RhoA Activation Decreases Phagocytosis of Trabecular Meshwork Cells.

PURPOSE: RhoA signaling is important for the regulation of intraocular pressure through the trabecular meshwork (TM). However, the relationship between RhoA signaling and phagocytosis in TM cells is unclear. The purpose of this study was to investigate the effects of RhoA signaling on the phagocytosis of TM cells. MATERIALS AND METHODS: TM cells were isolated from enucleated porcine eyes and treated with lysophosphatidic acid (LPA) or calpeptin to activate RhoA to determine phagocytic activity. To assess phagocytic activity, TM cells were incubated with pHrodo® Red S. aureus bioparticles, and the fluorescence intensity was measured using a cell sorter. The phagocytic activity of RhoA knockdown TM cells was also assessed using small interfering RNA (siRNA). To resolve the effects of dexamethasone on phagocytosis, TM cells were treated with dexamethasone for 72 h. The immunocytochemistry of vinculin and F-actin were evaluated in LPA- and dexamethasone-treated TM cells. RESULTS: RhoA activities after treatment with 10 µM LPA and 100 µM calpeptin were 1.38 ± 0.026-fold and 1.47 ± 0.070-fold higher, respectively, compared with the control. The phagocytic activity was reduced by LPA (0.67 ± 0.099) and calpeptin (0.57 ± 0.016), compared with the control. C3 transferase (Rho inhibitor) and Y-27632 (Rho-associated kinase inhibitor) prevented the effects of LPA on phagocytosis, and C3 partially inhibited the effects of calpeptin on phagocytosis. Knockdown of RhoA prevented the effect of LPA on phagocytosis. By immunostaining, LPA-induced stress fiber and focal adhesion formation was prevented by C3 and Y-27632 treatment. Moreover, RhoA knockdown prevented the effects of LPA on F-actin and focal adhesion. Dexamethasone treatment decreased phagocytic activity and increased stress fiber and focal adhesion. Y-27632 prevented the effects of dexamethasone on phagocytosis, and on stress fiber and focal adhesion fomation. CONCLUSIONS: These results suggest that the RhoA signal pathway regulates the phagocytic activity of TM cells.Abbreviations: TM: trabecular meshwork; LPA: lysophosphatidic acid; C3: C3 transferase; ROCK: Rho-associated kinase; siRNA: small interfering RNA.

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.

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.

The Influence of Phacoemulsification on Surgical Outcomes of Trabeculectomy with Mitomycin-C for Uveitic Glaucoma.

PURPOSE: To evaluate the influence of phacoemulsification after trabeculectomy on the postoperative intraocular pressure (IOP) in eyes with uveitic glaucoma (UG). SETTING: Kumamoto University Hospital, Kumamoto, Japan. DESIGN: A retrospective cohort study. METHODS: The medical records of patients with UG who had trabeculectomy with mitomycin-C (MMC) were reviewed. Complete and qualified surgical failures were defined by an IOP of ≥21 mmHg (condition A), ≥18 mmHg (condition B), or ≥15 mmHg (condition C) without and with glaucoma eye drops, respectively. Kaplan-Meier survival analysis, generalized by the Wilcoxon test, and the Cox proportional hazards model analysis were conducted. Post-trabeculectomy phacoemulsification was treated as a time-dependent variable. In 24 (30%) of the included 80 eyes, phacoemulsification was included, and they were divided into two groups: groups I (8 eyes with phacoemulsification within 1 year after trabeculectomy) and group II (16 eyes after 1 year following trabeculectomy). RESULTS: Multivariable Cox proportional hazards model analysis showed post-trabeculectomy phacoemulsification was a significant factor in both complete success and qualified success based upon condition C (P = 0.0432 and P = 0.0488, respectively), but not for the other conditions. Kaplan-Meier survival analyses indicated significant differences in success probabilities between groups I and group II for complete success and qualified success based upon condition C (P = 0.020 and P = 0.013, respectively). There was also a significant difference for qualified success based upon condition B (P = 0.034), while there was no significant difference for the other conditions. CONCLUSION: Post-trabeculectomy phacoemulsification, especially within 1 year, can cause poor prognosis of IOP control of UG eyes after trabeculectomy with MMC.

Factors Influencing Aqueous Proinflammatory Cytokines and Growth Factors in Uveitic Glaucoma.

PURPOSE: To analyze the effects of factors on aqueous humor proinflammatory cytokine and growth factor levels in patients with uveitic glaucoma (UG). METHODS: In this cross-sectional study, we enrolled 143 participants: 1) UG patients (n = 39); 2) primary open-angle glaucoma (POAG) patients (n = 36); and 3) cataract surgery patients, as a comparative group (n = 68). Aqueous humor samples were obtained at the start of surgery. Aqueous cytokine levels were determined using a multiplex immunoassay (xMAP and the Human Cytokine/Chemokine Panel I). RESULTS: In UG cases, mean interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, and VEGF levels were 171.1, 214.5, 2791.7, 3.5, 23.9, 5.4, and 168.9 pg/mL, respectively, and were higher than those in cataract (non-glaucomatous) cases except PDGF. Levels of IL-6, MCP-1, and VEGF were higher in UG cases than in POAG cases. UG cases with a history of phacoemulsification displayed significantly higher levels of IL-6 (P = 0.0164), IL-8 (P = 0.0003), MCP-1 (P = 0.0465), and PDGF-AB/BB (P = 0.0062) compared to the phakic cases. The presence of cells in the anterior chamber was related to higher levels of IL-8 (P = 0.0002), TNF-α (P = 0.0037), and PDGF-AB/BB (P = 0.0009). The level of PDGF-AB/BB was higher in infectious uveitis than in non-infectious uveitis (P = 0.0211). The level of transforming growth factor (TGF)-ß2 was negatively correlated with the levels of MCP-1 (adjusted R2 = 0.28, t = -2.45, P = 0.031) and TNF-α (adjusted R2 = 0.27, t = -2.43, P = 0.032). CONCLUSION: A history of phacoemulsification, the presence of cells in the anterior chamber, and infectious uveitis were related to aqueous proinflammatory cytokine levels in patients with UG. TGF-ß2 might be an anti-inflammatory factor in aqueous humor of UG patients.

Postoperative Changes in Aqueous Monocyte Chemotactic Protein-1 Levels and Bleb Morphology after Trabeculectomy vs. Ex-PRESS Shunt Surgery.

PURPOSE: To evaluate the postoperative changes in blebs and levels of aqueous monocyte chemotactic protein-1 (MCP-1) after trabeculectomy vs. Ex-PRESS tube shunt surgery. METHODS: Rabbits were subjected to trabeculectomy or Ex-PRESS tube shunt surgery and observed for up to 3 months. Intraocular pressure (IOP) was measured using a rebound tonometer. The MCP-1 level was measured by enzyme-linked immunosorbent assay (ELISA). Bleb morphology was evaluated using photos and anterior-segment optical coherence tomography (OCT). RESULTS: There were no differences in bleb appearance or IOP at any time between the groups. Bleb wall density in the anterior-segment OCT image was significantly lower 1 week after surgery in the Ex-PRESS group than the trabeculectomy group. The MCP-1 level in control eyes was 304.1 ± 45.2 pg/mL. In the trabeculectomy group, the mean aqueous MCP-1 level was 1444.9, 1914.3, 1899.8, 516.4, 398.3, 427.3, 609.5, 1612.7, 386.2, and 167.9 pg/mL at 3, 6, and 12 h, and 1, 2, 5, 7, 14, 30, and 90 days after surgery, respectively. In the Ex-PRESS group, the corresponding values were 1744.0, 1372.0, 932.5, 711.7, 396.1, 487.3, 799.5, 1327.9, 293.6, and 184.0 pg/mL. There were no significant differences in the aqueous MCP-1 level between the groups at any time point. CONCLUSION: The postoperative changes were similar in the Ex-PRESS and trabeculectomy groups, except for bleb wall density in the anterior-segment OCT image. The postoperative aqueous MCP-1 level had bimodal peaks in both groups.

Simultaneous increase in multiple proinflammatory cytokines in the aqueous humor in neovascular glaucoma with and without intravitreal bevacizumab injection.

PURPOSE: To investigate aqueous humor proinflammatory cytokine levels of patients with neovascular glaucoma (NVG), and to analyze the effects of background factors in the expression of these molecules. METHODS: This cross-sectional study enrolled 137 participants who were grouped into (1) primary open-angle glaucoma (POAG; n = 36) patients; (2) NVG patients (NVG; n = 33); and (3) cataract surgery patients as a comparative group (CG; n = 68). Aqueous humor samples were collected from the anterior chamber at the start of surgery, deposited in CryoTubes, registered, and stored at -80 °C until processing. Multiplex microparticle-based immunodetection was performed by using xMAP and the Human Cytokine/Chemokine Panel I. Bevacizumab was injected into the vitreous cavity 1 to 2 days before surgery in 22 NVG patients (IVB group), whereas 11 NVG patients received no antivascular endothelial growth factor (VEGF) therapy 3 months preoperatively (N group). The Wilcoxon rank sum test or Fisher's exact test for two variables and the Tukey-Kramer honestly significant difference test for multiple variables were used to compare the cytokine levels. RESULTS: The NVG patients had higher levels of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor-α (TNF-α), and platelet-derived growth factor (PDGF)-AA compared to both the CG and POAG groups. The levels of IL-6, IL-8, MCP-1, and PDGF-AB/BB were higher in the IVB group than the N group, whereas the VEGF level was significantly lower in the IVB group (P < 0.01). CONCLUSIONS: Intravitreal bevacizumab injection decreased VEGF levels, but not those of the other cytokines.