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

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Copyright © 2016 Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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EVs recovered from the subcutaneous tissue of obese patients are impaired in their pro-angiogenic potential as well. (a) Representative Oct4 content obtained from cell extracts of subcutaneous-derived ASCs (snASCs, n=6; soASCs, n=10). Protein levels were normalized to tubulin content. pos: positive control, human ASCs purchased from Lonza; neg: negative control, adipocytes derived from the NIH 3T3L1 cell line) (***P<0.001 neg vs pos control). (b) Representative images of NanoSight analyses performed on the 100 k fraction of subcutaneous ASC-derived EVs. Curve 1, relationship between particle distribution (left y axis) and particle size (x axis); curve 2, correlation between cumulative percentage distribution of particles (percentile in right y axis) and particle size (x axis). (c) Number of EV particles (mean±s.e.m.) per cell at isolation. Data refer to EVs from subcutaneous adipose tissue samples recovered from obese subjects (soEVs, n=10) and non-obese subjects (snEVs, n=6). (d) Representative photomicrographs of in vitro angiogenesis assays, showing tube-like structure formation by ECs treated with snEVs or soEVs. 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 (n=4) performed in triplicate (for length, ***P<0.001 soEVs vs snEVs; for % area, ***P<0.001 soEVs vs snEVs; for no. of branches, ***P<0.001 soEVs vs snEVs). Scale bars indicate 50 μm. (e) Migration assays performed on ECs untreated or treated with snEVs or soEVs. The results are representative of four different experiments (n=4) performed in triplicate (***P<0.001 snEVs vs none; ***P<0.001 soEVs vs snEVs). (f) miR-126 expression was evaluated by quantitative real-time PCR on snEVs and soEVs. Data normalized to RNU6B are representative of all samples (snEVs, n=6; soEVs, n=10) (***P<0.001 soEVs vs snEVs). (g) VEGF and MMP-2 content was evaluated in snEVs and soEVs. Protein levels were normalized to CD63 content. (**P<0.01 soEVs, n=10, vs snEVs, n=6).