Cell proliferation assay

Cell proliferation was analyzed using the MTT assay. VSMCs (1×10⁴/well) were seeded in a 96-well microplate and were cultured with 200 µl DMEM supplemented with 10% FBS. Once the cells had reached 60% confluence they were serum-starved for 16 h at 37°C in a humidified chamber containing 5% CO₂. VSMCs were then treated with 100 nM Ang II or 100 nM Ang II + LWDHF (3, 6 and 12 µg/ml) for 24 h; cells were incubated with MTT (5 mg/ml) for the last 4 h at 37°C in a humidified chamber containing 5% CO₂ and then dissolved into 150 µl DMSO. Untreated cells served as the control group. MTT was dissolved in PBS at a concentration of 5 mg/ml. Subsequently, optical density was measured at 490 nm using a Universal Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). All experiments were performed in triplicate.