Fig. 6
Selective localization of VEGF189 in sEVs is mediated by heparin-binding and increases ligand stability. a VEGF levels in conditioned media and sEVs of ES2 VEGF−/− and HCT116 VEGF−/− cells transfected with VEGF121, VEGF165, or VEGF189. TSG101 was assayed in sEVs as a control. Mean ± SD of n = 3 independent experiments are shown. b, c VEGF levels in whole and sEV-depleted conditioned media of VEGF−/− cells transfected with VEGF189 (b) and VEGF121 (c). Mean ± SD of n = 3 independent experiments are shown. d Conditioned media of non-transfected VEGF−/− cells was incubated with addition of recombinant VEGF189 (rVEGF189) at a concentration equivalent to the VEGF concentration in conditioned media of VEGF189-transfected VEGF−/− cells (2500 pg/mL for ES2; 1500 pg/mL for HCT116; see data in a). Thereafter, sEVs were isolated. Amounts of VEGF189 detected in these sEVs were compared with VEGF content in sEVs secreted by VEGF189-transfected VEGF−/− cells. Mean ± SD of n = 3 independent experiments are shown. e Levels of human VEGF189 in conditioned media and sEVs of CHO-K1 and pgsD-677 cells transfected with human VEGF189. Mean ± SD of n = 3 independent experiments are shown. f, g PKH67-labeled sEVs of parental ES2 cells were pretreated with heparinase, chondroitinase, or no enzyme, and then incubated with VEGF Ab coupled to microbeads. VEGF on sEVs was detected by flow cytometric analysis of PKH67 fluorescence in Ab-coupled microbeads (solid histograms). Dotted histograms show background fluorescence when sEVs were incubated with control Ig-coupled beads. As a negative control for enzymatic digestion, the same approach was used to detect the transmembrane protein CD63. In f, representative histogram plots. In g, MFI values of n = 3 independent experiments (mean ± SD). Contour plots are shown in Supplementary Fig. 12. h rVEGF189 and sEVs with an equivalent content of VEGF were added to healthy donor plasma. Following incubation at 37 °C for the indicated times, VEGF levels in plasma were assayed. Shown are mean of n = 2 independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Bonferroni’s corrections in a and g, by two-sided unpaired t-test in b–e. Source data used for graphs in a–e, g, and h can be found in Supplementary Data 5
