Inhibition of Rho Kinase Induces Antioxidative Molecules and Suppresses Reactive Oxidative Species in Trabecular Meshwork Cells

Purpose To investigate the effect of rho kinase inhibitors on oxidative stress in trabecular meshwork (TM) cells. Methods TM cells were isolated from the eyes of cynomolgus monkeys. Y-27632 and menadione were used to inhibit rho kinase and induce production of reactive oxygen species (ROS), respectively. The cynomolgus monkey array and 12,613 probes were used in DNA microarray analysis, and the affected genes were categorized using gene ontology analysis. The mRNA levels of the target genes were confirmed by real-time RT-PCR. Intracellular oxidative stress was detected using a fluorescent reagent sensitive to ROS. Cell viability was assessed by the WST-8 assay. Results Gene ontology analysis revealed upregulation of genes involved in antioxidant activity, and upregulation of catalase was confirmed by real-time RT-PCR after 30 min treatment with Y-27632. Production of ROS was increased by menadione, and the effect was partly suppressed by pretreatment with Y-27632. At a lower dose of menadione, Y-27632 stimulated TM cells and significantly increased their viability following menadione treatment compared to control cells. Conclusion Using microarray analysis, Y-27632 was shown to upregulate antioxidative genes including catalase and partially reduce ROS production and cell death by oxidative stress caused by menadione.


Introduction
Oxidative stress is a major physiological phenomenon, mediated through the production of reactive oxygen species (ROS), such as peroxides, superoxide, hydroxyl radical, and singlet oxygen. ROS play an important role in cell homeostasis and pathogen response and are therefore essential in biological processes. In contrast, increases in ROS are seen in various age-related diseases including glaucoma [1]. For instance, in the aqueous humor of glaucoma patients, the levels of oxidative stress markers are significantly increased [2][3][4][5]. Additionally, oxidative DNA damage is reportedly increased in the trabecular meshwork (TM) of glaucoma patients [6,7]. These findings indicate that the TM of glaucomatous eyes is continuously exposed to oxidative stress, and therefore, damage to TM may increase outflow resistance and the risk of glaucoma progression. In line with this, lower systemic antioxidant capacity is related to higher intraocular pressure (IOP) levels in open-angle glaucoma patients [8]. Moreover, glaucoma-related genes, such as CYP1B1 and FOXC1, are reportedly linked to oxidative stress in the eyes [9][10][11][12]. Taken together, control of oxidative stress in the eye may be a therapeutic target to slow glaucoma progression.
Rho-rho kinase (ROCK) signaling controls polymerization of actin and thereby mediates various cell functions, such as contraction, migration, phagocytosis, and mitosis. Inhibition of ROCK increases aqueous outflow by depolymerizing F-actin in TM cells and Schlemm's canal endothelial cells [13,14]. A ROCK inhibitor, ripasudil, has been approved as an IOP-lowering drug in Japan [15]. Ripasudil significantly reduces the IOP of glaucoma patients upon either single or multiple administration [16,17]. However, ROCK inhibitors have drawn attention as antioxidative drugs against cardiovascular diseases and chronic renal injury [18,19]. Indeed, ripasudil (also known as K-115) has been reported to have a neuroprotective effect on the optic nerve by suppressing oxidative stress in an animal model [20]. Thus, the effect of ROCK inhibitors on oxidative stress in TM cells is of interest from a therapeutic point of view against glaucoma.
Here, we show the results of an exhaustive investigation using a microarray, revealing that treatment with Y-27632, a well-known ROCK inhibitor, upregulates antioxidative molecules in TM cells, inhibits ROS production, and promotes cell survival.

Materials and Methods
2.1. Cell Culture. Trabecular meshwork (TM) cells were isolated from the eyes of cynomolgus monkeys (Shin Nippon Biomedical Laboratories, Kagoshima, Japan) according to the method described previously [21]. Primary TM cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan) 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% CO 2 . These cells were used after 2-5 passages. The character of the isolated cells in the present study was confirmed by expression of specific TM markers (caveolin 1, collagen 4α5, matrix gla protein, tissue inhibitor of metalloproteinase 3, and vascular cell adhesion protein 1), phagocytosis function, and myocilin induction by dexamethasone as described previously [22].

DNA Microarray Analysis.
Custom cDNA microarray analysis was performed using a CombiMatrix microarray (CombiMatrix, Mukilteo, WA) as described previously [23]. Briefly, the cynomolgus monkey array was designed to detect directly labeled mRNA from 12,613 probes. Confluent TM cells in 100 mm dishes were treated with 25 μM Y-27632 (Merck Millipore, Darmstadt, Germany) or vehicle (deionized water) for 30 min. Total RNA was extracted from the cells, and the integrity and concentration of total RNA was measured using an 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 SureLABEL Core kit (Takara Bio, Shiga, Japan). 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). Fluorescent images of hybridized microarrays were obtained with a GenePix 4000B Scanner (Molecular Devices, Sunnyvale, CA). Array-Pro Analyzer Ver4.5 (Media Cybernetics, Silver Spring, MD) was used to determine the signal intensity of each spot and its local background. Scanned images were analyzed using Microarray Data Analysis Tool Ver3.2 software (Filgen).
Signals from Y-27632 treated cells were compared with those from vehicle-treated cells, and genes that showed greater than 3/2-fold change in expression in at least one of the pairwise probe comparisons were considered upregulated, whereas those that showed a change of expression smaller than 2/3-fold were considered downregulated. These analyses were performed three times using TM 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 homology search of EST sequences available from the National Center for Biotechnology Information. Relative expression of catalase in the Y-27632-treated samples was compared to that in control samples using the comparative C T method (ΔΔC T method); 18S ribosomal RNA was used as an endogenous control. The threshold cycle, C T , was determined after setting the threshold in the linear amplification phase of the PCR reaction and ΔC T was defined as ΔC T = C T (target gene) − C T (18S rRNA). Relative expression of the target gene was calculated as: 2 −ΔΔCT , ΔΔC T = ΔC T (treated sample) − ΔC T (control).

Intracellular Oxidative Stress
Detection. The effects of Y-27632 on the production of ROS were evaluated using CellROX® green reagent (Life Technologies) in the TM cells. These cells were cultured on 6 cm dishes in DMEM containing 10% FBS and antibiotics at 37°C in 5% CO 2 . After cells had grown to confluence, they were pretreated with Y-27632 for 30 min and then stimulated with 100 μM menadione (Sigma, St. Louis, MO) for 1 h. CellROX reagent was then added to each dish to give a final concentration of 5 μM and incubated for 30 min at 37°C. After incubation, TM cells were washed in PBS and detached by trypsin/EDTA solution and centrifuged at 1200 rpm for 3 min. The supernatant was removed, and cells were fixed in 4% paraformaldehyde in PBS for 15 min and then centrifuged twice at 1200 rpm for 3 min, resuspending in PBS after each             Tables 3 and 4. Gene ontology analysis revealed that the upregulated genes were related to various cellular functions including antioxidant activity (P = 0 014), and downregulated genes were related to integrin complexes (P = 0 039), and calcium ion transport into the cytosol (P = 0 008). In the category of antioxidant activity, upregulated genes were homologous to human gene coding catalase (P = 0 046), thioredoxin domain-containing 2 (also known as spermatozoa; P = 0 032), nucleoredoxin (P = 0 017), albumin (probe 1, P = 0 002; probe 2, P = 0 021), and glutathione transferase zeta 1 (P = 0 004). Upregulation of the mRNA of catalase, an extensively investigated antioxidant, was confirmed by real-time RT-PCR and found to be 1.5 times higher in TM cells treated with Y-27632 compared to the control TM cells (P = 0 032; Figure 1(a)). In contrast, four other genes involved in antioxidant activity were not significantly affected after treatment with Y-27632 (data not shown).

Effects of Y-27632 on the Production of Reactive Oxygen
Species in TM Cells. To assess the effects of Y-27632 on the production of ROS in TM cells, we utilized a fluorogenic probe that exhibits bright fluorescence upon oxidation by ROS. In the absence of an oxidative reagent, the fluorescence   Figure 1(b)). In the presence of 100 μM menadione, the fluorescence intensity was significantly elevated (16097.7 ± 1133.0; P < 0 0001); this elevation was partly suppressed by treatment with Y-27632 (11443.6 ± 1332.2; P = 0 0182), suggesting that Y-27632 reduces ROS production in TM cells under oxidative stress.

Effects of Y-27632 on the Viability of TM Cells under
Oxidative Stress. Finally, we investigated the effects of Y-27632 on the viability of TM cells under oxidative stress. As shown in Figure 2(a), menadione reduced TM cell viability in a dose-dependent manner. At a lower dose of menadione, Y-27632-stimulated TM cells regained significant viability against menadione treatment compared to control cells (P = 0 0238). In contrast, the effects of Y-27632 on cell viability were not significant at a higher dose of menadione.

Direct Antioxidant Activity of Y-27632.
To confirm the extracellular antioxidant activity of Y-27632, we assessed xanthine oxidase-induced superoxide production using a luminescent reagent. As shown in Figure 2(b), there was no significant difference in ROS production between the control and Y-27632 treatment. Thus, Y-27632 does not appear to affect extracellular oxidants.

Discussion
In the present study, we have identified the antioxidative effect of Y-27632 in TM cells by microarray analysis, an exhaustive investigation of gene expression, and shown that Y-27632 partially suppresses ROS production and cell death induced by menadione. To the best of our knowledge, this is the first report to show the antioxidant effect of ROCK inhibitor on TM cells. Previously, we presented depolymerization of F-actin before morphometric recovery from oxidative stress in TM cells [24], suggesting a correlation between oxidative stress and regulation of the actin cytoskeleton in TM cells. In other tissues, rho-kinase was identified as a mediator of various diseases associated with inflammation and oxidative stress, and inhibition of rhokinase has been drawing attention as a promising therapeutic strategy. For instance, activation of the rho/rho-kinase pathway is related to the pathophysiology of chronic renal injury, and long-term fasudil treatment has renoprotective effects in this malignant hypertension model. The mechanism of the renoprotective effect of fasudil, a nonspecific ROCK inhibitor, was suggested to involve a combination of factors, including inhibition of the TGF-β-collagen cascade, control of inflammation, reduction of oxidative stress, and upregulation of eNOS [18]. Clinical studies with fasudil have suggested that it may be useful for the treatment of a wide range of cardiovascular diseases [19]. Importantly, rhokinase inhibitors block ROS production by suppressing CyPA secretion from vascular smooth muscle cells [25], suggesting the beneficial effect of rho-kinase inhibitors against cardiovascular diseases.
Recently, Yamamoto and colleagues demonstrated the neuroprotective effect of the ROCK inhibitor K-115, a novel IOP-lowering drug, using the mouse optic crush model [20]. They showed the effect was at least partially dependent on suppression of ROS production via inhibition of Nox1 expression in retinal ganglion cells. We also showed that ROCK inhibitors' antioxidant effects are indirect using monkey TM cells. However, in the present study using microarray analysis, Nox family genes were not identified as affected, but catalase was upregulated after treatment with Y-27632. This disagreement might be caused by differences in species and/or tissues. Thus, the precise molecular mechanisms of the antioxidative effect of ROCK inhibitors have not been clarified completely. On the other hand, a recent study     reported that Y-27632 induced p-53-mediated apoptosis in hemangioma [26]. In the present study, we indicated that ROCK inhibitor effected cell survival in TM cells. This is interesting point since ROCK inhibitor-induced effects such as cell death or cell protection were changed by differences of cell types. TM has a critical role in the maintenance of aqueous outflow resistance through the regulation of extracellular matrix metabolism, phagocytosis of debris, and empty space associated with tissue contraction [27,28]. Indeed, the number of TM cells is decreased in glaucomatous eyes [29], suggesting that functional TM cells are essential in controlling IOP. In this context, oxidative stress is a potential cause of cellular dysregulation in TM, both functionally and numerally, because it has been suggested that the TM of glaucomatous eyes is continuously exposed to oxidative  stress [2][3][4][5][6][7]. Thus, an antioxidant drug might reduce oxidative stress in TM cells, slowing progression of glaucomatous damage in outflow tissues. Though it remains unknown whether clinically used eye-drops containing ripasudil have significant antioxidative effects on TM cells in vivo, the present study's findings may be clinically relevant. The effect of Y-27632 on cell survival under oxidative stress was significant, but limited. Since glaucoma progresses chronically in the majority of the patients, the acute oxidative damage in the present study may not reflect pathological conditions in glaucomatous TM cells, which is one of the limitations of the present study. Another limitation is that the antioxidative effects of ROCK inhibition were not corroborated in vivo. Further studies are required to acquire more clinically relevant evidence of the effects of ROCK inhibitor on oxidative stress in TM.

Conclusion
Microarray analysis reveals that Y-27632 upregulates antioxidative genes including catalase and partially reduces the ROS production and cell death by oxidative stress induced by menadione.

Conflicts of Interest
Dr. Hidenobu Tanihara has received consulting fees from Kowa and MSD and board membership fees from Senju Pharmaceutical, Santen Pharmaceutical, Alcon Japan, and Pfizer Japan. Data are shown as the mean ± SE from six independent experiments. * P < 0 05 compared with control by Wilcoxon rank sum test.
(b) The effect of Y-27632 on extracellular antioxidative activity. The xanthine-oxidase-induced superoxide production was assessed using a superoxide-sensitive luminescent reagent. Data are shown as the mean ± SE from six independent experiments. * P < 0 05 compared with the control by Dunnett's test. Nac: n-acetyl cysteine.