Title: Mechanical Feed-Forward Loops Contribute to Idiopathic Pulmonary Fibrosis
Abstract: Idiopathic pulmonary fibrosis is a progressive scarring disease characterized by extracellular matrix accumulation and altered mechanical properties of lung tissue. Recent studies support the hypothesis that these compositional and mechanical changes create a progressive feed-forward loop in which enhanced matrix deposition and tissue stiffening contribute to fibroblast and myofibroblast differentiation and activation, which further perpetuates matrix production and stiffening. The biomechanical properties of tissues are sensed and responded to by mechanotransduction pathways that facilitate sensing of changes in mechanical cues by tissue resident cells and convert the mechanical signals into downstream biochemical signals. Although our understanding of mechanotransduction pathways associated with pulmonary fibrosis remains incomplete, recent progress has allowed us to begin to elucidate the specific mechanisms supporting fibrotic feed-forward loops. The mechanosensors discussed here include integrins, Piezo channels, transient receptor potential channels, and nonselective ion channels. Also discussed are downstream transcription factors, including myocardin-related transcription factor and Yes-associated protein/transcriptional coactivator with PDZ-binding motif. This review describes mechanosensors and mechanotransduction pathways associated with fibrosis progression and highlights promising therapeutic insights. Idiopathic pulmonary fibrosis is a progressive scarring disease characterized by extracellular matrix accumulation and altered mechanical properties of lung tissue. Recent studies support the hypothesis that these compositional and mechanical changes create a progressive feed-forward loop in which enhanced matrix deposition and tissue stiffening contribute to fibroblast and myofibroblast differentiation and activation, which further perpetuates matrix production and stiffening. The biomechanical properties of tissues are sensed and responded to by mechanotransduction pathways that facilitate sensing of changes in mechanical cues by tissue resident cells and convert the mechanical signals into downstream biochemical signals. Although our understanding of mechanotransduction pathways associated with pulmonary fibrosis remains incomplete, recent progress has allowed us to begin to elucidate the specific mechanisms supporting fibrotic feed-forward loops. The mechanosensors discussed here include integrins, Piezo channels, transient receptor potential channels, and nonselective ion channels. Also discussed are downstream transcription factors, including myocardin-related transcription factor and Yes-associated protein/transcriptional coactivator with PDZ-binding motif. This review describes mechanosensors and mechanotransduction pathways associated with fibrosis progression and highlights promising therapeutic insights. Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with a life expectancy shorter than that of lung cancer.1Baroke E. Gauldie J. Kolb M. New treatment and markers of prognosis for idiopathic pulmonary fibrosis: lessons learned from translational research.Expert Rev Respir Med. 2013; 7: 465-478Crossref PubMed Scopus (9) Google Scholar, 2Nathan S.D. Shlobin O.A. Weir N. Ahmad S. Kaldjob J.M. Battle E. Sheridan M.J. du Bois R.M. Long-term course and prognosis of idiopathic pulmonary fibrosis in the new millennium.Chest. 2011; 140: 221-229Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 3Schwartz D.A. Helmers R.A. Galvin J.R. Van Fossen D.S. Frees K.L. Dayton C.S. Burmeister L.F. Hunninghake G.W. Determinants of survival in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 1994; 149: 450-454Crossref PubMed Scopus (292) Google Scholar It is characterized in part by the accumulation of extracellular matrix (ECM) proteins in the interstitial space and impaired gas exchange, thereby decreasing lung function. The ECM composition in the lung plays an important role in regulating cell behavior and function. Wolff’s law, proposed in 1892, states4Wolff J. Das Gesetz der Transformation der Knochen. Verlag von August Hirschwald, Berlin1892Google Scholar: “Every change in the form and function of bone or of their function alone is followed by certain definite changes in their internal architecture, and equally definite alteration in their external conformation, in accordance with mathematical laws.” The law defines that bone adapts and optimizes strength in response to the loads under which it is placed. Although the principle of mechanical adaptation is most obvious with muscle and bones,5Fuchs R.K. Warden S.J. Turner C.H. Bone anatomy, physiology and adaptation to mechanical loading.in: Bone Repair Biomaterials. Elsevier Inc., New York, NY2009: 25-68Crossref Scopus (10) Google Scholar all tissues adapt to the mechanical loads they are under, including the lung.6Roan E. Waters C.M. What do we know about mechanical strain in lung alveoli?.Am J Physiol Lung Cell Mol Physiol. 2011; 301: L625-L635Crossref PubMed Scopus (99) Google Scholar Structure–function relationships can be observed by differences in ECM architecture and are accompanied by distinct mechanical properties in anatomic compartments of the lung.7Sicard D. Haak A.J. Choi K.M. Craig A.R. Fredenburgh L.E. Tschumperlin D.J. Aging and anatomical variations in lung tissue stiffness.Am J Physiol Lung Cell Mol Physiol. 2018; 314: L946-L955Crossref PubMed Scopus (46) Google Scholar, 8Suki B. Stamenović D. Hubmayr R. Lung parenchymal mechanics.Compr Physiol. 2011; 1: 1317-1351PubMed Google Scholar, 9Knudsen L. Ochs M. The micromechanics of lung alveoli: structure and function of surfactant and tissue components.Histochem Cell Biol. 2018; 150: 661-676Crossref PubMed Scopus (101) Google Scholar It experiences several principal mechanical forces. It is under pressure from inhaled air, and it is subjected to cyclic tensile stretching with respiration cycles, as well as to the stiffness of the underlying ECM. These forces are sensed by fibroblasts that synthesize and modify the ECM to structurally stabilize the tissue and shield other cells from mechanical stimulation.10Herum K.M. Choppe J. Kumar A. Engler A.J. McCulloch A.D. Mechanical regulation of cardiac fibroblast profibrotic phenotypes.Mol Biol Cell. 2017; 28: 1871-1882Crossref PubMed Google Scholar In healthy lung tissue, this is a homeostatic process involving synthesis, modification, and degradation of matrix, collectively called matrix remodeling. In pathologic conditions, an imbalance in this process can lead to many pathologies, including increased ECM stiffness (fibrosis), decreased elasticity (emphysema), and airway remodeling (asthma). The imbalanced matrix buildup in fibrosis creates a self-fulfilling feed-forward loop of enhanced matrix deposition and stiffening.11Liu F. Mih J.D. Shea B.S. Kho A.T. Sharif A.S. Tager A.M. Tschumperlin D.J. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.J Cell Biol. 2010; 190: 693-706Crossref PubMed Scopus (459) Google Scholar Recent and ongoing work in IPF supports the hypothesis that diseased matrix is both a consequence and a driver of fibrosis.12Santos A. Lagares D. Matrix stiffness: the conductor of organ fibrosis.Curr Rheumatol Rep. 2018; 20: 2Crossref PubMed Scopus (69) Google Scholar, 13Huang X. Yang N. Fiore V.F. Barker T.H. Sun Y. Morris S.W. Ding Q. Thannickal V.J. Zhou Y. Matrix stiffness-induced myofibroblast differentiation is mediated by intrinsic mechanotransduction.Am J Respir Cell Mol Biol. 2012; 47: 340-348Crossref PubMed Scopus (285) Google Scholar, 14Tschumperlin D.J. Ligresti G. Hilscher M.B. Shah V.H. Mechanosensing and fibrosis.J Clin Invest. 2018; 128: 74-84Crossref PubMed Scopus (102) Google Scholar IPF is characterized by accumulation of fibroblasts and myofibroblasts within fibroblastic foci, deposition and cross-linking of the ECM, and dysregulation of proteases. Cumulatively, this action results in a buildup of ECM, increased stiffness (loss of compliance), thickened alveolar septae, and impairment of gas exchange, leading to progressive dyspnea, supplemental oxygen dependence, and eventually death. This stiffened matrix changes the forces sensed by fibroblasts, which drive myofibroblast differentiation, expression and cross-linking of ECM proteins, and other profibrotic effector functions, leading to a progressive feed-forward loop centered on the matrix mechanics.11Liu F. Mih J.D. Shea B.S. Kho A.T. Sharif A.S. Tager A.M. Tschumperlin D.J. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.J Cell Biol. 2010; 190: 693-706Crossref PubMed Scopus (459) Google Scholar,13Huang X. Yang N. Fiore V.F. Barker T.H. Sun Y. Morris S.W. Ding Q. Thannickal V.J. Zhou Y. Matrix stiffness-induced myofibroblast differentiation is mediated by intrinsic mechanotransduction.Am J Respir Cell Mol Biol. 2012; 47: 340-348Crossref PubMed Scopus (285) Google Scholar Current therapeutic options slow the progression of IPF but do not halt or reverse the scarring in the lung.15Maher T.M. Strek M.E. Antifibrotic therapy for idiopathic pulmonary fibrosis: time to treat.Respir Res. 2019; 20: 205Crossref PubMed Scopus (70) Google Scholar Given the role of the ECM in the initiation and progression of fibrosis, therapeutic strategies that target the matrix or cellular mechanical sensing may be an essential target to combine with current therapeutic options. The current review describes how lung fibroblasts sense and respond to mechanical stresses, as well as how altered mechanical properties of the fibrotic lung drive the progression of fibrosis. Possible therapeutic insights are also discussed. The matrix is bioactive and plays a role in the progression of IPF as a regulator of cellular phenotype and behavior. In the lung, the ECM is composed of a basement membrane with thin sheets of glycoproteins and the interstitial matrix, creating a fiber network that provides structure and the bulk of the mechanical integrity of the tissue.16Burgstaller G. Oehrle B. Gerckens M. White E.S. Schiller H.B. Eickelberg O. The instructive extracellular matrix of the lung: basic composition and alterations in chronic lung disease.Eur Respir J. 2017; 50: 1601805Crossref PubMed Scopus (151) Google Scholar The ECM contains >300 different proteins, including collagens, proteoglycans, and glycoproteins.16Burgstaller G. Oehrle B. Gerckens M. White E.S. Schiller H.B. Eickelberg O. The instructive extracellular matrix of the lung: basic composition and alterations in chronic lung disease.Eur Respir J. 2017; 50: 1601805Crossref PubMed Scopus (151) Google Scholar There is interest in identifying the physical aspects, including ECM composition and mechanical properties, that contribute to cellular activation and disease initiation and progression. Examples include barrier function and activation of endothelial and epithelial cells in response to stretch and shear, as well as fibroblast activation in response to stiffness and stretch of the ECM. The biomechanical properties of lung tissue are strongly influenced by the biochemical composition of the ECM. The ECM in IPF is compositionally different from that in healthy lung tissue, with expanded collagens and glycosaminoglycans.17Booth A.J. Hadley R. Cornett A.M. Dreffs A.A. Matthes S.A. Tsui J.L. Weiss K. Horowitz J.C. Fiore V.F. Barker T.H. Moore B.B. Martinez F.I. Niklason L.E. White E.S. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.Am J Respir Crit Care Med. 2012; 186: 866-876Crossref PubMed Scopus (377) Google Scholar, 18Raghu G. Striker L.J. Hudson L.D. Striker G.E. Extracellular matrix in normal and fibrotic human lungs.Am Rev Respir Dis. 1985; 131: 281-289PubMed Google Scholar, 19Kuhn 3rd, C. Boldt J. King T.E. Crouch E. Vartio T. McDonald J.A. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.Am Rev Respir Dis. 1989; 140: 1693-1703Crossref PubMed Scopus (361) Google Scholar Immunohistochemical analysis shows that fibroblastic foci are rich in collagen and EDA-fibronectin.19Kuhn 3rd, C. Boldt J. King T.E. Crouch E. Vartio T. McDonald J.A. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.Am Rev Respir Dis. 1989; 140: 1693-1703Crossref PubMed Scopus (361) Google Scholar Periostin is a glycoprotein that is secreted by monocytes and fibrocytes, is overexpressed in IPF, and promotes fibrogenesis.20Naik P.K. Bozyk P.D. Bentley J.K. Popova A.P. Birch C.M. Wilke C.A. Fry C.D. White E.S. Sisson T.H. Tayob N. Carnemolla B. Orecchia P. Flaherty K.R. Hershenson M.B. Murray S. Martinez F.J. Moore B.B. COMET InvestigatorsPeriostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol. 2012; 303: L1046-L1056Crossref PubMed Scopus (178) Google Scholar The accumulation of additional ECM is due to enhanced myofibroblast activation and production of ECM, coupled with inhibited ECM turnover and degradation.21Kadler K.E. Hill A. Canty-Laird E.G. Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators.Curr Opin Cell Biol. 2008; 20: 495-501Crossref PubMed Scopus (473) Google Scholar, 22Branton M.H. Kopp J.B. TGF-β and fibrosis.Microbes Infect. 1999; 1: 1349-1365Crossref PubMed Scopus (495) Google Scholar, 23Chanda D. Otoupalova E. Smith S.R. Volckaert T. De Langhe S.P. Thannickal V.J. Developmental pathways in the pathogenesis of lung fibrosis.Mol Aspects Med. 2019; 65: 56-69Crossref PubMed Scopus (94) Google Scholar Furthermore, posttranslational modification such as enzymatic and chemical cross-linking are altered in IPF and contribute to tissue stiffening. Increased mechanical forces within tissues activate latent transforming growth factor-β (TGF-β) through integrin-dependent mechanisms,24Buscemi L. Ramonet D. Klingberg F. Formey A. Smith-Clerc J. Meister J.J. Hinz B. The single-molecule mechanics of the latent TGF-[beta]1 complex.Curr Biol. 2011; 21: 2046-2054Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 25Hinz B. Tissue stiffness, latent TGF-beta1 activation, and mechanical signal transduction: implications for the pathogenesis and treatment of fibrosis.Curr Rheumatol Rep. 2009; 11: 120-126Crossref PubMed Scopus (255) Google Scholar, 26Hinz B. Phan S.H. Thannickal V.J. Prunotto M. Desmoulière A. Varga J. De Wever O. Mareel M. Gabbiani G. Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.Am J Pathol. 2012; 180: 1340-1355Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar driving profibrotic effector functions, including ECM deposition and cross-linking that increase tissue stiffness and thereby further activates TGF-β (Figure 1). Cross-linking enzymes include lysyl oxidase (LOX), lysyl oxidase-like (LOXL), and transglutaminases (TGs). Pathologic cross-linking due to up-regulation of LOXL and TG enzyme activity enhances fibroblast accumulation and increases resistance to ECM turnover.27Philp C.J. Siebeke I. Clements D. Miller S. Habgood A. John A.E. Navaratnam V. Hubbard R.B. Jenkins G. Johnson S.R. Extracellular matrix cross-linking enhances fibroblast growth and protects against matrix proteolysis in lung fibrosis.Am J Respir Cell Mol Biol. 2018; 58: 594-603Crossref PubMed Scopus (54) Google Scholar TG2 knockout mice are protected from bleomycin-induced pulmonary fibrosis. Serum LOXL2 levels are elevated in patients with IPF and are associated with increased risk of disease progression.28Chien J.W. Richards T.J. Gibson K.F. Zhang Y. Lindell K.O. Shao L. Lyman S.K. Adamkewicz J.I. Smith V. Kaminski N. O’Riordan T. Serum lysyl oxidase-like 2 levels and idiopathic pulmonary fibrosis disease progression.Eur Respir J. 2014; 43: 1430-1438Crossref PubMed Scopus (93) Google Scholar Despite promising preclinical studies,29Roberts J. Tear V. Lunn K. Cao L. Murphy J. Shimbori C. Imani J. Tandon K. Ayaub E. Jones M. Davies D. Jarolimek W. Ask K. Gauldie J. Kolb M. Monk P. Evaluation of novel LOXL2-selective inhibitors in models of pulmonary fibrosis.Eur Respir J. 2017; 50: PA3476Google Scholar a Phase II clinical trial with the LOXL2 inhibitor simtuzumab showed no effect on progression-free survival for patients with IPF.30Raghu G. Brown K.K. Collard H.R. Cottin V. Gibson K.F. Kaner R.J. Lederer D.J. Martinez F.J. Noble P.W. Song J.W. Wells A.U. Whelan T.P.M. Wuyts W. Moreau E. Patterson S.D. Smith V. Bayly S. Chien J.W. Gong Q. Zhang J.J. O’Riordan T.G. Efficacy of simtuzumab versus placebo in patients with idiopathic pulmonary fibrosis: a randomised, double-blind, controlled, phase 2 trial.Lancet Respir Med. 2017; 5: 22-32Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar More in-depth details on the ECM of the lung in healthy and diseased states have been provided in several previously published review articles.16Burgstaller G. Oehrle B. Gerckens M. White E.S. Schiller H.B. Eickelberg O. The instructive extracellular matrix of the lung: basic composition and alterations in chronic lung disease.Eur Respir J. 2017; 50: 1601805Crossref PubMed Scopus (151) Google Scholar,31Thannickal V.J. Henke C.A. Horowitz J.C. Noble P.W. Roman J. Sime P.J. Zhou Y. Wells R.G. White E.S. Tschumperlin D.J. Matrix biology of idiopathic pulmonary fibrosis: a workshop report of the National Heart, Lung, and Blood Institute.Am J Pathol. 2014; 184: 1643-1651Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar,32Upagupta C. Shimbori C. Alsilmi R. Kolb M. Matrix abnormalities in pulmonary fibrosis.Eur Respir Rev. 2018; 27: 180033Crossref PubMed Scopus (71) Google Scholar Multiple mechanical forces contribute to pathologic tissue remodeling and fibrosis. Prior studies of lung tissue mechanics (in the normal and diseased states) have been limited to the characterization of matrix stiffness.11Liu F. Mih J.D. Shea B.S. Kho A.T. Sharif A.S. Tager A.M. Tschumperlin D.J. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.J Cell Biol. 2010; 190: 693-706Crossref PubMed Scopus (459) Google Scholar,17Booth A.J. Hadley R. Cornett A.M. Dreffs A.A. Matthes S.A. Tsui J.L. Weiss K. Horowitz J.C. Fiore V.F. Barker T.H. Moore B.B. Martinez F.I. Niklason L.E. White E.S. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.Am J Respir Crit Care Med. 2012; 186: 866-876Crossref PubMed Scopus (377) Google Scholar,26Hinz B. Phan S.H. Thannickal V.J. Prunotto M. Desmoulière A. Varga J. De Wever O. Mareel M. Gabbiani G. Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.Am J Pathol. 2012; 180: 1340-1355Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar,33Southern B.D. Grove L.M. Rahaman S.O. Abraham S. Scheraga R.G. Niese K.A. Sun H. Herzog E.L. Liu F. Tschumperlin D.J. Egelhoff T.T. Rosenfeld S.S. Olman M.A. Matrix-driven myosin II mediates the pro-fibrotic fibroblast phenotype.J Biol Chem. 2016; 291: 6083-6095Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Tissue stiffness, measured in kilopascals (kPa), measures the force required for deformation. Normal lung tissue has a stiffness in the range of 0.5 to 5 kPa, whereas fibrotic lung tissue is in the range of 15 to 100 kPa.11Liu F. Mih J.D. Shea B.S. Kho A.T. Sharif A.S. Tager A.M. Tschumperlin D.J. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.J Cell Biol. 2010; 190: 693-706Crossref PubMed Scopus (459) Google Scholar,17Booth A.J. Hadley R. Cornett A.M. Dreffs A.A. Matthes S.A. Tsui J.L. Weiss K. Horowitz J.C. Fiore V.F. Barker T.H. Moore B.B. Martinez F.I. Niklason L.E. White E.S. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.Am J Respir Crit Care Med. 2012; 186: 866-876Crossref PubMed Scopus (377) Google Scholar,26Hinz B. Phan S.H. Thannickal V.J. Prunotto M. Desmoulière A. Varga J. De Wever O. Mareel M. Gabbiani G. Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.Am J Pathol. 2012; 180: 1340-1355Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar,33Southern B.D. Grove L.M. Rahaman S.O. Abraham S. Scheraga R.G. Niese K.A. Sun H. Herzog E.L. Liu F. Tschumperlin D.J. Egelhoff T.T. Rosenfeld S.S. Olman M.A. Matrix-driven myosin II mediates the pro-fibrotic fibroblast phenotype.J Biol Chem. 2016; 291: 6083-6095Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Lung fibroblasts also experience mechanical forces from stretching, as the lungs expand and contract during breathing. Unlike either normal or fibrotic lung tissue, solid plastic tissue culture dishes have a substrate stiffness of approximately 100,000 kPa, and there is evidence that these forces induce nonphysiological cellular responses to mechanical strain.34Solon J. Levental I. Sengupta K. Georges P.C. Janmey P.A. Fibroblast adaptation and stiffness matching to soft elastic substrates.Biophys J. 2007; 93: 4453-4461Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar, 35Achterberg V.F. Buscemi L. Diekmann H. Smith-Clerc J. Schwengler H. Meister J.J. Wenck H. Gallinat S. Hinz B. The nano-scale mechanical properties of the extracellular matrix regulate dermal fibroblast function.J Invest Dermatol. 2014; 134: 1862-1872Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 36Caliari S.R. Perepelyuk M. Cosgrove B.D. Tsai S.J. Lee G.Y. Mauck R.L. Wells R.G. Burdick J.A. Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation.Sci Rep. 2016; 6: 21387Crossref PubMed Scopus (109) Google Scholar To better recapitulate the native physiological environment, researchers have developed in vitro model systems using silicone membranes or polyacrylamide gels with tunable substrate stiffness.37Syed S. Karadaghy A. Zustiak S. Simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses.J Vis Exp. 2015; 25: 52643Google Scholar,38Tse J.R. Engler A.J. Preparation of hydrogel substrates with tunable mechanical properties.Curr Protoc Cell Biol. 2010; (Chapter 10:16)Crossref PubMed Scopus (516) Google Scholar There are now commercially available systems for tunable stiffness (Advanced BioMatrix, Carlsbad, CA), as well as application of cyclic stretch (Flexcell International, Burlington, NC; Strex, San Diego, CA). Using custom and commercially available systems, investigators have begun to elucidate the effects of mechanical cues on fibroblast activation as well as mechanotransduction mechanisms. Increased stiffness both enhances recruitment of fibroblasts (durotaxis)39Lo C.M. Wang H.B. Dembo M. Wang Y.L. Cell movement is guided by the rigidity of the substrate.Biophys J. 2000; 79: 144-152Abstract Full Text Full Text PDF PubMed Scopus (2392) Google Scholar and myofibroblast differentiation.11Liu F. Mih J.D. Shea B.S. Kho A.T. Sharif A.S. Tager A.M. Tschumperlin D.J. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression.J Cell Biol. 2010; 190: 693-706Crossref PubMed Scopus (459) Google Scholar,25Hinz B. Tissue stiffness, latent TGF-beta1 activation, and mechanical signal transduction: implications for the pathogenesis and treatment of fibrosis.Curr Rheumatol Rep. 2009; 11: 120-126Crossref PubMed Scopus (255) Google Scholar,32Upagupta C. Shimbori C. Alsilmi R. Kolb M. Matrix abnormalities in pulmonary fibrosis.Eur Respir Rev. 2018; 27: 180033Crossref PubMed Scopus (71) Google Scholar,40Balestrini J.L. Chaudhry S. Sarrazy V. Koehler A. Hinz B. The mechanical memory of lung myofibroblasts.Integr Biol (Camb). 2012; 4: 410-421Crossref PubMed Scopus (191) Google Scholar Excitingly, reducing stiffness can partially reverse a stiffness-induced myofibroblast phenotype.36Caliari S.R. Perepelyuk M. Cosgrove B.D. Tsai S.J. Lee G.Y. Mauck R.L. Wells R.G. Burdick J.A. Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation.Sci Rep. 2016; 6: 21387Crossref PubMed Scopus (109) Google Scholar,41Caliari S.R. Perepelyuk M. Soulas E.M. Lee G.Y. Wells R.G. Burdick J.A. Gradually softening hydrogels for modeling hepatic stellate cell behavior during fibrosis regression.Integr Biol (Camb). 2016; 8: 720-728Crossref PubMed Google Scholar,42Marinković A. Liu F. Tschumperlin D.J. Matrices of physiologic stiffness potently inactivate idiopathic pulmonary fibrosis fibroblasts.Am J Respir Cell Mol Biol. 2013; 48: 422-430Crossref PubMed Scopus (92) Google Scholar This action supports the hypothesis that restoraton of healthy ECM will result in cells responding and reverting from their diseased phenotype to restore homeostasis. Lung tissue, as with other tissues in the body, has more complex mechanical properties, as they are viscoelastic, exhibiting stress–strain relationships dependent on time and history. This presents an opportunity to more comprehensively analyze the mechanical properties of lung tissue in healthy and diseased states, and then implement more complex in vitro systems to model the mechanical microenvironment. IPF results in heterogeneous distribution of fibrotic lesions adjacent to normal-appearing areas within the lung. The boundary between the presumed healthy tissue and the fibrotic lesion introduces stress concentrations in which abrupt changes of mechanical properties are present, leading to high cellular and matrix strains at the transition. Furthermore, new techniques have revealed that the normal-appearing tissue adjacent to fibrotic lesions is not actually normal. Second harmonic generation imaging has shown that the fibrillar collagen microstructure of the “normal”-appearing tissue adjacent to fibrotic foci matches the second harmonic generation signature within the fibroblastic foci themselves.43Kottmann R.M. Sharp J. Owens K. Salzman P. Xiao G.Q. Phipps R.P. Sime P.J. Brown E.B. Perry S.W. Second harmonic generation microscopy reveals altered collagen microstructure in usual interstitial pneumonia versus healthy lung.Respir Res. 2015; 16: 61Crossref PubMed Scopus (24) Google Scholar A similar phenomenon has been observed in liver fibrosis in which there is an observed increase in tissue stiffness before fibrotic matrix deposition.44Georges P.C. Hui J.J. Gombos Z. McCormick M.E. Wang A.Y. Uemura M. Mick R. Janmey P.A. Furth E.E. Wells R.G. Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis.Am J Physiol Gastrointest Liver Physiol. 2007; 293: G1147-G1154Crossref PubMed Scopus (386) Google Scholar These data indicate that the ECM microstructure at the leading edge of a fibrotic lesion is altered before the alveolus is destroyed. The mechanical properties at the leading edge will be particularly important to characterize with extreme cellular mechanical forces experienced by it. There is a prime need to understand how lung cells, particularly fibroblasts, sense and respond to these mechanical signals to determine cellular responses within the transition region. The matrix is bioactive and communicates with cells. Cells sense changes in matrix mechanical properties through force-induced conformation change of mechanically sensitive molecules; this leads to the activation of downstream biochemical signaling pathways, a process defined as mechanotransduction.45Balaban N.Q. Schwarz U.S. Riveline D. Goichberg P. Tzur G. Sabanay I. Mahalu D. Safran S. Bershadsky A. Addadi L. Geiger B. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.Nat Cell Biol. 2001; 3: 466-472Crossref PubMed Scopus (1693) Google Scholar,46Sun Z. Guo S.S. Fässler R. Integrin-mediated mechanotransduction.J Cell Biol. 2016; 215: 445-456Crossref PubMed Scopus (403) Google Scholar Mechanosensors reside on the cell membrane and include integrins, stretch-activated ion channels, and G protein coupled–receptors (Figure 1). Integrin-mediated mechanotransduction is the primary mechanism for cells to sense ECM mechanics. One critical mechanotransduction feed-forward loop in fibrosis is integrin-mediated mechanical activation of latent TGF-β47Henderson N.C. Sheppard D. Integrin-mediated regulation of TGF[beta] in fibrosis.Biochim Biophys Acta. 2013; 1832: 891-896Crossref PubMed Scopus (128) Google Scholar (Figure 1). The integrins αvβ3, αvβ5, and αvβ6 have been shown to bind the RGD sequence (arginine-glycine-aspartate) in the latency-associated peptide of TGF-β1 to activate latent TGF-β.48Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1565) Google Scholar, 49Asano Y. Ihn H. Yamane K. Jinnin M. Tamaki K. Increased expression of integrin alphavbeta5 induces the myofibroblastic differentiation of dermal fibroblasts.Am J Pathol. 2006; 168: 499-510Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 50Wipff P.J. Rifkin D.B. Meister J.J. Hinz B. Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix.J Cell Biol. 2007; 179: 1311-1323Crossref PubMed Scopus (908) Google Scholar Integrins are up-regulated in fibrotic disease.48Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibr