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Glaucoma  |   October 2012
Involvement of RhoA/Rho-Associated Kinase Signal Transduction Pathway in Dexamethasone-Induced Alterations in Aqueous Outflow
Author Affiliations & Notes
  • Tomokazu Fujimoto
    From the Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; the
  • Toshihiro Inoue
    From the Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; the
  • Takanori Kameda
    From the Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; the
    Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; and the
  • Nanako Kasaoka
    From the Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; the
  • Miyuki Inoue-Mochita
    From the Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; the
  • Naoko Tsuboi
    From the Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; the
    Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan.
  • Hidenobu Tanihara
    From the Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; the
  • Corresponding author: Toshihiro Inoue, Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan; noel@da2.so-net.ne.jp
Investigative Ophthalmology & Visual Science October 2012, Vol.53, 7097-7108. doi:10.1167/iovs.12-9989
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      Tomokazu Fujimoto, Toshihiro Inoue, Takanori Kameda, Nanako Kasaoka, Miyuki Inoue-Mochita, Naoko Tsuboi, Hidenobu Tanihara; Involvement of RhoA/Rho-Associated Kinase Signal Transduction Pathway in Dexamethasone-Induced Alterations in Aqueous Outflow. Invest. Ophthalmol. Vis. Sci. 2012;53(11):7097-7108. doi: 10.1167/iovs.12-9989.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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 COL4A1and fibronectin expression.

Conclusions.: Activation of the Rho/ROCK pathway in SCE cells contributes to the mechanism of DEX-induced changes in aqueous outflow.

Introduction
Steroid-induced ocular hypertension is an important clinical problem related to the use of glucocorticoid therapy for many types of disorders. Already in 1963, Armaly reported that the widely used glucocorticoid dexamethasone (DEX) induces changes in intraocular pressure (IOP) and fluid dynamics in normal eyes, and that DEX-induced IOP elevation is observed more frequently in the patients with glaucoma. 13 IOP levels often are quite high in patients with steroid-induced ocular hypertension, and prolonged IOP elevation sometimes results in the loss of visual function. Topical administration of triamcinolone acetonide (TA), a synthetic corticosteroid, is considered a useful therapeutic modality for the management of intraocular inflammatory and/or proliferative diseases. The increasing number of patients treated with TA by intravitreous and/or sub-Tenon injections, however, has attracted the attention of ophthalmologists because of the potential risk of steroid-induced ocular hypertension. 4,5  
The mechanisms underlying steroid-induced ocular hypertension are associated with abnormal changes in the conventional outflow pathway caused by steroid administration. Some researchers attribute the decreased outflow to an accumulation of extracellular matrix (ECM) components, such as glycosaminoglycans, collagens, fibronectin, and elastin. 69 Others have proposed that the decreased outflow is due to decreased phagocytic function by trabecular meshwork (TM) cells, 10,11 which results in the accumulation of trabecular debris. Clark et al. recently reported that exposure of cultured TM cells to steroids induces a reorganization of the cytoskeleton to form cross-linked actin networks. 12,13 Thus, it is likely that cytoskeletal rearrangement at least partially underlies the aberrant metabolism of the ECM, resulting in decreased outflow and IOP elevation. 
Recent studies, including ours, suggest that cytoskeletal drugs are promising candidates for the development of novel IOP-reducing drugs against glaucoma. 1416 Among the known cytoskeletal drugs, accumulating data suggest that Rho-associated kinase (ROCK) inhibitors are a useful medical treatment for the management of glaucoma. Recent clinical trials demonstrated clearly the efficacy and safety of ROCK inhibitors in reducing IOP in human eyes. 14,15 Laboratory studies indicate that ROCK inhibitors alter basic cellular properties, such as cytoskeletal arrangement, cell shape, motility, and attachment in human TM cells, 1719 and that the number of giant vacuoles in Schlemm's canal endothelial (SCE) cells increases after perfusion of a ROCK inhibitor. 18,20 In addition, physiologic studies revealed that ROCK inhibitors induce an increase in total outflow facility. 17,2022 Thus, it is likely that the IOP-reducing and outflow facility-increasing effects of ROCK inhibitors are due to changes in the conventional outflow facility. A better understanding of the interactions between ROCK inhibition and steroid-induced changes in aqueous outflow, therefore, is important potentially for the management of steroid-induced glaucoma. In cells of conventional aqueous outflow tissues, such as TM and Schlemm's canal, however, the molecular mechanisms underlying this interaction remain unknown. 
We reported that activation of RhoA/ROCK signaling transduction is involved in DEX-induced changes in aqueous outflow. 
Materials and Methods
Cell Culture
Experiments were conducted according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Porcine TM cells were isolated by collagenase as described previously. 23 Porcine TM cells were cultured in Dulbecco's modified Eagle medium (DMEM; WAKO Pure Chemical Industries, Osaka, Japan) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.5 μg/mL amphotericin B at 37°C in 5% CO2, and TM cells between passages 3 and 5 were used for all the experiments in our study. 
The monkey TM and SCE cells were isolated from eyes of 6- to 12 month-old cynomolgus monkeys obtained from Shin Nippon Biomedical Laboratories (SNBL; Kagoshima, Japan) according to the method described previously. 20,24,25 Schlemm's canal was identified by cannulating its lumen with a 6-0 nylon suture under microscopic observation. The TM tissues were picked up with fine forceps and placed on plates coated with collagen gel. After completely removing TM tissues, explants of the inner wall of Schlemm's canal were placed on plates coated with collagen gel. Primary TM and SCE cells were expanded in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.5 μg/mL amphotericin B at 37°C in 5% CO2. We used TM cells between passages 2 and 5, and SCE cells between passages 3 and 8 in our study. 
RhoA Activation Assay
TM and SCE cells were cultured on 10 cm dishes. After the cells had grown to confluence, the culture medium was replaced with serum-free DMEM 24 hours before DEX stimulation. DEX was dissolved in dimethyl sulfoxide, and the final concentration of dimethyl sulfoxide was 0.1% in culture medium. The cells were treated with 10, 100, or 1000 nM DEX for 5 minutes. The effect of DEX on RhoA activation was evaluated by a pull-down assay using the Rho Activation Assay Biochem Kit (#BK036; Cytoskeleton, Denver, CO) according to the manufacturer's instructions. Active RhoA (GTP binding form) immunoreactive bands were visualized using an ECL Advance Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and determined using a luminescent image analyzer (LAS-4000mini; Fujifilm, Tokyo, Japan). Densitometry of immunoreactive bands was analyzed by Image J software (NIH, Bethesda, MD). 
Myosin Light Chain (MLC) Phosphorylation
Phosphorylated MLC of TM and SCE was determined by Western blotting. The cells were cultured on 6-cm dishes, and treated with 100 nM DEX and 10 μM Y-27632 (Merck KGaA, Darmstadt, Germany) for 5, 15, or 30 minutes. As the positive control, monkey TM cells were treated with 20 μM lysophosphatidic acid (LPA; Sigma-Aldrich, St. Louis, MO) for 30 minutes. Cells then were washed with ice-cold PBS and lysed in 300 μL RIPA buffer (Thermo Fisher Scientific, Waltham, MA) containing a protease inhibitor (Thermo Fisher Scientific) and a phosphatase inhibitor (Nacalai Tesque, Kyoto, Japan). The cell lysates were sonicated and centrifuged at 15,000 revolutions per minute (rpm) for 10 minutes at 4°C. Protein concentrations of the cell lysates were measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The cell lysate was mixed with NuPAGE LDS sample buffer and 50 mM dithiothreitol (Life Technologies, Carlsbad, CA) and heated at 70°C for 10 minutes. Equal amounts of the protein samples were loaded onto a 10% or 4% to 12% gradient polyacrylamide gel (Life Technologies), and proteins were fractioned by SDS-PAGE. The separated proteins were transferred to PVDF membranes. Membranes were blocked with 2% blocking reagent (GE Healthcare) in Tris-buffered saline (137 mM NaCl, 20 mM Tris, pH 7.4) containing 0.1% Tween-20 (TBS-T) for 60 minutes at room temperature, and then incubated overnight at 4°C with rabbit polyclonal antibody phospho-MLC or total MLC (1:1000 dilution in 5% bovine serum albumin/TBS-T; Cell Signaling Technology, Danvers, MA). Membranes were washed 3 times for 10 minutes each with TBS-T, incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000 dilution in TBS-T; Cell Signaling Technology) for 30 minutes at room temperature and visualized using ECL Advance Western Blotting Detection Reagent (GE Healthcare). All membranes were stripped of antibodies using WB Stripping solution (Nacalai Tesque) and incubated with mouse monoclonal antibody β-actin (1:10,000 dilution in blocking solution; Sigma-Aldrich), and subsequently with HRP-conjugated goat anti-mouse IgG (1:20,000 dilution in TBS-T; GE Healthcare) for a loading control. β-Actin immunoreactive bands were visualized with ECL Western Blotting Detection Reagent (GE Healthcare) and determined using a luminescent image analyzer. 
Anterior Segment Organ Culture Perfusion
The perfusion protocol was similar to that described by Johnson et al. 26 and Song et al. 23 with several modifications. Fresh paired porcine eyes were obtained from a local abattoir. Eyes were bisected at the equator, and the vitreous, lens, iris, and ciliary body were removed. The anterior segments of the porcine eyes were placed onto specially designed chambers and perfused with DMEM containing 0.1% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), gentamicin (36 μg/mL), and amphotericin (0.25 μg/mL) at a constant flow (3 μL/min) using an infusion pump (#KDS101; Muromachi Kikai, Tokyo, Japan). The anterior segments were cultured at 37°C under 5% CO2. After the initial baseline was recorded for at least 24 hours, the test eyes were perfused with DMEM containing 100 nM DEX or 100 nM DEX + 50 μM Y-27632 for 7 days. IOP was monitored continuously with a pressure transducer connected to the chamber and the data were recorded on a computer at a rate of 1 Hz. Aqueous humor outflow facility (μL/min/mm Hg) was calculated as the ratio between the inflow rate (μL/min) and the IOP (mm Hg). Outflow facility values were calculated as the mean of 10 minutes and expressed as percent change from baseline. The baseline values of outflow facility were determined during the last 10 minutes before changing the perfusion medium. 
Measurement of Monolayer Transendothelial Electron Resistance
SCE cells were cultured on a Transwell polyester membrane insert (0.4 μm pore size and 6.5 mm diameter; Corning, Corning, NY) on 24-well culture plates in DMEM containing 10% FBS and antibiotics at 37°C in 5% CO2. The volume of the apical side (inside the membrane inserts) was 0.2 mL and that of the basal side (outside the membrane insert) was 1.2 ml. Transendothelial electron resistance (TEER) was recorded using MILLICELL-ERS (Millipore, Billerica, MA) according to the manufacturer's instructions. To evaluate the effect of DEX and Y-27632 against SCE barrier function, we performed two examinations. First, after the cells had grown to confluence, SCE cells were treated with 100 nM DEX and/or 10 μM Y-27632, and TEER was measured at 24, 48, and 72 hours after stimulation. Second, SCE cells were treated with DEX or vehicle for 72 hours, and then Y-27632 was treated with DEX for 24 hours. TEER values were normalized by subtracting the background resistance from the filter alone. Time-dependent changes after each treatment were followed and compared as percent change from baseline values. Each experiment was repeated at least three times. 
Hoechst 33342 and Propidium Iodide (PI) Dual Staining
Hoechst 33342 (Invitrogen, Carlsbad, CA) and PI (Invitrogen) double staining method was used to evaluate cell death in porcine TM and monkey SCE cells. These cells were cultured on 12-well plates in DMEM containing 10% FBS and antibiotics at 37°C in 5% CO2. After the cells had grown to confluence, cells were treated with DEX and/or Y-27632 for 72 hours. As the positive control 1 mM H2O2 was treated for 4 hours. Then, Hoechst 33342 and PI were added in each well at the final concentration of 1 μg/mL, and incubated for 30 minutes. Viable cells and dead/damaged cells were checked under a fluorescence microscope (IX71; Olympus, Tokyo, Japan). The viable cells were Hoechst 33342-positive and PI-negative, whereas dead cells were both Hoechst 33342-positive and PI-positive. 
Cell Viability Assay
The effects of DEX and Y-27632 on viability of porcine TM and monkey SCE cells were evaluated using WST-8 assay (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan). 27 These cells were seeded on 96-well plates (1 × 104 cells/well) and incubated at 37°C under 5% CO2 overnight. Cells were treated with DEX and/or Y-27632 for 72 hours. The CCK-8 reagents were added in each well and incubated for 2 hours at 37°C. The absorbance at 450 nm was determined using a microplate reader (Multiskan FC; Thermo Fisher Scientific). Cell viability was expressed as a percentage of the control (vehicle-treated) cells. 
Immunocytochemistry
SCE cells were cultured on gelatin-coated glass coverslips until confluence and then treated with DEX (100 nM) and/or Y-27632 (10 μM). After 72 hours of treatment, cells were fixed in 4% paraformaldehyde in PBS for 15 minutes, and washed with cytoskeletal buffer (10 mM 2- morpholinoethansulfonic acid potassium salt, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 5 mM glucose, pH 6.1) and serum buffer (10% FBS and 0.2 mg/mL sodium azide in PBS). The cells were permeabilized with 0.5% Triton X-100 in PBS for 12 minutes at room temperature and blocked with serum buffer at 4°C for at least 2 hours. After blocking, the cells were incubated overnight at 4°C with the following primary antibodies: rabbit anti-ZO-1 (1:100 dilution; Invitrogen), anti-β-catenin (1:1000 dilution; Sigma-Aldrich), and anti-pan cadherin (1:100 dilution; Sigma-Aldrich). The cells were rinsed with serum buffer and then incubated with anti-rabbit IgG secondary antibody Alexa Fluor 488 (Invitrogen) and Phalloidin-TRITC (Sigma-Aldrich) for F-actin stained at room temperature for 30 minutes. After cells were washed with PBS, they were mounted with VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and observed using a fluorescence microscope (BX51; Olympus). 
Real-Time RT-PCR
Total RNA was isolated from cultured TM cells treated with DEX and/or Y-27632 for 24 hours using NucleoSpin RNA II (MACHEREY-NAGEL, Düren, Germany). The total RNA was reverse transcribed (Prime Script RT Master Mix; Takara Bio Inc., Shiga, Japan) according to the manufacturer's protocol. Quantitative real-time RT-PCR was performed using an ABI Prism 7000 (Life Technologies). Reactions were performed in 20 μL of reaction mixture containing 10 μL PCR master mix (SYBR Premix Ex Taq II; Takara Bio Inc.), 0.4 μM primer pairs (Table 1), and 2 μL cDNA samples. The thermal cycling conditions were 95°C for 30 seconds, 40 cycles of 95°C for 5 seconds, and 60°C for 31 seconds. All PCR reactions were performed in duplicate. 
Table 1. 
 
Primers for Real Time RT-PCR
Table 1. 
 
Primers for Real Time RT-PCR
Gene Primer Sequences (5′ to 3′) Product Size, bp
COL1A1 F: TGA CCG AGA CGT GTG GAA AC 81
R: CAG ATC ACG TCA TCG CAC AA
COL4A1 F: CAG CAA CGA ACC CCA GAA AT 120
R: CAG CAA GAA GAG GCC AAC AAG
Fibronectin F: ACC CAC AAG CCT GAC TGC CCA 91
R: TGG CCC GGT CTT CTC CTT GGG G
ITGA5 F: CGG ATC CTG GAG TCC TCG CCA 122
R: AGG GGA GCA CAG GCC AAG ATG
ITGB1 F: TGG GAC ACG GGT GAA AAT CCG A 150
R: ACG CTG CCC TGA AGC TAC CTC
18S rRNA F: GCC CGA AGC GTT TAC TTT GA 93
R: CCG CGG TCC TAT TCC ATT ATT
Relative expression levels of collagen type I α1 (COL1A1), collagen type IV α1 (COL4A1), fibronectin, integrin α5 (ITGA5), and integrin β1 (ITGB5) of the DEX and/or Y-27632–treated samples were compared to that of the control sample using the comparative CT method (ΔΔCT method). The 18S ribosomal RNA was used as an endogenous control. The threshold cycle, CT , was determined after setting the threshold in the linear amplification phase of the PCR reaction and ΔCT was defined as ΔCT = CT (target gene) – CT (18S rRNA). The relative expression level of the target gene was calculated as: 2−ΔΔCT , ΔΔCT = ΔCT (treated sample) – ΔCT (control). 
Statistical Analysis
Data are presented as means ± SE. Statistical comparisons of multiple groups were performed using ANOVA followed by Dunnett's multiple comparison test. Comparisons of two groups were performed using Student's t-test. Differences were considered statistically significant at P < 0.05. 
Results
Effect of DEX on RhoA Activation and MLC Phosphorylation
First, to elucidate the effects of DEX on the activation of RhoA/ROCK signal transduction, we conducted a pull-down assay for binding the active form of RhoA (GTP-RhoA). Relative RhoA activities were increased significantly at 5 minutes by stimulation with 100 nM DEX in porcine TM, and monkey SCE and TM cells, but not other concentrations. Mean (± SE) relative RhoA activities following treatment with 100 nM DEX were 1.64 ± 0.14, 1.58 ± 0.06, and 1.57 ± 0.06 times higher than that in the control porcine TM cells, and monkey SCE and TM cells, respectively (Figs. 1A, 1B, 1C). Statistical analysis revealed that these experimental results were significantly different from those in controls (P = 0.045, n = 6; P = 0.018, n = 4; and P = 0.018, n = 4, respectively). In addition, Western blot analysis showed that MLC phosphorylation was increased in SCE cells (P = 0.018, n = 6), but not in porcine and monkey TM cells (porcine P = 0.552, n = 6; monkey P = 0.528, n = 3), following DEX treatment for 5 minutes (Fig. 2). The addition of Y-27632 significantly inhibited the DEX-induced increase in MLC phosphorylation in SCE cells (P = 0.035, n = 4, Fig. 3). 
Figure 1. 
 
DEX-induced Rho activation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with DEX (10, 100, or 1000 nM) for 5 minutes, and the amount of activated Rho was determined by a pull-down assay. Data shown in upper panel are results of an immunoblot analysis against GTP-binding Rho (GTP-Rho) and total Rho. Data shown in lower panel are relative change in GTP-Rho. Data are shown as mean ± SE, n = 6 (A), n = 4 (B, C). *P < 0.05, compared to control by Dunnett's test.
Figure 1. 
 
DEX-induced Rho activation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with DEX (10, 100, or 1000 nM) for 5 minutes, and the amount of activated Rho was determined by a pull-down assay. Data shown in upper panel are results of an immunoblot analysis against GTP-binding Rho (GTP-Rho) and total Rho. Data shown in lower panel are relative change in GTP-Rho. Data are shown as mean ± SE, n = 6 (A), n = 4 (B, C). *P < 0.05, compared to control by Dunnett's test.
Figure 2. 
 
Effect of DEX on MLC phosphorylation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with 100 nM DEX for 5, 15, or 30 minutes (AC) or 20 μM LPA for 30 minutes (C). Data shown in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data shown in lower panels are relative change in MLC phosphorylation. Data are shown as mean ± SE, n = 6 (A, B) and n = 3 (C). *P < 0.05, compared to control by Student's t-test.
Figure 2. 
 
Effect of DEX on MLC phosphorylation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with 100 nM DEX for 5, 15, or 30 minutes (AC) or 20 μM LPA for 30 minutes (C). Data shown in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data shown in lower panels are relative change in MLC phosphorylation. Data are shown as mean ± SE, n = 6 (A, B) and n = 3 (C). *P < 0.05, compared to control by Student's t-test.
Figure 3. 
 
Effect of Y-27632 on DEX-induced MLC phosphorylation in SCE cells. Cells treated with 100 nM DEX and/or 10 μM Y-27632 for 5 minutes. Data in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data in lower panel are relative change in MLC phosphorylation. Data are mean ± SE, n = 4. *P < 0.05, compared to control by Student's t-test. #P < 0.05, compared to DEX by Student's t-test.
Figure 3. 
 
Effect of Y-27632 on DEX-induced MLC phosphorylation in SCE cells. Cells treated with 100 nM DEX and/or 10 μM Y-27632 for 5 minutes. Data in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data in lower panel are relative change in MLC phosphorylation. Data are mean ± SE, n = 4. *P < 0.05, compared to control by Student's t-test. #P < 0.05, compared to DEX by Student's t-test.
Effect of Y-27632 on DEX-Induced Ocular Outflow Resistance
We conducted a perfusion study to assess the effects of Y-27632 and DEX on outflow facility in the organ-cultured anterior ocular segments. Perfusion with 100 nM DEX for 24 hours significantly decreased the outflow facility by 31.9 ± 14.3% compared to controls (P = 0.036, n = 5, Fig. 4A), but not perfusion with 50 μM Y-27632 in addition to DEX (P = 0.884, n = 4, Fig. 4B). Additionally, perfusion with 50 μM Y-27632 in addition to DEX for 72 hours significantly increased the outflow facility by 29.0 ± 6.9% compared to DEX perfusion (P = 0.029, n = 3, Fig. 4C). The outflow facility remained decreased with DEX treatment during the 7-day experimental period (Fig. 4, Table 2). 
Figure 4. 
 
Effect of perfusion with DEX and Y-27632 on outflow facility in organ cultured porcine eye anterior segments. After measuring the baseline value, eyes were perfused with 100 nM DEX ± 50 μM Y-27632 at a constant flow of 3 μL/min at 37°C. (A) After 100 nM DEX perfusion, percent change of outflow facility from the baseline value decreased significantly over control eyes. Data are shown as mean values (n = 5). (B) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value did not decrease over control eyes. Data are shown as mean values from four independent experiments. (C) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value increased significantly over 100 nMDEX perfused contralateral eyes. Data are shown as mean values (n = 3).
Figure 4. 
 
Effect of perfusion with DEX and Y-27632 on outflow facility in organ cultured porcine eye anterior segments. After measuring the baseline value, eyes were perfused with 100 nM DEX ± 50 μM Y-27632 at a constant flow of 3 μL/min at 37°C. (A) After 100 nM DEX perfusion, percent change of outflow facility from the baseline value decreased significantly over control eyes. Data are shown as mean values (n = 5). (B) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value did not decrease over control eyes. Data are shown as mean values from four independent experiments. (C) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value increased significantly over 100 nMDEX perfused contralateral eyes. Data are shown as mean values (n = 3).
Table 2. 
 
Effects of 100 nM DEX and 50 μM Y-27632 on Outflow Facility in Porcine Eyes
Table 2. 
 
Effects of 100 nM DEX and 50 μM Y-27632 on Outflow Facility in Porcine Eyes
Time, h Outflow Facility (μL/min/mm Hg), Mean ± SE P Value, t-Test
Control vs. DEX (n = 5): Control 100 nM DEX
Baseline 0.514 ± 0.066 0.538 ± 0.021 0.673
24 0.626 ± 0.086 0.415 ± 0.070 0.036
48 0.623 ± 0.059 0.412 ± 0.032 0.042
72 0.606 ± 0.067 0.391 ± 0.054 0.025
168 0.725 ± 0.131 0.451 ± 0.078 0.068
Control vs. Y-27632 + DEX (N = 4, *n = 3): Control 50 μM Y-27632 + 100 nM DEX
Baseline 0.557 ± 0.119 0.562 ± 0.116 0.884
24 0.675 ± 0.154 0.678 ± 0.148 0.954
48 0.624 ± 0.140 0.653 ± 0.123 0.614
72 0.615 ± 0.157 0.653 ± 0.137 0.536
168 0.985 ± 0.611* 0.833 ± 0.172* 0.762
DEX vs. Y-27632 + DEX (n = 3): 100 nM DEX 50 μM Y-27632 + 100 nM DEX
Baseline 0.514 ± 0.066 0.538 ± 0.021 0.673
24 0.626 ± 0.086 0.415 ± 0.070 0.036
48 0.623 ± 0.059 0.412 ± 0.032 0.042
72 0.606 ± 0.067 0.391 ± 0.054 0.025
168 0.725 ± 0.131 0.451 ± 0.078 0.068
Effects of DEX and Y-27632 on Barrier Function of SCE Cell Monolayer
To evaluate the barrier function of the SCE cell monolayer, we measured TEER. Following treatment with 100 nM DEX, TEER increased in a time-dependent manner (Fig. 5). Mean (± SE) relative levels in TEER were 119.8 ± 6.1% (P = 0.036, n = 6), 137.7 ± 4.7% (P < 0.0001, n = 6), and 148.6 ± 6.4% (P = 0.0008, n = 6) that of the baseline levels, respectively, on days 1, 2, and 3 following treatment with 100 nM DEX. In contrast, treatment with 10 μM Y-27632 alone and 10 μM Y-27632 + 100 nM DEX induced no significant difference in TEER. These observations suggested that simultaneous administration of Y-27632 inhibited the DEX-induced increase in barrier function of the cultured SCE cell monolayer. 
Figure 5. 
 
Effect of DEX on TEER in SCE cell monolayer. SCE cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days. TEER was measured every 24 hours after drug treatment. Data are shown as mean values ± SE from six separate filters. *P < 0.05, compared to control based on Dunnett's test.
Figure 5. 
 
Effect of DEX on TEER in SCE cell monolayer. SCE cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days. TEER was measured every 24 hours after drug treatment. Data are shown as mean values ± SE from six separate filters. *P < 0.05, compared to control based on Dunnett's test.
Next, we evaluated the effects of the addition of a ROCK inhibitor to the culture media on an SCE cell monolayer pretreated with 100 nM DEX for 3 days. On day 3 (before Y-27632 treatment), we observed increased resistance in the SCE cell monolayer following the DEX treatment. On day 4, the addition of 10 μM Y-27632 to 100 nM DEX for 24 hours led to a significant recovery of the TEER, but not in the absence of Y-27632 (Fig. 6). 
Figure 6. 
 
Effect of Y-27632 on DEX-induced TEER increase in SCE cell monolayer. SCE cells were treated with 100 nM DEX for 3 days. Three days after DEX treatment, TEER values were increased significantly. After TEER measurement at day 3, Y-27632 (B) or vehicle (A) was added to the culture medium and incubated for 24 hours. Data are shown as mean ± SE from eight separate filters. *P < 0.05, compared to control by Dunnett's test. #P < 0.05, compared day 3 to day 4 by Student's t-test.
Figure 6. 
 
Effect of Y-27632 on DEX-induced TEER increase in SCE cell monolayer. SCE cells were treated with 100 nM DEX for 3 days. Three days after DEX treatment, TEER values were increased significantly. After TEER measurement at day 3, Y-27632 (B) or vehicle (A) was added to the culture medium and incubated for 24 hours. Data are shown as mean ± SE from eight separate filters. *P < 0.05, compared to control by Dunnett's test. #P < 0.05, compared day 3 to day 4 by Student's t-test.
Effects of Y-27632 on TM and SCE Cell Viability
We checked cell death and cell viability under the same conditions of TEER studies (treated with 100 nM DEX and 10 μM Y-27632 for 3 days). Hoechst 33342 and PI double staining method was used to evaluate cell death in SCE cells. PI positive cells were not observed after treatment of 100 nM DEX and 10 μM Y-27632 for 3 days in SCE cells (Fig. 7). We also evaluated the cell viability using WST-8 assay. Cell viability was not changed significantly after treatment of 100 nM DEX and 10 μM Y-27632 for 3 days compared to vehicle-treated controls in SCE cells (Fig. 8). We confirmed treatment of DEX and Y-27632 for 3 days didn't induce cell death, at least under the TEER study conditions. Additionally, we checked the effect of high concentration (50 μM) Y-27632 on cell viability and cell death, and found no harmful effects on cells under the condition (see Supplementary Material and Supplementary Figs. S1, S2, S3). 
Figure 7. 
 
Effects of DEX and Y-27632 on cell death in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were stained for Hoechst 33342 (blue, middle panels) and PI (red, bottom panels). The top panels show the phase contrast images. Scale bar: 100 μm.
Figure 7. 
 
Effects of DEX and Y-27632 on cell death in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were stained for Hoechst 33342 (blue, middle panels) and PI (red, bottom panels). The top panels show the phase contrast images. Scale bar: 100 μm.
Figure 8. 
 
Effects of DEX and Y-27632 on cell viability in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were evaluated using WST-8 assay. Data are shown as mean ± SE from three independent experiments.
Figure 8. 
 
Effects of DEX and Y-27632 on cell viability in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were evaluated using WST-8 assay. Data are shown as mean ± SE from three independent experiments.
Effects of DEX and Y-27632 on F-Actin and Junctional Proteins
The findings described demonstrated increases in outflow facility in the anterior ocular segments and in the permeability in the SCE cell monolayer. Thus, to investigate the mechanisms related to such changes in outflow, we examined immunoreactivity for the actin cytoskeleton proteins and some junctional complex proteins (i.e., ZO-1, cadherin, and β-catenin) in cultured SCE cells (Fig. 9). Following treatment with 100 nM DEX for 3 days, immunoreactivity for cortical actin, ZO-1, cadherin, and β-catenin increased. In contrast, following treatment with 10 μM Y-27632, immunostaining for the ZO-1 was decreased, and the staining of F-actin, β-catenin, and cadherin tended to be broad in the SCE cells. Further, in cultured SCE cells treated with 10 μM Y-27632 + 100 nM DEX, no DEX-induced increase in immunoreactivity was observed (Fig. 9). 
Figure 9. 
 
Effects of DEX and Y-27632 on junctional proteins and actin stress fibers in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were immunostained for molecules relating to cell-cell contact: ZO-1, β-catenin, and pan-cadherin (green). The right image of the ZO-1 images is a merged image with F-actin staining (red). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Scale bar: 50 μm.
Figure 9. 
 
Effects of DEX and Y-27632 on junctional proteins and actin stress fibers in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were immunostained for molecules relating to cell-cell contact: ZO-1, β-catenin, and pan-cadherin (green). The right image of the ZO-1 images is a merged image with F-actin staining (red). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Scale bar: 50 μm.
Changes in Extracellular Matrix and Integrin mRNA Expression by DEX and Y-27632
Previous studies reported that the production of collagen and fibronectin was increased by stimulation with DEX. 8,9 Therefore, we next quantified the expression levels for COL1A1, COL4A1, and fibronectin mRNAs in cultured porcine TM cells following treatment with DEX and/or Y-27632. Expression of COL4A1 mRNA was increased in cells stimulated with 100 nM DEX, but not in cells treated with 10 μM Y-27632 alone or in cells treated with 10 μM Y-27632 + 100 nM DEX. Mean relative levels (±SE) of COL4A1 mRNA expression induced by treatment with 100 nM DEX, 10 μM Y-27632, and 10 μM Y-27632 + 100 nM DEX were 2.07 ± 0.24-, 1.20 ± 0.28-, and 1.12 ± 0.15-fold higher than that of the controls, respectively (P = 0.006, 0.81, and 0.95, n = 4, Fig. 10B). Also, expression of fibronectin mRNA was increased in cells stimulated with 100 nM DEX (2.60 ± 0.41, P = 0.012, n = 4, Fig. 10C), and Y-27632 partially inhibited DEX-induced expression of fibronectin mRNA (2.03 ± 0.48, P = 0.048, n = 4). In contrast, stimulation with 100 nM DEX, 10 μM Y-27632, or 10 μM Y-27632 + 100 nM DEX induced no significant changes in the COL1A1 mRNA expression (Fig. 10A). Furthermore, we evaluated the expression level of integrin mRNAs. Expression of ITGA5 was slightly increased when stimulated with 100 nM DEX, but not significantly (1.27 ± 0.15, P = 0.419, Fig. 10D). ITGB1 did not change when stimulated with DEX and/or Y-27632 (Fig. 10E). 
Figure 10. 
 
Quantitative PCR analysis of collagen mRNA. The porcine TM cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 24 hours. Relative expression level of COL1A1 (A), COL4A1 (B), fibronectin (C), ITGA5 (D), and ITGB1 (E) of samples treated with DEX and/or Y-27632 was compared to that of the control sample using the comparative CT method (ΔΔCT method). The 18S ribosomal RNA was used as an endogenous control. Data are shown as mean ± SE from four independent experiments. *P < 0.05, compared to control by Student's t-test. #P < 0.05 compared to DEX by Student's t-test.
Figure 10. 
 
Quantitative PCR analysis of collagen mRNA. The porcine TM cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 24 hours. Relative expression level of COL1A1 (A), COL4A1 (B), fibronectin (C), ITGA5 (D), and ITGB1 (E) of samples treated with DEX and/or Y-27632 was compared to that of the control sample using the comparative CT method (ΔΔCT method). The 18S ribosomal RNA was used as an endogenous control. Data are shown as mean ± SE from four independent experiments. *P < 0.05, compared to control by Student's t-test. #P < 0.05 compared to DEX by Student's t-test.
Discussion
Glucocorticoid-induced ocular hypertension is a form of secondary open-angle glaucoma associated commonly with the topical administration of steroids, but it also results from other forms of steroid administration, such as sub-Tenon, intravitreal, and systemic injection. 15 Many risk factors, such as age, 4,5,28 steroid administration technique, 4 and diagnosis of open-angle glaucoma, 1,2,2931 are associated with the IOP elevation in response to steroid administration. These findings imply that changes in cellular behaviors caused by steroids may differ between patients. We reported previously that, in most cases of steroid-induced ocular hypertension, IOP tends to return to basal levels spontaneously within several months. 5 In some cases, however, IOP is not controllable with the usual medical treatments, and surgical treatment, such as external trabeculotomy or trabeculectomy, is required. 32 Thus, it is quite important to develop a better understanding of the mechanisms related to the responsiveness of the aqueous outflow to steroids and effective therapeutic modalities for steroid-induced ocular hypertension. 
To the best of our knowledge, this is the first report that RhoA is activated by DEX treatments in TM and SCE cells based on the results of a pull-down assay. The activation of RhoA, however, occurred only at a certain DEX concentration, and the upregulation of MLC phosphorylation, a downstream reaction of RhoA activation, was observed only in SCE cells. Although the underlying mechanism for this difference is unknown, regulation of the cytoskeletal structure might be context-dependent. In addition, RhoA activation generally is observed transiently, and returns spontaneously to basal levels. 33,34 Thus, the instability of RhoA activity might be another factor related to this difference. Even after transient activation of RhoA, the prolonged effects of activation of ROCK and MLC could cause changes in aqueous outflow. In support of this notion, the results of our study demonstrated that, in SCE cells, DEX treatments induced MLC phosphorylation, and the addition of a ROCK inhibitor inhibited the upregulation of MLC phosphorylation induced by DEX. We reported previously that activation of the RhoA/ROCK signaling pathway is associated with cytoskeletal rearrangement, and changes in cell shape, motility, and attachment. 17,19 Additional studies demonstrated that the RhoA/ROCK signaling pathway has an important role in the regulation of IOP, as revealed by the significant reduction in IOP following the administration of a selective ROCK inhibitor. 14,17,21 Based on these findings, we hypothesized that the RhoA/ROCK signaling pathway is involved in the pathophysiology of steroid-induced ocular hypertension. 
In our perfusion studies using anterior ocular segments, treatment with DEX, but not 50 μM Y-27632 + DEX, decreased outflow facility by 32% compared to controls at 24 hours. The decreased outflow facility caused by DEX treatment is consistent with findings from previous studies. 35,36 In contrast, to our knowledge our observation of the inhibitory effects of ROCK inhibitor on the DEX-induced decrease in outflow facility is the first report to suggest the close relationship between the RhoA/ROCK signaling pathway and steroid-induced ocular hypertension. There are two possible mechanisms underlying this finding. The first is that the inhibition is caused by the summation of the decrease in outflow facility induced by DEX and the increase in outflow induced by ROCK inhibition, and the second is that selective ROCK inhibition is associated significantly with molecular mechanisms related to decreasing the effects of DEX in outflow facility. Because DEX inhibits the expression of Rho GDI, 37 a Rho GDP dissociation inhibitor that activates RhoA, we considered the latter possibility more likely. This hypothesis also is supported by the subsequent results in our experimental series of TEER, alterations in the junctional protein expression, and collagen mRNA expression, in addition to the perfusion study as described below. 
The SCE is an important ocular component producing outflow resistance against aqueous humor in the conventional outflow route, and junctional protein complexes in SCE cells create a barrier against aqueous humor outflow. 20,24 In our study, to evaluate the barrier function of SCE, we measured TEER in a confluent SCE cell monolayer, and observed that treatment with DEX induced a 36% increase in TEER over control levels. Selective ROCK inhibition blocked the DEX-induced increase of TEER, suggesting that the RhoA/ROCK signaling pathway is involved in regulating SCE permeability. This observation may be important for clinical practice because it implies that selective ROCK inhibitors may have therapeutic value against steroid-induced ocular hypertension. Furthermore, in our study, immunoreactivity for junctional proteins, including ZO-1 and cadherin, was enhanced by DEX, consistent with a previous study, 24 and the effect was diminished by treatment with Y-27632. These findings also suggested that the changes in cell-cell contact are associated with alterations in the outflow pathway, which is consistent with the aforementioned changes in TEER following treatment with DEX and/or Y-27632. These findings suggested that, in the pathogenesis of glucocorticoid-induced ocular hypertension, SCE cells are involved in the early increase in the resistance to aqueous outflow by the conventional route. 
In TM cells treated with DEX for 24 hours, COL4A1 and fibronectin mRNA expression was more than 2-fold higher than that of controls, consistent with previous reports. 8,9 Our results showed that selective ROCK inhibition blocked the increased COL4A1and fibronectin mRNA expression, suggesting that the RhoA/ROCK signaling pathway is related to the DEX-induced changes in aqueous outflow. Abnormal accumulation of ECM components, such as glycosaminoglycans, collagens, fibronectin, and elastin, is hypothesized to cause resistance against aqueous humor outflow. 69 Thus, our results suggested that DEX-induced alterations in ECM production may be partially affected by the RhoA/ROCK signaling pathway. 
In conclusion, our results demonstrated that activation of the Rho/ROCK signaling pathway in SCE cells contributes to the mechanisms of DEX-induced changes in aqueous outflow. These findings suggested that a ROCK inhibitor may be effective to reduce IOP in eyes with steroid-induced ocular hypertension. 
Supplementary Materials
References
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Footnotes
 Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Tokyo, Japan, and from the Ministry of Health, Labor and Welfare, Tokyo, Japan.
Footnotes
 Disclosure: T. Fujimoto, None; T. Inoue, None; T. Kameda, None; N. Kasaoka, None; M. Inoue-Mochita, None; N. Tsuboi, None; H. Tanihara, None
Figure 1. 
 
DEX-induced Rho activation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with DEX (10, 100, or 1000 nM) for 5 minutes, and the amount of activated Rho was determined by a pull-down assay. Data shown in upper panel are results of an immunoblot analysis against GTP-binding Rho (GTP-Rho) and total Rho. Data shown in lower panel are relative change in GTP-Rho. Data are shown as mean ± SE, n = 6 (A), n = 4 (B, C). *P < 0.05, compared to control by Dunnett's test.
Figure 1. 
 
DEX-induced Rho activation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with DEX (10, 100, or 1000 nM) for 5 minutes, and the amount of activated Rho was determined by a pull-down assay. Data shown in upper panel are results of an immunoblot analysis against GTP-binding Rho (GTP-Rho) and total Rho. Data shown in lower panel are relative change in GTP-Rho. Data are shown as mean ± SE, n = 6 (A), n = 4 (B, C). *P < 0.05, compared to control by Dunnett's test.
Figure 2. 
 
Effect of DEX on MLC phosphorylation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with 100 nM DEX for 5, 15, or 30 minutes (AC) or 20 μM LPA for 30 minutes (C). Data shown in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data shown in lower panels are relative change in MLC phosphorylation. Data are shown as mean ± SE, n = 6 (A, B) and n = 3 (C). *P < 0.05, compared to control by Student's t-test.
Figure 2. 
 
Effect of DEX on MLC phosphorylation in porcine TM (A), and monkey SCE (B) and TM (C) cells. Cells treated with 100 nM DEX for 5, 15, or 30 minutes (AC) or 20 μM LPA for 30 minutes (C). Data shown in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data shown in lower panels are relative change in MLC phosphorylation. Data are shown as mean ± SE, n = 6 (A, B) and n = 3 (C). *P < 0.05, compared to control by Student's t-test.
Figure 3. 
 
Effect of Y-27632 on DEX-induced MLC phosphorylation in SCE cells. Cells treated with 100 nM DEX and/or 10 μM Y-27632 for 5 minutes. Data in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data in lower panel are relative change in MLC phosphorylation. Data are mean ± SE, n = 4. *P < 0.05, compared to control by Student's t-test. #P < 0.05, compared to DEX by Student's t-test.
Figure 3. 
 
Effect of Y-27632 on DEX-induced MLC phosphorylation in SCE cells. Cells treated with 100 nM DEX and/or 10 μM Y-27632 for 5 minutes. Data in upper panels are results of a representative immunoblot analysis against phosphorylated MLC (p-MLC) and total MLC. Data in lower panel are relative change in MLC phosphorylation. Data are mean ± SE, n = 4. *P < 0.05, compared to control by Student's t-test. #P < 0.05, compared to DEX by Student's t-test.
Figure 4. 
 
Effect of perfusion with DEX and Y-27632 on outflow facility in organ cultured porcine eye anterior segments. After measuring the baseline value, eyes were perfused with 100 nM DEX ± 50 μM Y-27632 at a constant flow of 3 μL/min at 37°C. (A) After 100 nM DEX perfusion, percent change of outflow facility from the baseline value decreased significantly over control eyes. Data are shown as mean values (n = 5). (B) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value did not decrease over control eyes. Data are shown as mean values from four independent experiments. (C) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value increased significantly over 100 nMDEX perfused contralateral eyes. Data are shown as mean values (n = 3).
Figure 4. 
 
Effect of perfusion with DEX and Y-27632 on outflow facility in organ cultured porcine eye anterior segments. After measuring the baseline value, eyes were perfused with 100 nM DEX ± 50 μM Y-27632 at a constant flow of 3 μL/min at 37°C. (A) After 100 nM DEX perfusion, percent change of outflow facility from the baseline value decreased significantly over control eyes. Data are shown as mean values (n = 5). (B) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value did not decrease over control eyes. Data are shown as mean values from four independent experiments. (C) After 100 nM DEX + 50 μM Y-27632 perfusion, percent change of outflow facility from the baseline value increased significantly over 100 nMDEX perfused contralateral eyes. Data are shown as mean values (n = 3).
Figure 5. 
 
Effect of DEX on TEER in SCE cell monolayer. SCE cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days. TEER was measured every 24 hours after drug treatment. Data are shown as mean values ± SE from six separate filters. *P < 0.05, compared to control based on Dunnett's test.
Figure 5. 
 
Effect of DEX on TEER in SCE cell monolayer. SCE cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days. TEER was measured every 24 hours after drug treatment. Data are shown as mean values ± SE from six separate filters. *P < 0.05, compared to control based on Dunnett's test.
Figure 6. 
 
Effect of Y-27632 on DEX-induced TEER increase in SCE cell monolayer. SCE cells were treated with 100 nM DEX for 3 days. Three days after DEX treatment, TEER values were increased significantly. After TEER measurement at day 3, Y-27632 (B) or vehicle (A) was added to the culture medium and incubated for 24 hours. Data are shown as mean ± SE from eight separate filters. *P < 0.05, compared to control by Dunnett's test. #P < 0.05, compared day 3 to day 4 by Student's t-test.
Figure 6. 
 
Effect of Y-27632 on DEX-induced TEER increase in SCE cell monolayer. SCE cells were treated with 100 nM DEX for 3 days. Three days after DEX treatment, TEER values were increased significantly. After TEER measurement at day 3, Y-27632 (B) or vehicle (A) was added to the culture medium and incubated for 24 hours. Data are shown as mean ± SE from eight separate filters. *P < 0.05, compared to control by Dunnett's test. #P < 0.05, compared day 3 to day 4 by Student's t-test.
Figure 7. 
 
Effects of DEX and Y-27632 on cell death in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were stained for Hoechst 33342 (blue, middle panels) and PI (red, bottom panels). The top panels show the phase contrast images. Scale bar: 100 μm.
Figure 7. 
 
Effects of DEX and Y-27632 on cell death in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were stained for Hoechst 33342 (blue, middle panels) and PI (red, bottom panels). The top panels show the phase contrast images. Scale bar: 100 μm.
Figure 8. 
 
Effects of DEX and Y-27632 on cell viability in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were evaluated using WST-8 assay. Data are shown as mean ± SE from three independent experiments.
Figure 8. 
 
Effects of DEX and Y-27632 on cell viability in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were evaluated using WST-8 assay. Data are shown as mean ± SE from three independent experiments.
Figure 9. 
 
Effects of DEX and Y-27632 on junctional proteins and actin stress fibers in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were immunostained for molecules relating to cell-cell contact: ZO-1, β-catenin, and pan-cadherin (green). The right image of the ZO-1 images is a merged image with F-actin staining (red). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Scale bar: 50 μm.
Figure 9. 
 
Effects of DEX and Y-27632 on junctional proteins and actin stress fibers in SCE cells. SCE cells in culture treated with 100 nM DEX and/or 10 μM Y-27632 for 3 days were immunostained for molecules relating to cell-cell contact: ZO-1, β-catenin, and pan-cadherin (green). The right image of the ZO-1 images is a merged image with F-actin staining (red). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Scale bar: 50 μm.
Figure 10. 
 
Quantitative PCR analysis of collagen mRNA. The porcine TM cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 24 hours. Relative expression level of COL1A1 (A), COL4A1 (B), fibronectin (C), ITGA5 (D), and ITGB1 (E) of samples treated with DEX and/or Y-27632 was compared to that of the control sample using the comparative CT method (ΔΔCT method). The 18S ribosomal RNA was used as an endogenous control. Data are shown as mean ± SE from four independent experiments. *P < 0.05, compared to control by Student's t-test. #P < 0.05 compared to DEX by Student's t-test.
Figure 10. 
 
Quantitative PCR analysis of collagen mRNA. The porcine TM cells were treated with 100 nM DEX and/or 10 μM Y-27632 for 24 hours. Relative expression level of COL1A1 (A), COL4A1 (B), fibronectin (C), ITGA5 (D), and ITGB1 (E) of samples treated with DEX and/or Y-27632 was compared to that of the control sample using the comparative CT method (ΔΔCT method). The 18S ribosomal RNA was used as an endogenous control. Data are shown as mean ± SE from four independent experiments. *P < 0.05, compared to control by Student's t-test. #P < 0.05 compared to DEX by Student's t-test.
Table 1. 
 
Primers for Real Time RT-PCR
Table 1. 
 
Primers for Real Time RT-PCR
Gene Primer Sequences (5′ to 3′) Product Size, bp
COL1A1 F: TGA CCG AGA CGT GTG GAA AC 81
R: CAG ATC ACG TCA TCG CAC AA
COL4A1 F: CAG CAA CGA ACC CCA GAA AT 120
R: CAG CAA GAA GAG GCC AAC AAG
Fibronectin F: ACC CAC AAG CCT GAC TGC CCA 91
R: TGG CCC GGT CTT CTC CTT GGG G
ITGA5 F: CGG ATC CTG GAG TCC TCG CCA 122
R: AGG GGA GCA CAG GCC AAG ATG
ITGB1 F: TGG GAC ACG GGT GAA AAT CCG A 150
R: ACG CTG CCC TGA AGC TAC CTC
18S rRNA F: GCC CGA AGC GTT TAC TTT GA 93
R: CCG CGG TCC TAT TCC ATT ATT
Table 2. 
 
Effects of 100 nM DEX and 50 μM Y-27632 on Outflow Facility in Porcine Eyes
Table 2. 
 
Effects of 100 nM DEX and 50 μM Y-27632 on Outflow Facility in Porcine Eyes
Time, h Outflow Facility (μL/min/mm Hg), Mean ± SE P Value, t-Test
Control vs. DEX (n = 5): Control 100 nM DEX
Baseline 0.514 ± 0.066 0.538 ± 0.021 0.673
24 0.626 ± 0.086 0.415 ± 0.070 0.036
48 0.623 ± 0.059 0.412 ± 0.032 0.042
72 0.606 ± 0.067 0.391 ± 0.054 0.025
168 0.725 ± 0.131 0.451 ± 0.078 0.068
Control vs. Y-27632 + DEX (N = 4, *n = 3): Control 50 μM Y-27632 + 100 nM DEX
Baseline 0.557 ± 0.119 0.562 ± 0.116 0.884
24 0.675 ± 0.154 0.678 ± 0.148 0.954
48 0.624 ± 0.140 0.653 ± 0.123 0.614
72 0.615 ± 0.157 0.653 ± 0.137 0.536
168 0.985 ± 0.611* 0.833 ± 0.172* 0.762
DEX vs. Y-27632 + DEX (n = 3): 100 nM DEX 50 μM Y-27632 + 100 nM DEX
Baseline 0.514 ± 0.066 0.538 ± 0.021 0.673
24 0.626 ± 0.086 0.415 ± 0.070 0.036
48 0.623 ± 0.059 0.412 ± 0.032 0.042
72 0.606 ± 0.067 0.391 ± 0.054 0.025
168 0.725 ± 0.131 0.451 ± 0.078 0.068
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