Abstract
Purpose :
Age-related macular degeneration (AMD) involves formation of lipid peroxidation and insufficient levels of antioxidants. Due to high metabolic demand and postmitotic nature, retinal pigment epithelial cells (RPEC) and neurons are susceptible to oxidative stress damage. Oxidized phospholipids (OxPLs) cause detrimental perturbations to photoreceptor cells (PRC) and to the RPEC. Elovanoids (ELVs), which are oxygenated derivatives of very long-chain polyunsaturated fatty acids (VLC-PUFAs) such as C32:6 n3 and C34:6 n3, represent a distinctive class of lipid mediators essential for maintaining the integrity of photoreceptor cells. Here, we have studied ELV biosynthesis and enhanced cell survival under uncompensated oxidative stress conditions in RPEC. We have identified OxPL species in RPEC under UOS conditions by combining bioinformatics and advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Methods :
RPEC were exposed to UOS, +/- ELV-N34 precursor (FFA 34:6), and lipids were analyzed by LC-MS/MS. For OxPLs, automatic data processing for compound identification, which includes peak assignments and deconvolution of MS/MS data was performed using MS-DIAL software. Final data were matched with a reference library of known oxidized lipid compounds. Cell viability was assessed using an Incucyte Live-Cell Analysis system.
Results :
Both ELV and mono hydroxylated intermediate were upregulated in RPEC with FA supplementation. Both ELV and their respective mono-hydroxylated intermediate decreased under UOS when compared to non-UOS conditions. UOS exhibited elevated concentrations of oxidized phospholipids (OxPLs), primarily oxidized phosphatidylethanolamine, followed by oxidized phosphatidylcholine. Notably, oxidized lipid profiles of RPEC treated with the precursor C34:6 displayed reduced OxPL levels compared to UOS samples. Following exposure to UOS ± ELV, there is a decline in the percentage of dead cells as compared to conditions involving UOS alone.
Conclusions :
Our data shows a protective role of ELVs on RPEC by preventing the oxidation of phospholipid molecular species. Therefore ELV functions would likely include a regulatory role of lipid peroxidation that may involve targeting of ferroptosis. Thus, ELVs, by this additional property of their bioactivity, would protect PRC integrity.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.