Adult human aortic smooth muscle cells (Thermo Fisher Scientific Inc.) were seeded at a density of 20,000 cells per well. The next day, HUVECs were plated at density of 5000 cells per well on the confluent smooth muscle monolayer in a 96-well plate as previously described [51 (link)]. GSK429486 (5 μM) or DMSO as a control was added one day after the plating of HUVECs. The co-cultures were sustained under full growth media (EGM-2) for seven days. Endothelial tubes were visualized via immunofluorescence using a mouse anti-human CD31 monoclonal antibody (R&D systems) at 1:400 dilution and goat Alexa 488-conjugated anti-mouse IgG secondary antibody (Thermo Fisher Scientific Inc.) at 1:500 dilution. Actin fibers were stained with Alexa 568-conjugated Phalloidin (Thermo Fisher Scientific Inc.). Nuclei were visualized via DAPI staining. The endothelial tubes were imaged automatically at 10× magnification using the high-content imaging system (Operetta). Nine identically positioned fields were acquired from each well in each experiment. The following quantification of endothelial network features, total tube length, branching points and mesh number, was performed automatically using an NIH Image J-based tool (angiogenesis analyzer) [94 (link)]. Meanwhile, the tube thickness feature was quantified automatically using Harmony image analysis software (Perkin Elmer).
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