Abstract
Here, we describe highly reproducible methods to investigate human EC invasion and sprouting behavior in 3D collagen matrices. Two assay models are presented whereby ECs are induced to sprout from a monolayer surface or from aggregated ECs suspended within a collagen gel matrix. In each case, the assays are performed using serum-free defined media containing a combination of five growth factors (Factors): FGF-2, SCF, IL-3, SDF-1α, and insulin. In both models, marked EC sprouting occurs with leading EC tip cells over a 12–24 h period. To illustrate their utility, we present data showing the influence of various pharmacologic inhibitors directed to membrane-type matrix metalloproteinases (MT-MMPs), protein kinase C alpha (PKCα), Src family kinases, and Notch-dependent signaling. Marked inhibition of sprouting is observed after blockade of MT-MMPs and PKCα, while strong increases in sprouting and EC tip cell number is observed following blockade of Src kinases, Notch signaling or both. Interestingly, the increased sprouting behavior observed following Src or Notch blockade directly correlates with a loss in the ability of ECs to form lumens. These defined in vitro assay models allow for a genetic and signaling dissection of EC tip cells vs. lumen forming ECs, which are both necessary for the formation of branching networks of tubes during vascular morphogenic events.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Davis GE, Stratman AN, Sacharidou A, Koh W (2011) Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting. Int Rev Cell Mol Biol 288:101–165. https://doi.org/10.1016/B978-0-12-386041-5.00003-0
Bowers SL, Norden PR, Davis GE (2016) Molecular signaling pathways controlling vascular tube morphogenesis and pericyte-induced tube maturation in 3D extracellular matrices. Adv Pharmacol 77:241–280. https://doi.org/10.1016/bs.apha.2016.04.005
Koh W, Stratman AN, Sacharidou A, Davis GE (2008) In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis. Methods Enzymol 443:83–101
Nakatsu MN, Hughes CC (2008) An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods Enzymol 443:65–82
Aplin AC, Fogel E, Zorzi P, Nicosia RF (2008) The aortic ring model of angiogenesis. Methods Enzymol 443:119–136
Iruela-Arispe ML, Davis GE (2009) Cellular and molecular mechanisms of vascular lumen formation. Dev Cell 16(2):222–231
Davis GE, Norden PR, Bowers SL (2015) Molecular control of capillary morphogenesis and maturation by recognition and remodeling of the extracellular matrix: functional roles of endothelial cells and pericytes in health and disease. Connect Tissue Res 56(5):392–402. https://doi.org/10.3109/03008207.2015.1066781
Sacharidou A, Stratman AN, Davis GE (2012) Molecular mechanisms controlling vascular lumen formation in three-dimensional extracellular matrices. Cells Tissues Organs 195(1-2):122–143. https://doi.org/10.1159/000331410
Stratman AN, Davis MJ, Davis GE (2011) VEGF and FGF prime vascular tube morphogenesis and sprouting directed by hematopoietic stem cell cytokines. Blood 117(14):3709–3719. https://doi.org/10.1182/blood-2010-11-316752
Smith AO, Bowers SL, Stratman AN, Davis GE (2013) Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions. PLoS One 8(12):e85147. https://doi.org/10.1371/journal.pone.0085147
Stratman AN, Malotte KM, Mahan RD, Davis MJ, Davis GE (2009) Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation. Blood 114(24):5091–5101
Stratman AN, Schwindt AE, Malotte KM, Davis GE (2010) Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization. Blood 116(22):4720–4730. https://doi.org/10.1182/blood-2010-05-286872
Stratman AN, Davis GE (2012) Endothelial cell-pericyte interactions stimulate basement membrane matrix assembly: influence on vascular tube remodeling, maturation, and stabilization. Microsc Microanal 18(1):68–80. https://doi.org/10.1017/S1431927611012402
Davis GE, Kim DJ, Meng CX, Norden PR, Speichinger KR, Davis MT, Smith AO, Bowers SL, Stratman AN (2013) Control of vascular tube morphogenesis and maturation in 3D extracellular matrices by endothelial cells and pericytes. Methods Mol Biol 1066:17–28. https://doi.org/10.1007/978-1-62703-604-7_2
Kim DJ, Norden PR, Salvador J, Barry DM, Bowers SLK, Cleaver O, Davis GE (2017) Src- and Fyn-dependent apical membrane trafficking events control endothelial lumen formation during vascular tube morphogenesis. PLoS One 12(9):e0184461. https://doi.org/10.1371/journal.pone.0184461
Sainson RC, Aoto J, Nakatsu MN, Holderfield M, Conn E, Koller E, Hughes CC (2005) Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis. FASEB J 19(8):1027–1029
Holderfield MT, Hughes CC (2008) Crosstalk between vascular endothelial growth factor, notch, and transforming growth factor-beta in vascular morphogenesis. Circ Res 102(6):637–652
Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8(6):464–478
Stratman AN, Saunders WB, Sacharidou A, Koh W, Fisher KE, Zawieja DC, Davis MJ, Davis GE (2009) Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices. Blood 114(2):237–247
Acknowledgments
This work was supported by NIH grants HL126518, HL128584, and HL136139 to G.E. Davis.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Salvador, J., Davis, G.E. (2018). Evaluation and Characterization of Endothelial Cell Invasion and Sprouting Behavior. In: Oliver, G., Kahn, M. (eds) Lymphangiogenesis. Methods in Molecular Biology, vol 1846. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8712-2_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8712-2_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8711-5
Online ISBN: 978-1-4939-8712-2
eBook Packages: Springer Protocols