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

Abstract

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.

Revealing Early Steps of α2β1Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

“…At moderate force loading (ϳ500 pN/s), the ␣ 2 ␤ 1 /collagen unbinding force was 47 Ϯ 13 pN, similar to forces measured for other integrin-ligand interactions (for example, 39 Ϯ 8 pN for ␣ 5 ␤ 1 -fibronectin; Sun et al, 2005). The strength of ␣ 2 ␤ 1 integrin binding to collagen type I locates at a lower to middle position within the spectrum of known receptor-ligand unbinding forces (Weisel et al, 2003).…”

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

“…Assuming 10 fibers are placed under tension to hold the cell in place against this drag, the load on a single fiber/cell junction would be a mere 1.6 pN. By comparison, the bond-strength of a typical integrin-ligand interaction is 40 pN (Lehenkari and Horton, 1999;Sun et al, 2005), and fibroblasts appear to be capable of exerting 10 nN of force with a single focal adhesion on fibronectin (Tan et al, 2003). However, matrix tension from fluid drag on the matrix itself, which may in turn be transmitted to integrins, might be much larger than the direct effect on the cell.…”

The Plastic Nature of the Vascular Wall: A Continuum of Remodeling Events Contributing to Control of Arteriolar Diameter and Structure

“…One example of these extracellular components is fibronectin. The content of fibronectin in blood vessels is particularly important not only because it modifies the mean stress and elastic modulus of the wall (24) but because recent evidence suggests that fibronectin is closely associated with the capacity of arteriolar smooth muscle cells to detect and react to mechanical forces via integrin receptors (159,160) and with the capacity of contracting skeletal muscle to induce vasodilation of its adjacent vasculature (79). Thus changes in the amount of fibronectin in the vascular wall have the potential to significantly impact two functional characteristics of resistance vessels.…”