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.

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.

Fornix-based versus limbal-based conjunctival flaps in trabeculectomy with mitomycin C in high-risk patients.

PURPOSE: To compare the surgical outcomes of limbal-based and fornix-based trabeculectomy in eyes with a history of ocular incisional surgery. METHODS: Twenty-six eyes underwent limbal-based trabeculectomy (group LB), and were condition matched with 26 eyes that received fornix-based trabeculectomy (group FB). Surgical failure was recorded retrospectively if the intraocular pressure value was either ≥21, ≥18, and ≥15 mmHg (conditions A, B, and C, respectively) or <4 mmHg or if the patient required additional glaucoma surgery. Kaplan-Meier survival curve analysis was used to assess surgical failure. RESULTS: For condition A, the 2 year surgical success probabilities were 75.0% and 63.9% in groups FB and LB, respectively (P=0.124). The corresponding values were 55.0% and 61.7% (P=0.638) in condition B, and 55.0% and 57.0% (P=0.454) in condition C. The rates of bleb leakage, hypotony, choroidal detachment, and bleb-related infection were 11.5%, 26.9%, 50.0%, and 7.7% in group LB, respectively. The corresponding values in group FB were 30.8%, 23.1%, 46.2%, and 0.0%, which were not statistically different between the two groups. CONCLUSION: No significant differences in surgical outcomes were observed between limbal-based and fornix-based trabeculectomy for patients with a history of incisional ocular surgeries.

Prognostic factors in trabeculectomy with mitomycin C having history of previous glaucoma surgery.

PURPOSE: To evaluate the prognostic factors for surgical outcomes of subsequent trabeculectomy with mitomycin C (MMC) after prior incisional glaucoma surgery. METHODS: We reviewed medical records of a total cohort of 781 trabeculectomies with MMC, and selected 125 patients (125 eyes). The primary endpoints included persistent intraocular pressure (IOP) of ≥21 or <5 mmHg, the need for additional glaucoma surgery and deterioration of visual acuity to no light perception. Univariate and Multivariate analyses were performed by using the Cox proportional hazards model. RESULTS: The mean follow-up period was 26.8 months. The probabilities of success at 1, 2, and 3 years were 80.6, 72.2, and 70.6 %, respectively. Multivariate analysis showed that a shorter time interval between prior glaucoma surgery and subsequent trabeculectomy [relative risk (RR), 0.8867/year; P = 0.0090] and the number of prior trabeculectomies (RR, 2.2645; P = 0.0029) were significant prognostic factors for subsequent failure of trabeculectomy with MMC. CONCLUSION: A short time period between prior glaucoma surgery and subsequent trabeculectomy and the number of prior trabeculectomies are associated with surgical failure of subsequent trabeculectomy with MMC.

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.

Frequency and risk factors for neovascular glaucoma after vitrectomy in eyes with proliferative diabetic retinopathy.

PURPOSE: To investigate the rate and risk factors for neovascular glaucoma (NVG) after vitrectomy in proliferative diabetic retinopathy (PDR). METHODS: Five hundred and twelve patients (512 eyes) with PDR who underwent vitrectomy between January 1, 2003 and June 30, 2009 at Kumamoto University Hospital, Japan, were retrospectively evaluated. Postoperative NVG was defined as neovascularization in the anterior segment and intraocular pressure (IOP) ≥ 22 mm Hg after vitrectomy. Kaplan-Meier survival analysis was applied to calculate the rate of NVG after vitrectomy. Risk factors for NVG after vitrectomy were identified by multivariable analysis using the Cox proportional hazards model. RESULTS: The mean follow-up period was 422 days. Twenty-seven of 512 patients (5.3%) developed postoperative NVG after vitrectomy. The probability of NVG occurrence at 6 and 12 months after vitrectomy was 6.0% and 7.1%, respectively. Male sex [relative risk (RR)=4.247; P=0.0032), younger age (RR=0.956/y; P=0.0237), higher baseline IOP (RR=1.203/mm Hg; P=0.0335), preoperative neovascularization in the anterior chamber angle (RR=8.899; P<0.0001), and presence of NVG in the fellow eye (RR=5.355; P=0.0013) were significant risk factors for postoperative NVG. CONCLUSIONS: The frequency of NVG in PDR eyes within 1 year after vitrectomy was estimated as 7.1%. The risk is independently associated with male sex, younger age, higher baseline IOP, preoperative neovascularization in the angle, and NVG in the fellow eye.

[A prospective study of postoperative aqueous flare in trabeculectomy alone versus phacotrabeculectomy].

PURPOSE: To compare trabeculectomy alone with phacotrabeculectomy in postoperative aqueous flare. SUBJECTS AND METHODS: Aqueous flare was prospectively measured using a laser flare-cell meter in open-angle glaucoma patients with cataract undergoing trabeculectomy with either mitomycin C or phacotrabeculectomy with mitomycin C. Flare was measured before surgery and at 2 and 4 weeks after surgery. RESULTS: Twenty-four patients who underwent trabeculectomy and 26 patients who underwent phacotrabeculectomy completed the study. There were no significant differences in flare values between the two groups at any point. Logistic regression analysis demonstrated that exfoliation glaucoma (odds ratio = 8.978; p = 0.0028) and postoperatively shallow anterior chamber (odds ratio = 15.539 ; p = 0.0074) were risk factors for high values of aqueous flare at 2 weeks after surgery. CONCLUSIONS: Combined phacoemulsification does not enhance postoperative aqueous flare in trabeculectomy. Exfoliation glaucoma and postoperative shallow anterior chamber are factors in the elevation of aqueous flare.

The effect of monocyte chemoattractant protein-1/CC chemokine ligand 2 on aqueous humor outflow facility.

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.

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.

Prognostic risk factors for failure of trabeculectomy with mitomycin C after vitrectomy.

PURPOSE: To evaluate the prognostic risk factors for failure of trabeculectomy with mitomycin C (MMC) in vitrectomized eyes. METHODS: Retrospective cohort study. We reviewed the medical records of 116 patients (116 eyes) treated at Kumamoto University Hospital. The primary endpoints were persistent intraocular pressure of >21 mmHg, deterioration of visual acuity to no light perception, or additional glaucoma procedures. Multivariable analysis was performed with the Cox proportional hazards model. RESULTS: The mean follow-up period was 36.5 months (range, 0.5-134.1 months). The probability of success 1 year after trabeculectomy was 55.1 %, 2 years after was 45.3 %, and 3 years after was 43.1 %. The multivariable model showed that higher preoperative intraocular pressure (IOP) [relative risk (RR), 1.05/mmHg; P = 0.0077] and neovascular glaucoma (NVG) (RR, 1.88; P = 0.049) were prognostic factors for surgical failure. CONCLUSIONS: The prognostic factors for surgical failure of trabeculectomy with MMC in vitrectomized eyes are a higher preoperative IOP and NVG.

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 Schlemm's 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, ß-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.

Impact of phacoemulsification on failure of trabeculectomy with mitomycin-C.

PURPOSE: To evaluate whether phacoemulsification after trabeculectomy affects postoperative intraocular pressure (IOP). SETTING: Kumamoto University, Kumamoto, Japan. DESIGN: Cohort study. METHODS: The medical records of patients with primary open-angle glaucoma or exfoliation glaucoma who had trabeculectomy with mitomycin-C were reviewed. The primary endpoints were condition A (persistent postoperative IOP 21 mm Hg or higher or additional glaucoma procedures with or without medications) and condition B (postoperative IOP 18 mm Hg or higher or additional glaucoma procedures with or without medications). Multivariable analysis was performed using the Cox proportional hazards model. RESULTS: The records of 178 patients (178 eyes) were reviewed. The mean follow-up was 37.0 months. For condition A, the probability of treatment success at 1 year, 2 years, and 3 years was 97.9%, 95.0%, and 92.7%, respectively. For condition B, the corresponding probabilities of success were 92.3%, 84.1%, and 81.8%. Thirty-seven patients (37 eyes) had phacoemulsification after trabeculectomy; 10 of those patients had phacoemulsification within 1 year after trabeculectomy. Multivariate analysis showed that a higher IOP before trabeculectomy was a significant risk factor for condition A and condition B (P=.01 and P=.0006, respectively); phacoemulsification within 1 year after trabeculectomy was significantly associated with trabeculectomy failure for condition B (P=.04). CONCLUSION: Postoperative IOP in eyes with previous trabeculectomy may be affected by the IOP before trabeculectomy and phacoemulsification within 1 year after trabeculectomy. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Trabeculectomy with mitomycin for open-angle glaucoma in phakic vs pseudophakic eyes after phacoemulsification.

OBJECTIVE: To determine whether previous phacoemulsification adversely affects surgical prognosis of trabeculectomy with mitomycin for open-angle glaucoma. METHODS: The study is a retrospective, consecutive, comparative case series. At 2 clinical centers, we reviewed 226 medical records of eyes with open-angle glaucoma undergoing initial trabeculectomy, including 175 phakic eyes (phakic group) and 51 pseudophakic eyes that had previously undergone phacoemulsification with superior conjunctival incision (pseudophakic group). Primary outcome was the probability of success after trabeculectomy. Surgical failure was defined as an additional glaucoma surgery or 1 of the following 3 criteria: intraocular pressure (IOP) of 21 mm Hg or greater (A); IOP of 18 mm Hg or greater (B); and IOP of 15 mm Hg or greater (C). Multivariable analysis was performed using the Cox proportional hazards model. RESULTS: The mean follow-up period was 37.5 months. The probability of success for criteria A, B, and C at 1 and 3 years in the phakic vs the pseudophakic group was 97.8% and 92.6%, respectively, vs 78.6% and 65.1%, respectively, for criterion A (P < .001); 92.9% and 81.3%, respectively, vs 72.8% and 63.7%, respectively, for criterion B (P = .004); and 73.1% and 54.2%, respectively, vs 53.1% and 38.4%, respectively, for criterion C (P = .009). The multivariable model confirmed that pseudophakia independently contributes to surgical failure (criterion A relative risk, 4.59 [P < .001]; criterion B, 2.88 [P = .004]; and criterion C, 2.02 [P = .009]). The pseudophakic group required more postoperative laser suture lysis (P = .01). CONCLUSION: Previous phacoemulsification is a prognostic factor for surgical failure of trabeculectomy with mitomycin for open-angle glaucoma.