Vascular smooth muscle cell durotaxis depends on extracellular matrix composition

Abstract

Mechanical compliance has been demonstrated to be a key determinant of cell behavior, directing processes such as spreading, migration, and differentiation. Durotaxis, directional migration from softer to more stiff regions of a substrate, has been observed for a variety of cell types. Recent stiffness mapping experiments have shown that local changes in tissue stiffness in disease are often accompanied by an altered ECM composition in vivo. However, the importance of ECM composition in durotaxis has not yet been explored. To address this question, we have developed and characterized a polyacrylamide hydrogel culture platform featuring highly tunable gradients in mechanical stiffness. This feature, together with the ability to control ECM composition, allows us to isolate the effects of mechanical and biological signals on cell migratory behavior. Using this system, we have tracked vascular smooth muscle cell migration in vitro and quantitatively analyzed differences in cell migration as a function of ECM composition. Our results show that vascular smooth muscle cells undergo durotaxis on mechanical gradients coated with fibronectin but not on those coated with laminin. These findings indicate that the composition of the adhesion ligand is a critical determinant of a cell's migratory response to mechanical gradients.durotaxis | cell migration | extracellular matrix | polyacrylamide C ell migration is essential to numerous biological processes, including development, angiogenesis, wound healing, and cancer metastasis (1-4). The movement of cells in these processes is determined by a complex assessment of environmental cues that include soluble factors, ECM composition, orientation, and stiffness. Numerous experiments have demonstrated that directional cell migration can result from gradients in these environmental cues: for example, chemotaxis (cell migration in response to gradients of soluble signals) and haptotaxis (cell migration in response to gradients of bound ligands) have been established in both in vitro and in vivo experimental systems (5-7). More recently, it has been demonstrated that cells are also capable of directed migration in response to gradients in substrate stiffness, a process termed durotaxis (8). Though there have been limited reports of specifically measured in vivo gradients (9), a number of recent stiffness mapping measurements imply the presence of stiffness gradients in both healthy and diseased tissues spanning a wide range of stiffnesses (10-13). In vitro experiments have demonstrated that directed migration in response to stiffness gradients can be observed in numerous cell types, using various materials as substrates, and across various stiffness levels (8,(14)(15)(16)(17)(18)(19). However, the role of ECM composition in mediating this behavior has not been thoroughly investigated.The interplay between mechanical stiffness and matrix composition in normal and pathological physiology is only now becoming appreciated. Recent studies in which tissue stiffness was mapped by atomic fo...

Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels

“…Previous methods of reproducing in vivo stiffness gradients in culture systems are either complex (19,30,34,36,37) or lack the stiffness or gradient range (9,30,32) to interrogate the physiological mechanical landscape fully (Table S1). Therefore, we developed a method to fabricate planar PA hydrogels with linear stiffness gradients capable of spanning both biologically relevant ranges and disease conditions.…”

“…Indeed, studies performed with gradient photomasks for UV photopolymerization have shown poor reproducibility and gradient linearity (19). Recent work has demonstrated the ability to build stiffness gradients in PA based on diffusion-driven gradients, although these methods require photolithography and microfluidics (34). Thus, although simple methods exist to produce chemical gradients (4)(5)(6), protocols to create simple (i.e., not requiring a clean room or photolithography facility) and highly reproducible [i.e., not relying on photopolymerization or PDMS interfaces] linear mechanical gradients spanning all biologically relevant ranges remain elusive (Table S1) (15,35).…”

Extracellular matrix type modulates cell migration on mechanical gradients

“…Polyacrylamide gels featuring gradients in mechanical compliance between uniform stiffness control regions were prepared as previously described [37]. Briefly, gradient generator slides were prepared by micropatterning a hydrophobic silane boundary around an adhesive, hydrophilic silane region of defined geometry using maskless lithography [38].…”

“…Sacrificial slides were removed, and the resulting polyacrylamide gradient gels were stored in PBS adjusted to pH 6.0. The resulting gradient gels featured 1mm gradient regions with an 18.6 kPa/mm gradient between 1 and 25 kPa low- and high-stiffness control regions [37]. …”

“…Thus, it will be important to assess whether migratory responses of cells to mechanical gradients are also regulated by extracellular matrix composition. We recently reported an experimental system to generate polyacrylamide gels with highly reproducible linear mechanical gradients in substrate stiffness and used it to explore whether migration of vascular smooth muscle cells on mechanical gradients was extracellular matrix type-dependent [37]. However, the effect of combinations of extracellular matrix on the cellular response to mechanical gradients has yet to be explored.…”