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Francesco Giorgianni, Diwa Koirala, Sarka Beranova-Giorgianni; Early events in oxLDL-induced transcriptome alteration in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4019.
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Oxidized low-density lipoproteins (oxLDL) constitute major components of drusen, a hallmark of AMD. OxLDL is a known trigger of oxidative stress and tissue inflammation. We hypothesize that exposure to non-toxic levels of oxLDL in retinal pigment epithelium (RPE) cells triggers an early gene expression response that recapitulates early events in the aging RPE cells associated with the onset of AMD.
ARPE-19 cells were treated with oxLDL or non-oxidized low-density lipoprotein (LDL) for 2h and 4h. Experiments were performed in multiple biological replicates. RNA was extracted with Qiagen RNeasy mini kit. Samples were analyzed with an Affymetrix Human Clariom S array using Affymetrix WT Plus Amplification kit. The differentially expressed genes (DEGs) were identified based on fold change and Benjamini-Hochberg adjusted false discovery rate. Gene expression levels for some of the DEGs were confirmed by real-time qPCR and by Western blot analysis. All transcripts significantly up- or down-regulated were analyzed with STRING and DAVID bioinformatics tools.
In RPE cells, treatment with oxLDL generated an early gene expression response which affected more than 700 genes. Gene pathway analysis unveiled gene networks involved in the regulation of various cellular functions, including response to oxidative stress via up-regulation of Nrf2-regulated gene pathways, down-regulation of lipid metabolism gene pathways, up-regulation of genes involved in cholesterol efflux, regulation of cell cycle, and circadian rhythm. In contrast, LDL treatment triggered a very different gene expression response, which did not include the regulation of the same gene pathways as those affected by oxLDL.
In contrast to earlier studies that focused on oxLDL-induced transcriptome alterations at longer exposure times, our study shows that in the RPE cells many gene pathways are altered selectively by treatment with oxLDL as compared to LDL, within a 2-4 h time treatment window. These results support our hypothesis that the early physiological response to non-cytotoxic levels of oxLDL by RPE cells involves gene pathways known to be altered during AMD development. In addition, the observed autophagy and circadian rhythm gene pathways alterations were not anticipated and might represent novel findings that could be used to develop future, innovative testable hypotheses related to RPE physiology in response to oxidative damage.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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