Tocilizumab

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).

Cells derived from the LF specimens were harvested from the dishes using 0.25% trypsin for further passaging when they were grown to confluence in 60 mm culture dishes. P2 generation cells were used in the subsequent experiments. In order to analyze IL-6 and collagen expression regulated by leptin, P2 LF cells were cultured and stimulated with different concentrations of recombinant human leptin (Abcam, U.S.A.) ranging from 0 to 150 ng/ml for 24 h. Finally, cells and medium were then harvested for mRNA and protein analysis. IL-6 protein in supernatants of cell culture was measured using ELISA kit (Takara, China) according to the manufacturer’s instructions. For all cell inhibition experiments, 10⁵ cells were seeded in each well of 24-well plates and 10⁶ cells were seeded in each well of six-well plates. When cells were grown to 80% confluence, the indicated doses of different inhibitors (BAY11-7082 [14 (link)], Sigma, U.S.A. and Tocilizumab [15 (link)], Sigma, U.S.A.) under concentrations without cytotoxicity were used to stimulate cells. Forty eight hours later, cells were collected for RNA isolation, Western blot, and other subsequent experiments.