Targeting STAT3/GPX4 Alleviates Photoreceptor Ferroptosis Induced by Red Blood Cell Leakage Following Retinal Ischemia/Reperfusion

Abstract

Purpose : Retinal ischemia/reperfusion (I/R) is one of the most common pathologies of many vision-threatening diseases, such as diabetic retinopathy, glaucoma and retina artery occlusion. This study aims to find a new approach for retinal I/R related diseases.

Methods : Unilateral ligation and release of the internal carotid artery to construct retinal I/R model. Histological analysis and ELISA were employed for red blood cell (RBC) leakage and ferroptosis detection. Single-cell transcriptome, CHIP-pPCR and luciferase assay were for STAT3 binding to GPX4 promoter. To provide in vivo evidence of the protective role of the STAT3-GPX4 axis in functional recovery, adeno-associated virus (AAV) delivery and transgenetic mice were employed.

Results : We report blood-retinal barrier (BRB) breakdown and subsequent RBC leakage after retinal I/R. The lysis of RBC leads to iron overload in retina, triggering ferroptosis, especially in photoreceptors. Intriguingly, GPX4 levels initially increase 6h after I/R and subsequently decrease, in contrast to retinal ferroptosis levels, suggesting GPX4 expression regulation as a potential therapeutic target. Single-cell transcriptome analysis unveiled a significant increase of STAT3 in photoreceptors 6h post I/R, aligning with the time point of GPX4 expression increase. We further identified the binding site of STAT3 on GPX4 promoter. We also aim to determine whether GPX4 overexpression and STAT3 activation in vivo promote functional recovery and whether the protective role of STAT3 is nullified by deletion of GPX4 using sgRNA delivered by AAV in Rho-Cre::Rosa26^{CAG-LSL-Cas9-tdTomato} mice.

Conclusions : Our results identify BRB breakdown, RBC leakage and ferroptosis of photoreceptor as critical determinants of retinal I/R and demonstrate the potential for targeting STAT3-GPX4 axis in retinal I/R.

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

 

(a-c) Ter119⁺ RBC leakage and lysis levels.
(d-f) Iron, MDA, GSH levels.
(g) GPX4 and ACSL4 levels in retinas.
(h) GPX4 level is in contrast with ferroptosis progression.
(i-j) Cell death levels.
(k-l) Retinal cell subgroups.
(m) Ferroptosis regulation genes with significant expression differences in photoreceptors.
(n) STAT3 level in photoreceptors.
(o) Binding sequence prediction.
(p) Binding rate of STAT3 to GPX4 promoter in 661W cells.
(q-r) STAT3 binding sequences on the GPX4 promoter.
(s-t) The schematic diagram shows future in vivo experiments.