed these processes. Collectively, TGF-ß2-induced production of type 1 collagen is suppressed by p38 inhibition and accompanied by partial inactivation of Smad2/3, in human trabecular meshwork cells.

Oxidative stress response signaling pathways in trabecular meshwork cells and their effects on cell viability.

PURPOSE: To clarify the primary oxidative stress response signaling pathways in trabecular meshwork (TM) cells and their effects on cell viability. METHODS: Porcine TM cells were treated with 600 µM or 800 µM H2O2, and their time-dependent morphologic changes were observed. Phosphorylation of protein kinase B (Akt), extracellular regulated kinase (ERK)1/2, p38, and c-Jun NH2-terminal kinase (JNK) was evaluated by western blot analysis. The intracellular localization of NFκB was evaluated by western blot analysis. One-hour pretreatments with LY294002, U0126, and SB203580, with the inhibitors of PI3K, ERK1/2, and p38, respectively, were conducted to evaluate the roles of these molecules in the cellular reaction against H2O2. Cell viability was assessed using propidium iodide and anticleaved caspase-3 antibody. RESULTS: TM cells treated with 600 µM H2O2 showed morphologic changes at 2 h that were partially recovered at 8 h after treatment. TM cells treated with 800 µM H2O2 did not recover, and the viability was significantly decreased. Both doses of H2O2 activated Akt, ERK1/2, and p38 in TM cells at 20 min after treatment, but not JNK or NFкB until 1 h after treatment. Inhibitors of PI3K, ERK1/2, and p38 suppressed recovery from the morphologic changes induced by 600 µM H2O2. Of these three inhibitors, the PI3K and ERK1/2 inhibitors decreased TM cell viability under oxidative stress. CONCLUSIONS: In TM cells, the PI3K-Akt, ERK, and p38 signaling pathways are primary oxidative stress response pathways involved in the mechanism of recovery from cellular morphologic changes induced by H2O2 treatment accompanied by actin cytoskeletal changes.

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 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 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.