Depletion of Embryonic Macrophages Leads to a Reduction in Angiogenesis in the Ex Ovo Chick Chorioallantoic Membrane Assay

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.

Evaluation of Angiogenesis in an Acellular Porous Biomaterial Based on Polyhydroxybutyrate and Chitosan Using the Chicken Ex Ovo Chorioallantoic Membrane Model

“…Chicken embryonic macrophages play an important role in angiogenesis. Earlier study with the chicken CAM model indicated the importance of macrophages for neovascularization of implants on the CAM [ 28 ]. We conclude that their presence is a sign of new vessels formation in the surrounding area of implanted PHB/CHIT scaffold.…”

“…WGA bounds well to the luminal surface of vessels; therefore, it can be effectively used as a marker for the visualization of the vessels of early stages of the embryo development [ 58 ]. An important role in the body immune response and in developmental angiogenesis of the embryo is undertaken by macrophages, which have been demonstrated to express pro-angiogenic markers [ 28 ]. Macrophages are found when angiogenesis and inflammation occur [ 60 ].…”

“…Avian embryos, especially their chorioallantoic membrane (CAM), can be used as a valuable pre-screening assay to determine the angiogenic potential and initial tissue responses of graft materials before more extensive in vitro studies in mammals, especially in the case when multiple experimental conditions need to be tested [ 26 ]. The avian CAM is an extraembryonic membrane formed on the fourth embryonic day (ED4) by the fusion of two extraembryonic membranes, the chorion and the allantois [ 1 , 5 , 27 , 28 ]. The major functions of the CAM are to support extraembryonic respiratory capillaries, transport sodium and chloride to the allantoic cavity and calcium from the eggshell, and maintain the acid-base balance [ 29 , 30 ].…”

Identification of new markers of angiogenic sprouting using transcriptomics: New role for RND3

“…Angiogenesis is well-accepted to occur alongside inflammation. 80 Resident cells such as macrophages, dendritic cells, lymphocytes, and microglia promote angiogenesis and anastomosis during zebrafish development, 81 collateral vessel growth, 82 macular degeneration, 83 chick CAM assays, 84,85 retinal angiogenesis, 81 and developmental and pathological angiogenesis. 86,87 Leukocytes secrete angiogenic factors, including chemokines and VEGF.…”

Emerging role of exosomes in cancer progression and tumor microenvironment remodeling

“…Recruitment of macrophages can induce angiogenesis to enhance nerve regeneration [ 133 ]. In addition, reduction or depletion of macrophages suppresses angiogenesis [ 134 ]. Tumor-derived exosomes (TEX) are capable of carrying CD39/CD73 and adenosine, which are enzymatically active.…”