Human crp

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).

ELISA plates (Nunc MaxiSorp ™, ThermoFisher) were coated with human CRP (Sigma) or a preparation of human immunoglobulins (Pentaglobin^®, Biotest) overnight at 4°C and then blocked for 1 h with 3% BSA (Sigma). Afterward, the plates were incubated with 0.25% NHS, and the particular C2 variant was diluted in GVB++ buffer (5 mM veronal buffer, 0.1% gelatin, 145 mM NaCl, 1 mM MgCl₂, 0.15 mM CaCl₂) and incubated for 30 min at 37°C with mild shaking. C3b protein (Complement Technology) serially diluted in NHS and coated on the plate instead of CRP was used for the preparation of the standard curve. C3b detection was performed with polyclonal goat anti-human C3 antibody (Complement Technology), followed by rabbit anti-goat antibody conjugated with HRP (Dako, Glostrup, Denmark) diluted 1:10,000 and 1:5,000 in PBS, respectively. The assay was developed by using 3,3′,5,5′-Tetramethylbenzidine (Sigma Aldrich) according to the manufacturer’s instructions.