Mechanical properties of the interaction between fibronectin and α5β1-integrin on vascular smooth muscle cells studied using atomic force microscopy

The mechanical properties of integrin- extracellular matrix ( ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells ( VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin ( FN)-alpha(5)beta(1) interaction on the surface of VSMC using atomic force microscopy ( AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN- VSMC adhesions were selectively blocked by antibodies against alpha(5)- and beta(1)- integrins as well as RGD-containing peptides but not by antibodies against alpha(4)- and beta(3)- integrins, indicating that FN primarily bound to alpha(5)beta(1). A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha(5)beta(1) single- bond strength. The ability of FN to adhere to VSMC ( binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet- derived growth factor ( PDGF)BB, whereas lysophosphatidic acid ( LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time- dependent increase in number of adhesions and/ or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin- ECM interactions and their regulation. Our results indicate that the functional activity of alpha(5)beta(1) and focal contact assembly can be rapidly regulated.