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Enhua Zhou, W Daniel Stamer, Chan Young Park, Kristin Perkumas, James Butler, Mark Johnson, Jeffrey Fredberg; Monolayer traction forces of Schlemm’s canal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):380.
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The mechanism underlying primary open angle glaucoma is not completely understood, although impaired aqueous humor outflow is implicated. Recent data from our laboratories support the hypothesis that increased outflow resistance in glaucoma is a function of elevated stiffness and contraction of the Schlemm’s canal (SC) endothelial cells (Zhou et al. 2011, Journal of the Royal Society, Interface. DOI: 10.1098). However, contractile forces exerted by a confluent SC monolayer have not been studied previously, and the role of substrate stiffness is unknown.
Human SC cells were isolated from two human donor eyes as previously described (Stamer et al. 1998, IOVS); we cultured human SC endothelial cell monolayers on polyacrylamide gels of 3 stiffnesses, 1.1, 2.5 and 11.9 kPa in shear modulus. Gels were coated with type 1 collagen and labeled with fluorescent beads. Traction forces were obtained using Fourier transform traction microscopy. In a 96-well plate, the effect of TGF-β2 (1 ng/ml) and Y27632 (10 µM) on monolayer traction forces was monitored for over 4 days.
We tracked 48 samples simultaneously. TGF-β2 caused average traction forces to increase gradually over the course of 4 days; the intermediate stiffness gel (2.5 kPa) elicited the greatest increase, by over 3 fold. Y27632 caused average traction forces to decrease abruptly within 2 hours and remained decreased over 4 days; the relative reduction in monolayer traction decreased from about 70% on the softest substrate and to about 20% on the stiffest substrate.
Our data suggest that substrate stiffness significantly affects monolayer contractile responses to TGF-β2, a potential glaucoma-causing cytokine, and Y27632, a conventional outflow drug candidate. Our high throughput traction platform could be useful for conventional outflow-targeted drug discovery.
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