Ecgs h

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (BioWhittaker, CC‐2519) and cultured on 0.5% gelatin‐coated plates (unless otherwise indicated) in medium 199 supplemented with 20% FBS, 100 U/ml penicillin (Lonza), 100 μg/ml streptomycin (Lonza), 2 mM l‐glutamine (Lonza), 10 mM HEPES (Lonza), and ECGS/H (Promocell, C‐30120). Cells were used up to passage 5. Mouse aortic endothelial cells (MAEC) were obtained from aortas of 4‐week‐old mice. Briefly, after fat removal under a microscope, aortas were incubated for 5 min at 37°C in collagenase solution (collagenase type I, 3.33 mg/ml, Worthington, LS004194), thus allowing removal of the adventitia with forceps. The aortas were then cut into small pieces (1–2 mm), and a cell suspension was obtained by incubation for 45 min at 37°C in 6 mg/ml type I collagenase and 2.5 mg/ml elastase (Worthington, LS002290) diluted in DMEM. Once EC colonies were visible, they were subjected to two rounds of positive selection with anti‐ICAM2 and magnetic beads. MAEC were cultured on 0.2% gelatin‐coated plates in DMEM/F12 medium supplemented with 20% FBS, 100 U/ml penicillin (Lonza), 100 μg/ml streptomycin (Lonza), 2 mM l‐glutamine (Lonza), 10 mM HEPES (Lonza), and ECGS/H (Promocell, C‐30120).

Human umbilical vein endothelial cells (HUVECs), murine-derived RAW 264.7 macrophages (RAW) and mouse osteogenic precursor MC3T3-E1 cells were purchased from the American Type Culture Collection (ATCC). HUVECs were cultured with a mixture of endothelial cell medium (ECM, ScienCell, United States), 5% foetal bovine serum (FBS), 1% endothelial cell growth supplement/heparin (ECGS/H, Promocell) and 1% penicillin/streptomycin. RAW cells and MC3T3-E1 cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM, HyClone, United States) containing 5% FBS and 1% penicillin/streptomycin. The cells were cultured at 37°C in an atmosphere of 5% CO₂.