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Effects of K-115 (Ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm's canal endothelial cells.

ABSTRACT: Ripasudil hydrochloride hydrate (K-115), a specific Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was the first ophthalmic solution developed for the treatment of glaucoma and ocular hypertension in Japan. Topical administration of K-115 decreased intraocular pressure (IOP) and increased outflow facility in rabbits. This study evaluated the effect of K-115 on monkey trabecular meshwork (TM) cells and Schlemm's canal endothelial (SCE) cells. K-115 induced retraction and rounding of cell bodies as well as disruption of actin bundles in TM cells. In SCE-cell monolayer permeability studies, K-115 significantly decreased transendothelial electrical resistance (TEER) and increased the transendothelial flux of FITC-dextran. Further, K-115 disrupted cellular localization of ZO-1 expression in SCE-cell monolayers. These results indicate that K-115 decreases IOP by increasing outflow facility in association with the modulation of TM cell behavior and SCE cell permeability in association with disruption of tight junction.

SUBMITTER: Kaneko Y

PROVIDER: S-EPMC4725980 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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The effects of exosomes derived from trabecular meshwork cells on Schlemm's canal endothelial cells.

Project description:Trabecular meshwork (TM) and Schlemm's canal (SC) are the main structures within the conventional outflow pathway, and TM cells and SC endothelial (SCE) cells are essential for controlling intraocular pressure. To examine the interaction between TM cells and SCE cells, we investigated whether exosomes contribute to intercellular communication. Additionally, TM cells in glaucoma acquire mesenchymal characteristics in response to transforming growth factor (TGF)-β2 and extracellular matrix proteins such as collagen type 1 (Col-1); these changes result in increased resistance of aqueous outflow. In this study, we stimulated TM cells with TGF-β2 and Col-1 and characterized the exosomal miRNAs (exomiRs) released in response to each stimulus. Isolated exosomes were rich in miRNAs, with downregulated miR-23a-5p and upregulated miR-3942-5p and miR-7515 levels following Col-1 or TGF-β2 stimulation. Next, a miRNA-mRNA network under TGF-β2 stimulation was constructed. There were no connections among the 3 miRNAs and predicted genes under Col-1 stimulation. GO and KEGG analyses revealed that the identified miRNAs were associated with various signaling pathways, including the inflammatory response. Interestingly, SCE cells treated with miR-7515 mimic showed increased VEGFA, VEGFR2, PECAM, and Tie2 expression. Ultrastructures typical of exosomes and positive staining for exosomal markers were observed in human TM cells. Our data showed that TM cells may communicate with SCE cells via exomiRs and that miR-7515 may be important for SCE cell reprogramming.

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Effects of selective EP2 receptor agonist, omidenepag, on trabecular meshwork cells, Schlemm's canal endothelial cells and ciliary muscle contraction.

Project description:This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm's canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.

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The Effects of ROCK Inhibitor on Prevention of Dexamethasone-Induced Glaucoma Phenotype in Human Trabecular Meshwork Cells.

Project description:PurposeThis study investigated the effects of dexamethasone (Dex) on human trabecular meshwork (TM) cells, a model of glucocorticoid-induced glaucoma, and evaluated the impact of ripasudil (Rip) as a co-delivery or sequential dosing strategy.MethodsIn vitro experiments were conducted to assess the effects of Dex and Rip on TM cells. Confocal microscopy was used to evaluate the impact of Dex and Rip on F-actin staining signals. Contractility of the TM cells upon Dex and Rip treatment mimicking co-delivery and sequential delivery was quantified using collagen gel contraction assay. Transepithelial electrical resistance (TEER) values and fluorescein isothiocyanate (FITC)-dextran permeability were also measured to assess the impact of Dex and Rip on TM cells.ResultsDex and Rip did not exhibit cytotoxicity at the maximum tested concentration (20 µM). Dex-treated TM cells exhibited higher F-actin staining signals compared to controls, which were reduced when co-treated with Rip. Rip inhibited Dex-induced collagen gel contraction activity in both co-delivery and sequential treatments. Dex resulted in increased TEER values as the dose increased, whereas TEER values were maintained when co-treated with Rip.ConclusionsCo-delivery of Rip has the potential to prevent glaucoma symptoms when patients are treated with Dex. This study highlights the importance of identifying strategies to reduce the side effects of prolonged use of glucocorticoids, such as Dex, in the treatment of various diseases.Translational relevanceThis study demonstrates the potential of co-delivering ripasudil with dexamethasone to mitigate glucocorticoid-induced ocular hypertension and a secondary glaucoma that resembles primary open-angle glaucoma, providing insights for the development of novel preventive strategies in clinical care.

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Data on differentially expressed proteins in rock inhibitor-treated human trabecular meshwork cells using SWATH-based proteomics.

Project description:Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors represent a novel class of anti-glaucoma drugs because of their ocular hypotensive effects. However, the underlying mechanisms responsible for lowering intraocular pressure (IOP) are not completely clear. The protein profile changes in primary human trabecular meshwork (TM) cells after two days treatment with a ROCK inhibitor were studied using label-free SWATH acquisition. These results provided significant data of key protein candidates underlying the effect of ROCK inhibitor. Using the sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive TM proteome library. All raw data generated from IDA and SWATH acquisitions were uploaded and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01254).

| S-EPMC7322233 | biostudies-literature

Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix.

Project description:PurposeTreatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood.MethodsPrimary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined.ResultsTreatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of ?-smooth muscle actin (?SMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM.DiscussionThis integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated ?SMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.

| S-EPMC4509060 | biostudies-literature

Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal.

Project description:The purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues.A set of JCTM and SC cells was cultured from each of 2 donor eyes by an explant method, grown to passage 3, and frozen in liquid nitrogen. The cells were thawed, total RNA was extracted, and the probes made from total RNAs were hybridized to MICROMAX human cDNA microarray slides in 2 separate trials. Differentially expressed genes were analyzed using PubMed, Prosite, and IPA software, and the expression of several of the genes including intercellular adhesion molecule-1 (ICAM-1), tenascin, and ?-spectrin was assessed by immunofluorescence.Schlemm's canal cells differentially expressed ICAM-1, spectrin, complement, fibulin-1, and several genes consistent with an endothelial origin in both arrays, while the JCTM cells more often overexpressed genes consistent with contractile, matrix function, and neural character. At the same time, many genes highly expressed in the first array were not highly overexpressed in the second. One highly overexpressed gene in the JCTM in both arrays, that for heparan sulfate 3-O-sulfotransferase-1 precursor, is thought to be somewhat unique, and could affect the glycosaminoglycan functionality in the extracellular matrix (ECM).We found generally good agreement between the 2 array trials, but some contradictions as well. Many of the genes overexpressed in each cell type had been described in earlier work, but several were new. Tables of genes, grouped by cellular function, and the complete datasets are provided for the development of new hypotheses.

| S-EPMC3991976 | biostudies-literature

A new insight into the cellular regulation of aqueous outflow: how trabecular meshwork endothelial cells drive a mechanism that regulates the permeability of Schlemm's canal endothelial cells.

Project description:AimTo test the hypothesis that trabecular meshwork endothelial cells (TMEs) increase the permeability of Schlemm's canal endothelial cells (SCEs) by actively releasing ligands that modulate the barrier properties of SCEs.MethodsThe TMEs were first irradiated with a laser light and allowed to condition the medium, which is then added to SCEs. The treatment response is determined by both measuring SCE permeability (flow meters) and the differential expression of genes (Affymetrix chips and quantitative polymerase chain reaction (PCR)). The cytokines secreted by the treated cells were identified using ELISA and the ability of these cytokines to increase permeability is tested directly after their addition to SCEs in perfusion experiments.ResultsSCEs exposed to medium conditioned by the light activated TMEs (TME-cm) respond by undergoing a differential expression (DE) of 1,120 genes relative to controls. This response is intense relative to a DE of only 12 genes in lasered SCEs. The TME-cm treatment of SCEs increased the SCE permeability fourfold. The role of cytokines in these responses is supported by two findings: adding specific cytokines established to be secreted by lasered TMEs to SCEs increases permeability; and inactivating the TME-cm by boiling or diluting, abrogates these conditioned media permeability effects.ConclusionThese experiments show that TMEs can regulate SCE permeability and that it is likely that TMEs have a major role in the regulation of aqueous outflow. This novel TME driven cellular mechanism has important implications for the pathogenesis of glaucoma and the mechanism of action of laser trabeculoplasty. Ligands identified as regulating SCE permeability have potential use for glaucoma therapy.

| S-EPMC1772935 | biostudies-literature

Endoplasmic Reticulum Stress Response of Trabecular Meshwork Stem Cells and Trabecular Meshwork Cells and Protective Effects of Activated PERK Pathway.

Project description:PurposeThis study aimed to investigate the differential responses of trabecular meshwork stem cells (TMSCs) and trabecular meshwork (TM) cells to endoplasmic reticulum (ER) stress inducers.MethodsHuman TM cells and TMSCs were exposed to tunicamycin, brefeldin A, or thapsigargin. Cell apoptosis was evaluated by flow cytometry. ER stress markers were detected by quantitative PCR, Western blotting, and immunostaining. Morphologic changes were evaluated by transmission electron microscopy. Cells were treated with the PERK inhibitor GSK2606414 or the elF2? dephosphorylation inhibitor Salubrinal together with tunicamycin to evaluate their effects on ER stress.ResultsBoth TMSCs and TM cells underwent apoptosis after 48- and 72-hour treatment with ER stress inducers. ER stress triggered the unfolded protein response (UPR) with increased expression of GRP78, sXBP1, and CHOP, which was significantly lower in TMSCs than TM cells. Swollen ER and mitochondria were detected in both TMSCs and TM cells. Neither GSK2606414 nor salubrinal alone activated UPR. GSK2606414 significantly reduced cell survival rates after tunicamycin treatment, and salubrinal increased cell survival rates. The increased expression of GRP78, sXBP1, CHOP, and GADD34 peaked at 6 or 12 hours and lasted longer in TM cells than TMSCs. Salubrinal treatment dramatically increased OCT4 and CHI3L1 expression in TMSCs.ConclusionsIn response to ER stress inducers, TMSCs activated a lower level of UPR and lasted shorter than TM cells. Inhibition of elF2? dephosphorylation had a protective mechanism against cell death. Stem cells combined with salubrinal may be a more effective way for TM regeneration in glaucoma.

| S-EPMC6340162 | biostudies-literature

A ROCK inhibitor suppresses the transforming growth factor-beta-2-induced endothelial-mesenchymal transition in Schlemm's canal endothelial cells.

Project description:In the normal eye, most of the aqueous humor drains through the trabecular meshwork (TM) and Schlemm's canal (SC). The concentration of transforming growth factor beta 2 (TGF-β2) is increased in the aqueous humor of primary open angle glaucoma patients. TGF-β2 increases outflow resistance by affecting the TM and SC, and endothelial-mesenchymal transition (EndMT) of SC cells is involved in these changes. Here, we investigated the effect of a ROCK inhibitor on TGF-β2-induced EndMT in SC cells. The ROCK inhibitor Y-27632 suppressed the TGF-β2-induced increase in the trans-endothelial electrical resistance (TER) and proliferation of SC cells. Y-27632 suppressed the expression of α-SMA, N-cadherin, and Snail, which are upregulated by TGF-β2. Moreover, TGF-β2 decreased mRNA levels of bone morphogenetic protein (BMP) 4 and increased those of the BMP antagonist gremlin (GREM1), but Y-27632 significantly suppressed these changes. Y-27632 also inhibited TGF-β2-induced phosphorylation of p-38 mitogen-activated protein kinase (MAPK). BMP4 and the p-38 MAPK inhibitor SB203580 suppressed the TGF-β2-induced TER elevation in SC cells. Moreover, SB203580 suppressed TGF-β2-induced upregulation of fibronectin, Snail, and GREM1. These results indicate that a ROCK inhibitor inhibited the TGF-β2-induced EndMT in SC cells, implying the involvement of p38 MAPK and BMP4 signaling.

| S-EPMC10267201 | biostudies-literature

Genome-wide DNA methylation in cultured human Schelmm's Canal endothelial cells and trabecular meshwork cells with or without glaucoma

Project description:Genome-wide DNA methylation profiling in cultured human Schelmm's Canal endothelial cells (SC) and trabecular meshwork (TM) cells derived from post-mortem eyes with or without glaucoma. Our study aimed to identify glaucoma-associated genes that were affected by DNA methylation.

2020-01-03 | GSE129183 | GEO

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