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.

Intraocular pressure-lowering effects of ripasudil in uveitic glaucoma, exfoliation glaucoma, and steroid-induced glaucoma patients: ROCK-S, a multicentre historical cohort study.

To evaluate the efficacy and safety of ripasudil for treatment of secondary glaucoma, a historical cohort study was conducted at 18 centres in Japan. Adults (age ≥20 years) who needed additional IOP reduction and received topical 0.4% ripasudil between 2014 and 2018 due to three secondary glaucoma subtypes, including uveitic glaucoma (UG), exfoliation glaucoma (EG) or steroid-induced glaucoma (SG) were assessed for mean IOP change from baseline prior to additional treatment with ripasudil. We further evaluated the IOP change in each glaucoma subtype, baseline characteristics of each cohort, course of uveitis-induced inflammation in UG eyes, and proportion of patients in each cohort with adverse events. In 332 eyes from 332 patients eligible for this study, the mean overall IOP reductions from baseline at 1, 3, and 6 months were -5.86 ± 9.04 mmHg (-19.4 ± 25.1%), -6.18 ± 9.03 mmHg (-20.0 ± 27.1%), and -7.00 ± 8.60 mmHg (-23.4 ± 25.6%), respectively. These changes were all statistically significant. Of 332 eyes, 109 eyes had UG, 181 had EG, and 42 eyes had SG. The IOP-lowering effects of ripasudil in UG and SG were significantly greater than those of EG at every time point. This finding could have been related to higher baseline IOP levels in UG and SG. UG patients exhibited significant decreases in mean cell score of the anterior segment after ripasudil treatment. No severe adverse events were reported. These findings suggest that treatment with ripasudil is a safe and effective therapeutic modality for IOP reduction in secondary glaucoma.

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.

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.

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.

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.