Human aortic endothelial cells haecs

LDL was isolated from human plasma obtained from a local blood bank—Lifesource (now Vitalant, Chicago, IL), as described in our previous studies (7 (link)). Briefly, LDL was isolated by sequential centrifugation in potassium bromide (KBr, Acros Organics; Thermo Fisher Scientific, Waltham, MA), and dialyzed to remove KBr and EDTA. Oxidized LDL (oxLDL) was prepared by Cu²⁺ as previously described (8 (link)). The degree of oxidation was assessed by the TBARS (thiobarbituric acid reactive substances) content of LDL and was measured using a TBARS Assay Kit (ZeptoMetrix, Buffalo, NY) as expressed with malondialdehyde (MDA) equivalent. Experiments were performed with Human Aortic Endothelial cells (HAECs; Lonza, Allendale, NJ), grown in standard conditions. Serum-starved cells were treated with either 10 μg/ml oxLDL, 50 mg/dl LDL, or 250 mg/dl LDL for 24 h, then lysed in methanol and stored at −80°C.

Human umbilical vein endothelial cells (HUVECs) and human acute monocytic leukemia (THP-1) cells were purchased from American Type Culture Collection. Human aortic endothelial cells (HAECs) were purchased from Lonza. HUVECs and HAECs were grown in Lonza’s EGM-2-MV medium on collagen-coated (20 μg/ml) tissue culture dishes. HUVECs and HAECs from fewer than 4 generations were used for all experiments. THP-1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS). A single-donor human peripheral blood buffy coat was purchased from the Gulf Coast Regional Blood Center (Houston, TX, USA) and used for isolation of peripheral blood monocytes.