*2.2. Cell Culture*

A rat VSMC cell line A7r5 was purchased from the Bioresource Collection and Research Center. A7r5 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin mixed antibiotics, 1% glutamine, and 1.5 g/<sup>L</sup> sodium bicarbonate (all regents from Hyclone, Logan, Utah, USA). All cell cultures were maintained at 37 ◦C under 95% moisturized air with 5% CO2. Before cell treatments, A7r5 cells were seeded onto each 60 mm Petri dish (Corning Inc, Corning, NY, USA) at a density of 10<sup>5</sup> for 24 h. For induction of VSMC dysfunction, A7r5 cells at 70% confluence were serum-starved for 24 h and treated with TNF-α (10 ng/mL; Sigma-Aldrich, St Louis, MO, USA) for 24 h.
