CD45⁺ cells isolated from the fetal liver bind VEGF₁₆₅ and phosphorylate VEGFR-2 on ECs.

(A) Cells obtained from an E12.5 fetal liver were stained with anti-CD45–PE and anti-B220–APC and analyzed by flow cytometry. Some cells (2.6%) were positively stained by both antibodies (i), and sorted CD45⁺B220⁺ cells were then stained with VEGF-biotin and streptavidin-FITC. Many of the cells shifted to the right (blue line; positive cells). Fifty percent (50%) of the cells over the negative gate (i; right histogram) expressed NP-1 alone among VEGF₁₆₅ receptors (ii). (B) E12.5 embryos were dissociated and stained with PE-conjugated anti–PECAM-1 and biotin-conjugated anti–VEGFR-2 mAbs. Biotin was developed to avidin-allophycocyanin. PECAM-1⁺VEGFR-2⁺ cells indicated by the box were sorted by using FACS Vantage. (C) PECAM-1⁺VEGFR-2⁺ cells were cultured with OP9 cells in the presence or absence of VEGF, NP-1⁺ HCs, and VEGFR-2–Fc. The number of PECAM-1⁺ endothelial clusters was scored. *P, **P < .05. (D) Cell lysates of PECAM-1⁺VEGFR-2⁺ cells that had been stimulated by various factors or cells were immunoprecipitated with anti–VEGFR-2 antibody and then subjected to Western blotting using an antiphosphotyrosine mAb (anti-PY). The PECAM-1⁺VEGFR-2⁺ cells were incubated with the CD45⁺ cells from fetal livers of NP-1 mutant (lanes 1-5) or wild-type (lanes 6-10) embryos with or without the indicated factors in each lane; no factor (lanes 1, 6); 10 ng/mL VEGF (lanes 2, 7); 10 ng/mL VEGF plus 20 μg/mL NP-1 Flag (lanes 3, 8); 10 ng/mL VEGF plus 300 ng/mL SemaIIIA (lanes 4, 9); 10 ng/mL VEGF plus 1 μg/mL anti-VEGF neutralizing antibody (lanes 5, 10). Phosphorylation of VEGFR-2 was induced by the addition of CD45⁺NP1⁺ cells mixed with 10 ng/mL VEGF (lane 7), and it was specifically blocked by NP-1 Flag or SemaIIIA or anti-VEGF neutralizing antibody (lanes 8-10). The addition of CD45⁺NP1⁻ cells mixed with VEGF barely induced phosphorylation of VEGFR-2 on ECs (lane 2). “wild” indicates cells from the wild type, and “mutant” indicates cells from the mutant embryo of NP-1 from the same litter.