Comparative mechanistic study of RPE cell death induced by different oxidative stresses

Abstract

Purpose : Age-related macular degeneration (AMD) is a degenerative disorder of the macula, the region of the central retina responsible for visual acuity. Geographic atrophy (GA) occurs in late dry AMD. Oxidative stress-induced retinal pigment epithelial (RPE) cell death is the major reason for AMD, especially in GA. However, the mechanism of RPE cell death is still controversial. The goal of this study is to compare the features of RPE cell death induced by different oxidative stresses, to identify potential universal therapeutic targets for GA.

Methods : RPE cells were treated with 4-HNE, a major product of lipid peroxidation; a ferroptosis inducer RSL3; a necroptosis inducer shikonin; or NaIO₃, a widely used stressor in dry AMD model. Cell viability was then tested. Effects of apoptosis, necroptosis, pyroptosis and ferroptosis inhibitors were tested. Pathway markers were also checked. Overexpression of cytoplasmic GPX4 (CytoGPX4) was performed to confirm ferroptosis involvement. An ex vivo RPE explant model was adopted to confirm the in vitro data.

Results : 1. 4-HNE induces RIPK1, RIPK3, MLKL activation and mildly increases oxidized lipid ROS in RPE cells; ferroptosis inhibitors (Lip-1, Fer-1) and necroptosis inhibitors (Nec-1, NSA) rescue 4-HNE-induced RPE cell death; 2. RSL3 induces RIPK1, RIPK3 but not MLKL activation in RPE cells; RSL3-induced RPE ferroptosis can be rescued by Nec-1 (RIPK1 inhibitor) but not NSA (MLKL inhibitor); 3. Lip-1 inhibits RSL3-induced RIPK3 activation while Nec-1 represses RSL3-induced oxidized lipid ROS accumulation; 4. Shikonin mildly increases oxidized lipid ROS accumulation in RPE cells; Lip-1 and Fer-1 partially rescue shikonin-induced RPE necroptosis; 5. Nec-1, NSA but not Lip-1 or Fer-1 rescue NaIO₃-induced RPE cell death; 6. CytoGPX4 overexpression only rescues RSL3-induced RPE cell death.

Conclusions : Some portion of necroptosis and ferroptosis pathways are activated during RPE cell death induced by different oxidative stresses. Both 4-HNE and NaIO₃-induced RPE cell death are likely through necroptosis. RSL3 induces necrosome activation in RPE cells. Lipid ROS exists both upstream and downstream of necrosome in RPE ferroptosis. Shikonin-induced RPE necroptosis is associated with mild lipid ROS accumulation. Nec-1 could be a better therapeutic compound for GA since it inhibits both necrosome activation and lipid ROS accumulation in different cell death pathways.

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