Abstract
Endothelial cell tube formation assay is one of the most widely used and reliable methods for studying in vitro angiogenesis. Endothelial cells plated over a basement membrane extract and subjected to angiogenic factors in conditioned medium, form a rapid and quantifiable tube network within hours. Tube formation is sustained for 18–24 h, after which time apoptosis occurs and tube networks disintegrate. The tube network can be imaged using a phase contrast microscope, or upon Calcein-AM treatment, a fluorescence/confocal microscope. This assay has several advantages, namely: ease of set up, the ability to test numerous angiogenic/anti-angiogenic factors simultaneously, quick network formation, ability to view live or fixed tube networks, and quantifiability. To ensure successful results and limit variability, proper selection of basement membrane extracts and endothelial cells is necessary, and conditions must be optimized. In summary, this assay is a useful method for screening potential angiogenic/anti-angiogenic factors as well as identifying critical mechanisms and signaling pathways underlying angiogenic-related pathologies.
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References
Eelen G, Treps L, Li X, Carmeliet P (2020) Basic and therapeutic aspects of angiogenesis updated. Circ Res 127:310–329
Yoo SY, Kwon SM (2013) Angiogenesis and its therapeutic opportunities. Mediat Inflamm 2013:127170
Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1:27–31
Krüger-Genge A, Blocki A, Franke RP, Jung F (2019) Vascular endothelial cell biology: an update. Int J Mol Sci 20:4411
Zhao Y, Adjei AA (2015) Targeting angiogenesis in cancer therapy: moving beyond vascular endothelial growth factor. Oncologist 20:660–673
Folkman J, Haudenschild C (1980) Angiogenesis in vitro. Nature 288:551–556
Folkman J, Haudenschild C (1980) Angiogenesis by capillary endothelial cells in culture. Trans Ophthalmol Soc U K 100:346–353
Maciag T, Kadish J, Wilkins L, Stemerman MB, Weinstein R (1982) Organizational behavior of human umbilical vein endothelial cells. J Cell Biol 94:511–520
Kubota Y, Kleinman HK, Martin GR, Lawley TJ (1988) Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 107:1589–1598
DeCicco-Skinner KL et al (2014) Endothelial cell tube formation assay for the in vitro study of angiogenesis. J Vis Exp (91):e51312
Carpentier G (2012) ImageJ contribution: angiogenesis analyzer. ImageJ News
Acknowledgments
This work was supported by grant #2R15CA152907-02. U.S. Department of Health & Human Services, National Institutes of Health (NIH).
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Kelley, M., Fierstein, S., Purkey, L., DeCicco-Skinner, K. (2022). Endothelial Cell Tube Formation Assay: An In Vitro Model for Angiogenesis. In: Fiedler, L.R., Pellet-Many, C. (eds) VEGF Signaling. Methods in Molecular Biology, vol 2475. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2217-9_12
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DOI: https://doi.org/10.1007/978-1-0716-2217-9_12
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