Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice
Serena Zacchigna, … , Gianfranco Sinagra, Mauro Giacca
Serena Zacchigna, … , Gianfranco Sinagra, Mauro Giacca
Published May 15, 2008
Citation Information: J Clin Invest. 2008;118(6):2062-2075. https://doi.org/10.1172/JCI32832.
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Research Article Vascular biology

Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice

Abstract

Experimental and clinical evidence indicate that bone marrow cells participate in the process of new blood vessel formation. However, the molecular mechanisms underlying their recruitment and their exact role are still elusive. Here, we show that bone marrow cells are recruited to the sites of neoangiogenesis through the neuropilin-1 (NP-1) receptor and that they are essential for the maturation of the activated endothelium and the formation of arteries in mice. By exploiting adeno-associated virus vector–mediated, long-term in vivo gene expression, we show that the 165-aa isoform of VEGF, which both activates the endothelium and recruits NP-1+ myeloid cells, is a powerful arteriogenic agent. In contrast, neither the shortest VEGF121 isoform, which does not bind NP-1 and thus does not recruit bone marrow cells, nor semaphorin 3A, which attracts cells but inhibits endothelial activation, are capable of sustaining arterial formation. Bone marrow myeloid cells are not arteriogenic per se nor are they directly incorporated in the newly formed vasculature, but they contribute to arterial formation through a paracrine effect ensuing in the activation and proliferation of tissue-resident smooth muscle cells.

Authors

Serena Zacchigna, Lucia Pattarini, Lorena Zentilin, Silvia Moimas, Alessandro Carrer, Milena Sinigaglia, Nikola Arsic, Sabrina Tafuro, Gianfranco Sinagra, Mauro Giacca

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Figure 4

Fundamental role of NP-1 in CD11b+ cell recruitment by VEGF165 and Sema3A.

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Fundamental role of NP-1 in CD11b+ cell recruitment by VEGF165 and Sema3...
(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.