Huvecs

Human umbilical vein cells (HUVECs) obtained from the Bena Culture Collection (Cat. No. ATCCPCS-100-010, ATCC, Manassas, VA, USA) were cultured in Endothelial Cell Medium (ScienCell, USA) with 5% (v/v) fetal bovine serum (FBS) (HyClone, South Logan, UT, USA), 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA), and 1% ECGF (Invitrogen). A human acute monocytic leukemia cell line (THP-1) was obtained from the National Infrastructure of Cell Line Resource (resource No. 3111C0001CCC000057), and then, the cell was cultured in RPMI-1640 medium (Gibco BRL) with 10% (v/v) FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were incubated in a 5% CO₂ incubator at 37°C. At 70-80% confluence, cells were splitting according to standard procedures. THP-1 cells were harvested in RPMI-1640 medium treated with 50 μg/ml ox-LDL (oxidized low-density lipoprotein) after being induced by 160 nM for 24 h. The in vitro atherosclerosis cell model was constructed using ox-LDL. For the coculture of THP-1 cells (monocytes) and human umbilical vein ECs (HUVECs), 5 × 10⁵ HUVEC cells were seeded into six-well plates, and then, 1 × 10⁶ ox-LDL-treated THP-1 cells were added onto the HUVEC layers. A transwell chamber was prepared for an indirect coculture.

4T1, Panc02, B16-F10 cancer cells, and HUVECs were purchased from BeNa Culture Collection, China. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin under humidified conditions with 5% CO₂ and 37 ℃.
BALB/c (female, SPF, 5 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. All animal handing procedures were performed in accordance with the internationally accepted principles and Guidelines for the Care and Use of Laboratory Animals of Huazhong University of Science and Technology. Experimental protocols were approved by the Institutional Animal Ethical Committee of the Huazhong University of Science and Technology. The mice were kept in specific pathogen-free environment, and had access to food and water ad libitum.

The HUVECs were obtained from BeNa Culture Collection (BNCC363216). HUVECs were cultured in an ECM complete medium supplemented with 10% FBS, 1% penicillin, 1% streptomycin, and 1% ECGS in a 5% CO₂ atmosphere at 37 °C. VEGF165 (vascular endothelial growth factor-165), is a member of the VEGF family that promotes angiogenesis1 by stimulating endothelial cell proliferation and migration [33 (link)]. A model group was treated with VEGF165 (200 ng/mL) and the ECM medium (10% FBS, 1% penicillin, and 1% streptomycin). On this basis, the experimental group was incubated with various concentrations of components from CA. The NS group was only cultured with the ECM medium.

HUCMSCs were acquired from Union Stem Cell & Gene Engineering Co. Ltd. (Tianjin, China). The cells were cultured in α-modified essential medium (α-MEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 10% antibiotic–antifungal agent (Gibco, USA). HUCMSCs between Passages 4 and 6 were used for subsequent experiments. HUVECs were obtained from BeNa Culture Collection Co. Ltd. (Beijing, China) and expanded in endothelial growth medium-2 (EGM-2) (Lonza, Morristown, NJ, USA). All incubations were performed at 37 °C with 5% CO₂. All experimental procedures received approval from the Ethics Committee of Tianjin Stomatological Hospital.