Endothelial cell culture and functional assays

HCAECs were grown in Nunc T75 flasks (Thermo Fisher Scientific) in endothelial cell growth medium (EGM, Lonza, #CC-3162) supplemented with 10% FCS. For functional assays, cells were switched to MCDB 131 medium (Thermo Fisher Scientific, #10372019) containing 2% FCS. Branching morphogenesis was assessed on growth factor-reduced Matrigel (Corning) in 48-well plates (2.5 × 10⁴ cells/well). After stimulation, cells were stained with the membrane-permeable fluorescent dye BCECF-AM (Sigma-Aldrich) for 2 h. Digital images were acquired with an Axio Observer.Z1 fluorescence microscope (Zeiss) and analyzed using AxioVision software (Zeiss). Branching points were defined as intersections of at least three tubes and closed tubes as circular structures surrounded by tubes. Cell migration after scratch injury was analyzed in endothelial cell monolayers grown in 24-well plates. Monolayers were scratched with a 200 μl pipet tip, washed and stimulated for 16 h. Before and after stimulation, digital phase-contrast images were captured with the Axio Observer.Z1 microscope and analyzed using AxioVision software. Recovery (%) was calculated as [(cell free area at 0 h – cell free area at 16 h)/cell free area at 0 h] × 100. For signaling analysis, HCAECs were stimulated with recombinant proteins for 0, 5, 15 and 30 min, lysated in RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% Triton X, 0.5% IGEPAL containing protease and phosphatase inhibitors (Roche, #4693132001, #4906845001)) and subjected to immunoblot analysis. Immunoblots were densitometrically analyzed using ImageJ v1.48.