Role of PMC (2,2,5,7,8-pentamethyl-6-chromanol), a sterically hindered phenol, in rescuing oxidized low-density lipoprotein (ox-LDL) induced cytotoxicity in human retinal pigment epithelial cells

Abstract

Purpose : Late stage dry age-related macular degeneration (AMD) is characterized by irreversible vision loss due to retinal pigment epithelium (RPE) degeneration and death. An early sign of AMD involves drusen formation, which is associated with dysfunctional lipid metabolism. In vitro and in vivo studies show that RPE degeneration can be induced with ox-LDL, reflecting AMD-associated dysregulation in lipid handling. Previously we have demonstrated a significant role for sterically hindered phenols in protecting RPE from ox-LDL-induced damage. Specifically, PMC has shown significant protection against oxidative stress. The goal of the current study is to determine the molecular mechanism of action of PMC in preventing ox-LDL-induced cytotoxicity in RPE.

Methods : Primary human fetal RPE (HRPE) cells were cultured in RPE cell growth medium (RtEGM) supplemented with RtEGM SingleQuots. Half of the media was replenished every 2-3 days for 4 weeks to allow maturation. Serum-starved cells were treated with ox-LDL (100 µg/ml) with or without PMC (1.3 µM) for 24 and 48 hr. Cytotoxicity was assessed using CyQUANT-LDH assay. Oxygen consumption rate was measured using Seahorse XFe96 Mito Stress Test. Total RNA was bulk RNA sequenced and DESeq2 was used to identify differentially expressed genes.

Results : Treatment of HRPE cells with ox-LDL (100 µg/ml) induced a 21% cell death at 24 hr and 47% cell death at 48 hr, which was reduced to 15% and 7% at 24 hr and 48 hr, respectively, in comparison to control cells. PMC alone did not influence RPE cell viability. Interestingly, ox-LDL caused a decrease in the rate of oxygen consumption, an effect that was not rescued by treatment with PMC. Gene ontology enrichment analysis of RNA sequencing data indicated upregulation of apoptosis, autophagy, and cell death-associated pathways with ox-LDL treatment, which were all downregulated by PMC treatment.

Conclusions : Our findings validate the effect of PMC on ox-LDL-induced RPE cytotoxicity and RNA sequencing analysis is helping to unravel the mechanisms involved in PMC mediated rescue of AMD-associated pathologies.

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