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Yuan Lei, W Daniel Stamer, Jihong Wu, Xinghuai Sun; Oxidative Stress Impacts On Barrier Function of Porcine Angular Aqueous Plexus Cell Monolayers. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3567. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our goal was to model aging and to investigate the effects of oxidative stress on angular aqueous plexus (AAP) endothelial cells isolated from porcine eyes. AAP is the porcine equivalent of human Schlemm’s canal (SC). Our hypothesis is that oxidative stress would impact AAP cell barrier function.
Cells were differentially isolated from porcine outflow tissues using puromycin treatment. Cultures of porcine AAP cells were grown for 2 weeks in physiological (5% O2) or hyperoxic conditions (40% O2). Cell growth rate, size, transendothelial electrical resistance (TEER), hydraulic conductivity (HC) were measured. The expression of ageing marker SA-galactosidase was monitored, and the expression levels of cytoskeletal and barrier proteins such as F-actin, phospho-myosin light chain (phospho-MLC), occludin, claudin, ZO-1, beta-catenin and VE-cadherin were measured by immunofluorescence staining and western blot analysis.
Our data showed that chronic hyperoxia inhibited cell growth rate from day 3 onward, the cell size increased by 18.2% ± 5.1%, and cells stained positive for SA-gal. Hyperoxia resulted in a significant increase of TEER compared with the control group from day 10 onward (p<0.05, n=6). When perfused in the basal-to-apical direction, at 4 mmHg, HC of AAP cells was 1.97±0.12 and 1.54±0.13 uL/mmHg/min/cm2 in control and hyperoxia group, respectively (p<0.05, n=6). Upon examining cells more closely by immunofluorescence staining, stressed cells expressed a greater abundance of F-actin, phospho-MLC, occludin, claudin-5, beta-catenin and VE-cadherin comparing to the control group (n=6). At the site of cell-cell contact, we also observed increased expression of F-actin, beta-catenin and VE-cadherin. Western blot confirmed an overall higher level of expression of phospho-MLC, occludin, claudin-5, ZO-1, beta-catenin and VE-cadherin in the high oxygen group compared with control.
Chronic exposure of AAP cells to oxidative stress decreased cell monolayer permeability and up-regulated cytoskeletal and cell-cell adhesion protein expression; suggesting that with age and increased oxidative stress, resistance at the level of Schlemm's canal in humans also increases.
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