The role of Proteoglycan 4 in retinal pigment epithelial cell homeostasis in in vitro and in vivo oxidative stress models.

Abstract

Purpose : 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 propagate cellular and tissue damage. Proteoglycan 4 (PRG4) has been shown to bind and affect downstream signaling for a number of cell surface receptors implicated in regulating cellular stress responses, including those induced by oxidative stress. In this study, the protective ability of recombinant human PRG4 (rhPRG4) during oxidative stress-mediated RPE cell death in vitro via hydrogen peroxide and in vivo using a well-established sodium iodate (NaIO₃) model was explored.

Methods : ARPE-19 cells were treated with 400 µM hydrogen peroxide (H₂O₂), and/or rhPRG4 (50 µg/mL, Lubris BioPharma). ROS generation was determined by H2DCFDA assay and live/dead cell staining was performed and quantified using ImageJ. 40 mg/kg NaIO₃ was injected intravenously in 10-week-old Brown Norway rats. Rats received 2 µL intravitreal injections of rhPRG4 (150 μg/mL) or saline 3 days after NaIO₃ induction. Optical coherence tomography (OCT) and electroretinograms (ERG) were performed at baseline, 7 and 14 days post-NaIO₃ injection to evaluate retinal structure and functional changes. Rats were then euthanized and enucleated for immunohistochemistry or RPE isolation for further RNA-Seq analysis.

Results : The addition of rhPRG4 to H₂O₂-treated ARPE-19 cells led to a significant reduction in ROS generation (p<0.0001) and cell death (p<0.01). Two weeks post-NaIO₃ induction, significant subretinal changes and retinal thickness loss (p<0.01) were observed. In NaIO₃ animals treated with rhPRG4, there were less substantial subretinal changes noted, including a reduction in retinal thickness loss measured via OCT imaging. ERG analyses demonstrated a protective effect of rhPRG4 for a- and b-wave amplitudes which were diminished in NaIO₃-induced animals.

Conclusions : Addition of rhPRG4 demonstrated a protective effect for RPE cells after oxidative stress challenges both in vitro and in vivo. Future studies will further elucidate PRG4 function in the RPE and the role it plays in inflammation and oxidative stress to support rhPRG4 as a potential therapeutic agent for treatment of RPE and photoreceptor loss in ocular disease.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.