ECM Stiffness Research Articles

Mechanical signals regulate and activate SNAIL1 protein to control the fibrogenic response of cancer-associated fibroblasts.

Increased deposition of collagen in extracellular matrix (ECM) leads to increased tissue stiffness and occurs in breast tumors. When present, this increases tumor invasion and metastasis. Precisely how this deposition is regulated and maintained in tumors is unclear. Much has been learnt about mechanical signal transduction in cells, but transcriptional responses and the pathophysiological consequences are just becoming appreciated. Here, we show that the SNAIL1 (also known as SNAI1) protein level increases and accumulates in nuclei of breast tumor cells and cancer-associated fibroblasts (CAFs) following exposure to stiff ECM in culture and in vivo SNAIL1 is required for the fibrogenic response of CAFs when exposed to a stiff matrix. ECM stiffness induces ROCK activity, which stabilizes SNAIL1 protein indirectly by increasing intracellular tension, integrin clustering and integrin signaling to ERK2 (also known as MAPK1). Increased ERK2 activity leads to nuclear accumulation of SNAIL1, and, thus, avoidance of cytosolic proteasome degradation. SNAIL1 also influences the level and activity of YAP1 in CAFs exposed to a stiff matrix. This work describes a mechanism whereby increased tumor fibrosis can perpetuate activation of CAFs to sustain tumor fibrosis and promote tumor metastasis through regulation of SNAIL1 protein level and activity.

The effect of Young’s modulus on the neuronal differentiation of mouse embryonic stem cells

There is substantial evidence that cells produce a diverse response to changes in ECM stiffness depending on their identity. Our aim was to understand how stiffness impacts neuronal differentiation of embryonic stem cells (ESC’s), and how this varies at three specific stages of the differentiation process. In this investigation, three effects of stiffness on cells were considered; attachment, expansion and phenotypic changes during differentiation. Stiffness was varied from 2kPa to 18kPa to finally 35kPa. Attachment was found to decrease with increasing stiffness for both ESC’s (with a 95% decrease on 35kPa compared to 2kPa) and neural precursors (with a 83% decrease on 35kPa). The attachment of immature neurons was unaffected by stiffness. Expansion was independent of stiffness for all cell types, implying that the proliferation of cells during this differentiation process was independent of Young’s modulus. Stiffness had no effect upon phenotypic changes during differentiation for mESC’s and neural precursors. 2kPa increased the proportion of cells that differentiated from immature into mature neurons. Taken together our findings imply that the impact of Young’s modulus on attachment diminishes as neuronal cells become more mature. Conversely, the impact of Young’s modulus on changes in phenotype increased as cells became more mature.

Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy.

Tumor vessels are characterized by abnormal morphology and hyper-permeability that together cause inefficient delivery of chemotherapeutic agents. Although VEGF has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here, we show that the mechanosensitive ion channel TRPV4 regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TEC exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards ECM stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 KO mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anti-cancer drug Cisplatin, significantly reduced tumor growth in WT mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

N-Cadherin Induction by ECM Stiffness and FAK Overrides the Spreading Requirement for Proliferation of Vascular Smooth Muscle Cells.

SUMMARYIn contrast to the accepted pro-proliferative effect of cell-matrix adhesion, the proliferative effect of cadherin-mediated cell-cell adhesion remains unresolved. Here, we studied the effect of N-cadherin on cell proliferation in the vasculature. We show that N-cadherin is induced in smooth muscle cells (SMCs) in response to vascular injury, an in vivo model of tissue stiffening and proliferation. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated activation of a focal adhesion kinase (FAK)-p130Cas-Rac signaling pathway induces N-cadherin. Additionally, by culturing paired and unpaired SMCs on microfabricated adhesive islands of different areas, we found that N-cadherin relaxes the spreading requirement for SMC proliferation. In vivo SMC deletion of N-cadherin strongly reduced injury-induced cycling. Finally, SMC-specific deletion of FAK inhibited proliferation after vascular injury, and this was accompanied by reduced induction of N-cadherin. Thus, a stiffness-and FAK-dependent induction of N-cadherin connects cell-matrix to cell-cell adhesion and regulates the degree of cell spreading needed for cycling.

Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis.

Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new analytical tool to analyze both population based and individual cell responses. Endothelial cells on soft substrates, 4 kPa, were the most responsive to VEGF, whereas cells on the 125 kPa substrates exhibited an attenuated response. Magnitude of activation, not the quantity of cells responding or the number of local maximums each cell experienced distinguished the responses. Individual cell analysis, across all treatments, identified two unique cell clusters. One cluster, containing most of the cells, exhibited minimal or slow calcium release. The remaining cell cluster had a rapid, high magnitude VEGF activation that ultimately defined the population based average calcium response. Interestingly, at low doses of VEGF, the high responding cell cluster contained smaller cells on average, suggesting that cell shape and size may be indicative of VEGF-sensitive endothelial cells. This study provides a new analytical tool to quantitatively analyze individual cell signaling response kinetics, that we have used to help uncover outcomes that are hidden within the average. The ability to selectively identify highly VEGF responsive cells within a population may lead to a better understanding of the specific phenotypic characteristics that define cell responsiveness, which could provide new insight for the development of targeted anti- and pro-angiogenic therapies.

Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing

Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear. We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness. Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.

Pivotal role of pervasive neoplastic and stromal cells reprogramming in circulating tumor cells dissemination and metastatic colonization.

Reciprocal interactions between neoplastic cells and their microenvironment are crucial events in carcinogenesis and tumor progression. Pervasive stromal reprogramming and remodeling that transform a normal to a tumorigenic microenvironment modify numerous stromal cells functions, status redox, oxidative stress, pH, ECM stiffness and energy metabolism. These environmental factors allow selection of more aggressive cancer cells that develop important adaptive strategies. Subpopulations of cancer cells acquire new properties associating plasticity, stem-like phenotype, unfolded protein response, metabolic reprogramming and autophagy, production of exosomes, survival to anoikis, invasion, immunosuppression and therapeutic resistance. Moreover, by inducing vascular transdifferentiation of cancer cells and recruiting endothelial cells and pericytes, the tumorigenic microenvironment induces development of tumor-associated vessels that allow invasive cells to gain access to the tumor vessels and to intravasate. Circulating cancer cells can survive in the blood stream by interacting with the intravascular microenvironment, extravasate through the microvasculature and interact with the metastatic microenvironment of target organs. In this review, we will focus on many recent paradigms involved in the field of tumor progression.

Force engages vinculin and promotes tumor progression by enhancing PI3K activation of phosphatidylinositol (3,4,5)-triphosphate.

Extracellular matrix (ECM) stiffness induces focal adhesion assembly to drive malignant transformation and tumor metastasis. Nevertheless, how force alters focal adhesions to promote tumor progression remains unclear. Here, we explored the role of the focal adhesion protein vinculin, a force-activated mechanotransducer, in mammary epithelial tissue transformation and invasion. We found that ECM stiffness stabilizes the assembly of a vinculin-talin-actin scaffolding complex that facilitates PI3K-mediated phosphatidylinositol (3,4,5)-triphosphate phosphorylation. Using defined two- and three-dimensional matrices, a mouse model of mammary tumorigenesis with vinculin mutants, and a novel super resolution imaging approach, we established that ECM stiffness, per se, promotes the malignant progression of a mammary epithelium by activating and stabilizing vinculin and enhancing Akt signaling at focal adhesions. Our studies also revealed that vinculin strongly colocalizes with activated Akt at the invasive border of human breast tumors, where the ECM is stiffest, and we detected elevated mechanosignaling. Thus, ECM stiffness could induce tumor progression by promoting the assembly of signaling scaffolds, a conclusion underscored by the significant association we observed between highly expressed focal adhesion plaque proteins and malignant transformation across multiple types of solid cancer. See all articles in this Cancer Research section, "Physics in Cancer Research."

Stroma as an Active Player in the Development of the Tumor Microenvironment.

The stroma is a considerable part of the tumor microenvironment. Because of its complexity, it can influence both cancer and immune cells in their behavior and cross-talk. Aside from soluble products released by non-cancer and cancer cells, extracellular matrix components have been increasingly recognized as more than just minor players in the constitution, development and regulation of the tumor microenvironment. The variations in the connective scaffold architecture, induced by transforming growth factor beta, lysyl oxidase and metalloproteinase activity, create different conditions of ECM density and stiffness. They exert broad effects on immune cells (e.g. physical barriers, modulation by release of stored TGF-β1), mesenchymal cells (transition to myofibroblasts), epithelial cells (epithelial-to-mesenchymal transition), cancer cells (progression to metastatic phenotype) and stem cells (activation of differentiation addressed by the microenvironment characteristics). Physiological mechanisms of the wound healing process, as well as mechanisms of fibrosis in some chronic pathologies, closely recall aspects of cancer deregulated biology. Their elucidation can provide a better understanding of tumor microenvironment immunobiology. In the following short review, we will focus on some aspects of the fibrous stroma to highlight its active participation in the tumor microenvironment constitution, tumor progression and the local immunological network.

Influence of Extracellular Matrix Stiffness on Micro-RNA Expression in Human Trabecular Meshwork Cells

Glaucoma is a chronic neurodegenerative eye disease characterized by a loss of retinal ganglion cells associated with structural changes in the ocular outflow tract, disturbed intraocular pressure regulation and changes in tissue biomechanics. Earlier studies revealed a strong influence of ECM stiffness on protein expression patterns and growth factor signaling in cells of the ocular outflow tract (trabecular meshwork). To gain further insight into possible regulatory mechanisms, we asked whether ECM stiffness would alter expression of short non-coding microRNAs.Human trabecular meshwork cells were plated on tissue culture plastic or polyacrylamide gel substrata (E-moduli approx. 40 or 120kPa) of different stiffness and allowed to adjust for 7 days. Subsequently, miRNA expression levels were studied by Affymetrix arrays and qPCR. Genes targeted by at least five mechanoresponsive miRNAs (stiffness-related change >2 fold) were determined using R software. Protein expression of selected targets was assessed by western blot. At least 26 miRNAs were differentially expressed and 24 genes were predicted targets of 5 or more mechanoresponsive miRNAs. ECM stiffness-dependent protein expression changes were confirmed for the predicted targets NF-kB, AKT, E2F1, p53, p21 and cyclin-D1. These data suggest a mechanoresponsive regulation of miRNA levels with a possible role in cell differentiation and disease.

Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation

Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for the regulation of bone formation and resorption. ECM stiffness and cell seeding density have been shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast–osteocyte differentiation are not yet understood. In this study, we cultured MC3T3-E1 cells on (A) substrates of different chemical compositions and stiffnesses, as well as, (B) substrates of identical chemical composition but different stiffnesses. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP-1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP-1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro.

Pan-neuronal maturation but not neuronal subtype differentiation of adult neural stem cells is mechanosensitive

Most past studies of the biophysical regulation of stem cell differentiation have focused on initial lineage commitment or proximal differentiation events. It would be valuable to understand whether biophysical inputs also influence distal endpoints more closely associated with physiological function, such as subtype specification in neuronal differentiation. To explore this question, we cultured adult neural stem cells (NSCs) on variable stiffness ECMs under conditions that promote neuronal fate commitment for extended time periods to allow neuronal subtype differentiation. We find that ECM stiffness does not modulate the expression of NeuroD1 and TrkA/B/C or the percentages of pan-neuronal, GABAergic, or glutamatergic neuronal subtypes. Interestingly, however, an ECM stiffness of 700 Pa maximizes expression of pan-neuronal markers. These results suggest that a wide range of stiffnesses fully permit pan-neuronal NSC differentiation, that an intermediate stiffness optimizes expression of pan-neuronal genes, and that stiffness does not impact commitment to particular neuronal subtypes.

Control of VEGF‐Endothelial Response by ECM Stiffness

Vascular endothelial growth factor (VEGF) drives both endothelial cell maintenance and normal/pathological angiogenesis. Recent research has suggested that mechanical signaling due to the stiffness of the cell contact surface may also have profound effects on endothelial cell behavior. To study VEGF signaling as a function of substrate stiffness, endothelial cells were cultured on fibronectin linked polyacrylamide gels and treated with VEGF. To analyze the response to VEGF we developed a custom MATLAB software script to analyze calcium flux movies. The script is able to individually identify cells, normalize each calcium signal value to the cell's individual background, and analyze the data. A variety of outputs are produced such as the magnitude of response, number of local maximums each cell experiences and clusters of coordinated cells. Utilizing this tool, we observed that VEGF‐stimulated intracellular calcium signaling varies in both magnitude and signaling kinetics depending on the stiffness of the substrate. At low VEGF concentrations we found maximal response (2–3.5 fold increases) at moderate stiffness whereas at high concentrations of VEGF the softest substrates showed the most intense response, but the response dissipated the fastest. We show that extracellular matrix stiffness can differentially drive endothelial response to VEGF. Supported by HL088572 and AHAF M2012014.

Host epithelial geometry regulates breast cancer cell invasiveness

Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment.

Abstract SY28-03: The dynamic interplay between extrinsic and intrinsic force regulates cancer progression

Abstract All cells experience force and possess mechanosensory mechanisms that enable them to detect mechanical stimuli and transduce these cues into biochemical signals and gene expression changes that influence cellular behavior. Thus embryogenesis, tissue development and homeostasis are all tightly regulated by mechanics and governed by spatially and temporally-regulated tensional homeostasis. By contrast, tumors characteristically exhibit increased tissue level forces across multiple length scales and transformed cells have typically acquired perturbations in mechanosensing. The challenge is to clarify how chemical and mechanical cues collaborate to regulate cellular and tissue level behaviors and to determine when and how these aberrant forces are acquired and if and how they regulate cancer initiation, progression and treatment. The Weaver group has been studying how cells sense and transduce mechanical cues to regulate their behavior and how altered mechanical force compromises tissue homeostasis to drive tumor formation and metastasis and modulate treatment response (reviewed in Butcher et al., Nature Cancer Reviews 2010; Dufort et al., Nature Mol Biol Reviews 2011). Using an array of in vitro and in vivo models we found that the ECM progressively stiffens in an array of tumor types including breast, pancreas, glioblastoma and skin and that ECM tension critically modulates transition to invasion and promotes a metastatic phenotype. We demonstrated that even soft tumors such as aggressive glioblastomas are characterized by a stiffened ECM that is associated with higher levels of ECM proteins that are cross-linked and linearized. We showed that abnormally elevated ECM stiffness compromises morphogenesis and destabilizes tissue architecture largely by promoting the assembly of integrin focal adhesions which potentiate growth factor receptor signaling and induce cytoskeletal remodeling and actomyosin contractility (Paszek et al., Cancer Cell 2005; Paszek et al., PLOS Computational 2009). ECM stiffness potentiates the tumorigenic effects of oncogenes such as Ras, EGFR and ErbB2 by increasing and modifying ERK, wnt and PI3 kinase activity and inducing tissue fibrosis and ECM tension enhances tumor aggression by compromising expression of critical tumor suppressors including PTEN. Consistently, inducing ECM tension promotes the malignant transformation of pre malignant oncogenically-primed epithelial cells, whereas inhibiting ECM stiffening prevents tissue fibrosis and tumor progression (Levental et al., CELL 2009; unpublished findings). ECM tension also regulates tumor metastasis by inducing hypoxic reprogramming and modifying the chemokine microenvironment through pleiotrophic effects on cells of the vascular and immune systems. Finally, and most importantly, we found that when tumor cells are oncogenically transformed or loss expression of critical tumor suppressors per se this corrupts their cellular mechanoresponsiveness and they become hypersensitized or desensitized to extrinsic force and acquire inappropriately elevated cellular tension. Indeed, we could show that oncogenic signaling through Ras to ROCK induces ECM remodeling and stiffening which are necessary for wnt-mediated skin tumor invasion and induction of an epithelial to mesenchymal transition through ECM remodeling and stiffening. Similarly, loss of TGFβ RII in breast and pancreatic tumor cells enhances their contractility to increase chemokine expression thereby fostering tissue inflammation and driving ECM remodeling and stiffening to promote invasion and metastasis. Through collaborations with colleagues at UCSF, Vanderbilt and Harvard the molecular mechanisms and physiological relevance of these observations are being further investigated. Moreover, the clinical relevance of our observations to breast and brain tumors are being explored with colleagues in Oregon, Duke and UCSF. (Supported by DOD BCRP W81XWH-05-1-0330 and NIH U54CA163155-01, 1U01CA151925-01, RO1CA138818-01A1, 2RO1CA085492-11A1, and R01CA140663-01A2 to VMW, NIH 1U01ES019458-01 to ZW and U54CA143836-01 to J.L., NCI P50CA058207 to VMW, CP, LC & SH and KG110560 to S.H. & L.C.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY28-03. doi:1538-7445.AM2012-SY28-03

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc. The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) < 3000 Pascal and stiff substrata/tissues as those with E > 20,000 Pascal.

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc. The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) <3000 Pascal and stiff substrata/tissues as those with E >20,000 Pascal.

Methods for investigating integrin‐matrix interactions as a function of matrix mechanics and composition

To investigate the role of mechanics in cell signaling, we have engineered polyacrylamide (PAAM) gels with precisely tunable stiffness and fibronectin (FN) content. Varying the acrylamide / bis ratio alters PAAM stiffness, as determined with tensile testing and atomic force microscopy (AFM). To promote cell attachment, FN is covalently linked throughout the gel, and can then be quantified with an ELISA. Analyzing AFM data with the Hertz and Oliver‐Pharr models, we calculated gel elastic moduli ranging from 11 – 344 kPa and 7.5 – 95 kPa. We also found that stiffer gels incorporate more FN than softer gels, and that adding FN reduces the modulus of softer gels by ~10%. Controlling for these factors allowed for precise and independent changes in gel mechanics and FN content.To study integrin‐FN interactions on PAAM gels, we adapted published methods for quantifying integrin binding. After vascular smooth muscle cells (VSMCs) were grown on gels or FN‐coated plastic, bound FN‐alpha5 integrin units were cross‐linked and assayed with an ELISA. Preliminary data suggest that integrin activity per cell is reduced with decreasing stiffness or higher cell density. Future studies using the methods presented herein will provide insight into how VSMCs recognize and respond to changes in ECM composition and stiffness, aiding the rational design of new treatment for vascular disease. Supported by NIH grants HL56200 and HL72900.