1. Exogenous clustered neuropilin 1 enhances vasculogenesis and angiogenesis
by Yoshihiro Yamada, Nobuyuki Takakura, Hirofumi Yasue, Hisao Ogawa, Hajime Fujisawa, and Toshio Suda
Blood
Volume 97(6):
March 15, 2001
©2001 by American Society of Hematology

2. P-Sp culture system reflects vascular defects in np-1 mutant embryos.
P-Sp culture system reflects vascular defects in np-1 mutant embryos. Spinal cord (A-B) and heart region (C-D) of E12.0 np-1 +/+ (A, C) andnp-1 −/− (B, D) murine embryos were subjected to immunohistochemical staining with anti–PECAM-1 antibody to visualize all ECs. Capillaries showed little branching and were of large caliber (B, D; arrows). P-Sp explants derived from E9.5 wild-type embryos (E) and mutant embryos (F) were cultured on OP9 stromal cells. Vascular network (vn) formation was defective innp-1 −/− P-Sp explants (F), as observed in the CNS and pericardium. The scale bar indicates 100 μm (E-F); vb, vascular bed.
Yoshihiro Yamada et al. Blood 2001;97:
©2001 by American Society of Hematology

3. The structure of recombinant NP-1 proteins
The structure of recombinant NP-1 proteins.(A) Schematic representation of the Fc-tagged NP-1 extracellular segment (NP-1–Fc) and Flag-tagged NP-1 extracellular segment (NP-1–Flag).
The structure of recombinant NP-1 proteins.(A) Schematic representation of the Fc-tagged NP-1 extracellular segment (NP-1–Fc) and Flag-tagged NP-1 extracellular segment (NP-1–Flag). (B) SDS-PAGE analysis of the recombinant proteins stained with Coomassie brilliant blue G-250. (C) Immunoblot of the recombinant proteins with the antirabbit NP-1 antibody.14 Lanes 1-4 correspond to NP-1–Flag (1), NP-1–Fc (2), NP-1–Flag (3), and NP-1–Fc (4) recombinant proteins; 1 and 2 were under nonreducing conditions and 3 and 4 were under reducing conditions.
Yoshihiro Yamada et al. Blood 2001;97:
©2001 by American Society of Hematology

4. Effect of soluble NP-1 in P-Sp cultures. P-Sp explants derived from E9
Effect of soluble NP-1 in P-Sp cultures.P-Sp explants derived from E9.5 wild-type embryos (A, C, E, G) and mutant embryos (B, D, F, H) were cultured on OP9 stromal cells.
Effect of soluble NP-1 in P-Sp cultures.P-Sp explants derived from E9.5 wild-type embryos (A, C, E, G) and mutant embryos (B, D, F, H) were cultured on OP9 stromal cells. NP-1–Flag (50 μg/mL; C,D,G,H), anti-Flag IgG (50 μg/mL; G-H), NP-1–Fc (50 μg/mL; E-F), or the same amount of control Flag (A-B) was added to this culture. In wild-type embryos, vb formation was suppressed when NP-1–Flag was added (C); however, on addition of NP-1–Fc (E) or NP-1–Flag plus anti-Flag IgG (G), which forms a dimer of NP-1, the vb was expanded compared with that on addition of control Flag (A). In homozygous mutants, vascular formation was slightly suppressed on addition of NP-1–Flag (D; arrow) compared with that on addition of control Flag (B); however, suppressed EC development was rescued by addition of NP-1–Fc (F) or NP-1–Flag plus anti-Flag IgG (H). Similar results were obtained in 4 independent experiments. The scale bar indicates 100 μm.
Yoshihiro Yamada et al. Blood 2001;97:
©2001 by American Society of Hematology

5. Effect of dimer of soluble NP-1 on sorted ECs
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.
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.
Yoshihiro Yamada et al. Blood 2001;97:
©2001 by American Society of Hematology

6. Synergistic effect of VEGF and dimer of soluble NP-1 in P-Sp cultures
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.
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 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.
Yoshihiro Yamada et al. Blood 2001;97:
©2001 by American Society of Hematology

7. Rescue of defective vascularity of np-1 mutant embryos in vivo by dimer of soluble NP-1.
Rescue of defective vascularity of np-1 mutant embryos in vivo by dimer of soluble NP-1. Injection of CD4-Fc as a control had no effect (A, C). On injection of NP-1–Fc, some np-1 −/− embryos showed a substantial recovery of vascularity, and interestingly, the caliber of the rescued capillaries (B, D) was larger than those of capillaries in wild-type embryos (Figure 1A,C). Similar results were obtained in 3 independent experiments. The scale bar indicates 150 μm (A-B) or 300 μm (C-D).
Yoshihiro Yamada et al. Blood 2001;97:
©2001 by American Society of Hematology

8. Effect of exogenous NP-1 on EC development
Effect of exogenous NP-1 on EC development.(A) When NP-1 was expressed on ECs, signaling by VEGF165was enhanced through dimerization of NP-1 and VEGFR-2 (ii) compared with situations in which VEGFR-2 alone was expressed on ECs (i).
Effect of exogenous NP-1 on EC development.(A) When NP-1 was expressed on ECs, signaling by VEGF165was enhanced through dimerization of NP-1 and VEGFR-2 (ii) compared with situations in which VEGFR-2 alone was expressed on ECs (i). This may have been caused by conformational changes produced by VEGFR-2 on ECs. (B) A monomer of soluble NP-1 inhibited binding of VEGF165 to VEGFR-2 and prevented phosphorylation of VEGFR-2 (i); however, a dimer of soluble NP-1 bound to VEGF and enhanced phosphorylation of VEGFR-2 exogenously (ii). Candidate cells that express NP-1 and affect activation of VEGFR-2 on EC exogenously are neuronal cells, stromal cells in the bone marrow, tumor cells, and so on (iii). However, it is not clear whether the NP-1 on such cell types forms a dimer.
Yoshihiro Yamada et al. Blood 2001;97:
©2001 by American Society of Hematology