Axitinib

HUVECs were plated at a density of 2 × 10³ cells/well in 96-well plates and cultured for 24 h in 200 μl/well of the EGM. The cells were then washed twice with HBSS and serum-starved for 18 h in the EBM supplemented with 0.1% FBS. The serum starvation medium was replaced with equal volumes of EGM, control medium, CM, CM containing anti-VEGF neutralizing antibody (10 μg/ml) or IgG isotype control (10 μg/ml), SFM or Axitinib (0.25 nM, 0.29 nM and 1.2 nM) (Cayman Chemical, MI, USA). HUVECs were allowed to proliferate for 72 h. Then 5-bromo-2′-deoxyuridine (100 μM) was added (1:1000 in SFM) at the 48th hour, and its uptake by the cells was measured using a colorimetric cell proliferation ELISA kit (Merck-Millipore, NJ, USA).
To determine the effect of CM on VEGFR1, VEGFR2 and VEGFR3 gene expression, HUVECs were plated at a density of 5 × 10³ cells/cm² in six-well dishes (BD Bioscience) in EGM. The following day, HUVECs were serum starved for 18 h in EBM supplemented with 0.1% FBS. CM from two batches of phBMMSCs were incubated alone or with anti-VEGF antibody (10 μg/ml) on HUVECs, and with EGM, which served as control. HUVECs were allowed to proliferate for 72 h. The cells were then harvested and processed further for RT-PCR analysis.

LDL, HDL, and VLDL were obtained from Lee Biosolutions (Maryland Heights, MO, USA). Cabozantinib, sunitinib, axitinib, pazopanib, U18666A, and posaconazole were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Antibody against AR was obtained from Cell Signaling Technology (Danvers, MA, USA).