Ox papc

Human aortic endothelial cells (HAECs) were purchased from Lonza (Basel, Switzerland) and cultured according to the manufacturer’s instructions. All experiments were performed using HAECs between the 2^th and the 5^th passage. HAECs were treated with: LPS (Sigma Aldrich, St. Louis, MO) at a concentration of 100 ng/mL for 6 hours; ox-PAPC (oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine, Hycult Biotech, Uden, The Netherlands), an antigenic epitope of oxidized LDL, at a concentration of 100 µg/mL for 6 hours; or long chain free fatty acids (FFA, oleic acid 500 µM+palmitic acid 500 µM) for 24 hours. Cells were incubated with 10 nM Eritoran for 30 minutes prior to treatments where indicated. Eritoran (Eisai Inc., Woodcliff Lake, NJ) is a synthetic analog of lipid A and a potent and specific antagonist of LPS action, which inhibits lipid A binding to MD2 and terminates MD2/TLR4-mediated signaling [17] (link). At the end of treatments, HAECs where collected and used for molecular analysis.

OxPAPC, PAPC and DMPC were purchased from Hycult Biotech (Plymouth Meeting, PA, USA); Complete Freund’s adjuvant (CFA) was purchased from Rockland Immunochemicals (Gilbertsville, PA, USA); Ionomycin was from Invitrogen (Grand Island, NY, USA). AH6809 and PF04418948 were obtained from Tocris (Minneapolis, MN, USA). Capsaicin, mustard oil and other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA)