June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Exosomal miRNAs in primary polarized Retinal Pigmented Epithelium
Author Affiliations & Notes
  • Belinda Judith Hernandez
    Ophthalmology, Duke University, Durham, North Carolina, United States
  • Yutao Liu
    Augusta University Medical College of Georgia, Augusta, Georgia, United States
  • Catherine Bowes Rickman
    Ophthalmology, Duke University, Durham, North Carolina, United States
    Department of Cell Biology, Duke University, Durham, North Carolina, United States
  • Footnotes
    Commercial Relationships   Belinda Hernandez None; Yutao Liu None; Catherine Bowes Rickman None
  • Footnotes
    Support  NEI R01 EY031748, P30 EY005722 (to Duke Eye Center); Research to Prevent Blindness (Duke Eye Center)
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2319. doi:
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    • Get Citation

      Belinda Judith Hernandez, Yutao Liu, Catherine Bowes Rickman; Exosomal miRNAs in primary polarized Retinal Pigmented Epithelium. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2319.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We previously described polarized proteomes of high-quality exosomes isolated from the basal and apical sides of differentiated primary porcine retinal pigmented epithelial cells (pRPE) grown on Transwell cell culture inserts. Using this same pRPE system we analyzed the miRNA cargo in RPE-derived exosomes isolated from the basal and apical media to determine the role of these miRNAs in RPE function.

Methods : We used two different exosome isolation methods: differential ultracentrifugation (DUC), and cushioned iodixanol buoyant density gradient ultracentrifugation (C-DGUC) to compare RPE-specific miRNAs isolated from pRPE exosomes. Exosomes were isolated from apical and basal conditioned media collected from established pRPE cell cultures grown on Transwell inserts. miRNAs were isolated from these exosomes with a Qiagen miRNeasy micro kit and miRNA libraries were prepared using a QIAseq miRNA library kit. miRNAs were sequenced at the Duke Sequencing and Genomic Technologies center using Illumina MiSeq v3 kits. The resulting miRNA datasets were curated using the Qiagen GeneGlobe RNA Data Analysis program.

Results : The yield of exosomes and resulting miRNA from apical and basal secreted exosomes isolated by C-DGUC was lower than those isolated by DUC, as expected since this method yields higher purity exosomes. A total of 250 miRNAs were identified from the RNA-Seq data from exosomes isolated by each isolation method, including RPE-specific miRNAs important for development, the visual cycle, and barrier function. We identified miR-204, miR-125b, and miR-9, which are important for RPE development and present in both apical and basal datasets. There was a 10-fold increase in apical compared to basal secreted exosomal miRNAs involved in oxidative stress and neovascularization. These differences were consistent when using DUC or C-DUGC. Exosomes isolated by the C-DUGC method had a 3-fold increase in basal secreted miRNAs associated with RPE development compared to DUC.

Conclusions : We have identified a set of miRNAs associated with RPE cellular health and are currently repeating these studies in pRPE cells under chronic oxidative stress. Our findings support that miRNAs in exosomes contribute to RPE homeostasis and function in a polarized manner.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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