Role of MCP-1 and IL-8 in viral anterior uveitis, and contractility and fibrogenic activity of trabecular meshwork cells.
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SUBMITTER: Lee J
PROVIDER: S-EPMC8298573 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.
| S-EPMC8266857 | biostudies-literature
Project description:Glaucoma, a prevalent blinding disease is commonly associated with increased intraocular pressure due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although increased TM tissue contraction and stiffness in association with accumulation of extracellular matrix (ECM) are believed to be partly responsible for increased resistance to AH outflow, the extracellular cues and intracellular mechanisms regulating TM cell contraction and ECM production are not well defined. This study tested the hypothesis that sustained activation of Rho GTPase signaling induced by lysophosphatidic acid (LPA), TGF-?, and connective tissue growth factor (CTGF) influences TM cell plasticity and fibrogenic activity which may eventually impact resistance to AH outflow. Various experiments performed using human TM cells revealed that constitutively active RhoA (RhoAV14), TGF-?2, LPA, and CTGF significantly increase the levels and expression of Fibroblast Specific Protein-1 (FSP-1), ?-smooth muscle actin (?SMA), collagen-1A1 and secretory total collagen, as determined by q-RT-PCR, immunofluorescence, immunoblot, flow cytometry and the Sircol assay. Significantly, these changes appear to be mediated by Serum Response Factor (SRF), myocardin-related transcription factor (MRTF-A), Slug, and Twist-1, which are transcriptional regulators known to control cell plasticity, myofibroblast generation/activation and fibrogenic activity. Additionally, the Rho kinase inhibitor-Y27632 and anti-fibrotic agent-pirfenidone were both found to suppress the TGF-?2-induced expression of ?SMA, FSP-1, and collagen-1A1. Taken together, these observations demonstrate the significance of RhoA/Rho kinase signaling in regulation of TM cell plasticity, fibrogenic activity, and myofibroblast activation, events with potential implications for the pathobiology of elevated intraocular pressure in glaucoma patients.
| S-EPMC3965649 | biostudies-literature
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
Project description:PurposeTo explore the role of inducible focal adhesion (FA) protein Hic-5 in actin cytoskeletal reorganization, FA formation, fibrogenic activity, and expression of myocilin in trabecular meshwork (TM) cells.MethodsUsing primary cultures of human TM (HTM) cells, the effects of various external factors on Hic-5 protein levels, as well as the effects of recombinant Hic-5 and Hic-5 small interfering RNA (siRNA) on actin cytoskeleton, FAs, myocilin, α-smooth muscle actin (αSMA), and collagen-1 were determined by immunofluorescence and immunoblot analyses.ResultsHic-5 distributes discretely to the FAs in HTM cells and throughout the TM and Schlemm's canal of the human aqueous humor (AH) outflow pathway. Transforming growth factor-β2 (TGF-β2), endothelin-1, lysophosphatidic acid, hydrogen peroxide, and RhoA significantly increased Hic-5 protein levels in HTM cells in association with reorganization of actin cytoskeleton and FAs. While recombinant Hic-5 induced actin stress fibers, FAs, αv integrin redistribution to the FAs, increased levels of αSMA, collagen-1, and myocilin, Hic-5 siRNA suppressed most of these responses in HTM cells. Hic-5 siRNA also suppressed TGF-β2-induced fibrogenic activity and dexamethasone-induced myocilin expression in HTM cells.ConclusionsTaken together, these results reveal that Hic-5, whose levels were increased by various external factors implicated in elevated intraocular pressure, induces actin cytoskeletal reorganization, FAs, expression of fibrogenic markers, and myocilin in HTM cells. These characteristics of Hic-5 in TM cells indicate its importance in regulation of AH outflow through the TM in both normal and glaucomatous eyes.
| S-EPMC4553930 | biostudies-literature
Project description:PurposeSecreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors.MethodsA lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively.ResultsSuppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography.ConclusionsSuppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.
| S-EPMC9187959 | biostudies-literature
Project description:Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular hypertension patients. To test the hypothesis that dysregulated Rho GTPase signaling and a resulting fibrotic activity within the trabecular meshwork may result in ocular hypertension, we investigated the effects of expressing a constitutively active RhoA GTPase (RhoAV14) in the AH outflow pathway in Sprague-Dawley rats by using lentiviral vector-based gene delivery. Rats expressing RhoAV14 in the iridocorneal angle exhibited a significantly elevated intraocular pressure. Elevated intraocular pressure in the RhoAV14-expressing rats was associated with fibrotic trabecular meshwork and increased levels of F-actin, phosphorylated myosin light chain, α-smooth muscle actin, collagen-1A, and total collagen in the trabecular AH outflow pathway. Most of these changes were ameliorated by topical application of Rho kinase inhibitor. Human autopsy eyes from patients with glaucoma exhibited significant increases in levels of collagen-1A and total collagen in the trabecular AH outflow pathway. Collectively, these observations indicate that increased fibrogenic activity because of dysregulated RhoA GTPase activity in the trabecular AH outflow pathway increases intraocular pressure in a Rho kinase-dependent manner.
| S-EPMC4305183 | biostudies-literature
Project description:PurposeTo establish a consistent and affordable, high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork.MethodsPorcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion. Twenty-two control eyes (CO) with a constant flow rate were compared to eight gravity perfused eyes (COgr, 15 mm Hg). We established gene delivery to the TM using eGFP expressing feline immunodeficiency virus (FIV) vector GINSIN at 108 transducing units (TU) per eye (GINSIN_8, n = 8) and 107 TU (GINSIN_7, n = 8). Expression was assessed for 14 days before histology was obtained.ResultsPig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12%. Control eyes had an intraocular pressure (IOP) of 15.8 ± 1.9 mm Hg at fixed pump perfusion with 3 μL/min compared to gravity perfused COgr with imputed 3.7 ± 1.6 μL/min. Vector GINSIN_8 eyes experienced a transient posttransduction IOP increase of 44% that resolved at 48 hours; this was not observed in GINSIN_7 eyes. Expression was higher in GINSIN_8 than in GINSIN_7 eyes. Trabecular meshwork architecture was well preserved.ConclusionsCompared with previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies of TM bioengineering. Trabecular meshwork could be observed directly. Despite significant anatomic differences, effects of transduction replicate the main aspects of previously explored human, feline and rodent models.
| S-EPMC4811178 | biostudies-literature
Project description:Polymorphisms in the CAV1/2 gene loci impart increased risk for primary open-angle glaucoma (POAG). CAV1 encodes caveolin-1 (Cav1), which is required for biosynthesis of plasma membrane invaginations called caveolae. Cav1 knockout mice exhibit elevated intraocular pressure (IOP) and decreased outflow facility, but the mechanistic role of Cav1 in IOP homeostasis is unknown. We hypothesized that caveolae sequester/inhibit RhoA, to regulate trabecular meshwork (TM) mechanosensing and contractile tone. Using phosphorylated myosin light chain (pMLC) as a surrogate indicator for Rho/ROCK activity and contractile tone, we found that pMLC was elevated in Cav1-deficient TM cells compared to control (131 ± 10%, n = 10, p = 0.016). Elevation of pMLC levels following Cav1 knockdown occurred in cells on a soft surface (137 ± 7%, n = 24, p < 0.0001), but not on a hard surface (122 ± 17%, n = 12, p = 0.22). In Cav1-deficient TM cells where pMLC was elevated, Rho activity was also increased (123 ± 7%, n = 6, p = 0.017), suggesting activation of the Rho/ROCK pathway. Cyclic stretch reduced pMLC/MLC levels in TM cells (69 ± 7% n = 9, p = 0.002) and in Cav1-deficient TM cells, although not significantly (77 ± 11% n = 10, p = 0.059). Treatment with the Cav1 scaffolding domain mimetic, cavtratin (1 μM) caused a reduction in pMLC (70 ± 5% n = 7, p = 0.001), as did treatment with the scaffolding domain mutant cavnoxin (1 μM) (82 ± 7% n = 7, p = 0.04). Data suggest that caveolae differentially regulate RhoA signaling, and that caveolae participate in TM mechanotransduction. Cav1 regulation of these key TM functions provide evidence for underlying mechanisms linking polymorphisms in the Cav1/2 gene loci with increased POAG risk.
| S-EPMC8969750 | biostudies-literature
Project description:PurposeTo determine the temporal effects of dexamethasone (DEX) and glucocorticoid-induced matrix (GIM) on integrins/integrin adhesomes, caveolins, cytoskeletal-related proteins, and stiffness in human trabecular meshwork (hTM) cells.MethodsPrimary hTM cells were plated on plastic dishes (TCP), treated with vehicle (Veh) or 100 nM DEX in 1% serum media for 1, 3, 5, and 7 day(s). Concurrently, hTM cells were also plated on vehicle control matrices (VehMs) and GIMs for similar time points; VehMs and GIMs had been generated from chronic cultures of Veh-/DEX-stimulated hTM cells and characterized biochemically. Subsets of cells prior to plating on TCP or VehMs / GIMs served as baseline. Protein expression of mechanoreceptors, cytoskeletal-related proteins, and elastic moduli of hTM cells were determined.ResultsCompared with Veh, DEX temporally overexpressed αV, β3, and β5 integrins from day 3 to day 7, and integrin linked kinase at day 7, in hTM cells. However, DEX decreased β1 integrin at day 1 and day 7, while increasing Cavin1 at day 7, in a time-independent manner. Further, DEX temporally upregulated α-smooth muscle actin(α-SMA) and RhoA at day 7 and day 5, respectively; while temporally downregulating Cdc42 at day 3 and day 7 in hTM cells. Conversely, GIM showed increased immunostaining of fibronectin extra-domain A and B isoforms. Compared with VehM, GIM temporally increased αV integrin, Cavin1, and RhoA from day 3 to day 7, at day 3 and day 7, and at day 5, respectively, in hTM cells. Further, GIM overexpressed α-SMA at day 3 and day 7, and stiffened hTM cells from day 1 to day 7, in a time-independent fashion.ConclusionsOur data highlight crucial mechanoreceptors, integrin adhesomes, and actin-related proteins that may temporally sustain fibrotic phenotypes precipitated by DEX and/or GIM in hTM cells.
| S-EPMC7686803 | biostudies-literature
Project description:Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open-angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho-kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor-?2 (TGF-?2), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM-based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties.
| S-EPMC6501821 | biostudies-literature