Hasmc

HASMCs, purchased as cryopreserved tertiary cultures (Cascade Biologics Inc., Portland, OR, USA), were grown in culture flasks in smooth muscle cell growth medium (M231, Cascade Biologics Inc.) supplemented with 5% smooth muscle growth supplement (Cascade Biologics Inc.). The growth medium was changed every other day until confluence, and then the cells were passaged every 3–5 days. Cells between passages 3 and 8 were used for the subsequent experiments. Before conducting experiments, HASMCs were pre-cultured in serum-starved medium (M231 alone) for 24 h.

HASMCs (Cascade Biologics, OR, USA) were grown and passaged as described previously [23 (link), 24 (link)]. Cells were used at passages 3–8. The purity of HASMC cultures was verified by immunostaining with a monoclonal antibody directed against smooth-muscle-specific α-actin (R&D Systems, MN, USA). Before treatment or stimulation with reagents, the cells were serum starved for 24 h.

HASMCs, purchased as cryopreserved tertiary cultures from Cascade Biologics (OR, USA), were grown in culture flasks in smooth muscle cell growth medium (M231, Cascade Biologics Inc.). The cells were used between passages 4 and 8. All cells were synchronized in serum-free medium for 24 h prior to experiments. For all data shown, the experiments were repeated at least 3 times in duplicate with different preparations of HASMCs. For Western blot analysis, the graphical analysis represents the results from three independent experiments and quantification by densitometry.

Human smooth muscle cell growth medium (M231), smooth muscle growth supplement, trypsin/EDTA solution, trypsin neutralizer solution and HASMCs were obtained from Cascade Biologics (Portland, OR). Fetal bovine serum (FBS), antibiotic-antimycotic mixture, mice TNF-α kits, oligofectamine and 2’,7’-dichlorofluorescein diacetate (DCFDA) were obtained from Life technology (Grand Island, NY). Small interfering RNA (siRNA) oligonucleotides against TNFR 1(sc-29507), p65 (sc-29410), and p47 (sc-76032) and antibodies against human TNFR1 (sc-8436), MSX-2(sc-17729), BMP2(sc-6895), RUNX2(sc-10758), CD68 (sc-9139), β-actin(sc-47778), and anti-hnRNA c1/c2 (sc-32308)were obtained from SantaCruz Biotechnology (Santa Cruz, CA). Antibodies against human NF-kB subunit P65(#558421), NADPH oxidase subunit p47(#610354), and caveoli-1 (610406) were obtained from BDBiosciences (San Jose, CA). Unless otherwise specified, all other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO).

HUVECs were freshly isolated from human umbilical cord veins of newborn babies with previously described methods [41 (link)]. Informed consent was obtained from all the participating mothers prior to sample collection. Each patient provided written informed consent. The protocol for this study (ER-100-072) was approved by the Institutional Review Board of National Cheng Kung University Hospital. Cells were cultured in medium 199 containing 20% FBS, 25 pg/mL EC growth factor, 10 units/mL heparin, and 100 units/mL penicillin at 37 °C in a 5% CO₂ and 95% humidity incubator. Cells cultured up to the third passage were used in all experiments.
Primary passage cryopreserved HASMCs were obtained from Cascade Biologics (Portland, OR, USA). Cells were plated in 75-cm² flasks and incubated in M199 supplemented with 10% FBS and 5% smooth muscle growth supplement (Thermo Fischer Scientific, Waltham, MA, USA). Cells cultured up to passages 6 to 9 were used for experiments.
For this experiment, both cell lines were cultured with 25 mM of glucose to induce HG conditions.

Human aortic smooth muscle cells (HASMCs; Cascade Biologics, Portland, OR) were maintained in medium 231 containing 5% smooth muscle growth supplement (SMGS) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. HASMCs (between passages 3 and 5) were grown to 80–90% confluence and were made quiescent by serum starvation (0.1% SMGS) for 24 hours. Subsequently, HASMCs were cultured in fresh medium without stimulation or were treated with indicated concentrations of recombinant human HMGB1 (R&D systems) for 24 hours.