Transferrin protects RPE cells from oxidative damages

Abstract

Purpose : Age-related Macular Degeneration (AMD) is a multifactorial disease involving oxidative stress, inflammation, complement activation and ferroptosis. Elevated retinal iron levels in AMD patients have been well documented and could contribute to all these pathophysiological hallmarks suggesting that iron chelation could benefit AMD. We previously demonstrated that local administration of Transferrin (TF), an endogenous iron chelator, is effective for reducing iron retinal content and oxidative stress and slowing down photoreceptor loss in various animal models of retinal degeneration. Here, we further explored the protective mechanism of action of TF in oxidative stress conditions at the retinal pigment epithelium (RPE) level.

Methods : Differentiated ARPE-19 (dARPE-19) and hiPSC-derived RPE (iRPE) cells were exposed for 24 hours to FeCl₃NTA or 4HNE in presence of TF. Viability of RPE cells was assessed by quantification of mitochondrial activity (CellTiter) and LDH release. Changes in the expression pattern of genes involved in iron homeostasis (TFR1, H-FT, L-FT, LCN2), oxidative stress (HMOX, SOD1), complement activation (C3), lipid metabolism (ABCA1), and ferroptosis (GPX4) was defined by RT-qPCR.

Results : Both FeCl₃NTA and 4HNE induced a dose-dependent decrease in dARPE-19 and iRPE cell viability. Incubation with TF significantly protected dARPE-19 and iRPE against FeCl₃NTA-induced toxicity (+101.1% and +91.2% viability, respectively), and against 4HNE-induced oxidative stress (+57.3% and +71.3% viability, respectively). 24h exposure of iRPE cells to FeCl₃NTA drastically decreased the expression of TFR1 (0.41-fold change) and increased the expression of H-FT and L-FT (2.47- and 3.39-fold change, respectively) and LCN2 (1.982-fold change). Moreover, a significant increase in C3, ABCA1, SOD1 and GPX4 genes was highlighted. Co-treatment with TF restored almost control levels of all genes impacted by FeCl₃NTA. Exposure to 4HNE drastically increased the expression of HMOX1 (11.77-fold change) which was significantly prevented by TF (2.39-fold change, p < 0.0001).

Conclusions : By controlling iron homeostasis, TF has the potential to counteract oxidative stress, complement activation and other pathways involved in AMD pathogenesis. The protective effect of TF in iron-independent stress condition suggests TF has activities beyond iron chelation.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.