Effect of dimer of soluble NP-1 on sorted ECs.

(A) Murine embryos of E12.5 wild-type and np-1 homozygous mutants were dissociated and stained with PE-conjugated anti–PECAM-1 and biotin-conjugated anti–VEGFR-2 mAbs. Biotin was developed to avidin-allophycocyanin. The stained cells were analyzed and sorted using FACSvantage. Approximately 0.3% of cells derived from wild-type embryos and 0.5% of cells derived from mutant embryos were double positive. (B) Sorted VEGFR-2⁺ PECAM-1⁺ cells from E12.5 wild-type (i) and np-1 homozygous mutants (ii-iii) were cultured on OP9 cells. The vascular structure was defective in the ECs from mutant embryos (ii) compared with those from wild-type litter mates (i). NP-1–Fc (50 μg/mL) or CD4-Fc (50 μg/mL) was added to the culture as described above. Vascular formation was rescued in np-1 homozygous mutants by addition of NP-1–Fc (iii), whereas the same amount of CD4-Fc did not have any effect (data not shown). Scale bar indicates 100 μm. (C) Cell lysates of VEGFR-2⁺ PECAM-1⁺ ECs fromnp-1^+/+ or np-1^−/−murine embryos were immunoprecipitated with an anti–VEGFR-2 antibody and subjected to Western blotting using an antiphosphotyrosine mAb (anti-PY). Lane 1 shows results with VEGF (1 ng/mL; wild type); 2, no factor (mutant); 3, VEGF (1 ng/mL; mutant); and 4, VEGF (1 ng/mL plus NP-1–Fc [50 μg/mL]; mutant). The VEGFR-2⁺PECAM-1⁺ ECs from np-1^+/+ ornp-1^−/− embryos were challenged by using VEGF with or without NP-1–Fc. In mutant embryos, phosphorylation of VEGFR-2 was induced by addition of a low dose of VEGF and NP-1–Fc (lane 4; arrowhead), whereas no factor (lane 2) or VEGF alone (lane 3) did not induce phosphorylation of VEGFR-2. Lane 1 was used as a positive control (wt). The lower panel shows the amount of immunoprecipitated VEGFR-2 confirmed by Western blotting using anti–VEGFR-2 mAb.