Huvecs

HUVECs are widely used to study endothelial injury in vitro [15 (link)]. HUVECs (Lifeline Cell Technology, Walkersville, MD, USA) were cultured in VascuLife complete medium (Lifeline Cell Technology) at 37 °C in a humidified 5% CO₂ incubator. The cells at passages 4–10 were used in these experiments. For hypoxic exposure, the cells were maintained in VascuLife basal medium supplemented with 2% fetal bovine serum and exposed to hypoxic conditions (1% O₂) in a humidified 5% CO₂ hypoxic chamber (Astec, Fukuoka, Japan).

Human umbilical vein endothelial cells (HUVECs) were obtained from LIFELINE Cell Technology. Replicative senescent EC was prepared as previously described^{21 (link), 31 (link)}. Briefly, HUVECs were passaged at 1:4 ratio for 15–17 times (P18-20) until no obvious growth was observed. P3-5 HUVECs were used as control young cells. Cells were treated with 5 ng/ml LPS or 1 ng/ml TNF-α for 24 h to induce inflammatory responses. In some experiments, cells were treated with TNF-α in the presence of 100 μM L-NAME or 1 μM Helenalin.

A549 cells were obtained from the Cell Resource Center, Peking Union Medical College (Beijing, China). A549 cells have been authenticated using STR profiling within the last three years and all experiments were performed with mycoplasma-free cells. A549 cells and c-Jun-KO A549 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. HUVECs were obtained from Lifeline Cell Technology (Frederick, MD, USA). Cell cultures were maintained in a humidified atmosphere with 5% CO₂ at 37°C.

HUVECs were purchased from Lifeline cell technology (C-12200, cat. no. 10171-906). HAECs were a kind gift from Dr. Lusis (UCLA, David Geffen School of Medicine). HUVECs and HAECs were cultured in Petri dishes or flasks coated with 0.2% gelatin type A (cat. no. 901771; MP Biomedicals, Santa Ana, CA, USA), in Endothelial Cell Medium (ECM, Cat.no. 1001, ScienCell, Carlsbard, CA. USA) containing 465 mL of basal medium, 25 mL of fetal bovine serum (FBS, Cat. no. 0025, ScienCell, Carlsbard, CA, USA), 5 mL of Endothelial Cell Growth Supplement (ECGS, Cat. no. 1052, ScienCell, Carlsbard, CA, USA) and 5 mL of penicillin/streptomycin solution (P/S, Cat. no. 0503, ScienCell, Carlsbard, CA, USA). Only HUVECs with less than 6 passages and HAECs with <15 passages were used in this study.

HUVECs were purchased from Lifeline Cell Technology and cultured in completed endothelial cell medium (Lifeline^® Cell Technology, Frederick, MD). CRC cell lines HCT116 and CT26 were purchased from Cell Bank of Shanghai institute of Cell Biology, Chinese Academy of Sciences, and cultured in McCoy's 5A (Gibco, 16600082) and 1640 (Gibco, 2192717) medium, respectively, with 10% fetal bovine serum (FBS, Gibco, 10091148) and 1% penicillin-streptomycin (HyClone, SV30010). All cell lines used in the study were maintained in a humidified incubator (Thermo Fisher, USA) containing 5% CO₂ at 37 ℃.

This study was approved by the Medical Research Ethics Committee of Tokyo Medical and Dental University (M2017-142), and all subjects provided informed consent. The human synovium was harvested from the knees of six female donors (55–81 years) with osteoarthritis, during total knee arthroplasty. The synovial cells were cultured as previously reported [24 (link)]. Briefly, the synovium was digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Co. LLC, Merck KGaA, Darmstadt, Germany) at 37 °C. After 3 h, the digested cells were filtered through a 70 µm cell strainer (Greiner Bio-One GmbH, Frickenhausen, Germany). The cells were cultured in α-minimum essential medium (α-MEM; Thermo Fisher Scientific, MA, USA) supplemented with 1% antibiotic–antimycotic (Thermo Fisher Scientific) and 10% fetal bovine serum in a cell culture incubator (Astec Co., Ltd., Fukuoka, Japan) at 37 °C under 5% CO₂.
HUVECs from four donors were purchased from Lifeline® Cell Technology (CA, USA) and maintained in Endothelial Cell Growth Medium 2 (PromoCell GmbH, Heidelberg, Germany).