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Methods for investigating integrin‐matrix interactions as a function of matrix mechanics and composition
Author(s) -
Sazonova Olga,
Nugent Matthew A,
Wong Joyce Y
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1122.1
Subject(s) - fibronectin , integrin , stiffness , elastic modulus , biophysics , polyacrylamide , matrix (chemical analysis) , chemistry , extracellular matrix , cell mechanics , materials science , cell , composite material , polymer chemistry , biochemistry , cytoskeleton , biology
To investigate the role of mechanics in cell signaling, we have engineered polyacrylamide (PAAM) gels with precisely tunable stiffness and fibronectin (FN) content. Varying the acrylamide / bis ratio alters PAAM stiffness, as determined with tensile testing and atomic force microscopy (AFM). To promote cell attachment, FN is covalently linked throughout the gel, and can then be quantified with an ELISA. Analyzing AFM data with the Hertz and Oliver‐Pharr models, we calculated gel elastic moduli ranging from 11 – 344 kPa and 7.5 – 95 kPa. We also found that stiffer gels incorporate more FN than softer gels, and that adding FN reduces the modulus of softer gels by ~10%. Controlling for these factors allowed for precise and independent changes in gel mechanics and FN content. To study integrin‐FN interactions on PAAM gels, we adapted published methods for quantifying integrin binding. After vascular smooth muscle cells (VSMCs) were grown on gels or FN‐coated plastic, bound FN‐alpha5 integrin units were cross‐linked and assayed with an ELISA. Preliminary data suggest that integrin activity per cell is reduced with decreasing stiffness or higher cell density. Future studies using the methods presented herein will provide insight into how VSMCs recognize and respond to changes in ECM composition and stiffness, aiding the rational design of new treatment for vascular disease. Supported by NIH grants HL56200 and HL72900.