Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells
Authors: Zhou Mi , Zhao Yuanjun , Weber Sarah R. , Chen Han , Ford Michael , Swulius Matthew T. , Barber Alistair J. , Grillo Stephanie L. , Sundstrom Jeffrey M. 
Published: October 2020

ABSTRACTPurposePrevious studies in our lab found that expression of R345W-Fibulin-3 induces retinal pigment epithelial (RPE) cells to undergo epithelial-mesenchymal transition (EMT). The purpose of the current study was to investigate the size, cargo and function of extracellular vesicles (EVs) derived from RPE cells expressing wild-type (WT)-Fibulin-3 compared to RPE cells expressing the R345W-Fibulin-3 mutation, and to determine the role of these EVs in regulating RPE cell dysfunction.MethodsARPE-19 cells were infected with luciferase-tagged wild-type Fibulin-3 (WT)- or luciferase-tagged R345W-Fibulin-3 (mutant) using lentivirus. EVs were isolated from the media of ARPE-19 cells by conventional ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo-EM) were performed to study the morphology of the EVs. 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 transforming growth factor beta 1 (TGF-β1) protein was measured by Enzyme-linked immunosorbent assay (ELISA). The EV transplant study was conducted and migration ability was evaluated in ARPE-19 cells with or without exposure to EVs by conducting scratch assays.ResultsTEM imaging revealed concave-appearing vesicles, and 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. There were no significant differences in EV protein concentrations per EV between WT and mutant groups. Proteomic studies showed that EVs derived from ARPE-19 cells expressing WT-Fibulin-3 contain critical members of sonic hedgehog signaling (SHH) signaling and ciliary tip components, whereas EVs derived from RPE cells expressing R345W-Fibulin-3 contain EMT mediators, including TGF-β-induced protein (TGFBI), vimentin, and mothers against decapentaplegic homolog 4 (SMAD4), indicating that the EV cargo reflects the phenotypic status of their parental cells. Subsequent studies revealed enhanced activity of TGF-β1 associated with mutant EVs compared to WT EVs. Critically, EV transplant studies showed that treatment of recipient RPE cells with mutant RPE cell-derived EVs was sufficient to induce an enhanced migration ability and elevated EMT marker expression of RPE cells.ConclusionsThe expression of R345W-Fibulin-3 alters the size, cargo and autocrine function of EVs. Notably, EVs derived from RPE cells expressing R345W-Fibulin-3 are sufficient to induce EMT in uninfected RPE cells.