Purpose: Oxidative stress in the retinal pigmented epithelium (RPE) has been implicated in age-related macular degeneration by impacting endocytic trafficking, including the formation, content, and secretion of extracellular vesicles (EVs). Using our model of polarized primary porcine RPE (pRPE) cells under chronic subtoxic oxidative stress, we tested the hypothesis that RPE miRNAs packaged into EVs are secreted in a polarized manner and contribute to maintaining RPE homeostasis.
Methods: Small EVs (sEVs) enriched for exosomes were isolated from apical and basal conditioned media from pRPE cells grown for up to four weeks with or without low concentrations of hydrogen peroxide using two sEV isolation methods, leading to eight experimental groups. The sEV miRNA expression was profiled using miRNA-Seq with Illumina MiSeq, followed by quality control and bioinformatics analysis for differential expression using the R computing environment. Expression of selected miRNAs were validated using qRT-PCR.
Results: We identified miRNA content differences carried by sEVs isolated using two ultracentrifugation-based methods. Regardless of the sEV isolation method, miR-182 and miR-183 were enriched in the cargo of apically secreted sEVs, and miR-122 in the cargo of basally secreted sEVs from RPE cells during normal homeostatic conditions. After oxidative stress, miR-183 levels were significantly decreased in the cargo of apically released sEVs from stressed RPE cells.
Conclusions: We curated RPE sEV miRNA datasets based on cell polarity and oxidative stress. Unbiased miRNA analysis identified differences based on polarity, stress, and sEV isolation methods. These findings suggest that miRNAs in sEVs may contribute to RPE homeostasis and function in a polarized manner.