Epidermal growth factor (egf)

Pooled HUVECs (C-12208, lot 447Z015; Promocell, Heidelberg, Germany) were cultivated in endothelial cell growth medium MV2 supplemented with 5% (v/v) fetal bovine serum, 5 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 20 ng/mL insulin-like growth factor-1, 0.5 ng/mL vascular endothelial growth factor 165, 1 µg/mL ascorbic acid, and 0.2 µg/mL hydrocortisone (Promocell, Heidelberg, Germany). HSVSMCs were isolated from surplus vein tissue from consenting subjects undergoing coronary artery bypass graft surgery as previously described [32 (link)] and cultivated in smooth muscle cell growth medium 2 supplemented with 5% (v/v) fetal bovine serum, 0.5 ng/mL epidermal growth factor, 2 ng/mL basic fibroblast growth factor, and 5 µg/mL insulin (Promocell, Heidelberg, Germany). SMC integrity was verified by the presence of SMC markers myosin heavy chain and α-actin and the absence of the endothelial marker PECAM1 (CD31). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂.

PBMNCs were incubated for 7 days on fibronectin-coated dishes using endothelial cell growth medium MV2 supplemented with 10% fetal bovine serum, hydrocortisone, ascorbic acid, heparin sulfate, 2% penicillin/streptomycin, 50 ng/mL human recombinant VEGF, insulin-like growth factor 1, basic fibroblast growth factor, and epidermal growth factor (all material from PromoCell). To confirm the cultured cells as EPC, we fixed them with 4% paraformaldehyde and stained them using FITC-conjugated Ulex europeaus agglutinin-I (UEA-1, Sigma). Additionally, the uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine (DiI)-labeled acetylated low-density lipoprotein (acLDL, Life Technologies) was monitored, as cultured-EPCs are known to be positive for both UEA-1 and acLDL. Double-positive cells that adhered to the fibronectin-coated dishes were counted in five randomly selected fields using an inverted fluorescent microscope. Nuclei were visualized using Hoechst 33342 (Lonza). To assess the adhesion ability of the cultured-EPCs, the number of adherent EPCs was compared to the initial number of PBMNCs plated.

Primary cultures of HA-SMCs (PromoCell) were maintained in SMC growth medium 2 containing 5% foetal calf serum (FCS), 0.5 ng/ml epidermal growth factor, 2.0 ng/ml basic fibroblast growth factor and 5μg/ml insulin (PromoCell). The cells were grown in 5% CO2 at 37°C in medium renewed every 3 days. Confluent cells were detached by trypsin/EDTA and subcultured with a split ratio 1:2. HAoSMC were used between 2 and 5 passages.