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.
Methods Mol Biol
; 2152: 401-416, 2020.
Article
em En
| MEDLINE
| ID: mdl-32524568
ABSTRACT
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 ß1 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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
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Tipos_de_cancer
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Outros_tipos
Base de dados:
MEDLINE
Assunto principal:
Endotélio Vascular
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Comunicação Celular
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Hemangioma Cavernoso do Sistema Nervoso Central
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Células Endoteliais
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Matriz Extracelular
Limite:
Animals
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Humans
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
França