β ecgf

Primary human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins according to a method by the author of [39 (link)] with slight modifications. Briefly, the cells were firstly isolated by 10% collagenase I and then cultured in M199 medium (HyClone, South Logan, UT, USA) containing 20% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel), 3.2 ng/mL β-ECGF (Sigma, Billerica, MA, USA), 0.108 mg/mL heparin sodium (Solarbio, Tongzhou, Beijing, China), 2.5 ng/mL thymidine (Solarbio, Beijing, China), and 100 U/mL penicillin/streptomycin. For all cell experiments, early passages (passages 2~7) were used. All cells were maintained at 37 °C in an atmosphere of 95% O₂/5% CO₂.

Primary human umbilical vein endothelial cells (HUVECs) were cultured in M199 Medium (HyClone, South Logan, UT) supplemented with 3.2 ng/ml β-ECGF (Sigma, Billerica, MA), 0.108 mg/ml heparin sodium (Solarbio, Tongzhou, BJ), 2.5 ng/ml thymidine (Solarbio, Beijing), and 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel). The osmolality of M199 medium (control) was about 270 mosmol/kg. To elevate the osmolality of M199 medium, NaCl (Sigma, Billerica, MA) was added into the control medium, with a final osmolality at 290, 310, 330, and 350 mosmol/kg. For all experiments except the long-term proliferation experiments, the logarithmically growing HUVECs between passages 2 and 6 were used. Human THP-1 monocytes were purchased from YiYuan (Guangzhou, China), and grown in RPMI 1640 medium supplemented with 10% FBS (Gibco, Grand Island, NY). Cells were maintained at 37 °C with 5% CO₂ in a humidified atmosphere.
Eight-week male apolipoprotein E-deficient (ApoE^−/−) mice weighed at 20~25 g were purchased from Research Institute of Surgery at Daping Hospital, the Third Military Medical University. The mice were fed with a normal salt diet (0.8% NaCl) for 2 weeks, and then divided into two groups. Group 1 was kept on with a normal salt diet (0.8% NaCl), whereas group 2 was fed with a high-salt diet (8% NaCl). All mice had free access to water.

For the experiments, cells were trypsinized, washed with PBS and cultivated in M199 medium supplemented with 1% penicillin–streptomycin solution (Sigma), 4 mM L-glutamine (Sigma), 50 μg/mL heparin (Sigma) and 20 μg/mL βECGF (Sigma), to avoid the effect of culture milieu on metabolic profile. The E4⁺EC cells were seeded in the T25 flask 24 h prior the co-culture with cancer cells, to form a dense monolayer. The cancer cells were seeded on the top of E4+EC monolayer. Samples were collected by trypsinization at different time points (6 h, 18 h, 24 h, and 48 h) and washed with PBS. Cells were resuspended in 50 μL of staining media (PBS1X/FBS 5%/EDTA 2 mM) and incubated with fluorochrome-conjugated antibody (CD326, EpCAM, alexa fluor, Biolegend) for 30 minutes at 4 °C and washed with PBS. The E4⁺EC and cancer cell suspension was separated using Fluorescence Activated Cell Sorting (FACS). Cancer cells cultivated in the absence of E4⁺EC, were used as control (T0). Five replicates for each conditions were prepared. The experimental design is illustrated in Fig. 1A.