Effect of Rap1GAP1 overexpression on in vitro angiogenesis. (A) HUVECs were transfected with Rap1GAP1-FLAG or empty vector. After 24 hours, cells were lysed and subjected to Western blot analysis using a Flag-tag specific antibody. An antibody directed against tubulin was used as loading control. (B) Three-dimensional in vitro angiogenic sprouting in a spheroidal culture system with collagen-embedded spheroids of Rap1GAP1-FLAG- versus mock-transfected endothelial cells in the presence or absence (control) of 50 ng/mL bFGF. The mean cumulative length of sprouts per spheroid was assessed after 24 hours (*P < .05 vs empty vector, **P < .05 vs empty vector + bFGF, n = 9). (C) Statistical analysis and representative micrographs of the tube-forming activity. HUVECs were seeded on Matrigel Basement Membrane Matrix 24 hours after transfection with the indicated plasmids (n = 3). The length of capillary-like structures/networks was measured in 5 different high-power fields by light microscopy after 24 hours (*P < .05 vs empty vector). Bar represents 200 μm. The wells were viewed with an Axiovert 100M inverted microscope using as objective a Plan-NEOFLUAR (at 10×/0.30). Images were acquired using an Axiocam MR digital camera and were processed using AxioVision Rel 4.5 digital imaging software (all from Carl Zeiss, Jena, Germany). (D) Three-dimensional in vitro angiogenesis with collagen-embedded spheroids of Rap1a-, Rap1aV12-, or mock-transfected HUVECs (n = 7; *P < .05 vs empty vector). (E) Three-dimensional in vitro angiogenic sprouting in a spheroidal culture system with collagen-embedded endothelial spheroids of Rap1aV12- or mock-transfected HUVECs. The sprouting assay was performed in the presence of blocking monoclonal β₁-integrin antibodies or murine isotype control antibodies. Data are presented as mean plus or minus SEM (n = 5, *P < .05 vs Rap1aV12 + IgG).