Abstract
Purpose :
Intraretinal hyperreflective foci (HRFs) appear on optical coherence tomographs (OCT) in retinal diseases such as Age-related Macular Degeneration (AMD). They are thought of as clinical indicators of disease severity. Sodium iodate (NaIO3) rapidly induces RPE degeneration in rodents, modeling characteristics of macular degeneration including HRFs. Recent studies from our lab and others have hypothesized that HRFs are RPE cells that have undergone epithelial-to-mesenchymal transition (EMT) and have migrated into the neural retina. The purpose of this study is to test whether NaIO3 alters the gene expression levels in human RPE-derived iPSC cells that drive them into the EMT process.
Methods :
Human iPSCs were differentiated into RPE cells according to published protocols (from PMCID: PMC8784963 with minor modifications). On Day 150, mature RPE cells were treated with NaIO3 (2.5mM) for 72 hours. Cells were then lysed and processed using an RNA isolation kit. qPCR was used to quantify key markers of EMT, ER-stress, cytoskeleton, junction proteins, extracellular matrix, RPE signature genes and NRF2 targets.
Results :
NaIO3 treatment significantly increased (p<0.05 to p<0.0001) selected EMT markers, including TGFB2, TGFBR1 and 2, SMAD 3 and 4, SNAI2, HES1, HEY1, HMGA2 and JAG1; Cytoskeleton marker, VIM; ER stress markers such as CANX; extracellular matrix markers such as FN1, MMP2, MMP9 and MMP14; and NRF2 targets (marker for oxidative stress) GCLM, HMOX1 and NQO1. NaIO3 treatment significantly reduced (p<0.01 to p<0.0001) RPE signature genes BEST1, CRALBP, RPE65, TRPM1, TYRP1 and tight junction proteins such as CDH1 and ZO-1.
Conclusions :
NaIO3 increased NRF2 targets in RPE cells, which suggests that the cells were undergoing oxidative stress. EMT markers were increased following NaIO3 treatment, while RPE signature genes and tight junction proteins were decreased. Additionally, extracellular matrix markers were increased. The hallmark of EMT is cell migration, proliferation and excretion of extracellular matrix. Therefore, our results suggest that NaIO3 treatment induces oxidative stress in RPE cells and drives them into EMT, with the consequent loss of the RPE phenotype.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.