Abstract
Macrophages play an important but poorly understood role in angiogenesis. To investigate their role in vessel formation, relevant in vivo models are crucial. Although the chick chorioallantoic membrane (CAM) model has been frequently used as an angiogenesis assay, limited data are available on the involvement of chicken macrophages in this process. Here, we describe a method to deplete macrophages in the ex ovo chick CAM assay by injection of clodronate liposomes and show that this depletion directly affects vascularisation of collagen onplants. Chicken embryos were injected intravenously with either clodronate or phosphate-buffered saline (PBS) liposomes, followed by placement of collagen type I plugs on the CAM to quantify angiogenic ingrowth. Clodronate liposome injection led to a significant 3.4-fold reduction of macrophages compared with control embryos as measured by immunohistochemistry and flow cytometry. Furthermore, analysis of vessel ingrowth into the collagen plugs revealed a significantly lower angiogenic response in macrophage-depleted embryos compared with control embryos, indicating that chicken embryonic macrophages play an essential function in the development of blood vessels. These results demonstrate that the chick CAM assay provides a promising model to investigate the role of macrophages in angiogenesis.
Highlights
Macrophages are best known as phagocytic cells that play a crucial role in the body’s immune response
To further enhance the potential of the chorioallantoic membrane (CAM) assay as angiogenesis model, we aimed to investigate the involvement of chicken embryonic macrophages in blood vessel development by means of macrophage depletion
On the third day after injection, a 3.4-fold decrease in the median count of KUL01-positive cells was observed in clodronate liposome-treated embryos compared with control embryos injected with phosphate-buffered saline (PBS) liposomes (** p < 0.001) (Figure 3a; Supplementary Table S1)
Summary
Macrophages are best known as phagocytic cells that play a crucial role in the body’s immune response. They act as antigen-presenting cells, produce inflammatory and antimicrobial cytokines and phagocytise bacteria and foreign objects. Macrophages possess a high plasticity and regulate processes such as angiogenesis, the development of new blood vessels from existing vasculature [1]. Accumulating evidence suggests that macrophages play an important but still not fully understood function during all the steps of the angiogenic cascade [3]. To study the involvement of macrophages in angiogenesis, relevant in vitro and in vivo models are required [4]
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