Mechanical properties of the interaction between fibronectin and alpha5beta1-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)-alpha5beta1 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 alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 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 alpha5beta1 and focal contact assembly can be rapidly regulated.

[1]  Kayla J Bayless,et al.  Sphingosine-1-phosphate markedly induces matrix metalloproteinase and integrin-dependent human endothelial cell invasion and lumen formation in three-dimensional collagen and fibrin matrices. , 2003, Biochemical and biophysical research communications.

[2]  M. Bryckaert,et al.  Platelet-derived Growth Factor Inhibits Smooth Muscle Cell Adhesion to Fibronectin by ERK-dependent and ERK-independent Pathways* , 2001, The Journal of Biological Chemistry.

[3]  Richard T. Lee,et al.  Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  G. Davis,et al.  Modulation of Calcium Current in Arteriolar Smooth Muscle by αvβ3 and α5β1 Integrin Ligands , 1998, The Journal of cell biology.

[5]  Harold P. Erickson,et al.  Force Measurements of the α5β1 Integrin–Fibronectin Interaction , 2003 .

[6]  H. Gaub,et al.  Unfolding forces of titin and fibronectin domains directly measured by AFM. , 2000, Advances in experimental medicine and biology.

[7]  M C Davies,et al.  The influence of epitope availability on atomic-force microscope studies of antigen-antibody interactions. , 1999, The Biochemical journal.

[8]  A. Clowes,et al.  Regulation and function of an activation-dependent epitope of the beta 1 integrins in vascular cells after balloon injury in baboon arteries and in vitro. , 1996, The American journal of pathology.

[9]  M. Ginsberg,et al.  Integrin Activation , 2001, Thrombosis and Haemostasis.

[10]  Daniel Choquet,et al.  Extracellular Matrix Rigidity Causes Strengthening of Integrin–Cytoskeleton Linkages , 1997, Cell.

[11]  H. Gaub,et al.  Intermolecular forces and energies between ligands and receptors. , 1994, Science.

[12]  Donald E Ingber,et al.  Mechanical properties of individual focal adhesions probed with a magnetic microneedle. , 2004, Biochemical and biophysical research communications.

[13]  G. Davis,et al.  Integrins and mechanotransduction of the vascular myogenic response. , 2001, American journal of physiology. Heart and circulatory physiology.

[14]  M. Pfaff,et al.  Integrin affinity modulation. , 1998, Trends in cell biology.

[15]  Andrés J. García,et al.  Distinct activation states of α5β1 integrin show differential binding to RGD and synergy domains of fibronectin , 2002 .

[16]  J. Trempe Molecular biology of the cell, 3rd edition Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts and James D. Watson, Garland Publishing, 1994, 559.95 (xiii + 1294 pages), ISBN 0-815-31619-4 , 1995, Trends in Endocrinology & Metabolism.

[17]  J. Weisel,et al.  Binding strength and activation state of single fibrinogen-integrin pairs on living cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M A Horton,et al.  Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy. , 1999, Biochemical and biophysical research communications.

[19]  Michael A. Hill,et al.  Integrins as Unique Receptors for Vascular Control , 2003, Journal of Vascular Research.

[20]  R M Hochmuth,et al.  Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane. , 1999, Biophysical journal.

[21]  Hermann E. Gaub,et al.  Discrete interactions in cell adhesion measured by single-molecule force spectroscopy , 2000, Nature Cell Biology.

[22]  R. Merkel,et al.  Energy landscapes of receptor–ligand bonds explored with dynamic force spectroscopy , 1999, Nature.

[23]  K. Burridge,et al.  Focal adhesions, contractility, and signaling. , 1996, Annual review of cell and developmental biology.

[24]  D. Ingber,et al.  Mechanotransduction across the cell surface and through the cytoskeleton , 1993 .