C 22062

The primary HCASMCs (ATCC® PCS-100-021™, American Type Culture Collection, Manassas, VA, USA) were cultured in smooth muscle cell growth medium 2 (#C-22062, PromoCell GmbH, Heidelberg, Germany), and all patient monocyte-derived macrophage cells were cultured in the RPMI-1640 culture media (Sigma-Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 μg/mL streptomycin, and 50 U/mL penicillin in 5% CO₂ humidified atmosphere incubator at 37°C to 98%-100% confluence. Cells were subcultured and culture media changed every 48 h. Human CRP (#C1617, Sigma-Aldrich Corporation, St. Louis, MO, USA), human IL-6 (#407652, purity ≥ 95% by SDA-PAGE, Sigma-Aldrich Corporation, St. Louis, MO, USA), human low-density lipoprotein (LDL) (#LP2, purity ≥ 95% by SDA-PAGE, Sigma-Aldrich Corporation, St. Louis, MO, USA), methyllycaconitine (MLA), a selective and potent antagonist of the α7-nAChR, (351344-10-0, caymanchem, USA), and anti-IL-6 receptor antibody (tocilizumab) were also obtained from Sigma-Aldrich. Stock solutions of tocilizumab were prepared at a concentration of 10 mM in double-distilled water (ddH₂O) and stored at -20 °C until use. Methylergonovine (Methergine®) was obtained from Novartis (Novartis Pharmaceuticals Corp., Basel, Switzerland) and nitroglycerin from G. Pohl-Boskamp (Millisrol®; G. Pohl-Boskamp, Hohenlockstedt, Germany).

Primary mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) and AOX-transgenic (AOX^Rosa26) [7 (link)] embryos (E13.5–15.5) in the C57BL/6 background and immortalized (generating iMEFs) as described elsewhere [35 (link), 36 (link)]. MEFs, iMEFs, and human lung carcinoma (A549) cells (86012804, Sigma-Aldrich/Merck Life Science) were grown in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 4.5 g/l glucose (BE12-614F, Lonza/BioNordika), 10% fetal bovine serum (FBS, 10270106, Gibco/ThermoFisher Scientific), 100 U/ml penicillin plus 100 μg/ml streptomycin (P0781, Sigma-Aldrich/Merck Life Science), and 2 mM L-glutamine (BE17-605E, Lonza/BioNordika). Mouse pulmonary artery smooth muscle cells (PASMCs) were isolated from precapillary pulmonary arterial vessels of WT C57Bl/6 mice as previously described [37 (link)]. PASMCs were cultured in Smooth Muscle Cell Growth Medium (C-22062, PromoCell) supplemented with 10% FCS (F7524, Sigma-Aldrich/Merck Life Science) and 0.002% Normocin (ant-nr-1, InvivoGen).

HAECs (CC-2535) from a 54-year-old male and HASMCs (CC-2571) from a 22-year-old male were purchased from Lonza. HAECs and HASMCs were cultured in EC growth media-2 (CC-3202, Lonza) and SMC growth medium-2 (C-22062, Promo Cell), respectively, at 37°C, 5% CO₂, in a humidified cell culture incubator. Both HAECs and HASMCs were used from passages 4 to 8 in all experiments. The human monocyte cell line THP-1 was purchased from ATCC and grown in RPMI 1640 containing 10% FBS (Thermo Fisher Scientific) and 50 mg/mL of a penicillin/streptomycin solution.

Human coronary smooth muscle cells from a young healthy donor were purchased from Promocell (C-12511) and cultured in growth medium (Promocell, C-22062), supplemented with 10% FBS and 1% penicillin-streptomicin (Sigma Aldrich). To achieve RS, cells were serially passaged (Bielak-Zmijewska et al., 2014 (link)). Cells were cultured until 70–80% confluence and re-seeded in T75 Flasks (Thermo Fisher Scientific, Nunc EasYFlask, #156499) at a density of 3.5 × 10³ cells/cm² until proliferative arrest (passage 6–8). Cumulative population doublings were counted using the formula cPD = X + 3.322^∗(log Y−log I), X representing the cumulative population doubling of the subculture used to initiate the culture, Y being the viable cell number on harvest, and I the cell number on inoculation. Low passage VSMCs (passage 2–3) were treated with bleomycin to induce DS, as previously reported (Gardner et al., 2015 (link)).