Abstract
Purpose :
Age-related macular degeneration is one of the leading causes of vision loss in the elderly and oxidative stress is believed to be the major cause of loss of retinal pigment epithelial (RPE) cells and photoreceptors in large zones of the macula. Proteoglycan-4 (PRG4) has been shown to bind and affect downstream signalling of a number of cell surface receptors implicated in regulating cellular stress responses, including those induced by oxidative stress. In the present study, we have explored the protective ability of recombinant human PRG4 (rhPRG4) during oxidative stress-mediated RPE cell death.
Methods :
Human ARPE-19 cells were treated with 400 µM hydrogen peroxide (H2O2), and/or rhPRG4 (75 µg/mL, Lubris BioPharma). PRG4 was visualized and localized using western blotting and immunofluorescence, respectively. Reactive oxygen species (ROS) generation was determined by H2DCFDA assay and live/dead cell staining was performed and quantified using ImageJ. Whole globes of C57/BL6 mice were fixed, embedded, and sectioned, followed by immunolabeling with anti-PRG4 Ab. Exclusion of the primary Ab was used as a negative control.
Results :
PRG4 expression is observed throughout the ocular tissues, including the RPE as seen in immunohistochemical staining of mouse eye sections. Human ARPE-19 cells subjected to oxidative stress demonstrates an up-regulation of PRG4 via western blot analysis. Pre-treatment of cells with rhPRG4 prevented H2O2-mediated ROS generation and oxidative stress induced cell death.
Conclusions :
PRG4 is expressed in RPE cells both in-vivo and in-vitro. Addition of rhPRG4 shows a protective effect for RPE cells under oxidative stress conditions. Future studies will further elucidate PRG4 function in the RPE and the role it plays in inflammation and oxidative stress conditions, such as AMD, and potential treatments.
This is a 2020 ARVO Annual Meeting abstract.