Dataset Information

Impaired Contracture of 3D Collagen Constructs by Fibronectin-Deficient Murine Fibroblasts.

ABSTRACT: Fibronectin (FN) is an extracellular matrix glycoprotein that is abundantly expressed by fibroblasts in contracting wounds, where it mediates cell adhesion, migration and proliferation. FN also efficiently binds to collagen. Therefore, we and others hypothesized that FN and its cellular receptor integrin ?5?1 might be involved in collagen matrix contracture by acting as linkers. However, there are conflicting reports on this issue. Moreover, several publications suggest an important role of collagen-binding integrin receptors ?2?1 and ?11?1 in collagen matrix contracture. Therefore, the aim of the present study was to determine the contributions of FN-integrin ?5?1 interactions relative to those of collagen receptors ?2?1 and ?11?1 in this process. To assess the role of cellular FN directly, we employed FN-deficient mouse fibroblasts, subjected them to a collagen gel contracture assay in vitro, and compared them to their wildtype counterparts. Exogenous FN was removed from serum-containing medium. For dissecting the role of major collagen receptors, we used two FN-deficient mouse fibroblast lines that both possess integrin ?5?1 but differ in their collagen-binding integrins. Embryo-derived FN-null fibroblasts, which express ?11- but no ?2-integrin, barely spread and tended to cluster on collagen gels. Moreover, FN-null fibroblasts required exogenously added FN to assemble ?5?1-integrin in fibrillar adhesion contacts, and to contract collagen matrices. In contrast, postnatal kidney fibroblasts were found to express ?2- but barely ?11-integrin. When FN expression was suppressed in these cells by shRNA transfection, they were able to spread on and partially contract collagen gels in the absence of exogenous FN. Also in this case, however, collagen contracture was stimulated by adding FN to the medium. Antibody to integrin ?5?1 or RGD peptide completely abolished collagen contracture by FN-deficient fibroblasts stimulated by FN addition. We conclude that although collagen-binding integrins (especially ?2?1) can mediate contracture of fibrillar collagen gels by murine fibroblasts to some extent, full activity is causally linked to the presence of pericellular FN and its receptor integrin ?5?1.

SUBMITTER: Beyeler J

PROVIDER: S-EPMC6413635 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Impaired Contracture of 3D Collagen Constructs by Fibronectin-Deficient Murine Fibroblasts.

Beyeler Joël J Katsaros Christos C Chiquet Matthias M

Frontiers in physiology 20190305

Fibronectin (FN) is an extracellular matrix glycoprotein that is abundantly expressed by fibroblasts in contracting wounds, where it mediates cell adhesion, migration and proliferation. FN also efficiently binds to collagen. Therefore, we and others hypothesized that FN and its cellular receptor integrin α<sub>5</sub>β<sub>1</sub> might be involved in collagen matrix contracture by acting as linkers. However, there are conflicting reports on this issue. Moreover, several publications suggest an ...[more]

PMID: 30890950

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Project description:Changes in extracellular matrix stiffness impact a variety of biological processes including cancer progression. However, cells also actively remodel the matrices they interact with, dynamically altering the matrix mechanics they respond to. Further, cells not only react to matrix stiffness, but also have a distinct reaction to matrix viscoelasticity. The impact of cell-driven matrix remodeling on matrix stiffness and viscoelasticity at the microscale remains unclear, as existing methods to measure mechanics are largely at the bulk scale or probe only the surface of matrices, and focus on stiffness. Yet, establishing the impact of the matrix remodeling at the microscale is crucial to obtaining an understanding of mechanotransduction in biological matrices, and biological matrices are not just elastic, but are viscoelastic. Here, we advanced magnetic probe-based microrheology to overcome its previous limitations in measuring viscoelasticity at the cell-size-scale spatial resolution within 3D cell cultures that have tissue-relevant stiffness levels up to a Young's modulus of 0.5 kPa. Our magnetic microrheometers exert controlled magnetic forces on magnetic microprobes within reconstituted extracellular matrices and detect microprobe displacement responses to measure matrix viscoelasticity and determine the frequency-dependent shear modulus (stiffness), the loss tangent, and spatial heterogeneity. We applied these tools to investigate how microscale viscoelasticity of collagen matrices is altered by fibroblast cells as they contract collagen gels, a process studied extensively at the macroscale. Interestingly, we found that fibroblasts first soften the matrix locally over the first 32 hours of culture, and then progressively stiffen the matrix thereafter. Fibroblast activity also progressively increased the matrix loss tangent. We confirmed that the softening is caused by matrix-metalloproteinase-mediated collagen degradation, whereas stiffening is associated with local alignment and densification of collagen fibers around the fibroblasts. This work paves the way for the use of measurement systems that quantify microscale viscoelasticity within 3D cell cultures for studies of cell-matrix interactions in cancer progression and other areas.

Dataset Information

Impaired Contracture of 3D Collagen Constructs by Fibronectin-Deficient Murine Fibroblasts.

Publications

Impaired Contracture of 3D Collagen Constructs by Fibronectin-Deficient Murine Fibroblasts.

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