Rescue of NRP1 binding and vessel branching by VEGF-A-NZ2. (A) PAE/VEGFR-2, NRP1 cells treated with different VEGF-A isoforms (Figure 5A) were subjected to immunoprecipitation (IP) of VEGFR-2, followed by immunoblotting (IB) for phosphotyrosine and NRP1 (as indicated). VEGFs were added at 2 nM; in addition, VEGF-E-R126E was added at 2 and 5 nM to saturate receptor activation. To control for loading, blotting for VEGFR-2 and NRP1 on total lysates is shown. Note that VEGF-A165, VEGF-A-NZ2, and VEGF-E-NZ2 induced efficient coprecipitation of NRP1 and VEGFR-2. The loss-of-function VEGF-E-R126E did not induce complex formation, even at the higher concentration of the factor. (B) EB cultures in 2D and 3D conditions as indicated were treated with VEGF-E-NZ2 or the loss-of-function VEGF-E-R126E. Only VEGF-E-NZ2 allowed formation of a capillary plexus in the 2D condition and sprouting angiogenesis in the 3D condition. Scale bars represent 100 μm. (C) Subcutaneous matrigel plugs containing VEGF-A121 or the gain-of-function VEGF-A-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. Microphotographs show z-stacks of 108 μm. VEGF-A-NZ2, but not VEGF-A121, showed formation of branched, pericyte-embedded vessels. Scale bar represents 50 μm. (D) Quantification of branch points in panel C by manual marking and counting of multiple matrigel plug samples (n = 5/group).