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Vascular Smooth Muscle Cell Durotaxis Depends on Extracellular Matrix Composition

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Vascular Smooth Muscle Cell Durotaxis Depends on Extracellular Matrix Composition Vascular smooth muscle cell durotaxis depends on extracellular matrix composition Christopher D. Hartmana, Brett C. Isenberga, Samantha G. Chuaa, and Joyce Y. Wonga,1 aDepartment of Biomedical Engineering, Boston University, Boston, MA 02215 Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved August 9, 2016 (received for review July 14, 2016) Mechanical compliance has been demonstrated to be a key de- by an increase in collagen I concentration (21), and in breast cancer terminant of cell behavior, directing processes such as spreading, an increase in stiffness from the tumor core to the periphery is migration, and differentiation. Durotaxis, directional migration from associated with increased levels of collagen I and laminin (12). In softer to more stiff regions of a substrate, has been observed for a atherosclerosis, a disease characterized by the thickening of the variety of cell types. Recent stiffness mapping experiments have intimal region of the arterial wall, changes in the mechanics and shown that local changes in tissue stiffness in disease are often composition of the intimal matrix occur in conjunction with the accompanied by an altered ECM composition in vivo. However, accumulation of smooth muscle and inflammatory cells (22–24). the importance of ECM composition in durotaxis has not yet been Stiffness mapping experiments have shown that plaque stiffness is explored. To address this question, we have developed and charac- spatially heterogeneous, and that these changes can be histo- terized a polyacrylamide hydrogel culture platform featuring highly logically related to the ECM composition of the plaque (20, 25). tunable gradients in mechanical stiffness. This feature, together with Given the increasing number of examples for which changes in the ability to control ECM composition, allows us to isolate the effects ECM composition in disease are coupled to changes in me- of mechanical and biological signals on cell migratory behavior. Using chanical properties of the diseased tissue, there is a need for this system, we have tracked vascular smooth muscle cell migration in vitro experimental systems that allow for systematic explora- in vitro and quantitatively analyzed differences in cell migration as a tion of how the cellular response to stiffness gradients is mod- function of ECM composition. Our results show that vascular smooth ulated by ECM composition. muscle cells undergo durotaxis on mechanical gradients coated with Recent experimental work has demonstrated that the effect of SCIENCES fibronectin but not on those coated with laminin. These findings matrix stiffness on cell behaviors such as differentiation, spreading, APPLIED BIOLOGICAL indicate that the composition of the adhesion ligand is a critical and motility is modulated by the composition of the ECM on which determinant of a cell’s migratory response to mechanical gradients. the cells are grown (26–30). Though the role of matrix composition has been investigated on uniformly stiff substrates, previous work durotaxis | cell migration | extracellular matrix | polyacrylamide investigating the cellular response to mechanical gradients has been limited to substrates coated with a single type of matrix protein, typically collagen or fibronectin (31), and the behavioral response of ell migration is essential to numerous biological processes, SCIENCES including development, angiogenesis, wound healing, and a given cell type to different matrix compositions has yet to be ex- C APPLIED PHYSICAL cancer metastasis (1–4). The movement of cells in these processes plored in the same study. Previous work in our laboratory has is determined by a complex assessment of environmental cues that demonstrated that vascular smooth muscle cell (VSMC) adhesion include soluble factors, ECM composition, orientation, and stiff- rate, spread area, cytoskeletal assembly, and focal adhesion signaling ness. Numerous experiments have demonstrated that directional undergo opposing responses to substrate stiffness depending on cell migration can result from gradients in these environmental whether they are seeded on fibronectin- or laminin- coated sub- cues: for example, chemotaxis (cell migration in response to gra- strates (27), and furthermore that VSMCs will preferentially migrate dients of soluble signals) and haptotaxis (cell migration in response toward stiffer regions (durotaxis) when exposed to mechanical gra- to gradients of bound ligands) have been established in both dients on fibronectin substrates (14, 16). Additionally, there is in vitro and in vivo experimental systems (5–7). More recently, it has been demonstrated that cells are also capable of directed mi- Significance gration in response to gradients in substrate stiffness, a process termed durotaxis (8). Though there have been limited reports of Many cell types have been observed to migrate toward stiffer specifically measured in vivo gradients (9), a number of recent regions of mechanical gradients in a process termed durotaxis. stiffness mapping measurements imply the presence of stiffness Tissue stiffness gradients are being discovered in an increasing gradients in both healthy and diseased tissues spanning a wide number of diseases, including cancer and fibrotic diseases. – range of stiffnesses (10 13). In vitro experiments have demon- However, the role of ECM composition, which often changes in strated that directed migration in response to stiffness gradients diseased tissues with stiffness, in directing this behavior has can be observed in numerous cell types, using various materials as not previously been investigated. To understand how stiffness – substrates, and across various stiffness levels (8, 14 19). However, gradients and changing matrix composition may affect cell the role of ECM composition in mediating this behavior has not migration in disease, we have designed a mechanical gradient been thoroughly investigated. platform that allows for independent control of absolute The interplay between mechanical stiffness and matrix compo- stiffness, gradient steepness, and ECM coating. We demon- sition in normal and pathological physiology is only now becoming strate that smooth muscle cells will undergo durotaxis on appreciated. Recent studies in which tissue stiffness was mapped mechanical gradients coated with fibronectin but not laminin. by atomic force microscopy (AFM) indentation have identified heterogeneities that indicate the presence of mechanical stiffness Author contributions: C.D.H., B.C.I., and J.Y.W. designed research; C.D.H., B.C.I., and S.G.C. gradients in both healthy and diseased tissues. These measurements performed research; C.D.H. analyzed data; and C.D.H. and J.Y.W. wrote the paper. indicate the presence of a wide range of absolute stiffnesses and The authors declare no conflict of interest. gradient strengths in vivo (9–13, 20). Importantly, such stiffness This article is a PNAS Direct Submission. gradients have been demonstrated to accompany changes in ECM 1To whom correspondence should be addressed. Email: [email protected]. composition in a number of diseases. For instance, in lung fibrosis, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. local increases in lung parenchymal tissue stiffness are accompanied 1073/pnas.1611324113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1611324113 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 evidence that both stiffness and ECM composition can modulate VSMCs between a quiescent, contractile phenotype typical of healthy tissue and a synthetic, proliferative, and migratory phenotype observed in vascular disease (32–34). Fibronectin and laminin have opposing effects on VSMC phenotype, with fibronectin driving cells away from the contractile phenotype in vitro, whereas laminin has been shown to conserve it (35–38). A loss of laminin and an increase in fibronectin surrounding smooth muscle cells has been observed during the progression of neointima formation in vascular disease, suggesting these proteins may play an important role in regulation of cell phenotype in disease (39). Additionally, increasing stiffness has been shown to drive smooth muscle cells toward the synthetic, mi- gratory phenotype observed in atherosclerosis (40, 41), This evidence that VSMC phenotype is modulated by both matrix stiffness and composition, coupled with our previous observations of matrix type- dependent responses to stiffness by VSMCs, led us to hypothesize that the durotactic migratory behavior of VSMC s on substrates with mechanical gradients may also be differentially modulated by fi- bronectin and laminin. To test this hypothesis, we have developed a method of gener- ating polyacrylamide hydrogels with tunable gradients in substrate stiffness and independent control of matrix composition. Previously described methods to fabricate polyacrylamide mechanical gradient gels have been limited due to the difficulty of independently con- Fig. 1. Schematic of gradient generator device preparation and use. (A)Glass trolling the absolute stiffness range and gradient steepness (8, 14–16, slide coated in S1818 photoresist (orange). (B) Maskless lithography with visible 19, 42–44). However, by carefully controlling hydrogel geometry, light exposes device pattern onto photoresist-coated slide. (C) Photoresist re- cross-linker diffusion, and UV photopolymerization (Fig. 1), we are moved by development in TMAOH. (D) Exposed glass is coated with OTS to form a able
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