Angiogenic Potential of HMVECs under VEGF Stimulation

HMVECs were grown to 80% confluency (1 × 10⁶ cells/ml) in 10-cm dishes. Cells were then transduced as described previously (m.o.i. = 20). The following day, transduced cells were serum-deprived by replacing complete growth medium with EBM-2MV for 12 h. Next, cells were resuspended in either EBM-2MV medium supplemented with 0.4% FBS or EBM-2MV medium supplemented with 0.4% FBS containing 20 ng/ml VEGF. For VEGF signaling inhibition, cells were preincubated with SU1498 (25 μm) for 2 h in EMB-2MV before seeding. Cells were seeded at a density of 2 × 10⁴ cells/, BD Biosciences). The plates were incubated for 18 h in a 37 °C incubator with 5% CO₂. After incubation, cells were stained with CalceinAM, and images were captured using fluorescent microscopy (Olympus). Tube-like network structures were analyzed using AngioTool analysis software (25 (link), 26 (link)). All treatments were performed in triplicate and repeated at least five times.