Endothelial cells lining cerebral cavernous malformations (CCM) present strong adhesive and mechanical defects. Increased cell contractility is a driver to the onset and the expansion of the CCM lesions. 2D in vitro endothelial models have been developed from either endothelial cells isolated from ccm1-3 knock-out mice or CCM1-3-silenced primary endothelial cells. These in vitro models faithfully recapitulate the adhesive and contractile defects of the CCM-deficient endothelial cells such as increased cell–extracellular matrix (ECM) adhesion through β1 integrin-anchored actin stress fibers, abnormal remodeling of the ECM, and destabilized VE-cadherin-dependent cell–cell junctions. Using such 2D in vitro CCM models, we have shown that the ECM remodeled by CCM-depleted endothelial cells can propagate CCM-like adhesive defects to wild-type endothelial cells, a process potentially pertinent to CCM lesion expansion. Here, we detail methods for studying the morphology of focal adhesions, actomyosin cytoskeleton, and VE-cadherin-dependent Adherens junctions by immunofluorescence and morphometric analyses. Moreover, we detail the protocols to produce and purify remodeled ECM and to test its effect on endothelial cell adhesion.
CITATION STYLE
Manet, S., Vannier, D., Bouin, A. P., Lisowska, J., Albiges-Rizo, C., & Faurobert, E. (2020). Immunofluorescence of cell–cell and cell–extracellular matrix adhesive defects in in vitro endothelial CCM model: Juxtacrine role of mutant extracellular matrix on wild-type endothelial cells. In Methods in Molecular Biology (Vol. 2152, pp. 401–416). Humana Press Inc. https://doi.org/10.1007/978-1-0716-0640-7_29
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