Vegf a

For analyzing of the antagonistic response to VEGF, tube formation, scratch wound migration, and cell proliferation assays were performed after the exposure of HUVECs to 50 ng/mL VEGF-A (R&D Systems, Minneapolis, MN), which induced a significant angiogenic response. For the cell proliferation assays, cells were incubated overnight in endothelial basal media (EBM; Lonza, Walkersville, MD, USA) containing 0.5% FBS or supplemented with VEGF-A and/or VEGF inhibitors. The cells were washed with PBS and counted in 4 random microscope fields. The tube formation assay was performed by seeding cells on Matrigel-coated plates (BD Bioscience, Bedford, MA, USA) and incubating in EBM containing 0.5% FBS or supplemented with VEGF-A and/or VEGF inhibitors. After overnight incubation, tubule networks were quantified by measuring the tubule length in 4 random microscope fields. For the analysis of scratch wound migration, confluent cell monolayers grown on 6-well plates were scratched using a micropipette tip. After the plates were washed with PBS to remove dislodged cells and media, they were incubated with EBM containing 0.5% FBS or supplemented with VEGF and/or VEGF inhibitors for 8 hours. Cell migration was observed by optical microscopy and quantified by measuring the number of cells that had migrated from the wound edges.

The capillary sprouting assay was performed as previously described previously (Schulz et al., 2012 (link)). HDLECs were incubated in a 15 ml conical tube in presence of cytodex 3 beads (Sigma-Aldrich) at a ratio of 400 cells per bead in EGM-2MV medium (Lonza) for 4 h at 37°C with shaking every 20 min. Beads were then incubated in a cell flask at 37°C for 48 h in EGM-2MV medium. The HDLEC-coated beads were subsequently collected and labelled with 2 μM Cell Tracker Green dye (Invitrogen) for 30 min, embedded in 1 mg/ml Type I collagen hydrogel (Advanced Bio-Matrix) containing 2 μM D-erythro-sphingosine-1-phosphate (Avanti Polar Lipids), and cultured in black clear-bottom 96-well plates (Perkin-Elmer) in the presence or absence of 10,000 human iPS-derived macrophages-RFP per well. After 60 min of incubation, a 1:1 mix containing EGM2-Incomplete (EGM2 media without supplementation with EGF, FGF2 and VEGF-A; Lonza) and macrophage media (v/v) was added and the beads incubated for 2 days at 37°C. The beads were then fixed with 4% PFA for 30 min and washed twice with PBS. Each well was imaged with a Zeiss LSM 780 scanning confocal microscope and the number of sprouts per bead calculated.