Figure 4.

Extracellular vesicle-produced VEGF-A is pro-angiogenic. (a) Endothelial cells were seeded for permeability assays (10⁵ cells, 4 days), treated for 6 h with semaxitinib (semax.; 5 µM) and sunitinib (100 nM) in serum-free media. The passage of FITC-dextran permeability was assessed in response to recombinant VEGF-A (50 ng/ml) and GSC#4 and GSC#9-prepared EVs (15 µg/ml), as previously described. **, p < 0.01. ***, p < 0.001. (b) GSC#4 were transfected with either non-silencing RNA (ns) or VEGF-A-targeting duplexes and knockdown efficiency was assessed by RT-PCR as compared to ACTB. Results were normalized to the expression levels of the ns condition, represented as the dashed red line. Relative expression levels of other angiogenic factors (VEGF-B, TGFβ, angiogenin (ANG) and FGF2) were also quantified (n = 3). ***, p < 0.001. (c) EVs were isolated from the conditioned media of GSC#4 transfected cells, and VEGF-A levels were measured by ELISA (n = 2). *, p < 0.05. (d) A confluent monolayer of ECs was exposed to control EV#1 (ns) or VEGF-A-depleted EVs (si#1) and permeability was assessed as described previously (n = 2). ***, p < 0.001. (e) Tubulogenesis and (f) sprouting were performed in ECs treated either with control (ns) or VEGF-A-depleted GSC#1-EVs. Representative pictures of EC-coated beads stained with phalloidin (actin, green) and DAPI (nucleus, blue) (n = 3). **, p < 0.01; ***, p < 0.001. Serum-free mitogen-free medium and VEGF-A (50 ng/ml) were used as negative and positive controls, respectively.