Sprouting angiogenesis and vessel formation in response to the different VEGF ligands. (A) Sprouting of EBs in 3D collagen cultures was induced in response to VEGF-A165 and VEGF-E-NZ2 (arrows) but not to VEGF-A121 or vehicle, as visualized by anti-CD31 immunostaining. The core of the EB is shown in the top left corner of each panel. Scale bar represents 100 μm. (B) Details of angiogenic sprouts formed in VEGF-A165–treated (top) or VEGF-E-NZ2–treated (bottom) EBs. Immunostaining shows expression of CD31 (red) and ASMA (green). Hoechst 33 342 (blue) staining indicates nuclei. Scale bar represents 100 μm. (C) Quantification of the number of EBs forming angiogenic sprouts in response to vehicle, VEGF-A165, VEGF-A121, or VEGF-E-NZ2. (D) Quantification of CD31-positive area in angiogenic sprouts in relation to total CD31 area. (E) Subcutaneous matrigel plugs containing VEGF-A165, VEGF-A121, or VEGF-E-NZ2, implanted for 7 days in nude mice. Whole-mount fixation was followed by staining for expression of CD31 (red) and ASMA (green) and analysis by confocal microscopy. Asterisks indicate branch points in VEGF-A165– and VEGF-E-NZ2–containing matrigel plugs. Microphotographs show projections of z-stacks of 50 μm. Bottom row of panels shows magnifications of areas indicated by white boxes in the merged panels above. Scale bars represent 50 μm. (F) Quantification of branch points in the different conditions by manual marking and counting of multiple samples (n = 4/condition).