Hasmcs

HAECs and HASMCs were purchased from PromoCell (UK) and maintained at 37°C in a humidified incubator supplemented with 5% CO_2. Cells were cultured in endothelial cell growth media and smooth muscle cell growth media, respectively, containing 1% penicillin–streptomycin (Sigma‐Aldrich, UK) and supplemental mix (PromoCell, UK). In all experiments, cells were used between passages 3 and 5. After experimental treatments, cell media was collected and cells were washed once with phosphate‐buffered saline (PBS; pH 7.4; Gibco™). Radioimmunoprecipitation buffer (Sigma‐Aldrich) with protease and phosphatase inhibitors (A32959; Thermo Fisher Scientific) was added to lyse the cells and the plates were shaken at 4°C for an hour. The cells were then collected and centrifuged at 14,000g for 5 min at 4°C and cell supernatants were frozen at −80°C.

Primary cultures of HA-SMCs (PromoCell) were maintained in SMC growth medium 2 containing 5% foetal calf serum (FCS), 0.5 ng/ml epidermal growth factor, 2.0 ng/ml basic fibroblast growth factor and 5μg/ml insulin (PromoCell). The cells were grown in 5% CO2 at 37°C in medium renewed every 3 days. Confluent cells were detached by trypsin/EDTA and subcultured with a split ratio 1:2. HAoSMC were used between 2 and 5 passages.

The HASMCs were purchased from PromoCell, and the HASMCs were cultured in a smooth muscle cell growth medium (Sigma) and maintained in a humidified incubator supplied with 5% CO₂ at 37°C.