Isolation and Culture of Vascular Smooth Muscle Cells

Vascular smooth muscle (VSM) cells were enzymatically isolated and cultured as previously described^{46 (link),47 (link)}. Briefly, F344XBN rat thoracic aortas were rinsed in Hanks balanced salt solution (HBSS) containing 50 μg/mL penicillin, 50 μg/mL streptomycin and 0.25 μg/mL amphotericin B (Gibco). After digestion for 30 min with 2 mg/mL collagenase I solution (Worthington Biomedical, Freehold, New Jersey) at 37 °C, the adventitia and intima layers were removed, and the vessel media was further digested with 2 mg/mL collagenase II/0.5 mg/mL elastase (Sigma) for 1 h at 37 °C. The isolated cells were washed and plated in complete medium. In all cases, >95% of cells stained positive for α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chains (SM1 and SM2) (Suppl. Fig. 1b,c). Cells were maintained with VSMC medium including: DMEM (Gibco), 10% FBS (Sigma), 1% NEAA and 1% penicillin/streptomycin (Gibco). For all studies, we used early passage cells (passages 1–3).