Effects of A2E-Induced Oxidative Stress on Retinal Epithelial Cells: New Insights on Differential Gene Response and Retinal Dystrophies

Abstract

Oxidative stress represents one of the principal inductors of lifestyle-related and genetic diseases. Among them, inherited retinal dystrophies, such as age-related macular degeneration and retinitis pigmentosa, are well known to be susceptible to oxidative stress. To better understand how high reactive oxygen species levels may be involved in retinal dystrophies onset and progression, we performed a whole RNA-Seq experiment. It consisted of a comparison of transcriptomes' profiles among human retinal pigment epithelium cells exposed to the oxidant agent N-retinylidene-N-retinylethanolamine (A2E), considering two time points (3h and 6h) after the basal one. The treatment with A2E determined relevant differences in gene expression and splicing events, involving several new pathways probably related to retinal degeneration. We found 10 different clusters of pathways involving differentially expressed and differentially alternative spliced genes and highlighted the sub- pathways which could depict a more detailed scenario determined by the oxidative-stress-induced condition. In particular, regulation and/or alterations of angiogenesis, extracellular matrix integrity, isoprenoid-mediated reactions, physiological or pathological autophagy, cell-death induction and retinal cell rescue represented the most dysregulated pathways. Our results could represent an important step towards discovery of unclear molecular mechanisms linking oxidative stress and etiopathogenesis of retinal dystrophies.

Keywords: A2E; RNA-Seq; RPE; Retinitis pigmentosa.

Figure 1

MTT determination of A2E treatment in retinal pigment epithelium (RPE) cells. Cell death was assessed at considered time points (3 h and 6 h) in A2E treated samples compared to basal untreated group. Results are shown as mean ± standard error of mean (n = 3). p-value < 0.05.

Figure 2

Summary figure of expressed genes and significant DE, DE + DAS, DE + DTU, DAS and DTU genes from analysis of the considered time points of RPE cell data. (A) Number of genes regulated only by transcription (DE), only by alternative splicing (DAS) and by both transcription and alternative splicing (DE + DAS); (B) number of transcripts regulated only by transcription (DE), only by alternative splicing (DAS) and by both transcription and alternative splicing (DE + DAS). DTU = Differential Transcript Usage. AS = Alternative Splicing.

Figure 3

Comparison of the gene lists generated during differential expression analyses. Euler diagrams of DE (A) and DAS (B) genes identified during expression analyses in considered conditions of experimental design, setting the contrast groups as 0 h.untreated versus 3 h.treated, 0 h.untreated versus 6 h.treated, 3 h.treated versus 6 h.treated, 0 h.untreated versus (3 h.treated + 6 h.treated)/2.

Figure 4

Hierarchical clustering and heatmap of DE genes and Key GO terms. DE genes show segregation into 10 coexpressed clusters, of which the main ones related to oxidative stress were highlighted (circled) and linked to GO specific terms. Full results of GO enrichment analyses are shown in Tables S6 and S7. The z-score scale represents mean-subtracted regularized log-transformed transcripts per million (TPMs).

Figure 5

Hierarchical clustering and heatmap of DE genes + DTU transcripts from DAS genes and Key GO terms. DE genes and DTU transcripts from DAS genes show segregation into 10 coexpressed clusters, of which the main ones related to oxidative stress were highlighted (circled) and linked to GO specific terms. Full results of GO enrichment analyses are shown in Tables S6 and S7. The z-score scale represents mean-subtracted regularized log-transformed TPMs.

Figure 6

Volcano plots of significant DE and DAS genes and of DE and DTU transcripts. Volcano plots of significant (adjusted p-value < 0.01) DE genes (A), DE transcripts (B), DAS genes (C) and DTU transcripts (D). The low expressed genes and transcripts were filtered. The top 15 considered elements with the smallest corrected p-values and bigger fold-changes are highlighted, and different colors refer to different contrast groups. DE genes: log2FC vs. −log10(FDR) at gene level; DAS genes: maximum ΔPS of transcript in a gene vs. −log10(FDR) at gene level; DE transcripts: log2FC vs. −log10(FDR) at gene level at transcript level and DTU transcripts: ΔPS vs. −log10(FDR) at transcript level.

Figure 7

Expression profiles of most important DE genes/transcripts; first cluster associated with newly discovered pathways linked to oxidative stress. Detailed gene/transcript expression profiles across the time course of the first cluster of most important DE genes (ACTG1, CAPZB, CCN2, FTL and RPS11) linked to newly identified candidate pathways linked to oxidative stress.

Figure 8

Expression profiles of most important DE genes/transcripts secondo cluster associated with newly discovered pathways linked to oxidative stress. Detailed gene/transcript expression profiles across the time course of the second cluster of most important DE genes (P4HB, RNA5-8SN2, RPL3 and RPL19) linked to newly identified candidate pathways linked to oxidative stress.