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

ABSTRACT: 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.

SUBMITTER: Fujimoto T

PROVIDER: S-EPMC5540245 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Fujimoto Tomokazu T Inoue Toshihiro T Ohira Saori S Awai-Kasaoka Nanako N Kameda Takanori T Inoue-Mochita Miyuki M Tanihara Hidenobu H

Journal of ophthalmology 20170719

<h4>Purpose</h4>To investigate the effect of rho kinase inhibitors on oxidative stress in trabecular meshwork (TM) cells.<h4>Methods</h4>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 ta ...[more]

PMID: 28804648

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Project description:PurposeContinuous reductions in trabecular meshwork (TM) cellularity inhibit aqueous humor (AH) outflow, which is the main cause of primary open-angle glaucoma. Rho-associated protein kinase inhibitor (ROCKi) targets the TM to reduce intraocular pressure (IOP) and increase AH outflow facility. However, the underlying mechanisms are not entirely clear. Here, we aimed to investigate the effect of a ROCKi (Y-27632) on TM cell proliferation and phagocytosis.MethodsImmortalized human TM (iHTM) cells, glaucomatous TM (GTM3) cells, and primary human TM (pTM) cells were cultured and identified. The effects of various concentrations of Y-27632 on F-actin cytoskeleton were assessed using immunofluorescence. Cell proliferation effects were evaluated using a cell counting kit-8 (CCK8), cell counting, and Ki67 immunostaining. Cell phagocytosis was evaluated using immunofluorescence and flow cytometry in immortalized TM cells. C57BL/6J and Tg-MYOC Y437H mice were used to investigate the proliferative effects of Y-27632 on TM cells in vivo. The effect of Y-27632 on IOP was monitored for 2 weeks, and the outflow facility was detected 2 weeks after IOP measurement. TM cells in mice were counted using immunohistochemistry.ResultsY-27632 (100 μM) significantly promoted the proliferation of both immortal TM cells and pTM cells. In GTM3 cells, phagocytosis was significantly greater in the Y-27632 group than in the control group, nearly reaching the level of phagocytosis in iHTM, as determined using immunofluorescence and flow cytometry. In Tg-MYOC Y437H mice, treatment with Y-27632 significantly decreased IOP and increased outflow facility, which greatly influenced the long-term IOP-lowering effect. The number of TM cells in Tg-MYOC Y437H mice was significantly improved after Y-27632 administration.ConclusionY-27632 promoted cell proliferation and phagocytosis of TM cells, and its proliferative effect was demonstrated in a transgenic mouse model. These results revealed a new IOP-lowering mechanism of Y-27632 through effects on TM cells, suggesting the potential for a correlation between TM cellularity and long-term recovery of IOP.

Project description:PurposeCells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity.MethodsPrimary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2-6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment.ResultsInnate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions.ConclusionsWe demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.

Project description:Interactions between trabecular meshwork (TM) cells and their extracellular matrix (ECM) are critical for normal outflow function in the healthy eye. Multifactorial dysregulation of the TM is the principal cause of elevated intraocular pressure that is strongly associated with glaucomatous vision loss. Key characteristics of the diseased TM are pathologic contraction and actin stress fiber assembly, contributing to overall tissue stiffening. Among first-line glaucoma medications, the Rho-associated kinase inhibitor (ROCKi) netarsudil is known to directly target the stiffened TM to improve outflow function via tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no in vitro studies have explored the effect of netarsudil on human TM (HTM) cell contractility and actin remodeling in a 3D ECM environment. Here, we use our bioengineered HTM cell-encapsulated ECM hydrogel to investigate the efficacy of different netarsudil-family ROCKi compounds on reversing pathologic contraction and actin stress fibers. Netarsudil and all related experimental ROCKi compounds exhibited significant ROCK1/2 inhibitory and focal adhesion disruption activities. Furthermore, all ROCKi compounds displayed potent contraction-reversing effects on HTM hydrogels upon glaucomatous induction in a dose-dependent manner, relatively consistent with their biochemical/cellular inhibitory activities. At their tailored EC50 levels, netarsudil-family ROCKi compounds exhibited distinct effect signatures of reversing pathologic HTM hydrogel contraction and actin stress fibers, independent of the cell strain used. Netarsudil outperformed the experimental ROCKi compounds in support of its clinical status. In contrast, at uniform EC50-levels using netarsudil as reference, all ROCKi compounds performed similarly. Collectively, our data suggest that netarsudil exhibits high potency to rescue HTM cell pathobiology in a tissue-mimetic 3D ECM microenvironment, solidifying the utility of our bioengineered hydrogel model as a viable screening platform to further our understanding of TM pathophysiology in glaucoma.

Dataset Information

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

Publications

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

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