Pooled human umbilical vein ECs (HUVECs) were purchased from Cell Systems/Clonetics and cultured refer to previous work [38 (link)–41 (link)]. In brief, HUVEC were cultured in endothelial basal medium supplemented with hydrocortisone (1 mg/ml), bovine brain extract (3 mg/ml), gentamicin (50 mg/ml), amphotericin B (50 mg/ml), epidermal growth factor (10 mg/ml), and 10% fetal calf serum until the third passage. All HUVECs were divided into 2 groups, cells in group 1 were incubated with recombinant human soluble CD40 ligand (rh-CD40L, 0.5ug/ml, Abcam) and cells in group 2 were not. After detachment with trypsin, cells were grown for at least 18 h. Confluent monolayers of HUVEC were grown onto 6-cm wells and exposed to laminar fluid flow in a cone-and-plate apparatus as previously described. A constant shear stress of 15 dynes/cm2 was used in all experiments to simulate physiological shear stress.
In vitro scratched wounds were created by scraping the cell monolayer with a sterile disposable cell scraper as previously described [41 (link)–43 (link)]. To detect competence of HUVECs migration, scratch area 24 hours after injury were observed with a computer-assisted microscope (Zeiss) at 3 distinct positions (every 5 mm).
In vitro scratched wounds were created by scraping the cell monolayer with a sterile disposable cell scraper as previously described [41 (link)–43 (link)]. To detect competence of HUVECs migration, scratch area 24 hours after injury were observed with a computer-assisted microscope (Zeiss) at 3 distinct positions (every 5 mm).