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PROTOCOL
Use of the mouse aortic ring assay to study angiogenesis
Marianne Baker1, Stephen D Robinson2, Tanguy Lechertier1, Paul R Barber3, Bernardo Tavora1, Gabriela DAmico4, Dylan T Jones1, Boris Vojnovic3 & Kairbaan Hodivala-Dilke1
1Adhesion and Angiogenesis Laboratory, Centre for Tumour Biology, Barts Cancer Institutea CR-UK Centre of Excellence, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London, UK. 2School of Biological Sciences, University of East Anglia, Norwich, UK. 3Department of Oncology, Gray Institute for Radiation Oncology & Biology, University of Oxford, Oxford, UK. 4Molecular/Cancer Biology Program, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland. Correspondence should be addressed to M.B. ([email protected]).
Published online 22 December 2011; doi:10.1038/nprot.2011.435
Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ringassay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodelingall without the need for cellular dissociationthus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 614 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.
2011 Nature America,Inc. All rights reserved.
INTRODUCTION
Angiogenesis, the growth of new blood vessels from pre-existing ones, is a highly complex process involving a range of cell types and signaling pathways. Angiogenic processes involve the stimulation of endothelial cells by several different pro-angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and others14. These induce changes in a multitude of downstream signaling pathways involved in the control of neo- vessel sprouting and branching. However, many of these pathways are poorly understood. As with many essential physiological processes, angiogenesis is normally tightly regulated but is disrupted in many pathological conditions, including cancer, diabetes, arthritis and ischemic heart disease5.
In vivo analysis of angiogenesis currently involves models...