2.4 Cell culture
VSMCs were prepared from thoracic aorta of 50-day-old female Sprague-Dawley rats. Aortic VSMCs were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) with 10% fetal bovine serum (BI, USA) and 1% penicillin/streptomycin at 37 °C in a 5% CO2humidified incubator. Immunohistochemical staining revealed positivity for smooth muscle α-actin (Wanleibio, China) and negativity for CD105 (Wanleibio, China), indicating that primary VSMCs of rats were successfully isolated. Cells in passages 3-9 were used for all experiments.