Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells.

Publisher: Wiley Country of Publication: United States NLM ID: 101610479 Publication Model: Print Cited Medium: Internet ISSN: 2001-3078 (Electronic) Linking ISSN: 20013078 NLM ISO Abbreviation: J Extracell Vesicles Subsets: MEDLINE

Publication: 2020- : [Hoboken, NJ] : Wiley
Original Publication: Järfälla : Co-Action Pub.

Epithelial-Mesenchymal Transition* ; Extracellular Vesicles*/metabolism; Chromatography, Liquid ; Hedgehog Proteins/metabolism ; Tandem Mass Spectrometry ; Epithelial Cells/metabolism ; Transforming Growth Factor beta/metabolism ; Luciferases/metabolism ; Retinal Pigments/metabolism

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFβ1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFβ was determined by pre-incubation of EVs with a pan-TGFβ blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFβ1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFβ-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFβ dependent manner. These results suggest that EV-bound TGFβ plays a critical role in the induction of EMT in RPE cells.
(© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

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Keywords: EMT; Fibulin-3; RPE; TGFβ; extracellular vesicles

0 (fibulin)
0 (Hedgehog Proteins)
0 (Transforming Growth Factor beta)
EC 1.13.12.- (Luciferases)
0 (Retinal Pigments)

Date Created: 20231019 Date Completed: 20231023 Latest Revision: 20231024

20231025

PMC10585439

10.1002/jev2.12373

37855063