Oxidative stress impact on barrier function of porcine angular aqueous plexus cell monolayers.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Duke Scholars

Author W Daniel Stamer Ophthalmology, Glaucoma