Huvecs

HUVECs purchased from Xinyuan Technology (Guangzhou, China) were grown in culture medium containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO₂ incubator. The heat-stressed HUVEC model was replicated as described previously (22 (link)). Briefly, cells were placed in a circulating water bath at 43°C for 2 h, then replaced with fresh medium, and further incubated at 37°C for 0, 2, 6, 12, and 24 h. In the NAC+HS group, cells were pretreated with 10 mM NAC for 1 h before heat stress. vWF expression in HUVECs was detected using anti-vWF antibody (Abcam) and anti-rabbit Alexa Fluor 488 IgG (Cell Signaling Technology, Veverly, MA, USA) by immunofluorescent staining as described previously (23 (link)). vWF levels in the culture supernatant of HUVECs were measured using a vWF ELISA kit (Abcam).

Primary HUVECs were purchased from AllCells (Shanghai, China) and cultured in low-glucose DMEM with 10% FBS (Gibco). After being in vitro cultured and passaged for 8~10 times, the cells were used for experiments. After treating the cells with or without GW9662 (5 μmol/L) for 1 hour followed by the induction with Rosiglitazone (50 μmol/L) for 24 hours, HUVECs were fixed by 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked with 3% BSA for 30 min, and then they were stained by anti-PPAR-γ (1:200, ab45036, Abcam) for 1 hour at 4°C overnight. After washing for 3 times, the cells were stained by Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1:1000).

Pooled human umbilical vein ECs (HUVECs) were purchased from Cell Systems/Clonetics and cultured refer to previous work [38 (link)–41 (link)]. In brief, HUVEC were cultured in endothelial basal medium supplemented with hydrocortisone (1 mg/ml), bovine brain extract (3 mg/ml), gentamicin (50 mg/ml), amphotericin B (50 mg/ml), epidermal growth factor (10 mg/ml), and 10% fetal calf serum until the third passage. All HUVECs were divided into 2 groups, cells in group 1 were incubated with recombinant human soluble CD40 ligand (rh-CD40L, 0.5ug/ml, Abcam) and cells in group 2 were not. After detachment with trypsin, cells were grown for at least 18 h. Confluent monolayers of HUVEC were grown onto 6-cm wells and exposed to laminar fluid flow in a cone-and-plate apparatus as previously described. A constant shear stress of 15 dynes/cm² was used in all experiments to simulate physiological shear stress.
In vitro scratched wounds were created by scraping the cell monolayer with a sterile disposable cell scraper as previously described [41 (link)–43 (link)]. To detect competence of HUVECs migration, scratch area 24 hours after injury were observed with a computer-assisted microscope (Zeiss) at 3 distinct positions (every 5 mm).