Figure 1.

ECs and SMCs communicate via EVs. (a) Fluorescence microscopy detection of the uptake of PHK67-labeled EVs (green) by ECs and SMCs (DAPI, blue), scale bars = 50 µm. Graph shows the percentage of positive cells for PKH67. (b) Overview of the experimental setup: miR-298 levels evaluated qPCR in SMCs cultured with ECs transfected with pre-miR-control or pre-miR-298 (blue bars). The purple bars show the reverse experiment in which the level of miR-298 was analyzed in EC cultured in presence of SMC transfected with pre-miR control or pre-miR-298. (c) Overview of the experimental setup. ECs and SMCs were cocultured for 48 h, separated using magnetic beads, and seeded as monoculture to produce EVs. microRNA content of cells and EVs were analyzed by microRNA qPCR profiling. (d) Western blot of CD31 and α-SMA in EVs isolated from EC and SMC cultures (left panels) or after coculture and separation of cells with CD31-coated magnetic bead (right panels) (e) Diagram of miRNAs common and specific to EC EVs before and after coculture. (f) Volcano plot of fold changes (log₂ values) and probability values (−log₁₀) for individual miRNAs in the EVs before and after coculture. (g) Diagram of miRNAs common and specific to SMC EVs before and after coculture. (h) Volcano plot of fold changes (log₂ values) and probability values (−log₁₀) for individual miRNAs in the SMC EVs before and after coculture. (i) Analyses of the expression of miR-143, miR-145, and miR-539 by qPCR in EC EVs before and after coculture with SMCs and the expression of miR-143, miR-145, and miR-582 in SMC EVs before and after coculture with ECs. (j) Analyses of the expression of miR-143, miR-145, and miR-539 by qPCR in ECs before and after coculture with SMCs and the expression of miR-143, miR-145, and miR-582 in SMCs before and after coculture with ECs. All data are the mean ± SEM (N = 3 independent experiments). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the respective control.