SIRT1 Regulation of Endothelial Cells

Endothelial cells were freshly isolated from human umbilical cord veins as previously described [13] (link) and cultured in M200 medium. Cells between the third and the sixth passages were grown in monolayers in a humidified atmosphere of 5% CO₂ at 37°C, and used for experiments at >80% confluence. Replication-defective adenoviral vectors expressing SIRT1 (Ad-SIRT1) or control green fluorescentprotein (Ad-GFP) were prepared using the AdEasy vector kit (Quantum Biotechnologies) in according to the manufacturer's instructions. The adenovirus-mediated knockdown of SIRT1 (Ad-SIRT1 RNAi) and control vector (Ad-U6) were generated using the same system. The SIRT1 RNAi sequence was reported previously [21] (link). HUVECs were infected with the above adenovirus for 24 h and were cultured in fresh M200 for further treatment. PMA, Ionomycin, cyclosporin A, resveratrol, sirtinol, NFAT inhibitor, and NF-κB inhibitor JSH-23 were purchased from Sigma-Aldrich.