Oxidative Stress Impact on Barrier Function of Porcine Angular Aqueous Plexus Cell Monolayers

Abstract

Our goal was to investigate the effect of chronic oxidative stress on angular aqueous plexus (AAP, functional equivalent to human Schlemm's canal) endothelial cells from porcine eyes. AAP cells were differentially isolated from porcine outflow tissues using puromycin selection. Confluent cultures of porcine AAP cells were grown for 2 weeks in physiological (5% O2) or hyperoxic conditions (40% O2) to model elevated oxidative stress associated with ageing. Cell growth rate, size, transendothelial electrical resistance (TEER), and hydraulic conductivity (HC) were measured. The expression of senescence-associated β-galactosidase and DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) was monitored, and the levels of cytoskeletal and cell-cell adhesion proteins such as F-actin, phospho-myosin light chain (phosphor-MLC), occludin, claudin-5, ZO-1, β-catenin, and VE-cadherin were measured by immunofluorescence staining and Western blot analysis. 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 β-galactosidase and 8-OHdG. Hyperoxia resulted in a significant 30% increase in TEER compared with the control group (P<0.05, n=6). When perfused in the basal-to-apical direction at 4 mm Hg, HC of AAP cells was 1.97±0.12 and 1.54±0.13 μL/mm Hg/min/cm2 in control and hyperoxia groups, respectively (P<0.05, n=6). Stressed cells expressed a significantly greater abundance of F-actin, phospho-MLC, occludin, claudin-5, β-catenin, and VE-cadherin compared to the control group by both immunofluorescence and Western blot analyses. 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 increases.