Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice
Serena Zacchigna, Lucia Pattarini, Lorena Zentilin, Silvia Moimas, Alessandro Carrer, Milena Sinigaglia, Nikola Arsic, Sabrina Tafuro, Gianfranco Sinagra, Mauro Giacca
Serena Zacchigna, Lucia Pattarini, Lorena Zentilin, Silvia Moimas, Alessandro Carrer, Milena Sinigaglia, Nikola Arsic, Sabrina Tafuro, Gianfranco Sinagra, Mauro Giacca
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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 2

Inhibition of VEGF165-induced angiogenesis by Sema3A.

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Inhibition of VEGF165-induced angiogenesis by Sema3A. (A) Staining with ...
(A) Staining with an α-CD31 antibody revealed massive angiogenic sprouting induced by VEGF165 (upper panel), the absence of new capillary formation in Sema3A-expressing muscles (middle panel), and a potent effect of Sema3A in counteracting VEGF165-induced angiogenesis (lower panel). Scale bars: 100 μm. (B) Immunohistochemistry using an anti–α-SMA antibody shows the progressive formation of arteries over time in muscles overexpressing VEGF165 (upper panels), whereas new arteries were generated in response to Sema3A (middle panels). The combined expression of VEGF165 with Sema3A resulted in an almost complete inhibition of the VEGF165-induced angiogenic effect (lower panels). Scale bars: 100 μm. (C) The capacity of Sema3A to counteract the increase in vascular volume driven by VEGF165 was assessed by fluorescent microspheres at both 1 and 3 months after vector delivery. Data are presented as a ratio between the emission value of the treated and the contralateral mock-treated muscle. Shown are means ± SD. *P < 0.05. (D) The area occupied by endothelial cells, as measured by anti-CD31 fluorescent labeling, was quantified in muscles injected with AAV- VEGF165 and AAV- VEGF121, either alone or in combination with AAV-Sema3A. Shown are means ± SD. (E) The presence of apoptotic endothelial cells (white arrowheads) is shown by double labeling for the endothelial marker CD31 and for the apoptotic marker cleaved caspase-3 in muscles injected with AAV- VEGF165 and AAV-Sema3A. Right panels show the blue/green (upper) and blue/red (lower) channels of the image. Insets show higher magnification. Scale bar: 100 μm.