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 2
Inhibition of VEGF165-induced angiogenesis by Sema3A. (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.