Ferroptosis in human retinal pigment epithelial cells induced by oxidative stress.

Abstract

Purpose : Oxidative stress is considered implicated in retinal pigment epithelial (RPE) cell death in age-related macular degeneration (AMD), though the cell death mechanism remains elusive. Previous studies have disclosed excess iron accumulates in RPE and Bruch’s membrane of patients with AMD. Here, we investigated whether ferroptosis, a form of regulated necrosis characterized by iron dependency and lipid peroxidation, plays a role in oxidative stress-induced RPE cell death in vitro.

Methods : Cell viability was evaluated in ARPE-19 and human fetal RPE (hf-RPE) cells exposed to different concentrations of tert-Butyl hydroperoxide (tBH), a stable and long-acting form of H₂O₂, with or without inhibitors for apoptosis, necroptosis, and ferroptosis (i.e. ferrostatin-1 (Fer-1), deferoxamine (DFO)). Apoptosis and necrosis in ARPE-19 cells exposed to 500μM tBH for different durations were determined by Annexin V/ propidium iodide (PI) staining. Intracellular reactive oxygen species (ROS), lipid peroxide, glutathione (GSH), and Fe²⁺ were evaluated in tBH-exposed ARPE-19 cells with or without Fer-1 or DFO. The iron overload to ARPE-19 cells was performed using 120µM ferric ammonium citrate (FAC) treatment for 2 days. mRNA levels were quantified using real-time RT-PCR.

Results : All of the tested inhibitors for apoptosis, necroptosis, and ferroptosis mitigated tBH-induced cell death in ARPE-19 cells as well as hf-RPE cells, however, the ferroptosis inhibitors rescued RPE cell death most effectively. Annexin V/PI staining showed both apoptotic and necrotic cells in tBH-exposed ARPE-19 cells. Increase in intracellular ROS, lipid peroxide, and Fe²⁺, as well as decrease in GSH were observed in tBH-exposed ARPE-19 cells, which were abolished by Fer-1 and DFO. Increased intracellular Fe²⁺ by FAC treatment led ARPE-19 cells to increased vulnerability to tBH-induced cell death, which was also significantly attenuated by Fer-1 or DFO. After tBH exposure to ARPE-19 cells, mRNA levels of genes for iron homeostasis, GSH synthesis, and lipid peroxidation were altered significantly; most of the changes appeared to compensationally decrease intracellular Fe²⁺ and lipid peroxide, and to increase intracellular GSH, although the change in mRNA levels of transferrin receptor 1, ferroportin, and hephaestin appeared to increase Fe²⁺.

Conclusions : Ferroptosis may play a role in oxidative stress-induced human RPE cell death.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.