For collection of conditioned media (CM), cells were grown 6 h in FCS-free DMEM then fresh medium (3 ml/T-25 flask) was added for 12 h before to be collected. The quantitative determinations of human VEGF concentrations in CM were made by enzyme-linked immunosorbent assays (ELISAs; Quantikine; R&D Systems), according to the manufacturer's instructions.