Abstract
Purpose :
Retinal pigment epithelial (RPE) cells are in an oxidative stress environment due to their high metabolic demands and its damage contributes to age-related macular degeneration and other retinal degenerations. ABC cells are a new human RPE cell model which recapitulates gene expression profiles as the native RPE cells. Elovanoids (ELVs) are lipid mediators that promote cell survival upon injury. Here, we have used ABC cells to study ELVs biosynthesis under uncompensated oxidative stress (UOS) +/- very long-chain polyunsaturated fatty acids (VLC-PUFAs). We explore how ELVs regulates the expression of the Ring finger protein 146 (Iduna), critical for proteasomal protein processing.
Methods :
ABC cell cultures were exposed to vehicle, UOS, and VLC-PUFAs 32:6n3 and 34:6n3, which are precursors of ELV32 and ELV34, respectively. Extracted lipids from culture media were analyzed by LC-MS/MS. Cell viability was assessed using an Incucyte Live-Cell Analysis system. Western blot analysis was performed to follow Iduna protein abundance.
Results :
UOS alone did not trigger ELVs synthesis. Both ELVs and mono-hydroxylated intermediates were upregulated with VLC-PUFAs supplementation. Both ELVs and their respective mono-hydroxylated intermediates decreased under UOS when compared to non-UOS conditions. Time-response of cell viability after UOS ± ELV shows a decrease in the ratio of dead cells compared to UOS conditions alone. Iduna expression was downregulated in cells undergoing UOS. Supplementation of fatty acids (FAs) 32:6n3 and 34:6n3 upregulated Iduna expression.
Conclusions :
The decrease in VLC-PUFAs pool size in response to UOS suggests that they are used for ELVs biosynthesis. The relative abundance of each species in FA-mono-di conversion did not change under UOS compared to the non-UOS conditions; suggesting high use of ELVs in the recovery process rather than a reduction in ELVs production. The decrease in the ratio of dead cells from cell viability data indicates strong in vitro cell protection induced by ELVs. Also, cell protection induced by ELVs against UOS can be attributed partly to the upregulation of Iduna expression with FAs treatment, indicating that the protective mediators synthesized on demand enhance the abundance of an important protein for cell recovery.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.