The Effect of Rho-Associated Protein Kinase Inhibitor on Monkey Schlemm's Canal Endothelial Cells

Abstract

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, β-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.

Introduction

The major route of drainage of the aqueous humor is the conventional pathway through the trabecular meshwork (TM) and Schlemm's canal in primates. ^1–3 Although discrepancies exist among previous studies between clinical observations and basic examinations, ^4,5 some experimental studies suggest that the aqueous outflow resistance is generated in the locus of the inner wall of Schlemm's canal and the juxtacanalicular tissue region in both normal and glaucomatous eyes. ^6–8 The properties of the cytoskeletal structures, adhesive interactions, permeability of Schlemm's canal endothelial (SCE) cells, and secretion from TM cells are suggested to play important roles in the regulation of aqueous outflow. ^1,9,10  

Previous studies, including ours, revealed that a selective Rho-associated coiled-coil–forming protein kinase (ROCK) inhibitor, Y-27632, caused retraction and rounding of human TM-cell bodies, disruption of filamentous actin (F-actin), impairment of focal adhesion formation, and decreased myosin light chain phosphorylation in TM cells and SCE cells, resulting in a significant intraocular pressure (IOP) decrease and an aqueous outflow facility increase. ^11–16 Another selective ROCK inhibitor, Y-39983, was reported to have similar effects on outflow facility, ^17,18 and reduced IOP in healthy volunteers in our previous clinical trial. ¹⁹ Thus, ROCK inhibitors are recognized as potential glaucoma drugs to lower IOP. Previous works, however, mainly focused on the molecular mechanisms underlying the effects of ROCK inhibitors on TM cells, and effects on SCE cells have not been clarified thoroughly.  

The aim of the present study was to investigate the effect of Y-27632 on monkey SCE cells. Permeability of the SCE-cell monolayer and properties of the intercellular junctions were observed. Furthermore, we used DNA microarrays and gene ontology analysis to investigate whether Y-27632 modulates broad functions in SCE cells. 

Materials and Methods

Perfusion and Histologic Examination

Fresh cynomolgus monkey eyes were obtained from a commercial company (Keari Co., Ltd., Osaka, Japan). The eyes were stored on ice in eye storage medium (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan) and perfused within 48 hours. Radial iridotomy was performed to prevent artificial deepening of the anterior chamber during perfusion. A 23-gauge perfusion needle was inserted through the cornea, placing the tip into the anterior chamber of the eye. The perfusion medium was Dulbecco's phosphate-buffered saline (DPBS), pH 7.4, with 5.5 mM glucose. Eyes were perfused at a constant pressure of 10 mm Hg at 25°C. After the initial baseline was recorded for 1 hour, the test eyes were perfused for 5 hours with DPBS containing 50 μM Y-27632 (Wako Pure Chemical Industries, Osaka, Japan). As controls, the contralateral fellow eyes were perfused with DPBS alone. To calculate the inflow rate, the weight of the perfusion medium reservoir was measured using a digital balance (Shimadzu, Tokyo, Japan) and the data were recorded on a computer every 30 seconds. The aqueous humor outflow facility (μL/min/mm Hg) was calculated as the ratio between the inflow rate (μL/min) and the perfusion pressure (mm Hg). Outflow facility values were calculated as the average over 10 minutes and expressed as percentage change from baseline. The baseline values of outflow facility were determined at least 10 minutes before changing the perfusion medium. Following drug perfusion, the eyes were fixed by perfusion with 2.5% glutaraldehyde in 0.1 M phosphate buffer at 10 mm Hg for 2 hours. After fixation, the eyes were dissected at the equator and the vitreous and lens were removed. The anterior segment of the eyes was immersed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4°C. For scanning electron microscopy (SEM), specimens were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer. After critical point drying, the anterior segment was mounted on an SEM stub, gold-coated, and examined with an SE microscope (JSM-6360LV; JEOL, Tokyo, Japan). All three pairs of eyes were examined under SEM, and 4 to 10 different sites in each eye were observed.  

Culture of Monkey Schlemm's Canal Endothelial Cells

The SCE explants were dissected from eyes of 6- to 12-month-old cynomolgus monkeys obtained from a commercial laboratory (Shin Nippon Biomedical Laboratories, Kagoshima, Japan) according to the method described previously by Alvarado et al. ^20,21 Briefly, Schlemm's canal was identified by cannulating the lumen with a 9-0 nylon suture under microscopic observation. The corneoscleral and uveoscleral TM tissues were removed completely with fine forceps, and explants of the inner wall of Schlemm's canal were cut out and placed on plates coated with collagen gel. Primary SCE cells were expanded in Dulbecco's modified Eagle's medium (DMEM; WAKO Pure Chemical Industries, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) and antibiotics at 37°C in 5% CO₂, and SCE cells between passages 6 and 8 were used in all experiments in the present study. 

Measurement of Monolayer Transendothelial Electron Resistance

SCE cells were seeded at 1 × 10⁵ cells/well and were grown until confluent on polycarbonate membrane inserts (0.4 μm pore size and 12 mm diameter; Corning Transwell, Sigma-Aldrich, St. Louis, MO) on 12-well culture plates (BD Falcon, Franklin Lakes, NJ) in DMEM supplemented with 10% FBS at 37°C in 5% CO₂. The volume of the apical side (inside of the membrane inserts) was 0.5 mL and that of the basal side (outside of the membrane inserts) was 1.5 mL. Two weeks after seeding, the medium was changed to DMEM supplemented with Y-27632 (0, 1, 5, or 25 μM) or A23187 (a calcium ionophore that increases intracellular calcium ion levels; 10 μM; Calbiochem, Darmstadt, Germany) for TEER experiments. The TEER was measured using an electrical resistance system (Millicell ERS; Millipore, Billerica, MA) according to the manufacturer's instructions 30, 60, and 90 min after treatment with Y-27632, and recorded as net values (Ωcm²). Time-dependent changes after treatment were monitored and compared as percentage change from baseline values. Each experiment was repeated at least three times. The effect of EGTA (2 mM; Dojindo Laboratories, Kumamoto, Japan) was confirmed as a positive control. 

Measurement of Monolayer Cell Permeability

SCE cells were prepared by the same method for TEER measurement as described above. SCE-cell monolayers were then stimulated with Y-27632 at concentrations of 0, 1, 5, or 25 μM. A tracer, 4 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich), was simultaneously applied at 50 μM to the basal compartment of the wells. The medium was collected from the apical side for fluorescence measurements at 30, 60, and 90 minutes after adding the tracer and the same volume of the culturing medium was added to replace the medium removed. The concentration of FITC-dextran in the collected medium was measured using a multimode plate reader (Multi Microplate Reader, MTP-800AFC; Corona Electric, Ibaragi, Japan), with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Fluorescence intensity of the normal medium was measured as the background concentration in each experiment, and each experiment was repeated at least three times. 

Immunocytochemistry

SCE cells were cultured on gelatin-coated glass coverslips until confluent and then treated with Y-27632 (25 μM) or latrunculin B (an F-actin depolymerizing drug, 400 nM; Merck KGaA, Darmstadt, Germany). When needed, SCE cells were pretreated with jasplakinolide (an F-actin stabilizing drug, 15 nM; Enzo Life Sciences, Farmingdale, NY) for 1 hour before treatment with Y-27632. After a 30-minute treatment, cells were fixed with 4% paraformaldehyde in PBS for 15 minutes, washed with cytoskeletal buffer (10 mM MES [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). Cells were permeabilized with 0.5% Triton X-100 in PBS for 12 minutes at room temperature and blocked with serum buffer for at least 2 hours at 4°C. After blocking, the cells were incubated overnight at 4°C with the following primary antibodies: rabbit anti-ZO-1 (1.25 μg/mL; Invitrogen, Carlsbad, CA), anti-claudin-5 (2.5 μg/mL; 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 secondary antibody (Alexa Fluoro 488; Invitrogen) and Phalloidin-TRITC (Sigma-Aldrich) for F-actin stained for 30 minutes at room temperature. After washing the cells with PBS, the cells were mounted with commercial mounting medium (VECTASHIELD; Vector Laboratories, Burlingame, CA), with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and observed via a fluorescence microscope (BX51; Olympus, Tokyo, Japan). 

DNA Microarray Analysis

Custom cDNA microarray analysis was performed using DNA microarray technology (CombiMatrix, Mukilteo, WA). The cynomolgus monkey array was designed to detect directly labeled mRNA from 12,613 probes. Confluent SCE cells in 100-mm dishes were treated with Y-27632 (25 μM) or vehicle for 30 minutes. Total RNA was then extracted from the cells, and the integrity and concentration of total RNA were measured using a bioanalysis unit (Agilent 2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). Fluorescence-labeled antisense RNA was synthesized by direct incorporation of Cy5-UTP or Cy3-UTP, using each RNA sample and an RNA transcript kit (RNA Transcript SureLABEL Core Kit; TaKaRa BIO, Shiga, Japan). The labeled antisense RNAs were hybridized simultaneously with the microarray chips. DNA microarray preparation, hybridization, processing, scanning, and analyses were performed according to the manufacturer's instructions (Filgen, Nagoya, Japan). Fluorescence images of hybridized microarrays were obtained with a commercial scanner (GenePix 4000B Scanner; Molecular Devices, Sunnyvale, CA). The analyzer (Array-Pro Analyzer Ver4.5; Media Cybernetics, Silver Spring, MD) was used to determine the signal intensity of each spot and its local background. Scan data images were analyzed using microarray data software (Microarray Data Analysis Tool Ver 3.2 software; Filgen). Signals from Y-27632–treated cells were compared with those from vehicle-treated cells, and genes that showed a >2-fold change in expression in at least one of the pairwise probe comparisons were considered upregulated, whereas those that showed a change of expression <0.5-fold were considered downregulated. These analyses were performed three times using SCE cells from three different monkeys independently, and genes with common differences in expression among the three experiments were identified as affected genes. The affected genes were further analyzed by gene ontology, in which putative functions of gene products were categorized as “biological process,” “cellular component,” or “molecular function” by a BLAST (Basic Local Alignment Search Tool) homology search of the expressed sequence tags available at the National Center for Biotechnology Information. 

Calcium Imaging

Confluent SCE cells were incubated with Fluo-4/AM (5 μM; Invitrogen) in serum-free DMEM for 60 minutes in the dark, followed by a wash with Tyrode's solution with 0.5 mM of calcium according to the manufacturer's instructions. Fluo-4 was then excited at 480 nm, and emission was recorded continuously with a band-pass filter between 505 and 550 nm using a fluorescence microscope system (IX71; Olympus). The archived photographs were converted to gray-scale images, and the signals were quantified using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). The percent change from baseline (before treatment) was compared among conditions. 

Statistical Analysis

Data are presented as means ± SEs. Statistical comparisons of multiple groups were performed using one-way or two-way repeated-measure ANOVA followed by Bonferroni/Dunn's post hoc test. Comparisons of two groups used Student's t-test with Bonferroni's correction. Differences were considered statistically significant at P < 0.05. 

Results

Effect of Y-27632 on Outflow Facility

Baseline values of outflow facility in the control eyes and drug-perfused eyes were 0.50 ± 0.15 and 0.48 ± 0.08, respectively (n = 3). After the drug perfusion, outflow facility increased progressively, and was significantly higher within 170 minutes after perfusion compared with control eyes. There was a 38% increase in outflow facility over control eyes at 3 hours (P = 0.026), and the corresponding value progressed to 61% (P = 0.008) at 4 hours after drug perfusion (Fig. 1). 

Figure 1. 

 

Effect of perfusion with Y-27632 on outflow facility in enucleated monkey eyes. After measuring the baseline value, eyes were perfused with 50 μM Y-27632 at a constant pressure (10 mm Hg) at 25°C. After drug perfusion, the percentage change of outflow facility from the baseline value increased significantly and time-dependently over control eyes. Values are mean ± SE, n = 3, *P < 0.05, ^# P < 0.01.

Effect of Y-27632 on Morphology of SCE

After drug perfusion, baseline values of outflow facility in the control eyes and drug-perfused eyes were subjected to SEM observations. As shown in a past study, ¹² Y-27632–perfused eyes tended to have more giant vacuoles (Fig. 2B) on the SCE compared with control eyes (Fig. 2A). Although intercellular detachments, possibly paracellular drainage routes, were observed in both control eyes and drug-perfused eyes, they were more frequently seen in the latter. 

Figure 2. 

 

Scanning electron micrographs of SCE. Compared with control eyes (left), SCE of Y-27632 perfused eyes tended to have more giant vacuoles (right). Although intercellular detachments were also observed in control eyes, they were more frequently observed in the drug-perfused eyes.

Effect of Y-27632 on SCE-Cell Monolayer Permeability

TEER of the SCE cell monolayer after adding 25 μM of Y-27632 was significantly lower compared with that of controls at 30 minutes. At 60 minutes after treatment, 1, 5, and 25 μM Y-27632 significantly decreased TEER (Fig. 3). Of interest, the TEER values were not significantly different among the three samples treated with Y-27632 at 1, 5, and 25 μM each 60 minutes after treatment. The permeability assays using a flux of FITC-dextran showed that treatment with Y-27632 increased the concentration of FITC-dextran in the apical side time-dependently and dose-dependently. One millimolar Y-27632 did not significantly change the concentration of the FITC-dextran, whereas 25 μM of Y-27632 increased the concentration nearly 2-fold (Fig. 4). 

Figure 3. 

 

Changes in TEER in the SCE-cell monolayer. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.

Figure 4. 

 

Changes in the SCE-cell monolayer permeability of 4 kDa fluorescein isothiocyanate-dextran. SCE cells were treated with a ROCK inhibitor, Y-27632, at concentrations of 1, 5, 25 μM for 30, 60, and 90 minutes. Mean values ± SE from three separate filters are presented. One-way ANOVA was followed by Dunnett's test. *P < 0.05.

Effects of Y-27632 on F-Actin and Molecules Associated with Cell–Cell Contact in SCE Cells

Immunocytochemical staining revealed a modest disruption of tight-junction–related proteins, such as ZO-1 and claudin-5 in Y-27632–treated cells in conjunction with the disappearance of longitudinal F-actin (Figs. 5A–D). Meanwhile, the staining of molecules related to adherence junction, such as β-catenin and pan-cadherin, tended to be broad and sparse after treatment with Y-27632 (Figs. 5E–H). Those changes in adherence junctions were less prominent, if any, compared with tight junctions. We also investigated effects of latrunculin B, a Rho-independent cytoskeletal drug. As shown in Fig. 6, ZO-1 proteins were disrupted by treatment with latrunculin B. Moreover, pretreatment with jasplakinolide, an actin filament–stabilizing drug, rescued in part the effect of subsequent treatment with Y-27632 on both F-actin structure and intracellular localization of ZO-1 (Figs. 6C–F). 

Figure 5. 

 

Effects of the ROCK inhibitor Y-27632 on cell–cell contact and actin stress fibers in SCE cells. SCE cells in culture treated with 25 μM Y-27632 for 30 minutes were immunostained for molecules relating to cell–cell contact: ZO-1, claudin-5, β-catenin, and pan-cadherin (green). The left image of each figure is a merged image with F-actin staining (red). Cell nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.

Figure 6. 

 

Effects of latrunculin, jasplakinolide, and Y-27632 on the polymerization of F-actin and intracellular localization of ZO-1. The top images show ZO-1 expression (green) in untreated SCE cells and latrunculin-treated SCE cells. The effects of Y-27632 with or without pretreatment with jasplakinolide are shown in the middle (F-actin, red) and bottom images (ZO-1, green). Scale bar: 50 μm.

Microarray Expression Profile in Y-27632–Treated SCE Cells

Affected genes analyzed by microarray are listed in Tables 1 and 2, in which 57 genes were upregulated and 15 genes were downregulated. Among those genes, 10 genes showed >5-fold expression changes, and one gene showed <0.2-fold expression change (Tables 1 and 2). Significantly upregulated and downregulated gene categories based on gene ontology analysis in Y-27632–treated SCE cells are listed in Tables 3 and 4. Gene ontology analysis revealed that affected genes were related to various cellular functions, including upregulation of a biological process, “regulation of extracellular matrix disassembly” (P = 0.023), downregulation of a cellular component, “integrin complex” (P = 0.039), and downregulation of a biological process, “calcium ion transport into cytosol” (P = 0.008). 

Table 1.  

 

Representative Genes That Are Upregulated in SCE Cells

Accession Number	Gene Name	Fold Change
CJ435051	Myelin basic protein isoform 2	16.42917
CJ440786	Pleckstrin homology domain interacting protein	15.84593
BB889989	Chondroadherin precursor	7.27175
AB170326	Human syndecan 4 (amphiglycan, ryudocan) (SDC4)	4.79868
AB171586	Human adenylate cyclase 2 (brain) (ADCY2)	4.44696
DW528016	Thyroid hormone receptor interactor 3	4.38051
CJ492124	Splicing factor, arginine/serine-rich 6 [Rattus norvegicus]	4.285
AB168674	Human RIKEN cDNA 1700016G05 (LOC402604)	4.25099
DC621007	L-Threonine dehydrogenase	4.07265
BB898986	Complement factor I preproprotein	3.73984
AB172153	Human CDW92 antigen (CDW92)	2.9576
AB170860	Human WD repeat domain 8 (WDR8)	2.66228
AB070180	Human hypothetical protein FLJ46088, mRNA, AK127973	2.59433
DK578068	Hypothetical protein LOC348262 [Homo sapiens]	2.58971
CJ470450	Splicing factor 3b, subunit 1 isoform 1 [Danio rerio]	2.58217
EF208807	MHC class II antigen DP alpha chain (Mafa-DPA) mRNA, Mafa-DPA1*0202 allele	2.56233
AB174415	Spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) (SPTAN1)	2.55997
AB169059	CD4 precursor	2.40463
DC645348	Sulfotransferase family, cytosolic, 1C, member 1 isoform a	2.40361
CJ446069	Protein phosphatase 3, catalytic subunit, alpha isoform	2.40133
CJ440484	Brain cDNA	2.38472
DK579312	Spliceosome RNA helicase BAT1	2.36141
AB168995	Human ring finger protein 36 (RNF36)	2.28256
DC850662	Fc gamma RIIIa	2.26894
AB179134	Human syntaxin binding protein 4 (STXBP4)	2.25772
AB071116	Human ankyrin repeat and SOCS box-containing 15 (ASB15)	2.22498
DC850740	Cytosolic phospholipase A2 gamma precursor (cPLA2-gamma) (Phospholipase A2 group IVC)	2.2098
AB174030	Human galactose mutarotase (aldose 1-epimerase) (GALM)	2.20885
AB168319	Human chromosome 6 open reading frame 10 (C6orf10)	2.1913
AB169280	Human hypothetical protein FLJ13305 (FLJ13305)	2.16347
CJ492687	USP6 N-terminal like	2.16298
AB169348	Human hypothetical protein MGC33600 (MGC33600)	2.15724
AB172168	Human protein tyrosine phosphatase, receptor type, fpolypeptide (PTPRF), interacting protein (liprin), alpha4 (PPFIA4)	2.12463
AB173609	Human cholinergic receptor, nicotinic, epsilon polypeptide (CHRNE)	2.12166
AB173849	Human zinc finger protein 24 (KOX 17) (ZNF24)	2.07481
AB168153	Human DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 (DDX43)	2.07426

Table 2.  

 

Representative Genes That Are Downregulated in SCE Cells

Accession Number	Gene Name	Fold Change
DK576163	Human H3 histone, family 3B (H3.3B) (H3F3B)	0.28442
AB174375	Human dual specificity phosphatase 14 (DUSP14)	0.35283
DC631004	Nucleophosmin (nucleolar phosphoprotein B23, numatrin)	0.42112
AB072781	Chromodomain Y-like protein 2 (CDYL2)	0.44636
CJ440707	Brefeldin A–inhibited guanine nucleotide-exchange protein 1	0.46621
AB070198	Human DNA-damage inducible protein 1 (DDI1)	0.4856
DK581802	Cytokine receptor-like factor 3 [Homo sapiens]	0.49076
AB173178	Human hepatic leukemia factor (HLF)	0.49856

Table 3.  

 

Gene Ontology of Upregulated Genes in Y-27632–Treated SCE Cells

Ontology	Term	Total Genes	P-Value
Molecular function	MHC class II protein binding	3(3)	0.00131
Biological process	Transmembrane receptor protein tyrosine kinase signaling pathway	104(27)	0.00169
Biological process	Negative regulation of Ras protein signal transduction	6(3)	0.0036
Biological process	Negative regulation of small GTPase-mediated signal transduction	6(0)	0.0036
Biological process	Regulation of cytoskeleton organization	60(2)	0.0064
Biological process	RNA processing	339(35)	0.00688
Biological process	tRNA processing	35(28)	0.00961
Biological process	Enzyme-linked receptor protein signaling pathway	152(1)	0.00999
Biological process	Cellular response to insulin stimulus	39(18)	0.01265
Molecular function	MHC protein binding	13(0)	0.01275
Biological process	Cellular response to endogenous stimulus	78(0)	0.01507
Biological process	Cellular response to hormone stimulus	78(12)	0.01507
Biological process	Cellular response to peptide hormone stimulus	42(0)	0.01525
Molecular function	Molecular transducer activity	621(0)	0.01768
Molecular function	Signal transducer activity	621(134)	0.01768
Biological process	Positive regulation of cytoskeleton organization	16(2)	0.01822
Biological process	Regulation of cellular component organization	229(0)	0.01957
Molecular function	1-Acylglycerophosphocholine O-acyltransferase activity	1(1)	0.02316
Molecular function	1-Alkylglycerophosphocholine O-acetyltransferase activity	1(1)	0.02316
Molecular function	Aldose 1-epimerase activity	1(1)	0.02316
Molecular function	Cholesterol 24-hydroxylase activity	1(1)	0.02316
Molecular function	Choline transmembrane transporter activity	1(1)	0.02316
Molecular function	tRNA isopentenyltransferase activity	1(1)	0.02316
Biological process	Elastin catabolic process	1(0)	0.02321
Biological process	Extracellular matrix disassembly	1(0)	0.02321
Biological process	Histolysis	1(0)	0.02321
Biological process	Induction by virus of host cell–cell fusion	1(1)	0.02321
Biological process	Negative regulation of Rac protein signal transduction	1(1)	0.02321
Biological process	Negative regulation of blood vessel remodeling	1(1)	0.02321
Biological process	Negative regulation of collagen catabolic process	1(1)	0.02321
Biological process	Negative regulation of elastin catabolic process	1(1)	0.02321
Biological process	Negative regulation of extracellular matrix disassembly	1(1)	0.02321
Biological process	Negative regulation of histolysis	1(1)	0.02321
Biological process	Positive regulation of centriole replication	1(1)	0.02321
Biological process	Positive regulation of centrosome cycle	1(0)	0.02321
Biological process	Regulation of blood vessel remodeling	1(0)	0.02321
Biological process	Regulation of collagen catabolic process	1(0)	0.02321
Biological process	Regulation of elastin catabolic process	1(0)	0.02321
Biological process	Regulation of extracellular matrix disassembly	1(0)	0.02321
Biological process	Regulation of histolysis	1(0)	0.02321
Biological process	Tissue death	1(0)	0.02321
Biological process	Trophoblast giant cell differentiation	1(1)	0.02321
Biological process	Cellular response to organic substance	96(0)	0.02891
Biological process	Insulin receptor signaling pathway	21(12)	0.02903
Molecular function	Receptor activity	425(266)	0.0338
Cellular component	Exon–exon junction complex	2(2)	0.03402
Molecular function	3′,5′-Cyclic-GMP phosphodiesterase activity	2(1)	0.03454
Molecular function	O-Acetyltransferase activity	2(0)	0.03454
Molecular function	Actinin binding	2(0)	0.03454
Molecular function	alpha-Actinin binding	2(1)	0.03454
Molecular function	Amine oxidase activity	2(2)	0.03454
Molecular function	Arginase activity	2(2)	0.03454
Molecular function	Isocitrate dehydrogenase (NADP+) activity	2(2)	0.03454
Biological process	Factor XII activation	2(2)	0.03462
Biological process	Cell differentiation involved in embryonic placenta development	2(1)	0.03462
Biological process	Choline transport	2(2)	0.03462
Biological process	Elastin metabolic process	2(1)	0.03462
Biological process	Glyoxylate cycle	2(2)	0.03462
Biological process	Kinin cascade	2(0)	0.03462
Biological process	ncRNA catabolic process	2(0)	0.03462
Biological process	Negative regulation of Rho protein signal transduction	2(2)	0.03462
Biological process	Negative regulation of collagen metabolic process	2(0)	0.03462
Biological process	Negative regulation of multicellular organismal metabolic process	2(0)	0.03462
Biological process	Plasma kallikrein-kinin cascade	2(0)	0.03462
Biological process	Positive regulation of focal adhesion assembly	2(2)	0.03462
Biological process	rRNA catabolic process	2(2)	0.03462
Biological process	Regulation of Rac GTPase activity	2(2)	0.03462
Biological process	Regulation of centriole replication	2(1)	0.03462
Biological process	Response to insulin stimulus	59(21)	0.03533
Biological process	Cellular component disassembly	60(0)	0.0368
Biological process	Regulation of organelle organization	104(0)	0.03688
Cellular component	Nuclear speck	62(62)	0.03815
Biological process	Regulation of small GTPase- mediated signal transduction	106(20)	0.03906
Biological process	Nitrogen compound metabolic process	1974(14)	0.04265
Cellular component	Compact myelin	3(2)	0.04511
Cellular component	Spectrin	3(3)	0.04511
Molecular function	Calcium- and calmodulin-responsive adenylate cyclase activity	3(3)	0.04579
Molecular function	Histamine receptor activity	3(3)	0.04579
Molecular function	Structural constituent of myelin sheath	3(3)	0.04579
Molecular function	Thrombospondin receptor activity	3(3)	0.04579
Biological process	Regulation of catalytic activity	382(4)	0.0458
Biological process	Arginine catabolic process	3(3)	0.04589
Biological process	Cytoplasmic microtubule organization	3(2)	0.04589
Biological process	Positive regulation of fibrinolysis	3(3)	0.04589
Biological process	Positive regulation of protein maturation by peptide bond cleavage	3(0)	0.04589
Biological process	Regulation of cell-substrate junction assembly	3(0)	0.04589
Biological process	Regulation of centrosome cycle	3(0)	0.04589
Biological process	Regulation of centrosome duplication	3(1)	0.04589
Biological process	Regulation of focal adhesion assembly	3(0)	0.04589
Biological process	RNA splicing, via transesterification reactions with bulged adenosine as nucleophile	66(0)	0.04622
Biological process	Nuclear mRNA splicing, via spliceosome	66(30)	0.04622
Biological process	Regulation of cellular component biogenesis	67(0)	0.0479

Table 4.  

 

Gene Ontology of Downregulated Genes in Y-27632–Treated SCE Cells

Ontology	Term	Total Genes	P-Value
Molecular function	Hydroxymethylglutaryl-CoA synthase activity	2(2)	0.00033
Molecular function	Transferase activity, transferring acyl groups, acyl groups converted into alkyl on transfer	6(3)	0.00152
Molecular function	Exonuclease activity, active with either ribo- or deoxyribonucleic acids and producing  5′-phosphomonoesters	9(0)	0.00294
Biological process	Regulation of release of sequestered calcium ion into cytosol	11(0)	0.00395
Biological process	Negative regulation of sequestering of calcium ion	13(0)	0.00527
Biological process	Regulation of sequestering of calcium ion	13(0)	0.00527
Biological process	Release of sequestered calcium ion into cytosol	13(2)	0.00527
Molecular function	3′-5′ Exonuclease activity	13(7)	0.00551
Biological process	Isoprenoid biosynthetic process	14(10)	0.006
Biological process	Sequestering of calcium ion	15(2)	0.00677
Molecular function	Glutathione transferase activity	15(15)	0.00707
Biological process	Regulation of calcium ion transport into cytosol	16(0)	0.00758
Biological process	Calcium ion transport into cytosol	17(0)	0.00843
Biological process	Cytosolic calcium ion transport	17(0)	0.00843
Biological process	Sequestering of metal ion	17(0)	0.00843
Cellular component	Microtubule	122(101)	0.00956
Biological process	Cholesterol biosynthetic process	20(15)	0.01123
Biological process	DNA replication checkpoint	1(1)	0.01472
Biological process	Cysteine biosynthetic process from serine	1(1)	0.01472
Biological process	Cysteine biosynthetic process via cystathionine	1(1)	0.01472
Biological process	Homocysteine catabolic process	1(1)	0.01472
Biological process	Negative regulation of DNA-dependent DNA replication initiation	1(0)	0.01472
Molecular function	L-Gulonate 3-dehydrogenase activity	1(1)	0.01504
Molecular function	Bradykinin receptor activity	1(1)	0.01504
Molecular function	Cystathionine beta-synthase activity	1(1)	0.01504
Molecular function	Exodeoxyribonuclease III activity	1(1)	0.01504
Molecular function	Monodehydroascorbate reductase (NADH) activity	1(1)	0.01504
Molecular function	Polyribonucleotide nucleotidyltransferase activity	1(1)	0.01504
Molecular function	Ribonuclease III activity	1(1)	0.01504
Biological process	Sterol biosynthetic process	25(5)	0.01666
Biological process	Regulation of calcium ion transport	26(1)	0.01786
Molecular function	Exonuclease activity	26(19)	0.01864
Biological process	Regulation of metal ion transport	28(0)	0.02037
Molecular function	Nuclease activity	76(10)	0.02145
Biological process	L-Ascorbic acid biosynthetic process	2(2)	0.022
Biological process	L-Cysteine catabolic process	2(2)	0.022
Biological process	L-Cysteine metabolic process	2(1)	0.022
Biological process	Cysteine biosynthetic process	2(1)	0.022
Biological process	Cysteine catabolic process	2(0)	0.022
Biological process	Homocysteine metabolic process	2(0)	0.022
Biological process	Hydrogen sulfide biosynthetic process	2(2)	0.022
Biological process	Hydrogen sulfide metabolic process	2(0)	0.022
Biological process	Regulation of DNA-dependent DNA replication	2(0)	0.022
Biological process	Regulation of DNA-dependent DNA replication initiation	2(1)	0.022
Molecular function	Transferase activity, transferring alkyl or aryl (other than methyl) groups	31(2)	0.02542
Biological process	Isoprenoid metabolic process	32(0)	0.02578
Biological process	L-Serine catabolic process	3(3)	0.02923
Biological process	Positive regulation of ryanodine-sensitive calcium-release channel activity	3(3)	0.02923
Biological process	Positive regulation of survival gene product expression	3(3)	0.02923
Biological process	Regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum	3(3)	0.02923
Biological process	Release of sequestered calcium ion into cytosol by sarcoplasmic reticulum	3(0)	0.02923
Biological process	Sulfur amino acid catabolic process	3(0)	0.02923
Biological process	Sulfur compound catabolic process	3(0)	0.02923
Molecular function	N-Terminal myristoylation domain binding	3(3)	0.02987
Molecular function	Exodeoxyribonuclease activity	3(0)	0.02987
Molecular function	Exodeoxyribonuclease activity, producing 5′-phosphomonoesters	3(0)	0.02987
Molecular function	Interleukin-2 binding	3(1)	0.02987
Molecular function	Interleukin-2 receptor activity	3(3)	0.02987
Biological process	Regulation of ion transport	36(1)	0.03171
Biological process	Cell surface receptor linked signaling pathway	552(70)	0.03242
Molecular function	Ribonuclease activity	36(16)	0.03305
Biological process	L-Ascorbic acid metabolic process	4(2)	0.03641
Biological process	Negative regulation of ryanodine-sensitive calcium-release channel activity	4(4)	0.03641
Biological process	Sarcoplasmic reticulum calcium ion transport	4(1)	0.03641
Biological process	Cytokine-mediated signaling pathway	39(20)	0.03648
Biological process	Initiation of signal transduction	39(0)	0.03648
Biological process	Leukocyte migration	39(5)	0.03648
Biological process	Signal initiation by diffusible mediator	39(0)	0.03648
Biological process	Signal initiation by protein/peptide mediator	39(0)	0.03648
Cellular component	Barr body	5(5)	0.03939
Cellular component	Integrin complex	5(5)	0.03939
Cellular component	Receptor complex	47(4)	0.04198
Biological process	Elevation of cytosolic calcium ion concentration	43(26)	0.04323
Biological process	Ephrin receptor signaling pathway	5(5)	0.04353
Biological process	Positive regulation of ion transmembrane transporter activity	5(0)	0.04353
Biological process	Regulation of defense response to virus by virus	5(5)	0.04353
Biological process	Regulation of ryanodine-sensitive calcium-release channel activity	5(1)	0.04353
Biological process	Regulation of survival gene product expression	5(0)	0.04353
Molecular function	MAP kinase phosphatase activity	5(0)	0.04447
Molecular function	MAP kinase tyrosine/serine/threonine phosphatase activity	5(5)	0.04447
Biological process	Signaling pathway	1024(0)	0.04488
Cellular component	X chromosome	6(1)	0.0458
Biological process	Cytosolic calcium ion homeostasis	46(4)	0.04858

Effects of Y-27632 on the Calcium Concentration in SCE Cells

TEER of the SCE cell monolayer after adding 2 mM EGTA was significantly lower compared with that of control at 30 minutes or more (Fig. 7A). Meanwhile, treatment with 10 μM A23187, a calcium ionophore, increased the TEER significantly (Fig. 7B). A23187 increased the intracellular concentration of calcium ions in SCE cells, and Y-27632, interestingly, partially diminished the A23187-induced increase of intracellular calcium ions (Fig. 7C). 

Figure 7. 

 

(A, B) Changes in TEER in the SCE cell monolayer. SCE cells were treated with EGTA (A) or A23187 (B), and TEER was measured at 30, 60, and 90 minutes after administration. White bars indicate the values in control condition, and black bars indicate the values with drug treatment. Mean values ± SEs from three separate filters are presented. *P < 0.05, Student's t-test comparing control and drug treatment at each time point. (C) Effects of A23187 with or without pretreatment by Y-27632 on the intracellular concentration of calcium ions estimated by Fluo-4 AM The mean ± SEs are presented as the ratio of F (value after treatment) to F0 (value before treatment). *P < 0.05, Student's t-test with Bonferroni correction comparing control and drug treatment at each time point.

Discussion

In the present study, a specific inhibitor of Rho kinase, Y-27632, increased outflow facility (Fig. 1), decreased TEER (Fig. 3), increased permeability of FITC-dextran (Fig. 4), and decreased ZO-1 and claudin-5 expression in conjunction with depolymerization of F-actin (Fig. 5) in the SCE-cell monolayer compared with controls, suggesting that these changes are related to a decrease in IOP and an increase in aqueous outflow facility by Y-27632. ^11,12 Moreover, microarray and subsequent gene ontology analyses first revealed that Y-27632 changed expression patterns of various genes, and affected broad range of cellular functions in SCE cells (Tables 1–4). Above all, the inhibitory effect on the increase in intracellular calcium ions was confirmed functionally (Fig. 7). 

It is widely believed that RhoA activation leads to macrovascular endothelial barrier breakdown. ²² Using intestinal epithelial cells, however, revealed that the dominant active RhoA increases TEER and decreases paracellular permeability. Of interest, Schlegel and colleagues ²² reported that both activation and inactivation of RhoA signaling increased paracellular permeability. Thus, the responses of cell monolayer against modulation of Rho signaling are assumed to be context dependent. In the present study, Y-27632 increased SCE-cell monolayer permeability and decreased TEER (Figs. 3 and 4), consistent with the study using human SCE cells, revealing that Y-27632 increased permeability of cell monolayer with cell–cell detachment in SCE cells. ¹² Although SCE cells have properties similar to those of vascular endothelial cells, ^23–25 their responses to Y-27632 were different from those of other vascular endothelial cells. Breaks were found in the endothelial lining of the Schlemm's canal and aqueous plexus after perfusion with certain cytoskeletal drugs, including latrunculin-B, ethacrynic acid, and wiskostatin. ^26–28 Moreover, our examination with SEM in the present study revealed that perfusion with Y-27632 increased giant vacuoles accompanied by opening of the intercellular routes of SCE (Fig. 2). In a previous study, however, the endothelial lining was intact after perfusion with Y-27632, as assessed by transmission electron microscopy. ¹² Taken together, although the results were reasonable to explain how Y-27632 increased aqueous outflow facility, ^11,12 further exploration will be required to determine the full effect of ROCK inhibitors against paracellular permeability in SCE cells in vivo. Additionally, SCE cells have transcellular pores accompanied by giant vacuoles, ²⁹ a different property from vascular endothelial cells, which were not evaluated in the present study. 

Tight junctions play an important role as a barrier to the movement of water, solutes, and immune cells, and their dysfunction leads to increased paracellular permeability. ZO-1 is a universal component of tight junctions and interacts with multiple other tight junction proteins. ³⁰ In the present study, ROCK inhibition resulted in modest disruption of the cell–cell contact along with decreased staining of ZO-1, claudin-5 (Fig. 5), suggesting that Y-27632 induced paracellular permeability through changes in the tight junctions. In support, Walsh et al. ³¹ reported that an inhibitor inhibited redistribution of ZO-1 to the intercellular junction upon calcium repletion. Because microarray analysis did not detect changes in RNA expression of the genes coding these molecules, ROCK did not regulate transcription of the molecules associated with tight junction; rather, it regulated protein degradation or intracellular localization in SCE cells. These changes in the intracellular localization of tight junction–associated proteins were linked to the depolymerization of F-actin, because latrunculin B caused similar changes to those of Y-27632. Moreover, treatment with Y-27632 in the presence of jasplakinolide, which potentially inhibited ROCK with polymerized F-actin, showed less effect on ZO-1 localization compared with Y-27632 treatment only. Thus, the changes in the tight junction are thought to be secondary to the disassembly of F-actin. 

Gene ontology grouping of the affected genes based on microarray analysis revealed that ROCK inhibition changed expressions of genes that were related to various cellular functions in SCE cells (Tables 3 and 4). Identified ROCK-related function included regulation of the extracellular matrix disassembly and expression of the integrin complex, which may be involved in the regulation of aqueous outflow because the interaction between SCE and extracellular matrix is important for aqueous outflow. ⁹ These changes might explain the mechanism underlying the inner wall/juxtacanalicular tissue separation between the basal lamina of the inner wall and the extracellular matrix of the juxtacanalicular tissue after perfusion with ROCK inhibitors in eyes of various species. ^12–14 Moreover, these data indicate that ROCK inhibitors potentially affect not only aqueous outflow but also other unknown cell properties, providing insights into possible pharmacologic reactions after clinical application of ROCK inhibitors. 

Microarray analysis using TM cells revealed that dominant active Rho, which activates Rho-ROCK signaling, led to the changes in gene expression. ³² This study revealed greater expression of potential glaucoma-related genes, such as myocilin, fibronectin, and collagen, encoding the extracellular matrix and increased expression of genes, including gremlin-2, IL-1, TGF-β, BMP, Wnt, and CTGF, encoding secreted bioactive factors. Although ROCK inhibitors have opposite effects to dominant active Rho generally, affected genes were different from those in the present study. This may be explained by the difference of cell properties between TM cells and SCE cells, the difference of species, or the difference of methods between pharmacologic treatment and gene induction. Another possibility is that the gene upregulated by activation of an intracellular signal is not always downregulated by inhibition of the same signal. 

It is well known that changes in the intracellular calcium ions can affect the monolayer permeability of the endothelial cells, including SCE cells. ³³ The present study, however, is the first to report the involvement of Rho-ROCK signaling in the regulation of intracellular calcium ions. This finding was unexpectedly derived from our data in microarray and gene ontology analyses. In rat arteries, a previous study reported that Rho-kinase is involved in noradrenaline activation of calcium ion entry. ³⁴ Although those authors found that the involvement was distinct from voltage- or store-operated channels in arteries, the data in the present study are limited, and further studies are required to clarify the underlying mechanisms in SCE cells. In addition, it remains unknown whether the effect on the calcium ion transport of Y-27632 is specific to ROCK inhibition or is a nonspecific side effect. 

In the present study, the effect of Y-27632 on outflow facility was significant at 2.5 hours or later, which was relatively slow compared with results of previous studies. ^12,35 The contradiction is likely due to the difference in study techniques. In the previous studies, the anterior chambers were washed with Y-27632–containing buffer just before switching from baseline buffer to Y-27632–containing buffer, whereas no wash was conducted in the present study. The absence of the wash may delay the time to reach the threshold amount of the drug for the increase of outflow facility. 

There is a limitation in SEM studies to compare the morphologic changes between control and Y-27632–treated eyes, because a previous study in which monkey eyes were perfused with Y-27632 showed that aqueous outflow differed by location and that some inactive areas exist. 

In summary, exposure to the ROCK inhibitor Y-27632 results in Rho/ROCK-dependent F-actin reorganization and disruption of proteins associated with tight junction, which may lead to increased aqueous humor outflow facility. Additionally, the current study revealed that Y-27632 induced changes in the expression of various genes in SCE cells, providing insight into possible pharmacologic reactions after clinical application of ROCK inhibitors. 

Footnotes

 Disclosure: T. Kameda, None; T. Inoue, None; M. Inatani, None; T. Fujimoto, None; M. Honjo, None; N. Kasaoka, None; M. Inoue-Mochita, None; N. Yoshimura, None; H. Tanihara, None