The Role of Proteoglycan 4 in Retinal Pigment Epithelial Cell Homeostasis in Oxidative Stress Conditions

Abstract

Retinal pigment epithelial (RPE) cell dysfunction and photoreceptor degeneration are hallmarks of late stage dry age-related macular degeneration, also known as geographic atrophy. Oxidative stress from accumulated reactive oxygen species (ROS) and inflammation propagates cellular and tissue damage. Proteoglycan 4 (PRG4) has been shown to bind and affect downstream signalling for several cell surface receptors implicated in regulating cellular stress responses, including those induced by oxidative stress. The protective ability of recombinant human PRG4 (rhPRG4) during oxidative stress-mediated RPE cell death in vitro and in vivo was explored.

To induce oxidative stress conditions in vitro, human retinal pigment epithelial cells were treated with hydrogen peroxide and/or rhPRG4. ROS production was determined by H2DCFDA assay and cell viability analysed. The effects of rhPRG4 on antioxidant enzyme activity and antioxidant regulatory pathways was determined via colorimetric assay and qRT-PCR. To further understand the effects of rhPRG4 in oxidative stress conditions, siRNA was used to knock down PRG4 expression and antioxidant capacity evaluated.

Sodium iodate and laser-induced choroidal neovascularization models of dry and wet AMD were used to determine the efficacy of rhPRG4 as a potential therapeutic for AMD. The animal models were induced and then treated with a single intravitreal injection of rhPRG4 and monitored for one to three weeks. Retinal health and function were monitored with imaging and electroretinography. RNA from the RPE of these animals were used for NanoString analysis to better understand the mechanisms and genetic pathways that may be regulated by the addition of rhPRG4.

Addition of rhPRG4 demonstrated a protective effect for RPE cells after oxidative stress challenges both in vitro and in vivo.