FN matrix stimulates sEV secretion by VSMCs In Vitro.
A, Immobilised FN but not a soluble FN promotes sEV secretion. Cells were cultured for 24h and sEVs in conditioned media were measured by CD63-beads assay. N=4 with technical triplicates. ANOVA, ****p<0.0001 B, Micrograph showing VSMC plated onto plastic or 3D matrix. C, VSMCs were plated on the 3D matrix for 24h, fixed and labelled for Syndecan-4 (green), F-actin (phalloidin, red) and fibronectin (blue) Size bar, 10µm. D, 3D matrix promote sEV secretion. Cells were cultured for 24h and conditioned media was collected and sEV secretion was measured by CD63-beads assay. N=3, T-test E, FN matrix does not affect sEV mode size. VSMCs were plated on non-coated or FN-coated flasks and incubated for 24h. Isolated sEVs were analysed by Nanoparticle Tracking Analysis. Representative from N=3. F, sEV and EV markers distribution is not altered by FN matrix. Cells were plated on non-coated or FN-coated flasks and AB, EV and Apoptotic bodies (AB, 1.2K pellet), extracellular vesicles (EV, 10K pellet) and small extracellular vesicles (sEVs, 100K pellet) were isolated by differential ultracentrifugation and analysed by western blotting. Equal protein load. Representative image from N=3. G, FN induces secretion of sEVs by activating β1 integrin. VSMCs were plated on non-coated or FN-coated plates in the absence or presence of integrin activating (12G10) or inhibiting (4B4) antibodies for 24h and conditioned media was analysed by CD63-bead assay. N=3, ANOVA, ****p<0.0001 H, Src is required for the sEV secretion. Cells were plated and sEV secretion was measured as in 2A. N=3, ANOVA, **p<0.0001 I, Inhibition of FAK blocks FN-induced sEV secretion. Cells were plated and sEV secretion was measured as in 1A. N=3, ANOVA