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Jin Liang, Fan Yuan, Pedro Gonzalez, William Stamer; Signaling Pathways that mediate endoMT of Human Trabecular meshwork cells exposed to Cyclic Mechanical Stress. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3288.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we observed that contractility, stiffness and α-SMA, a marker of endothelial to mesenchymal transition (endoMT) increase in human trabecular meshwork (TM) cells after 24 hours of 15% cyclic mechanical stretch (CMS). Here we extend this observation and investigate the signaling pathways that mediate CMS-driven endoMT in human TM cells.
A Flexcell system was used to apply CMS (15%-16% at 1 Hz for 24 hours) to primary cultures of human TM cell monolayers under serum-free conditions. Just prior to stretch, the cells were treated with a Rho kinase inhibitor (25µM, H1125, Sigma-Aldrich, MO), a Phosphatidylinositol 3 kinase (PI3-K) inhibitor (10µM, W1628, Sigma-Aldrich, MO), a P38 inhibitor (5µM, SB20025, Sigma-Aldrich, MO) and/or a Lim kinase (LIMK1) inhibitor (1µM, 435930, Calbiochem, MA) for 24 hours.
The rho-kinase and LIMK1-kinase inhibitors blunted the CMS mediated increase in α-SMA expression in human TM cells by 23.1% and 31.5%, respectively (p < 0.05). In contrast, the p38 and pI3 kinase inhibitors insignificantly decreased CMS-stimulated α-SMA expression(p > 0.05, n = 5). Interestingly, basal (unstretched) levels of α-SMA expression were significantly reduced in the presence of the Rho-kinase inhibitor or the P38 kinase inhibitor by 54.8% and 75.1% respectively (p < 0.05), but not the Lim-kinase nor PI3 kinase inhibitors. When added in combination, all four inhibitors significantly decreased CMS-stimulated α-SMA expression to the control level (p<0.05) but did not the unstretched levels of α-SMA.
Experimental evidence suggests that prolonged CMS drives endoMT in human TM cells through activation of several key pathway. These kinases may provide therapeutic target opportunities for intervention and restoration of homeostasis in glaucomatous eyes where TM cells are exposed to chronic rounds of exaggerated stretching due to elevated intraocular pressure.
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