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. 2015 Jul 21;40(1):102–111. doi: 10.1038/ijo.2015.123

Figure 2.

Figure 2

nEV and oEV pro-angiogenic capability. (a) EVs recovered from the visceral ASCs of obese subjects (oEVs) and of non-obese subjects (nEVs) were analyzed by western blotting for VEGF and MMP-2 content and normalized to CD63 and β-actin content. IL-3-treated ECs were used as a positive control (c+). The results are representative of all nEVs and oEVs samples (n=6 and n=10, respectively) (**P<0.01 for MMP-2 and VEGF content in oEVs vs nEVs). (b) miR-126 and miR-130 expression was evaluated by quantitative real-time PCR on nEVs and oEVs recovered from nASCs and oASCs, respectively. Data normalized to RNU6B are representative of all samples (nEVs, n=6; oEVs, n=10) (***P<0.001 oEVS vs nEVs). (c) Representative images obtained by confocal microscope of ECs treated for 1 h without (none) or with PHK26-labeled nEVs or oEVs to evaluate EV internalization. EV internalization was also evaluated either in the presence or in the absence of the blocking antibody against CD29. The results are representative of four different experiments performed in triplicate (n=4). Scale bars indicate 10 μm. (d) Representative photomicrographs of in vitro angiogenesis assays, showing tube-like structure formation by ECs, either alone or in the presence of VEGF (10 ng ml−1) (positive control) or nEVs and oEVs. Quantitative analysis of the number and length of branches and percentage of vessel area (% area) of in vitro formed vessel-like structures is reported as mean±s.e.m. The results are representative of four different experiments performed in triplicate (n=4) (for length, *P<0.05 pos ctrl and nEVs vs neg ctrl; **P<0.01 oEVs vs nEVs; for % area, ***P<0.001 pos ctrl and nEVs vs neg ctrl, oEVs vs nEVs; for no. of branches, ***P<0.001 pos ctrl vs neg ctrl, **P<0.01 nEVs vs neg ctrl, ***P<0.001 oEVs vs nEVs). Scale bars indicate 50 μm. (e) Migration assays performed on ECs treated either without (none) or with nEVs or oEVs. The results are representative of four different experiments performed in triplicate (n=4) (**P<0.01, nEVs vs none, *P<0.05 oEVs vs nEVs).