Heparin

Human umbilical vein endothelial cells (HUVEC) were obtained from Kurabo Industries Ltd. and maintained in HuMedia-EG2 medium (HuMedia-EB2 supplemented with 2% FBS, 10 ng/mL hEGF, 1.34 μg/ mL hydrocortisone, 5 ng/mL hFGF-B, 10 μg/mL heparin, Kurabo) at 37 ℃ in humidified atmosphere with 5% CO2. HUVEC were seeded in plates and allowed to attach for 1 day. Next, HUVEC were exposed to PA, LPS, EPA, triacsin C (ACSL inhibitor), hypoestoxide (IκB kinase inhibitor), or myriocin [serine palmitoyltransferase long chain base subunit 1 (SPTLC1) inhibitor] in HuMedia-EG2 medium for 1 day. After treatment, HUVEC were harvested for quantitative real-time polymerase chain reaction (PCR) and western blotting, and conditioned medium was harvested for enzyme-linked immunosorbent assay (ELISA).

Recombinant human IL-17A homodimer, human IL-17A/F heterodimer, human basic fibroblast growth factor (bFGF), mouse antihuman IL-17RA monoclonal antibody (mAb) and goat anti-human IL-17RC polyclonal antibody (Ab) were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human vascular endothelial growth factor (VEGF) and Suramin were purchased from KURABO (Osaka, Japan). Specific chemical inhibitors LY294002, PD98059 and SP600125 were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary HMVECs were purchased from KURABO (Osaka, Japan), and maintained in HuMedia-EB2 with 10 ng/mL epidermal growth factor (EGF), 1 μg/ mL hydrocortisone, 5 ng/mL bFGF, 10 μg/mL heparin, 39.3 μg/mL dbcAMP, 50 μg/mL gentamycin, 50 ng/mL amphotericin-B and 5% fetal calf serum (FCS) (all from KURABO, Osaka, Japan). Cells were grown at 37°C in an atmosphere of 5% CO 2 and used between passages 4 and 5. Primary human dermal fibroblasts were purchased from Takara Bio (Kusatsu, Shiga, Japan), and maintained in Fibroblast Basal Medium supplemented with 1 ng/mL bFGF, 5 μg/mL Insulin and 2% FCS (Takara Bio).