Abstract
Purpose:
Oxidative stress has been documented as the pathogenesis of degenerative disease of the retinal pigment epithelium (RPE) and retina, such as AMD. However, details on the protective mechanism of the retina in oxidative environment are not well known. We recently found that the increased level of Wnt inhibitory factor 1 (WIF-1) in the aqueous humor of AMD patients by proteomic analysis. Here we investigate the potential correlation between the increase of WIF-1 and cell survival mechanism in light and oxidative stress-induced retinal degeneration.
Methods:
Primary retinal cell and primary Muller cell cultures were generated from the retinas of newborn or one-week-old Sprague-Dawley rats as described previously. Cell viability and death were monitored using CCK-8 assay. Western blot analysis of WIF-1, DKK-3, and β-catenin was performed in the retinal and Muller cells and their conditioned medium. SB216763 was used as a Wnt activator in cell cultures. Adult C57BL6 mice were exposed to bright light of 20,000 lux for 2h, or injected with NaIO3 into tail vein. Eyes were collected 2h, 1, 5, and 7 days after exposure and prepared for western blotting and immunofluorescence (IF) of the RPE and retina.
Results:
Exposure to H2O2 (50 µM, 24h) did not cause apoptosis or cell death in retinal cell cultures. Expression of WIF-1, DKK-3, and β-catenin were increased in retinal cells and conditioned medium by exposure to oxidative stress. Exposure of retinal cell cultures to 2 - 4 µM SB216763 with 50 µM H2O2 showed increased expression of WIF-1 and decreased cell death. Among mixed cells in retinal cell cultures, Muller cells were markedly expressed WIF-1 and DKK-3 proteins by IF. Prominent increase of above proteins upon exposure to oxidative stress was found in Muller cell cultures and its conditioned medium. Exposure to bright light or NaIO3 resulted in a marked increase in WIF-1 expression, along with β-catenin until 5 days after exposure. Increased expression of WIF-1 was co-localized with increased expression of Muller cell marker, glutamine synthetase.
Conclusions:
Increased Wnt signaling in the retina exposed to light and oxidative stress is protective response against cell death. Increased WIF-1 produced by Muller cells might be protective mechanism against light and oxidative stress-induced retinal degeneration.
Keywords: 688 retina •
634 oxidation/oxidative or free radical damage •
695 retinal degenerations: cell biology