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Matthew Petroll, Miguel Miron Mendoza, Pouriska Kivanany, Eric Graham; Differential Regulation of Corneal Fibroblast Spreading and Migration Mechanics by Collagen and Fibrin Extracellular Matrices. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4606.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we reported that cells interacting with collagen matrices develop dendritic processes and move independently, whereas cells interacting with fibrin matrices develop stress fibers, move more slowly and form an interconnected meshwork. In this study, we investigate the roles of cell contractility and fibronectin in mediating these differences.
To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen and fibrin matrices. To assess 3-D cell migration, compacted cell-populated collagen matrices were embedded inside cell-free collagen or fibrin matrices. Constructs were cultured in serum-free media containing PDGF, with or without thrombin, the Rho kinase inhibitor Y-27632 and/or the myosin II inhibitor blebbistatin. In some experiments, anti-fibronectin antibody HFN 7.1 or an RGD peptide were used to block fibronectin-alpha5beta1 integrin binding. Quantitative 3-D and 4-D imaging were used to assess cell mechanical behavior, connectivity and cytoskeletal organization.
Thrombin stimulated increased contractility of corneal fibroblasts, as indicated by the development of stress fibers, and increased matrix displacement and reorganization. Thrombin also induced clustering of cells plated on top of 3-D collagen matrices, which was blocked by Y-27632 and Blebbistatin. In contrast, cells on fibrin matrices coalesced into clusters under all conditions tested. In nested matrices both Y-27632 and blebbistatin reduced the 3-D migration rate. However, migration patterns did not change - cells migrated collectively in fibrin but moved independently in collagen, even in the presence of thrombin. Cells migrating into fibrin matrices produced tracks of extracellular fibronectin. Interfering with fibronectin-alpha5beta1 integrin binding blocked cell attachment to fibrin matrices, but had no effect on cells cultured on collagen.
The data suggest that while increased contractility can induce fibroblast clustering on compliant collagen matrices, it is not required for collective spreading and migration of cells interacting with fibrin. We propose a model in which secreted fibronectin mediates cell attachment to fibrin via alpha5beta1 integrin, which leads to the formation of fibronectin conduits for 3-D interconnected cell streaming, as well as areas of enhanced adhesivity that promote 2-D cell clustering.
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