Multi-level characterization of the hypoxic stress response in retinal pigment epithelial cells

Abstract

Purpose : With advancing age, choriocapillaris dropout, reduced choroidal blood flow and thickening of Bruch’s membrane cumulatively result in a chronic restriction of oxygen to the outer retina. Resulting activation of hypoxia-inducible factors (HIFs) triggers a metabolic stress response in the retinal pigment epithelium (RPE) that consequently leads to RPE and photoreceptor dysfunction. Genetic deletion of HIF-2α can rescue the structural and visual defects in mice. Thus, learning how HIF-2α transcriptional activity affects the hypoxia-driven stress response in human RPE cells could give insights into hypoxia-induced retinal pathologies and identify strategies to prevent vision loss.

Methods : Mature pigmented retinal pigment epithelial cells (hRPE) isolated from human donors were cultured in normoxic (21% O₂) or hypoxic (4% O₂) conditions for up to 3 days. The hypoxic stress response was assessed by sequential transcriptomic, proteomic and metabolomic analyses followed by functional metabolic flux analysis. Transcriptomic analysis of HIF-2α-deficient hiPSC-derived RPE cells (hiRPE) as well as phenotypic analysis of hRPE treated with the HIF-2α inhibitor PT2977 were used to assess the metabolic effect of interfering with HIF-2α transcriptional activity.

Results : Hypoxia altered hRPE identity and led to a severe disruption of central carbon metabolism. Increased glucose and glutamine uptake, but reduced glycolytic and mitochondrial activity suggest that hypoxic hRPE store energy as lipid droplets that massively accumulated in the cytosol. Lactate and proline accumulated in- and outside of hypoxic hRPE, suggesting increased production and release into the extracellular space. Despite structural mitochondrial defects, mitochondrial respiration could be restored by re-directing glucose to the TCA cycle. However, HIF-2α inhibition did not improve respiration but reduced the lipid load in hypoxic hRPE and improved RPE signature gene expression. Transcriptome comparison of HIF-2α knockout and WT hiRPE revealed an overall improved metabolic profile and RPE identity.

Conclusions : Our study provides a comprehensive multi-level analysis and adds to the understanding of the HIF-2α driven stress response in hypoxic human RPE cells. In the future, these findings can be used to develop more broadly applicable therapeutic strategies for hypoxia-induced retinal pathologies.

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