Baecs

Bovine aortic endothelial cells (BAECs) were obtained from the European collection (ECACC; Sigma-Aldrich, Spain). BAECs were cultured in complete medium (Dulbecco’s modified Eagle’s medium [DMEM] with 10% fetal bovine serum [FBS], L-glutamine [2·10⁻³M], penicillin [100 U/mL], and streptomycin [100 μ/mL] from LONZA, USA) and maintained at 37 °C in a humidified atmosphere consisting of 5% CO₂ and 95% air. All the experiments were performed in confluent cells (90%), between passages 3 and 6 and cultured in DMEM and 0.1% of bovine serum albumin (BSA), thus being deprived of serum for 20 h. Under these conditions, cells were treated with insulin (5·10⁻⁷M) for 10 min, with EAE (40 μg/mL) or MA (5·10⁻⁴M) for 15 min, or nothing as a control. After these incubations, cells were briefly washed with ice-cold PBS and were then scraped in lysis buffer containing 0.42 mM NaCl, 1 mM Na₄P₂O₇, 1 mM dithiothreitol, 20 mM HEPES, 20 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 2 mM phenylmethylsulfonyl fluoride, 1 µl/ml leupeptin, 1 µl/ml aprotinin, and 0.5 µl/ml Tosyl-L-lysyl-chloromethane hydrochloride. Cells lysates were centrifuged at 17.000xG for 20 min at 4 °C and the supernatants were used for Western blot studies.

Human mesenchymal stem cells (HMSCs) and bovine aortic endothelial cells (BAECs) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). HMSCs and BAECs were cultured in human mesenchymal stem cell growth medium (MSCGM, PT-3001, Lonza Walkersville, Inc., Walkersville, MD) and in Dulbecco's modified Eagle's medium (DMEM), respectively, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were cultured in a humidified incubator of 95% O₂ and 5% CO₂ at 37°C. The DNA plasmids were transfected into the cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the product instructions.