Pcs 100 012tm

RVSMCs were isolated from de-endothelialized thoracic aortas of 12-week-old male Sprague–Dawley rats (Charles River Labs, Wilmington, MA, USA) by enzymatic digestion after removal of endothelial layers, as described [23 (link)]. Cells were cultured in M199 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin antibiotics and 2.5 µg/mL plasmocin. Human VSMCs (HVSMCs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) (PCS-100-012^TM) and cultured in M231 medium with smooth muscle growth supplement (Gibco, Waltham, MA, USA). For all the experiments, RVSMCs and HVSMCs between passages 3 and 6 were used. For in vitro experiments, the complete medium was replaced with serum-free medium containing 0.2% BSA for 24 h prior to stimulation with AngII (100 nM, Bachem, Torrance, CA, USA), platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL), or tumor necrosis factor-alpha TNF-α (50 ng/mL, 210-TA, R&D Systems, Minneapolis, MN, USA).

For most in vitro experiments involving ECs, human umbilical vein ECs, i.e., HUVECs (Cell Applications Inc., San Diego, CA, Catalog # 200p-05n and lot # 3,363 and 2,914) between passages 5–8 pooled from multiple donors were used after testing negative for mycoplasma contamination. The cells were cultured in M199 (Sigma M2520) supplemented with 15% FBS (Hyclone, SH30910.02), β-endothelial cell growth factor (Sigma, E1388), and 100 units/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific). Human VSMC (HVSMC) were purchased from ATCC (Manassas, VA, USA) (PCS-100-012TM) and cultured in M231 medium with smooth muscle growth supplement (Gibco, Waltham, MA, USA) as previously described (31 (link)). All the cells were maintained at 37°C with 5% CO₂ in a hydrated incubator.