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.

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.