Abstract
The migration of endothelial cells (ECs) play an important role in embryonic vasculogenesis, angiogenesis, and post-angioplasty reendothelialization. ECs are constantly subjected to fluid shear stress (the tangential component of hemodynamic forces) from blood flow, but the effects of shear stress on EC migration have not been well characterized until recently. We have used an in vitro flow system to apply shear stress to EC cultures and used imaging and biochemical techniques to analyze EC migration under well-defined flow conditions. Time-lapse microscopy and cell tracing analysis generated quantitative information on EC migration under flow, and this assay was used to dissect the signaling events involved in shear stress-induced EC migration. By expressing green fluorescent protein-tagged molecules in EC, cell focal adhesions and cytoskeleton were visualized and quantified in living cells under flow, which provides temporal and spatial resolution for mechanotransduction at the molecular level. The studies on EC migration under flow will advance our understandings on vascular repair and remodeling in vivo, and provide a rational basis for the development of strategy to promote endothelialization and vascularization in tissue-engineered constructs.
Key Words
- Endothelial cell
- migration
- flow
- shear stress
- time-lapse microscopy
- green fluorescent protein (GFP)
- molecular dynamics
- focal adhesion
- cytoskeleton
This is a preview of subscription content, access via your institution.
Buying options
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Lauffenburger, D. A. and Horwitz, A. F. (1996) Cell migration: a physically integrated molecular process. Cell 84, 359–369.
Sheetz, M. P., Felsenfeld, D. P., and Galbraith, C. G. (1998) Cell migration: regulation of force on extracellular-matrix-integrin complexes. Trends Cell Biol. 8, 51–54.
Albuquerque, M. L., Waters, C. M., Savla, U., Schnaper, H. W., and Flozak, A. S. (2000) Shear stress enhances human endothelial cell wound closure in vitro. Am. J. Physiol. Heart Circ. Physiol. 279, H293–H302.
Hsu, P. P., Li, S., Li, Y. S., Usami, S., Ratcliffe, A., Wang, X., et al. (2001) Effects of flow patterns on endothelial cell migration into a zone of mechanical denudation. Biochem. Biophys. Res. Commun. 285, 751–759.
Hu, Y. L., Li, S., Miao, H, Tsou, T. C, del Pozo, M. A., and Chien, S. (2002) Roles of microtubule dynamics and small GTPase rac in endothelial cell migration and lamellipodium formation under flow. J. Vase. Res. 39, 465–476.
Li, S., Butler, P., Wang, Y., Hu, Y., Han, D. C, Usami, S., et al. (2002) The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells. Proc. Natl. Acad. Sci. USA 99, 3546–3551.
Wojciak-Stothard, B. and Ridley, A. J. (2003) Shear stress-induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases. J. Cell Biol. 161, 429–439.
Chen, K. D., Li, Y. S., Kim, M., Li, S., Yuan, S., Chien, S., et al. (1999) Mechano-transduction in response to shear stress-roles of receptor tyrosine kinases, integrins, and Shc. J. Biol. Chem. 274, 18,393–18,400.
Tzima, E., Del Pozo, M. A., Kiosses, W. B., Mohamed, S. A., Li, S., Chien, S., and Schwartz, M. A. (2002) Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression. EMBO J. 21, 6791–6800.
Jalali, S., del Pozo, M. A., Chen, K., Miao, H, Li, Y., Schwartz, M. A., Shyy, J. Y., et al. (2001) Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands. Proc. Natl. Acad. Sci. USA 98, 1042–1046.
Urbich, C, Dernbach, E., Reissner, A., Vasa, M., Zeiher, A. M., and Dimmeler, S. (2002) Shear stress-induced endothelial cell migration involves integrin signaling via the fibronectin receptor subunits alpha(5) and beta(1). Arterioscler. Thromb. Vase. Biol. 22, 69–75.
Albuquerque, M. L. and Flozak, A. S. (2001) Patterns of living beta-actin movement in wounded human coronary artery endothelial cells exposed to shear stress. Exp. Cell Res. 270, 223–234.
Helmke, B. P., Goldman, R. D., and Davies, P. F. (2000) Rapid displacement of vimentin intermediate filaments in living endothelial cells exposed to flow. Circ. Res. 86, 745–752.
Li, S., Kim, M., Hu, Y. L., Jalali, S., Schlaepfer, D. D., Hunter, T., et al. (1997) Fluid shear stress activation of focal adhesion kinase—Linking to mitogen-activated protein kinases. J. Biol. Chem. 272, 30,455–30,462
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Li, S. (2005). Analysis of Endothelial Cell Migration Under Flow. In: Guan, JL. (eds) Cell Migration. Methods in Molecular Biology™, vol 294. Humana Press. https://doi.org/10.1385/1-59259-860-9:107
Download citation
DOI: https://doi.org/10.1385/1-59259-860-9:107
Publisher Name: Humana Press
Print ISBN: 978-1-58829-382-4
Online ISBN: 978-1-59259-860-1
eBook Packages: Springer Protocols