In vitro RPE cell models for iron toxicity studies

Abstract

Purpose : Oxidative stress mediated-retinal pigmented cells (RPE) dysfunctions were commonly accepted to be a primary failures in AMD. Iron, main source of free radicals, accumulates in RPE, and is an hallmark of ferroptosis that is now considered to be involved in RPE cell death during AMD. Although studies have shown iron toxicity in RPE cells, variability in experimental procedures and RPE cells used made difficult to have a clear overview of the effect of iron on RPE cells. Here, we further explored 3 models of RPE cells regarding iron molecules, time of exposure, iron metabolism and pathogenesis.

Methods : Undifferentiated (unARPE), 2-month-differentiated ARPE-19 (dARPE) and iPSC-derived RPE (iRPE) cells were exposed to FeCl₃NTA (FeN) or FeSO₄ (FeS) form 1 to 7 days. Viability of RPE cells and iron accumulation was assessed. Changes in the expression pattern of genes and proteins involved in iron homeostasis (TFR1, H-FT, L-FT, LCN2, FPN), oxidative stress, inflammation and ferroptosis (GPX4) was defined by RT-qPCR and Western Blot.

Results : One day exposure with FeS only decreased cell viability in unARPE and a FeN dose-dependent decreased of cell viability was observed in the three RPE cell models, with a higher resistance for iRPE cells. Longer FeN exposure didn't led to a loss of cell viability in unARPE and dARPE, while cell viability was still low in iRPE. While all cell types accumulated iron after 1 day of FeN exposure, only iRPE maintained high intracellular iron level after longer exposure. Transferrin expression was almost undetectable in unARPE and very low in dARPE compared to iRPE, RTF1 was present in the 3 cell types and RTF2 was only observed in iRPE. One day exposure of dARPE and iRPE to FeN mediated oxidative stress and led to similar iron homeostasis regulation with only discordance for ferroportin expression. Two days exposure of iRPE to FeN induced oxidative stress, iron homeostasis regulation, loss of cell integrity and tight junctions, mitochondrial damage, pro-inflammatory process, complement activation, lipid peroxidation and ferroptosis.

Conclusions : We demonstrated that cell types, iron source and iron homeostasis machinery should be taken into consideration to investigate iron toxicity effect and ferroptosis in RPE cells. We considered that undifferentiated ARPE-19 is not to be preferred, differentiated ARPE-19 could be used for screening investigations and iPSC-derived RPE seems to be the most relevant model

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.