Fig. 4. Macrophage depletion prevents activation of the vasculogenic zone and generation of CD31⁺ endothelial cells in ARA.

a Cross-sections of murine aortae were immunostained for the progenitor cell marker CD34. In FIA, a large number of CD34⁺ progenitor cells was present within the vasculogenic zone of the adventitia. By performing ARA, these cells were activated within their adventitial stem cell niche and differentiated thereby losing their CD34 immunoreactivity. Depletion of macrophages preserved the number of CD34⁺ cells within the adventitia during ARA. b Statistical analysis of the number of adventitial CD34⁺ cells with and without liposome treatment. Cell counts were normalized to the total media area (n = 6–12). c Cross-sections of murine aortae were immunostained for the endothelial cell marker CD31. In FIA, CD31⁺ cells were present within the intima as expected (arrowhead), but were almost completely absent from the adventitia. After ARA, a substantial number of CD31⁺ cells appeared within the adventitia (arrows). Their generation was largely prevented by the depletion of macrophages. d Statistical analysis of the number of adventitial CD31⁺ cells with and without liposome treatment. Cell counts were normalized to total media area (n = 3–10). e Cross-section of a cultured aortic ring at day 3 immunostained for CD34 and CD31. CD34⁺/CD31⁺ cells represent a transitional stage in the differentiation of CD34⁺ adventitial progenitors towards CD31⁺ endothelial cells. *P < 0.05; ***P < 0.001. Scale bars: in a and c 100 µm, in e 50 µm. FIA fresh isolated aorta, ARA aortic ring assay, ARA + CL ARA with clodronate-containing liposomes, ARA + PBS ARA with PBS-containing liposomes.