To investigate whether the PF exosomes are functionally active, we tested them on cultured ECs focusing on responses that are conducive to angiogenesis. Patient-derived plasma exosomes were used for comparison. PF or plasma exosomes clusters, labeled with a green fluorescent marker, were added to cultured ECs, and their internalization was confirmed by confocal microscopy 3D and reconstruction of the confocal image z stacks (Figures 5A and S6A). Two negative controls were included to confirm the specificity of the exosome uptake derived from the PF (Figure S4). Next, exosomes were studied in cell biology assays developed on cells kept under hypoxia. In comparison to the PBS control, PF-derived exosomes inhibited EC apoptosis (Figure 5B) and increased EC proliferation (Figure 5C). Moreover, the PF exosomes, in comparison to PBS control and PF-derived exosomes, were also able to promote the formation of capillary-like cellular networks on Matrigel (Figures 5D, 5E, and S5), while the exosome-depleted PF fraction had no effect. Plasma exosomes did not affect any of the tested EC functions (Figures S6B–S6E). Exosome concentration-response curves confirmed the inactivity of plasma exosomes on ECs (Figures S6F and S6G) in the concentration range whereas PF exosomes increased EC survival and proliferation (Figures S6H and S6I). We speculate that the differential behavior of exosomes from PF and plasma might be in part related to differences in their miRNA cargos.Figure 5PF-Derived Exosomes Are Incorporated by Cultured ECs and Enhance Their Angiogenic Capacity(A) PF-derived exosomes (10 μg/ml) stained using carboxyfluorescein succinimidyl ester (CFSE, in green) were incubated with ECs for 24 hr. Next, cells were stained with phalloidin (red) and DAPI (blue) (the scale bar represents 25 μm). (B and C) Column graphs show (B) EC apoptosis (measured by caspase-3 activity assay) and (C) EC proliferation (measured by BrdU incorporation) after treatment with 10 μg/ml of either PF- exosomes (black columns) or exosome-depleted PF (gray columns). PBS was used as an additional control (open columns); n = 7. (D) Photomicrographs show endothelial network formation on Matrigel (the scale bar represents 200 μm); 2.5× magnification. (E) Bar graphs show Matrigel assay quantification (total length of EC tube-like structures). All values are mean + SEM. *p ≤ 0.05 and ***p ≤ 0.001 versus PBS, # # #p ≤ 0.001 versus PF exosome-free (one-way ANOVA, Dunnett’s post hoc test); n = 6.