Ec growth supplement

Human umbilical artery smooth muscle cells (UASMCs) (CC-2579, Lonza) were cultured on 0.05% Gelatin (from 2% Gelatin Solution in PBS, both from SIGMA) coated flasks in Smooth Muscle Cell Basal Media (SmBM, CC-3181, Lonza) supplemented with SmBM plus SingleQuots of Growth Supplements (CC-3182, Lonza) and maintained in a humidified incubator with 5% CO2 at 37 °C. Cells were passaged every other day at a ratio of 1:2. Cells used in this study were below passage 10. Human umbilical vein endothelial cells (HUVECs) were cultured on 0.5% Gelatin coated flasks in M199 medium (Gibco) supplemented with 10% fetal bovine serum and 1 ml EC supplement cocktail (contains 1ng/ml ECGF, 3 μg/ml EC growth supplement, 10unit/ml heparin, 1.25 μg/ml thymidine; all from Sigma-Aldrich). HEK 293 T (ATCC, CRL-11268) cells were cultured in DMEM media with 10% FBS in a humidified incubator with 5% CO2 at 37 °C. 293 T cells were passaged every other day at a ratio of 1:4.

HeLa cells (ATCC, CCL-2) were grown in DMEM-High Glucose (Euroclone, Pero, Italy) supplemented with 10% FBS (Euroclone), 4 mM L-glutamine (Lonza, Basel, Switzerland), and 100 U/L penicillin/streptomycin (Euroclone). Mouse endothelial cells (moEC), previously referred as vascular endothelial (VE) cadherin-positive ECs and described in [28 (link),31 (link),35 (link),36 (link)], were cultured in DMEM-High Glucose (Lonza) with 10% FBS, 2 mM L-glutamine (Lonza), 100 U/L penicillin/streptomycin (Euroclone), 1 mM sodium pyruvate (Sigma–Aldrich, Merck, Darmstadt, Germany), 25 mM HEPES (Sigma–Aldrich), 100 µg/mL heparin (from porcine intestinal mucosa, Sigma–Aldrich), and 50 μg/mL EC growth supplement (ECGS from bovine pituitary gland, Sigma–Aldrich). Before seeding, plates were coated with 0.1% porcine gelatin (Difco) and incubated overnight at 37 °C. Cells were maintained in a humidified, 5% CO₂ atmosphere at 37 °C. For VEGF stimulation, moEC were grown in a serum-starved (0.2% FBS) medium, without ECGS supplementation, for 2 h prior to treatment with recombinant murine VEGF-165 (100 ng/mL, PeproTech, EC Ltd., London, UK) for 24 h.

Mouse aorta ECs were isolated and cultured^{14 (link)}. In brief, thoracic aorta was isolated from 3-week-old Tie2-Cre;Pdgfrb^fl/fl and Pdgfrb^fl/fl mice. Aortic rings were embedded in Matrigel-coated dishes and cultured for 5 days. After rinsing with phosphate-buffered saline (PBS), the rings were removed, and remaining cells incubated with 2 U/ml Dispase I (Gibco, 17105-041) for 20 min at 37 °C. After centrifugation at 500 × g for 10 min, the cell pellets were washed with PBS, and cells cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium supplemented with 25 mg/ml EC growth supplement (Sigma) and 5% fetal bovine serum (FBS) at 37 °C in a humidified air atmosphere with 5% CO₂.