Hypoxia-Induced Endothelial Cell Responses

HCMECs were purchased from Sciencell Research Laboratories and maintained in EC medium (Sciencell, California, U.S.A.) as described recently [27 (link)]. A density of 10000 cells/cm² of passages 2–6 were seeded in a six-well plate. Cells were divided into five groups including – (i) control group: HCMECs were cultured under normoxia conditions; (ii) hypoxia group: HCMECs were cultured under hypoxia conditions (1% O₂) for 3 days; (iii) hypoxia + rapamycin group: HCMECs were pretreated with rapamycin (100 nM, Sigma–Aldrich, St. Louis, MO, U.S.A.) for 2 h and cultured under hypoxia conditions (1% O₂) for 3 days; (iv) hypoxia + 3-methyladenine (3-MA) group: HCMECs were pretreated with 3-MA (5 mM, Sigma–Aldrich, St. Louis, MO, U.S.A.) for 2 h and cultured under hypoxia conditions (1% O₂) for 3 days; and (v) hypoxia + chloroquine (CQ) group: HCMECs were pretreated with CQ (20 μM, Sigma–Aldrich, St. Louis, MO, U.S.A.) for 2 h and cultured under hypoxia conditions (1% O₂) for 3 days.