Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice
J. Clin. Invest. Serena Zacchigna, et al. 118:2062 doi:10.1172/JCI32832 [Go to this article.]

Figure 4
Fundamental role of NP-1 in CD11b⁺ cell recruitment by VEGF₁₆₅ and Sema3A. (A) The number of BM CD11b⁺ cells migrated in response to different concentrations of VEGF₁₆₅, VEGF₁₂₁, and Sema3A was counted in 8 fields per membrane. Histograms show the mean number of migrated cells ± SD. *P < 0.05 over control (no chemoattractant). (B) Cell supernatants containing fusion proteins between AP and VEGF₁₆₅, VEGF₁₂₁, or Sema3A were added to CD11b⁺ cells. Binding was detected upon addition of VEGF₁₆₅-AP and Sema3A-AP, but not VEGF₁₂₁-AP. AP-tag only was used as a negative control. Original magnification, ×20. (C) Expression of Flk-1, Flt-1, and NP-1 was analyzed in CD11b⁺ cells transfected with lipids alone or with siNP1bis and siNP1ter (see Supplemental Figure 4) and normalized to the housekeeping gene GAPDH. Shown are means ± SD of 3 independent experiments. *P < 0.05 over untreated cells. (D) Purified CD11b⁺ cells were treated with siNP1ter or with a control scrambled siRNA and tested for their ability to migrate in response to VEGF₁₆₅ (50 ng/ml) and Sema3A (700 ng/ml). Silencing of NP-1 (white bars) markedly impaired cell migration to both chemoattractants. Shown are means ± SD of 3 independent experiments. *P < 0.05 over untreated cells. (E) CD11b⁺ cells were lipofected with either siNP1ter or the control scrambled siRNA, labeled with the fluorescent dye PKH67, and reinjected i.v. into AAV-VEGF₁₆₅-treated syngeneic mice. Several PKH67-labeled cells (green) were recruited to VEGF₁₆₅-expressing muscles, close to α-SMA–labeled arterioles (red). Conversely, very few siNP1ter-treated cells were found at the site of VEGF₁₆₅-induced angiogenesis, as quantified in the graphs on the right. Shown are means ± SD. n = 6. *P < 0.05. Scale bar: 50 μm.