Synergistic effect of VEGF and dimer of soluble NP-1 in P-Sp cultures.

(A) P-Sp explants derived from E9.5 wild-type and np-1homozygous mutant murine embryo litter mates were cultured on OP9 stromal cells. Formation of the vb and vn was defective in mutant embryo explants (ii) compared with those from wild-type litter mates (i). NP-1–Fc (50 μg/mL), VEGF (50 ng/mL), or both were added to this culture system as described above. The vb was expanded innp-1^+/+ embryos on addition of VEGF (iii) or NP-1–Fc (v). Suppressed vasculature in the culture ofnp-1^−/− embryos (ii) was partly rescued on addition of 50 ng/mL VEGF (iv) and was completely rescued on addition of 50 μg/mL NP-1–Fc (vi). Simultaneous application of VEGF and NP-1–Fc enhanced the formation of the vb innp-1^+/+ (vii) andnp-1^−/− (viii) P-Sp cultures. Similar results were obtained in 3 independent experiments. The scale bar indicates 1 mm. (B) The vascular areas shown in Figure 5A were calculated using NIH Image software version 1.62. The mean ± SD vascular areas per explant obtained from 3 independent experiments were as follows: (i) 10.8 ± 1.48 mm², (ii) 1.1 ± 0.55 mm², (iii) 35.2 ± 2.39 mm², (iv) 7.7 ± 1.68 mm², (v) 18.3 ± 2.48mm2, (vi) 12.1 ± 3.03mm2, (vii) 49.7 ± 3.91 mm², and (viii) 33.5 ± 3.77 mm².