Determining Mechanisms by which Pathological Extracellular Matrix induces Ocular Hypertensive Phenotypes in Human Trabecular Meshwork Cells



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Purpose: Aberrant extracellular matrix (ECM) remodeling is prevalent in several sight-threatening diseases; in the trabecular meshwork (TM), it is implicated in elevated intraocular pressure (IOP), the only treatable risk factor for glaucoma. Herein, two different glaucoma-relevant cell-derived matrices (CDMs) were used to dissect the specific mechanisms by which diseased ECMs may precipitate ocular hypertensive phenotypes in human TM (hTM) cells. Methods: (1) To determine whether dexamethasone (DEX)/glucocorticoid-induced matrix (GIM) temporally modulate mechanoreceptors / contractility / stiffness in hTM cells, hTM cells were treated with DEX/vehicle or cultured on GIM/VehM for 1/3/5/7 days. (2) To determine whether GIM modulates transforming growth factor beta-2 (TGFβ2) signaling in hTM cells, hTM cells were cultured on GIM/VehM for 24h or 7d in the presence/absence of TGFβ2, with/without type I TGFβ receptor inhibitor (TGFβRi). (3) To determine whether genipin-induced crosslinked CDM (XCDM) stiffens hTM cells via dysregulating β-catenin and Yes-associated protein (YAP)/Transcriptional co-activator with a PDZ-binding motif (TAZ) signaling pathways, hTM cells were seeded on CDMs/XCDMs for 24h. Where applicable, immunocytochemistry, RT-qPCR, Western blotting, enzymatic assays, transmission/scanning electron and atomic force microscopies were performed. Results: (1) Whereas DEX temporally overexpressed αV, β3 and β5 integrins, integrin linked kinase, α-Smooth muscle actin(α-SMA) and RhoA in hTM cells, GIM temporally upregulated αV integrin, Cavin1, and RhoA; and increased α-SMA and cell stiffness independent of time. (2) GIM triggered non-Smad/TGFβ2 signaling in hTM cells, correlated with overexpression of key ECM structural/matricellular/crosslinking/turnover genes/enzymes; whereas GIM+TGFβ2 activated Smad and non-Smad/TGFβ2 signaling, associated with increased cell contractility and heightened upregulation of aforementioned and new ECM genes/enzymes. TGFβRi abrogated GIM-/GIM+TGFβ-mediated increased contractility or ECM deposition. (3) At the highest genipin concentration (10%XCDM), XCDM had increased crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, p<0.001). On 10%XCDM, hTM cells were 7.8-fold (p<0.001) stiffer, associated with impaired nucleocytoplasmic shuttling of inactive β-catenin, reduced Cadherin-11, and key Wnt target genes/proteins; and increased cytoplasmic TAZ levels, reduced YAP levels in the nucleus and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10%XCDM-induced stiffening correlated with increased nuclear β-catenin. Conclusions: These data highlight critical signaling pathways/mediators that may instruct design of novel therapeutics in ameliorating pathological ECM-mediated fibrotic phenotypes in hTM cells associated with elevated IOP.



Extracellular matrix, Trabecular meshwork, Mechanotransduction, Fibrotic phenotypes, Ocular hypertension, Glaucoma


Portions of this document appear in: Yemanyi F, Vranka J, Raghunathan VK. Glucocorticoid-induced cell-derived matrix modulates transforming growth factor β2 signaling in human trabecular meshwork cells. Sci Rep. 2020;10(1):15641.; Yemanyi F, Vranka J, Raghunathan VK. Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways. Investig Opthalmology Vis Sci. 2020;61(10):41.; Yemanyi F, Vranka J, Raghunathan VK. Generating cell-derived matrices from human trabecular meshwork cell cultures for mechanistic studies. Methods Cell Biol. 2020;156:271-307.