Abstract
Purpose :
One of the primary causes of age-related retinal degeneration is dysfunction of the retinal pigment epithelium (RPE) due to genetic and environmental triggers. Oxidative stress is known to cause epigenetic and transcriptomic changes to the RPE leading to degenerative changes. The purpose of this study was to identify common genes and pathways that are altered with parallel short-term exposure to two oxidants- cigarette smoke condensate (CSC) and ferrous sulfate (FS), using an induced pluripotent stem cell-derived RPE (iPS-RPE) model. Both oxidants have been associated with AMD pathogenesis.
Methods :
Four iPS-RPE lines generated from normal human donors were treated with CSC (100ug/ml) or FS (100uM) for 1 and 5 days respectively. The treated cells with their corresponding controls were processed for ATAC and bulk-RNA sequencing. Significantly different accessible region peaks were annotated with Homer (v4.11) based on their locations and proximity to the TSS of the nearest gene. Exploratory GSEA pathway analysis was done with fgsea against the hallmark pathway set from the Molecular Signatures Database (MSigDB), using the DESeq2 statistic as a ranking metric. The common top 20 differentially expressed genes were identified using the cutoff criteria of adjusted p-value (< 0.05) and log2 fold change (≥ 1.5 or ≤ −1.5).
Results :
The top upregulated pathways in both treatments included Bile acid (NES-2.01,1.87; padj<0.0005) and Heme metabolism (NES-1.61, 1.27; padj<0.005). Downregulated pathways included 19 pathways associated with cell metabolism, inflammation, DNA repair, TGF-β, etc. (Fig 1a). The common top 20 upregulated and downregulated genes are listed in Fig 1b. ATAC-seq analysis showed no common differential accessible chromatin regions among the two treatments.
Conclusions :
This study shows that oxidative stress alters several pathways which include cholesterol metabolism, oxidative stress, inflammation, and DNA repair. The pathways altered by both insults- bile acid production and heme metabolism are of interest for AMD pathogenesis. Bile acids, synthesized from cholesterol affect the levels and solubility of cholesterol. Heme metabolism is of interest because iron has been implicated in AMD pathogenesis and hemoglobin protein is abundant within RPE cells, potentially serving as an oxygen sink.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.