Extracellular vesicles from ARPE-19 cells expressing R345W-Fibulin-3 can induce epithelial-mesenchymal transition in recipient cells via a TGF-beta pathway

Abstract

Purpose : The purpose of the current study was to investigate the size, cargo, and function of extracellular vesicles (EVs) derived from healthy ARPE-19 cells expressing wild-type (WT)-Fibulin-3 (WT-RPE-EV) compared to diseased ARPE-19 cells expressing the R345W-Fibulin-3 mutation (R345W-RPE-EV).

Methods : ARPE-19 cells were infected with luciferase-tagged WT-Fibulin-3 or luciferase-tagged R345W-Fibulin-3 using lentiviruses. EVs were isolated from the media of ARPE-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. The amount and size distribution of EVs was determined by Nanoparticle Tracking Analysis (NTA). EV protein concentrations were quantified using the DC^TM Protein Assay (Bio-Rad). Transmission and cryogenic electron microscopy (EM) were used to image the morphology of the EVs. EV markers were validated using western blot analysis. EV cargo was analyzed by unbiased proteomics using LC-MS/MS with subsequent pathway analysis (Advaita). The EV-associated transforming growth factor-beta 1 (TGF-β1) protein was measured by enzyme-linked immunosorbent assay. EV uptake was investigated by using PKH67-labeled vesicles and was analyzed by confocal imaging. Migration ability was evaluated in ARPE-19 cells using scratch assays in the presence or absence of EVs. mRNA expression levels of endothelial-mesenchymal transition (EMT) markers were measured in ARPE-19 cells after EV treatment, by RT-PCR.

Results : NTA analysis showed that the particle size distributions of R345W-RPE-EV were smaller than those of the WT-RPE-EV, however, there was no difference between EV protein concentrations. Similarly, EM revealed spherical and concave-appearing EVs with two subpopulations of diameters: 30 nm and over 100 nm. Pathway analysis revealed that primary cilia and sonic hedgehog pathways were found to be 3- to 5-fold more abundant in WT-RPE-EV. In contrast, EMT drivers, lysosome components, and ribosome components were found to be 3- to 7-fold more abundant in R345W-RPE-EV. We also found higher levels of TGF-β1 associated with R345W-RPE-EVs compared to WT-RPE-EVs (p<0.001). Incubation with R345W-RPE-EVs caused enhanced migration and elevated EMT marker expression in ARPE-19 cells.

Conclusions : These results suggest that R345W-RPE-EVs potentially induce EMT in RPE cells, which may be significant to RPE disease progression.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.