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M. K. Schwinn, J. M. Gonzalez, Jr., D. M. Peters; Integrin Co-Signaling Influences Contractility in Trabecular Meshwork (TM) Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3223. doi: https://doi.org/.
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The aim of this study was to determine if co-signaling between α1β1, α2β1, α4β1, and α5β1integrins regulates the contractile properties of TM cells.
Integrin binding studies were performed using a colorimetric assay to determine the number of TM cells bound to type IV collagen or fibronectin in the presence or absence of 25 µg/ml β1, α1, α2, α5, and αVβ3 integrin blocking antibodies. To visually assess TM contractility, confluent or subconfluent cultures were incubated with or without 0.5 mg/ml of the Heparin II (HepII) binding domain of fibronectin and changes in actin filaments were observed by fluorescence microscopy with Alexa 488-phalloidin. In some experiments, cells were grown on varying concentrations of fibronectin or type IV collagen. In other experiments, cells were grown on glass coverslips with only endogenous matrix present. Contractility was quantitated by incubating TM cells grown on 1.25 mg/ml rat tail collagen I gels for 24 hrs with 0.5-4 mg/ml of HepII or the PPRARI and IDAPS peptides. Gels were then detached from the dishes and their diameters were measured at 1, 6, and 24 hrs.
Antibodies to α1β1 and α2β1 integrins blocked binding of TM cells to type IV collagen while antibodies to α5β1 integrins blocked binding to fibronectin. The α4β1-activating HepII domain disrupted actin filaments in both confluent TM cells plated on coverslips and subconfluent cultures plated on collagen IV. In contrast, TM cells plated on increasing concentrations of fibronectin or the III7-10 repeat did not respond to HepII treatment. TM cells treated with HepII and the PPRARI peptide also reduced collagen I gel contraction by nearly 40% compared to untreated controls.
Contractile properties of TM cells are determined by integrin co-signaling and the composition of the extracellular matrix. Activation of α4β1 and α1/α2/β1 integrins by the HepII domain and type IV collagen leads to a disruption of actin filaments. In contrast, activation of α4β1 and α5β1 integrins by HepII and fibronectin increases stress fiber formation. This suggests that upregulation of fibronectin expression could alter actin dynamics and contribute to glaucoma.
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