Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a fundamental process in developmental biology but also a pathological event that initiates or aggravates many diseases. In this complex multistep process, endothelial cells are activated by angiogenic stimuli; undergo specialization in response to VEGF/Notch signaling; degrade the basement membrane of the parent vessel; sprout, migrate, and proliferate to form capillary tubes that branch; and ultimately anastomose with adjacent vessels. Here we describe an assay that mimics the invasion step in vitro. Human microvascular endothelial cells are confronted by a VEGF-enriched basement membrane material in a three-dimensional environment that promotes endothelial cell sprouting, tube formation, and anastomosis. After a few hours, endothelial cells have become tip cells, and vascular sprouts can be observed by phase contrast, fluorescence, or time-lapse microscopy. Sprouting endothelial cells express tip cell markers, display podosomes and filopodia, and exhibit cell dynamics similar to those of angiogenic endothelial cells in vivo. This model provides a system that can be manipulated genetically to study physiological or pathological angiogenesis and that can be used to screen compounds for pro-/anti-angiogenic properties. In this chapter, we describe the key steps in setting up this assay.
CITATION STYLE
Dong, Y., Alonso, F., Jahjah, T., Fremaux, I., & Génot, E. (2023). Angiogenesis Invasion Assay to Study Endothelial Cell Invasion and Sprouting Behavior. In Methods in Molecular Biology (Vol. 2608, pp. 345–364). Humana Press Inc. https://doi.org/10.1007/978-1-0716-2887-4_20
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