In vitro proangiogenic effects of EPC-derived MVs. (A) HMECs (■) or HUVECs (□) (5 × 10⁴ cells/well) were plated on growth factor–reduced Matrigel in DMEM + 0.25% BSA and were challenged for 6 hours at 37°C with vehicle alone (M199 medium) as control or with 10 μg protein EPC-derived MVs/mL, or RNase- or DNase (1 U/mL)–treated MVs, or 10 ng/mL VEGF or 10 ng/mL VEGF in the presence of RNase-treated MVs. In selected experiments, HMECs were stimulated with 10 μg protein MSC-derived MVs/mL. Data show the mean (± 1 SD) of total length of capillary-like structures detected by Nikon Eclipse TE 200 inverted microscope (objective, 10×/0.25; Tokyo, Japan), analyzed by the Micro-Image system (Casti Imaging) and expressed as arbitrary units by the computer analysis system in 5 different files at × 100 magnification in duplicated wells of 4 different experiments. Analysis of variance with Newmann-Keuls multicomparison test was performed; §P < .05 different treatments versus unstimulated control; *P < .05 RNase MVs and MSC-derived MVs versus EPC-derived MVs. (B) Representative micrographs of capillary-like structure formation on Matrigel by HMECs unstimulated (Control) and stimulated with either 10 μg/mL MVs or with RNase(1 U/mL)–treated MVs (scale bar, 20 μm). (C) Representative FACS analysis of ανβ3 and α5 integrin expression by HMECs unstimulated (gray filled curves) or stimulated with 10 μg/mL MVs (open curves, dark lines). Dotted lines indicate the isotypic controls. Two MV preparations were analyzed with similar results. In each experiment the Kolmogorov-Smirnov statistical analysis between relevant antibodies and the isotypic control was significant (P < .001). (D) Effect of 10 μg/mL blocking antibodies anti-ανβ3 and anti-α5 integrins on capillary-like structure formation by HMECs cultured on Matrigel added 30 minutes after stimulation with 10 μg/mL MVs. Analysis of variance with Newmann-Keuls multicomparison test was performed; *P < .05 MV plus blocking antibodies versus MVs alone.