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

Abstract

Purpose : Our previous studies found that expression of R345W-Fibulin-3 induces retinal pigment epithelial (RPE) cells to undergo epithelial-mesenchymal transition (EMT). In the current study we investigated the effect of the R345W-Fibulin-3 mutation on the size, cargo and function of extracellular vesicles (EVs) derived from RPE cells, as well as the role of these EVs in regulating RPE cell dysfunction.

Methods : EVs were isolated from the media of ARPE-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. The amount and size distribution of EVs were determined by Nanoparticle Tracking Analysis (NTA). EV protein concentrations were quantified using the DCTM Protein Assay (Bio-Rad). EV cargo were analyzed by unbiased proteomics using LC-MS/MS with subsequent pathway analysis (Advaita). The EV-associated TGF-β1 protein was measured by ELISA. The effect of EV transplantation was on ARPE-19 migration was evaluated using a scratch-injury assay.

Results : TEM imaging revealed concave-appearing vesicles, while cryo-EM imaging showed spherical vesicles with two subpopulations of EVs: a small group with diameters around 30nm and a large group with diameters around 100nm. Imaging also indicated a greater number of small EVs (~30 nm) in the mutant group compared to the WT group. This result was further confirmed by NTA showing that, in the mutant group, the particle size distributions were smaller than those of the WT EVs. The protein concentration per EV in the mutant group was not significantly different from that of the WT group. Proteomics identified critical members of sonic hedgehog (SHH) signaling and ciliary tip components in the EVs derived from WT ARPE-19 cells , whereas EVs derived from mutant ARPE19 cells contained EMT mediators. ELISA confirmed the elevated TGF-β1 associated with mutant EVs compared to WT EVs. Critically, EV transplant studies showed that treatment of recipient cells with EVs derived from mutant cells was sufficient to increase migration and elevate EMT markers in RPE cells after scratch-injury.

Conclusions : The protein cargo of EVs is determined by the phenotype of their parental cells. Expression of R345W-Fibulin-3 mutation also alters the size and autocrine function of EVs. Notably, EVs derived from RPE cells expressing R345W-Fibulin-3 are sufficient to induce EMT in wild-type RPE cells.

This is a 2021 ARVO Annual Meeting abstract.