Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

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.

SummaryThe 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.

Marinković A, Mih JD, Park J-A, Liu F, Tschumperlin DJ. Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-␤ responsiveness. Am J Physiol Lung Cell Mol Physiol 303: L169 -L180, 2012. First published June 1, 2012; doi:10.1152/ajplung.00108.2012.-Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-␤1 (TGF-␤1) 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-␤1 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-␤1-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. rigidity; fibrosis; lung; extracellular matrix; forces PULMONARY FIBROSIS IS ASSOCIATED with aberrant tissue repair and persistent activation of myofibroblasts (29,37,60,72). Myofibroblasts are mechanically active cells (23) that interact with the extracellular environment by secreting matrix proteins and exerting forces necessary for matrix rearrangement (28, 60). Myofibroblast contractility also contributes to activation of transforming growth factor (TGF)-␤1 (68), a profibrotic cytokine whose liberation from a matrix-bound latent complex perpetuates fibroblast activation and matrix deposition (18, 39). Thus delineating cellular, molecular, and mechanical mechanisms regulating fibroblast force generation and matrix deformation is central to understanding the function of these pivotal fibrosis-promoting cells.Although fibroblast contractile status can be inferred from the formation of stress fibers ...

Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) have been shown to differ from normal lung fibroblasts in functional behaviors that contribute to the pathogenesis of IPF, including the expression of contractile proteins and proliferation, but how such behaviors vary in matrices with stiffness matched to normal and fibrotic lung tissue remains unknown. Here, we tested whether pathologic changes in matrix stiffness control IPF and normal lung tissue-derived fibroblast functions, and compared the relative efficacy of mechanical cues to an antifibrotic lipid mediator, prostaglandin E 2 (PGE 2 ). Fibroblasts were grown on collagen I-coated glass or hydrogel substrates of discrete stiffnesses, spanning the range of normal and fibrotic lung tissue. Traction microscopy was used to quantify contractile function. The CyQuant Cell Proliferation Assay (Invitrogen, Carlsbad, CA) was used to assess changes in cell number, and PGE 2 concentrations were measured by ELISA. We confirmed differences in proliferation and PGE 2 synthesis between IPF and normal tissue-derived fibroblasts on rigid substrates. However, IPF fibroblasts remained highly responsive to changes in matrix stiffness, and both proliferative and contractile differences between IPF and normal fibroblasts were ablated on physiologically soft matrices. We also confirmed the relative resistance of IPF fibroblasts to PGE 2 , while demonstrating that decreases in matrix stiffness and the inhibition of Rho kinase both potently attenuate contractile function in IPF-derived fibroblasts. We conclude that pathologic changes in the mechanical environment control important IPF fibroblast functions. Understanding how mechanical cues control fibroblast function may offer new opportunities for targeting these cells, even when they are resistant to antifibrotic pharmacological agents or biological mediators.Keywords: pulmonary fibrosis; lung; extracellular matrix; fibroblast contractility Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive fibrosing disease with no proven pharmacological therapy (1). The fibroblast is the end effector cell of fibrosis, and fibroblasts increase in number and activation status during IPF. Scattered aggregates of proliferating fibroblasts are consistently observed in fibrotic lungs (2, 3), and the progression of IPF is accompanied by an excessive activation of lung fibroblasts to a synthetic and contractile myofibroblast phenotype responsible for the deposition, contraction, and remodeling of the lung's extracellular matrix (ECM) (4, 5).To understand the pathogenic mechanisms at work in IPF, much effort has been devoted to identifying functional differences in IPF-derived fibroblasts compared with fibroblasts isolated from normal lung tissue. These efforts have demonstrated, among a host of changes, that IPF fibroblasts are more contractile (6), express higher concentrations of a-smooth muscle actin (a-SMA) (7, 8), Type I collagen (8-10), and tissue inhibitors of metalloproteinase (11) than do normal lung tissue-d...

We quantified the effects of airway wall remodeling upon airway smooth muscle (ASM) shortening. Isolated ASM from sheep was attached to a servo-controller that applied a physiologic load. This load could be altered to reflect specified changes of airway wall geometry, elasticity, parenchymal tethering, transpulmonary pressure (P L ), and fluctuations in P L associated with breathing. Starting at a reference length (L ref ), ASM was stimulated with acetlycholine and held at constant P L of 4 cm H 2 O for 2 h. When all compartments were thickened to simulate the asthmatic airway but P L was held fixed, ASM shortened much more than that in the normal airway (to 0. ). These findings confirm the long-held conclusion that increased muscle mass is the functionally dominant derangement, but mechanisms accounting for this conclusion differ dramatically from those previously presumed. Furthermore, increased ASM mass explained both hyperresponsiveness and the failure of a DI to relax the asthmatic airway.Keywords: remodeling; asthma; hyperresponsiveness Asthma is characterized by airways that constrict too easily (airway hypersensitivity) and too much (airway hyperresponsiveness, AHR) (1). It is the excessive airway narrowing associated with AHR, rather than the hypersensitivity, that accounts for the morbidity and the mortality that is attributable to the disease (2-4). AHR is thought to arise as a result of ongoing and irreversible remodeling of the airway wall (2, 5-10). Among the various factors that come into play, AHR might be accounted for either by increased mass of airway smooth muscle (ASM) or by decreased load against which the ASM must contract, but it is widely believed that increased ASM mass is the main culprit (11-13). This important conclusion is based mainly upon theoretical considerations, however, wherein structural evidence has been incorporated into detailed mathematical models of airway narrowing (11,12,14).While these mathematical models incorporated the best information then available, they rested upon pivotal assumptions that were shown subsequently to be erroneous (15-18). In particular, muscle was represented by its isometric force-length relationship. But being a characterization of muscle contraction in purely static circumstances, such a description is intrinsically incapable of broaching issues involving smooth muscle dynamics as occur during tidal breathing and deep inspirations (DIs). For example, as early as 1859 Salter (19) reported the following:. . .[bronchial] spasm may be broken through, and the respiration for the time rendered perfectly free and easy, by taking a long, deep, full inspiration. In severe asthmatic breathing this cannot be done; but in the slight bronchial spasm that characterizes hay asthma I have frequently witnessed it. It seems as if the deep inspiration overcame and broke through the contracted state of the air-tubes, which was not immediately re-established.Subsequently we came to learn that of all innate agencies of bronchodilation the most potent of all is a ...

Pulmonary arterial (PA) stiffness is associated with increased mortality in patients with pulmonary hypertension (PH); however, the role of PA stiffening in the pathogenesis of PH remains elusive. Here, we show that distal vascular matrix stiffening is an early mechanobiological regulator of experimental PH. We identify cyclooxygenase-2 (COX-2) suppression and corresponding reduction in prostaglandin production as pivotal regulators of stiffness-dependent vascular cell activation. Atomic force microscopy microindentation demonstrated early PA stiffening in experimental PH and human lung tissue. Pulmonary artery smooth muscle cells (PASMC) grown on substrates with the stiffness of remodeled PAs showed increased proliferation, decreased apoptosis, exaggerated contraction, enhanced matrix deposition, and reduced COX-2–derived prostanoid production compared with cells grown on substrates approximating normal PA stiffness. Treatment with a prostaglandin I2 analog abrogated monocrotaline-induced PA stiffening and attenuated stiffness-dependent increases in proliferation, matrix deposition, and contraction in PASMC. Our results suggest a pivotal role for early PA stiffening in PH and demonstrate the therapeutic potential of interrupting mechanobiological feedback amplification of vascular remodeling in experimental PH.

When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signalling intermediates with poorly defined relationships to such a physiological endpoint. Using cellular force as the target, here we report a new screening technology and demonstrate its applications using human airway smooth muscle cells in the context of asthma and Schlemm's canal endothelial cells in the context of glaucoma. This approach identified several drug candidates for both asthma and glaucoma. We attained rates of 1000 compounds per screening day, thus establishing a force-based cellular platform for high-throughput drug discovery.