Matrix stiffness corresponding to strictured bowel induces a fibrogenic response in human colonic fibroblasts.

BACKGROUND: Crohn's disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression toward fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. METHODS: The stiffness of fresh ex vivo samples from normal human small intestine, Crohn's disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin staining, and gene expression. RESULTS: Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn's strictures and between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn's strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased α-smooth muscle actin protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase, and proinflammatory gene expression and was associated with nuclear localization of the transcriptional cofactor MRTF-A. CONCLUSIONS: Matrix stiffness, representative of the pathologic stiffness of Crohn's strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways, suggesting that the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to auto-propagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn's disease.

Matrix stiffness reverses the effect of actomyosin tension on cell proliferation.

The stiffness of the extracellular matrix exerts powerful effects on cell proliferation and differentiation, but the mechanisms transducing matrix stiffness into cellular fate decisions remain poorly understood. Two widely reported responses to matrix stiffening are increases in actomyosin contractility and cell proliferation. To delineate their relationship, we modulated cytoskeletal tension in cells grown across a physiological range of matrix stiffnesses. On both synthetic and naturally derived soft matrices, and across a panel of cell types, we observed a striking reversal of the effect of inhibiting actomyosin contractility, switching from the attenuation of proliferation on rigid substrates to the robust promotion of proliferation on soft matrices. Inhibiting contractility on soft matrices decoupled proliferation from cytoskeletal tension and focal adhesion organization, but not from cell spread area. Our results demonstrate that matrix stiffness and actomyosin contractility converge on cell spreading in an unexpected fashion to control a key aspect of cell fate.

Cell elasticity determines macrophage function.

Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function.

Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-ß responsiveness.

Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-ß1 (TGF-ß1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-ß1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-ß1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation.

A multiwell platform for studying stiffness-dependent cell biology.

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.

Matrix rigidity regulates cancer cell growth and cellular phenotype.

BACKGROUND: The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: "rigidity dependent" (those which show an increase in cell growth as extracellular rigidity is increased), and "rigidity independent" (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. CONCLUSIONS/SIGNIFICANCE: These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models.

Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.

Tissue stiffening is a hallmark of fibrotic disorders but has traditionally been regarded as an outcome of fibrosis, not a contributing factor to pathogenesis. In this study, we show that fibrosis induced by bleomycin injury in the murine lung locally increases median tissue stiffness sixfold relative to normal lung parenchyma. Across this pathophysiological stiffness range, cultured lung fibroblasts transition from a surprisingly quiescent state to progressive increases in proliferation and matrix synthesis, accompanied by coordinated decreases in matrix proteolytic gene expression. Increasing matrix stiffness strongly suppresses fibroblast expression of COX-2 (cyclooxygenase-2) and synthesis of prostaglandin E(2) (PGE(2)), an autocrine inhibitor of fibrogenesis. Exogenous PGE(2) or an agonist of the prostanoid EP2 receptor completely counteracts the proliferative and matrix synthetic effects caused by increased stiffness. Together, these results demonstrate a dominant role for normal tissue compliance, acting in part through autocrine PGE(2), in maintaining fibroblast quiescence and reveal a feedback relationship between matrix stiffening, COX-2 suppression, and fibroblast activation that promotes and amplifies progressive fibrosis.

IL-13 induces a bronchial epithelial phenotype that is profibrotic.

BACKGROUND: Inflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-beta2, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis. METHODS: Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22-25 or days 28-31. RESULTS: IL-13 induced increasing levels of MUC5AC protein, and TGF-beta2, while decreasing beta-Tubulin IV at day 22 and 28 in the NHBE. TGF-beta2, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-beta2 neutralizing antibody reversed the increase in collagen content and SHG signal. CONCLUSION: Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-beta2 at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-beta2 secretion from the airway epithelium.

Measurement of IL-13-induced iNOS-derived gas phase nitric oxide in human bronchial epithelial cells.

Exhaled nitric oxide (NO) is altered in numerous diseases including asthma, and is thought broadly to be a noninvasive marker of inflammation. However, the precise source of exhaled NO has yet to be identified, and the interpretation is further hampered by significant inter-subject variation. Using fully differentiated normal human bronchial epithelial (NHBE) cells, we sought to determine (1) the rate of NO release (flux, pl.s(-1.)cm(-2)) into the gas; (2) the effect of IL-13, a prominent mediator of allergic inflammation, on NO release; and (3) inter-subject/donor variability in NO release. NHBE cells from three different donors were cultured at an air-liquid interface and stimulated with different concentrations of IL-13 (0, 1, and 10 ng/ml) for 48 h. Gas phase NO concentrations in the headspace over the cells were measured using a chemiluminescence analyzer. The basal NO flux from the three donors (0.05 +/- 0.03) is similar in magnitude to that estimated from exhaled NO concentrations, and was significantly increased by IL-13 in a donor-specific fashion. The increase in NO release was strongly correlated with inducible nitric oxide synthase (iNOS) gene and protein expression. There was a trend toward enhanced production of nitrate relative to nitrite as an end product of NO metabolism in IL-13-stimulated cells. NO release from airway epithelial cells can be directly measured. The rate of release in response to IL-13 is strongly dependent on the individual donor, but is primarily due to the expression of iNOS.

Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy.

Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth ( approximately 1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4-37 degrees C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37-4 degrees C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G', from 23 +/- 3 Pa to 0.28 +/- 0.16 Pa, respectively, mean +/- SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 +/- 3.5 Pa before to 138 +/- 40 Pa after cross-linking, mean +/- SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.

Epithelial-derived TGF-beta2 modulates basal and wound-healing subepithelial matrix homeostasis.

The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-beta (TGF-beta) modulates these processes, we hypothesized that epithelial-derived TGF-beta also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-beta in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-beta2 at a concentration of 50-70 pg/ml, whereas TGF-beta1 is undetectable. TGF-beta2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both alpha-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-beta2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-beta2 (0-400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-beta2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

Expression of matrix proteins in an in vitro model of airway remodeling in asthma.

Acute asthma is characterized by a decrease in the pH of the exhaled breath condensate and bronchoconstriction. These perturbations may injure the epithelium in a chronic, intermittent pattern, leading to subepithelial fibrosis. We used an in vitro three-dimensional model of the bronchial mucosa to elucidate the response to a repeated chemical or physical insult to the epithelium in the postcontraction phase. We used enzyme-linked immunosorbent assay and reverse transcriptase--polymerase chain reaction to assess the production of the following proteins: matrix metalloproteinase (MMP) 3, MMP-9, tissue inhibitor of MMP-1, transforming growth factor beta 1, thrombospondin 1, tenascin, and fibronectin. The presence of the epithelium enhanced the degree of tissue contraction (50.1 +/- 4.4% of original area versus 75.4 +/- 2.3%). In the absence of injury, tenascin, fibronectin, MMP-3, and tissue inhibitor of MMP-1 are actively expressed. However, the chronic chemical wound markedly inhibited the expression of all proteins. We conclude that the epithelium, wound type, and age of the tissue (contracting versus postcontraction) impact the expression of key proteins in an in vitro model of subepithelial fibrosis in asthma.