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Enhua H Zhou, Rudolf Fuchshofer, W Daniel Stamer, Quynh N Dang, Jae H Kim, Kristin Marie Perkumas, Mark Johnson, Jeffrey J Fredberg; Mechanosensitivity of Schlemm’s canal endothelial cells from normal and glaucomatous eyes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5684.
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The aqueous humor mainly flows through the trabecular meshwork (TM) and across the Schlemm’s canal (SC) endothelium. In primary open angle glaucoma (POAG) eyes, resistance to flow increases and TM tissues are 20 times stiffer than that in normal eyes (Last et al. 2011, IOVS. DOI:10.1167). The extent to which elevated tissue stiffness influences SC cells is poorly understood (Zhou et al. 2012, J Royal Society, Interface. DOI: 10.1098). The goal of the current study is to evaluate changes in cell stiffness and gene expression of SC cells induced by stiffened ECM substrate and to compare the responses of normal to glaucomatous SC cells.
To mimic the spectrum of ECM stiffness from normal TM to glaucomatous TM, we casted polyacrylamide gels with 5 different crosslinker concentrations. Gels were coated with collagen I and seeded with SC cells isolated from normal (n=5) or glaucomatous (n=5) human donor eyes. To measure the stiffness of confluently plated SC cells, we used optical magnetic twisting cytometry (OMTC). The expression levels of 10 genes were examined, including connective tissue growth factor (CTGF), tissue growth factor (TGF-β2), MMP2, SPARC and collagen (col1A1), using real time quantitative PCR.
Cell stiffness increased with increasing substrate stiffness for both normal and glaucomatous SC cells. This increase in cell stiffness was significantly greater for glaucomatous SC cells than for normals (p = 0.02). CTGF, MMP2, SPARC and col1A1 all exhibited significantly increased expression level with increased substrate stiffness (p<0.05) with expression increased by up to 18 fold (col1A1). The increase in CTGF expression with increasing substrate stiffness was significantly greater in glaucomatous SC cells (11.3 fold) than in normals (3.9 fold) (p < 0.05). The average expression level of CTGF was also higher in glaucomatous SC cells.
Elevated substrate stiffness led to increases in SC cell stiffness and in ECM-related gene expression, both of which known to be associated with elevated outflow resistance. Importantly, glaucomatous SC cells were significantly more responsive to substrate stiffness as compared to controls. Mechanosensitivity of SC cells represents a therapeutic target for restoring the function of the conventional outflow pathway.
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