Biomechanical Force and Cellular Stiffness in Lung Fibrosis

Abstract

Lung fibrosis is characterized by the continuous accumulation of extracellular matrix (ECM) proteins produced by apoptosis-resistant (myo)fibroblasts. Lung epithelial injury promotes the recruitment and activation of fibroblasts, which are necessary for tissue repair and restoration of homeostasis. However, under pathologic conditions, a vicious cycle generated by profibrotic growth factors/cytokines, multicellular interactions, and matrix-associated signaling propagates the wound repair response and promotes lung fibrosis characterized not only by increased quantities of ECM proteins but also by changes in the biomechanical properties of the matrix. Importantly, changes in the biochemical and biomechanical properties of the matrix itself can serve to perpetuate fibroblast activity and propagate fibrosis, even in the absence of the initial stimulus of injury. The development of novel experimental models and methods increasingly facilitates our ability to interrogate fibrotic processes at the cellular and molecular levels. The goal of this review is to discuss the impact of ECM conditions in the development of lung fibrosis and to introduce new approaches to more accurately model the in vivo fibrotic microenvironment. This article highlights the pathologic roles of ECM in terms of mechanical force and the cellular interactions while reviewing in vitro and ex vivo models of lung fibrosis. The improved understanding of the fundamental mechanisms that contribute to lung fibrosis holds promise for identification of new therapeutic targets and improved outcomes.

Fibrosis is the consequence of an abnormal cellular response to tissue injury in which the wound repair response leads to the accumulation of excessive scar tissue that disrupts tissue architecture and function. From a teleological perspective, scar formation can be considered a life-preserving response to injury.¹ In modern times, however, the fibrotic response has become the disease, with up to 45% of deaths in the industrialized world attributable to diseases characterized by fibrosis.² The underlying mechanisms of fibrosis may become autonomous and self-propagating despite the resolution of the initial cause of injury. This is likely the case in the progressive fibrotic lung disease of idiopathic pulmonary fibrosis (IPF).

IPF is increasingly recognized as a disease of aging associated with underlying genetic and environmental risk factors. As denoted by the use of idiopathic, the proximate cause(s) of injury that initiates the progressive lung disease, with an estimated 50% mortality within 3 to 5 years of diagnosis, remains unclear. The pathologic hallmark of IPF, usual interstitial pneumonia, is characterized by temporal and spatial heterogeneity with areas of normal-appearing lung interspersed with areas of more advanced disruption. These areas range from thickening of the alveolar interstitium to more advanced honeycomb changes, with cystic dilation of terminal bronchioles within mature fibrotic scars associated with alveolar epithelial injury and focal collections of fibroblasts in lesions called fibroblastic foci (Figure 1).^3,4 The variegated histology suggests multifocal and recurrent injury; however, some data indicate the presence of an interconnected fibrotic reticulum linking spatially distinct fibroblastic foci, suggesting endogenous spread of this nonmalignant process.^3,5 IPF pathology shows a predilection for the lung periphery and bases, with relative sparing of the apices. This typical distribution has spurred interest in the idea that biomechanical forces, including cyclical stretch, may contribute to disease pathogenesis.^6,7 The role of biomechanical inputs is further supported by the physiological finding of decreased lung compliance in IPF coupled with increased tissue stiffness, as well as experimental evidence linking increased tissue stiffness to profibrotic cellular phenotypes.⁷ In fact, biomechanical signaling from the extracellular matrix (ECM) to cells is one potential mechanism that supports the autonomous propagation of the fibrotic response.^6,8

Figure 1.

Histologic and ultrastructural images of fibrotic lung tissue in idiopathic pulmonary fibrosis. A: Hematoxylin and eosin stain showing honeycomb change with cystic airway dilation corresponding to distended bronchioles in scarred, fibrotic lung.³B: Electron microscopy from idiopathic pulmonary fibrosis lung showing disruption of the alveolar–capillary barrier with fragments of the original alveolar basal lamina is displayed (arrows).⁴ Panel A reprinted from Visscher et al³ with permission of the American Thoracic Society. Panel B reprinted from Kuhn and McDonald⁴ with permission from The American Society for Investigative Pathology. Original magnification: ×40 (A); ×4000 (B).

The current article reviews some of the key cellular and extracellular features that contribute to physiological and pathologic repair. It discusses some of the established and emerging tools used to study cellular interactions in the context of biochemically and biomechanically relevant substrates and speculate how these models can be leveraged to advance our mechanistic understanding of lung repair and fibrosis.

ECM and Collagen

Four types of tissues—epithelial, connective, muscle, and nervous—have been described.⁹ Each tissue is composed of multiple cell types that interact to conduct necessary homeostatic functions. As in all solid tissues, lung cells are surrounded by, and constantly interact with, the ECM, a network of proteins, proteoglycans, and other multidomain macromolecules organized in a cell- and tissue-specific manner.¹⁰ Direct interactions with the ECM expose cells to the biomechanical properties of their microenvironment, which are transmitted to the cells via transmembrane receptors that bind to ECM components at specific sites known as focal adhesions.¹¹ Cells also interact with neighboring cells through cell–cell adhesions, and these interactions can affect ECM structure and mechanics.^7,12 Normally, the ECM forms a structurally stable fibrous network containing collagens, proteoglycans, elastin, and glycoproteins.¹² Beyond its structural contributions, the ECM is a highly dynamic entity that regulates fundamental cellular activities such as proliferation, adhesion, migration, polarity, differentiation, and survival.^13,14 The ECM is also a reservoir for growth factors that drive cellular responses in fibrosis, including latent transforming growth factor-β (TGF-β). Importantly, the mechanical properties of the ECM can affect the activation of, and cellular responses to, these bioactive molecules. Thus, ECM composition combined with biomechanical properties and cellular interactions has a broad regulatory impact on cell phenotype.¹⁵

ECM composition is controlled in a tissue-specific fashion, and changes to its composition influence the biomechanical properties and function of the tissue. The elastic fibrous component of ECM provides tensile strength, serves as an adhesive site for cell attachment, and facilitates cellular migration and tissue development.¹⁵ In lungs, elastin and microfibrils, including fibrillin-1, -2, and -3, are found in the inner core and the outer peripheral lung.¹⁶ Polysaccharide chains are covalently bound to transmembrane proteins and assemble into proteoglycans and the fibrillar proteins, including collagens, fibronectin, elastin, and laminins, allowing direct interactions with cells by serving as ligands for cell adhesion molecules.¹⁷ Collagen is the most abundant protein found in the ECM of vertebrates and plays a critical role in determining the biophysical properties of the lung tissues. Various types of collagen exist in different tissues,¹⁸ and these can be broadly categorized into fibrillar and nonfibrillar collagens. Fibrillar collagens such as type I, II, III, V, XI, XXIV, and XXVII provide the structural framework for tissues, whereas nonfibrillar collagens such as type IV are mainly found in the basement membrane. Among the collagens, type I collagen is the most abundant structural protein found in the lungs and in many different tissues and organs, including skin, tendons, vasculature, heart, and kidney.¹⁹ Beyond the presence or absence of individual ECM components, posttranslational modifications such as the addition of sugars by enzymatic (glycosylation) or nonenzymatic (glycation) processes affect ECM biomechanics. For example, the accumulation of advanced glycation end-products increases tissue stiffness.²⁰ Thus, the biomechanical properties of a tissue represent the complex interplay between synthesis and degradation of the individual ECM components, how these components are modified after synthesis, and the interactions between the ECM components.

Cell–ECM Interactions and Disease

Cell–ECM interactions greatly affect a cell’s susceptibility to stress and death signals. A study in normal human fibroblasts and tumor cells (eg, glioblastoma, pancreatic carcinomas, bronchial carcinomas, melanomas, breast cancers) documented that cell adhesion to the ECM enhances resistance to ionizing radiation, chemotherapy, and molecular therapies.²¹ These mechanisms are referred to as cell adhesion–mediated radioresistance and cell adhesion–mediated drug resistance.^21,22 In contrast, loss or inhibition of cell–matrix adhesions promotes a form of regulated cell death termed anoikis.^23,24 Consistently, fibroblasts from patients with fibrotic lung diseases, including IPF, have increased resistance to programed cell death induced by a variety of stimuli and attributed to several different mechanisms related to soluble signals and mechanotransduction-mediated signaling.^6,25 Various soluble mediators have been implicated in the pathobiology of fibrosis, including TGF-β, connective tissue growth factor, endothelin-1, and lysophosphatidic acid.²⁵ Among them, TGF-β is a multifunctional cytokine that plays a crucial role in myofibroblast differentiation and has been most strongly implicated in fibrosis. The potential interacting mechanisms involve kinases, transcription factors, mitochondrial regulators, caspase-binding proteins, and cell surface receptors, including phosphatidylinositol 3-kinase/Akt, FoxO3a, focal adhesion kinase (FAK), Nox4, inhibitors of apoptosis proteins, Fas, myocardin-related transcription factor-A, and Bcl-2 family proteins. These are all regulated by TGF-β and, in many cases, other soluble mediations implicated in fibrosis. The TGF-β–regulated signaling pathways are known to confer apoptosis resistance to fibroblasts and have been found to be active or expressed at increased levels in tissues from patients with IPF.25, 26, 27, 28 In addition, TGF-β regulates ECM production and stiffness, both of which are implicated in the progression of lung fibrosis (as described in ECM Stiffness, Cellular Stiffness, Cell Signaling Receptors, and Lung Fibrosis).

Decreased lung compliance is a well-recognized clinical manifestation observed in patients with lung fibrosis. Increased tissue stiffness reflecting the mechanical properties of the underlying ECM is likely to account for the decreased compliance. For example, the elastic modulus (similar to Young’s modulus, a measure of “stiffness” as described in ECM Stiffness, Cellular Stiffness, Cell Signaling Receptors, and Lung Fibrosis) of healthy lungs is about 1.96 kPa, and shear modulus (the material’s response to shear stress) is between 0.84 and 1.50 kPa.^29,30 In contrast, fibrotic lungs and other organs have stiffness that is 10- to 50-fold higher than that of healthy tissues.^31,32 Notably, tissue stiffness becomes altered in several human diseases. Therefore, the Young’s modulus (or elastic modulus) is frequently used as a method to quantify stiffness of healthy and diseased tissue.^31,33,34 It is important to note that there is a wide variability of the tissue stiffness reported in different fibrotic diseases, and topographic studies have identified significant variability in stiffness within a fibrotic microenvironment. This regional variation in stiffness within a fibrotic tissue may affect the accumulation of fibroblasts through a phenomenon known as durotaxis.^35,36

Fibroblast attachment to collagen-rich ECM is a well-defined cellular process in tissue homeostasis. After tissue injury, normal lung fibroblasts migrate to the site of injury and produce ECM to facilitate the repair process. Once the wound repair is completed, they undergo apoptosis, and tissue homeostasis is restored. In fibrotic repair, however, abnormally activated fibroblasts continuously produce ECM, leading to the destruction of lung parenchyma. Wound repair, whether physiological or pathologic, is a dynamic process in which multiple ECM components have critical roles. For example, early in the wound repair response, the provisional ECM is rich in fibrin and fibronectin. Fibronectin, a “cell-adhesive glycoprotein,” accumulates around activated fibroblasts and forms a bridge that facilitates fibroblast interactions with collagen and cross-linking of the ECM.³⁷ This can amplify fibroblast synthesis and secretion of other ECM molecules, including hyaluronan and proteoglycans, which promote ECM stabilization.^37,38 Thus, although fibronectin may not have a direct impact on the mechanical properties of the lung ECM, it is important for the adherence of a variety of pulmonary cell types to the matrix, and interacts with cells to direct their morphology, motility, and differentiation.³⁷ Interestingly, IPF lung tissues contain extra type III domain A fibronectin, a splice variant of which was found to be critical for TGF-β–mediated fibroblast differentiation and lung fibrosis.³⁹

Fibroblasts from patients with IPF have apoptosis-resistant properties, allowing their accumulation, persistence, and production of excessive amounts of ECM. Fibroblast apoptosis is greatly affected by collagen components in ECM. Prior studies support the concept that fibroblasts from patients with IPF cultured on a polymerized three-dimensional (3D) collagen matrix have increased resistance to the cell death signals derived from the collagen ECM.^26,40,41 Thus, it is plausible that aberrant interactions between fibroblasts and a collagen-rich ECM reflect the dysregulation of cellular homeostasis and contribute to the progression of fibrotic disease processes. Similarly, intact fibronectin in the ECM can promote fibroblast survival. Fibronectin proteolysis has been shown to trigger myofibroblast apoptosis⁴²; however, it is not known whether this is a direct result of fibronectin proteolysis or whether the fibronectin proteolysis disrupts interactions with collagen.

Biomechanical Forces and the Pathologic Role of a Stiff ECM

Cells interact with the ECM, and the biochemical and biophysical properties of the matrix directly or indirectly influence cell fate and function. Biomechanical forces are critical during development and shape the early embryo by driving tissues to move, strain, and deform.43, 44, 45 Mechanical forces from the ECM can manifest in several ways: tensional (alias tensile) stress, compressive stress, torsional stress, and shear stress.⁴⁶ The elasticity of materials is commonly represented by the Young’s modulus, which depicts the linear resistance of a material to elastic deformation under stress.⁴⁷ The Young’s modulus is also related to the stiffness or rigidity of a material; that is, the extent to which the material can resist to deformation or deflection in response to an applied force.^44,48 At the molecular level, mechanical forces generated by stiff ECM regulate gene expression and protein function. Increased ECM substrate stiffness, as seen in fibrotic lung tissues, is sufficient to induce myofibroblast activation and apoptosis resistance through the activation of well-established mechanotransduction signaling pathways.³⁵ This suggests that although activated fibroblasts can deposit and organize an ECM with increased stiffness, interactions with that ECM can perpetuate a profibrotic vicious cycle.

While a number of studies have focused on the effect of ECM stiffness on cellular responses, much less emphasis has been given to the role of tissue viscoelasticity. Viscoelasticity describes the behavior of materials that exhibit both viscous and elastic characteristics, and a material’s deformation may differ in a time-dependent manner. Certain ECM molecules, such as versican, are thought to play an important role in regulating tissue viscoelasticity. Versican is a large ECM proteoglycan found in human tissues, including lungs, and is implicated in remodeling in airway inflammatory disorders such as asthma and chronic obstructive pulmonary disease as well as in fibrosis.⁴⁹ Interestingly, versican expression is known to change with age, and IPF is an age-dependent disease. Moreover, versican has been identified in the type I collagen–rich core of fibroblastic foci and in association with type IV collagen in thickened alveolar septa in IPF tissue.⁵⁰ Consistent with this, a proteomic analysis of IPF fibroblast matrix stimulated with TGF-β showed increased deposition of versican that correlated with levels found in IPF lung tissue.⁵¹ Thus, it is plausible that alterations of viscoelasticity mediated by versican have a role in lung fibrogenesis. As with ECM stiffness, matrix viscoelasticity also reportedly affects cell behavior.⁵² Although matrix viscoelasticity regulates cellular behavior, and the changes in viscoelasticity are associated with disease progression,⁵² how matrix viscoelasticity changes during the evolution of fibrosis and how viscoelasticity affects the pathogenesis of fibrotic disease remain poorly understood.

ECM Stiffness, Cellular Stiffness, Cell Signaling Receptors, and Lung Fibrosis

ECM Stiffness

Increased matrix stiffness is sufficient to induce fibroblast activation and collagen production,⁴³ whereas more compliant matrices can reverse fibroblast activation.^53,54 It is important to note that the biomechanical properties of the ECM are directly influenced by the biochemical composition of the matrix and its individual components.⁵⁵ The concentration and organization of ECM components such as collagen, elastin, fibronectin, and glycosaminoglycans determine the mechanical properties of both healthy and diseased tissues.⁴⁸ Fibrous extracellular networks of collagen and elastin are the primary determinants of tissue mechanical strength.⁵⁶ These fibrous protein networks lie in a viscous interstitial milieu that is rich in glycoproteins, proteoglycans, glycosaminoglycans, and a complex composition of growth factors, cytokines, chemokines, and proteases.⁵⁷ Among ECM components, collagens (particularly type I collagen) have a dominant role in modulating ECM stiffness. Type I collagen forms thick, stiff, and long fibrils, which decrease in tissue compliance that can be altered by hundreds of megapascals in stiffness.^56,58 The wide range of substrate stiffness (from megapascals to gigapascals) that can be seen in different tissues also provides strong evidence that the stiffness of these tissues depends on much more than the biochemical composition of the matrix and that this property extends to posttranslational processing of the ECM components, including how macromolecules cross-link and interact with each other.⁵⁶

Stiffness is also influenced by the types of chemical bonds that exist within and between ECM components. For example, fibronectin and type IV collagen meshworks primarily interact through noncovalent protein–protein interfaces.⁵⁶ In the presence of pulling forces that can break these noncovalent bonds, meshwork-type matrices fail to transmit force. In contrast, covalently cross-linked type I collagen fibers support force propagation over long ranges because they have a much greater capacity to resist deformation.^56,59 These studies illustrate how type I collagen plays a critical role in transmitting mechanical forces across ECMs. This concept has important consequences in the context of fibrosis, as tissue stiffness is a critical factor in the activation of latent TGF-β that resides within the ECM.⁶⁰ Notably, this potent profibrotic cytokine is synthesized as the inactive pro–TGF-β that exists as the small latent complex. Latent TGF-β–binding protein 1 binds to the small latent complex, forming the large latent complex stored in large quantities within the ECM. Activation of TGF-β from the large latent complex is dependent on the deformation of the protein complex, which requires stiffness of the ECM itself. Once activated, TGF-β is free to bind to the TGF-β receptor and initiate profibrotic signaling cascades in fibroblasts.⁵⁵

From a physiological perspective, lung expansion is limited by elastance, and contraction is facilitated by increased elastance. Thus, abnormally increased elastance (decreases in lung compliance) can contribute to the sensation of dyspnea because additional work is required to expand the lungs during inhalation. In lung fibrosis, scar tissue impedes expansion and facilitates contraction of the lung, leading to a restrictive lung disease with decreased compliance. In addition, increased interstitial thickness and destruction of alveolar capillary units can impair diffusion and impede gas exchange (Figure 1).^61,62 Interestingly, fibrosis in patients with IPF is typically observed in the peripheral and basal regions of the lung. Physiologically, this distribution represents the areas of the lung that undergo the greatest degree of breath-to-breath deformation, leading to the hypothesis that increased mechanical stretch and distention is a critical aspect of this fibrotic lung disease. Thus, the lung matrix stiffness observed in patients with IPF is hypothesized to be associated with the peripheral and basal fibrosis and the distribution of IPF lesions corresponds to the areas of greatest mechanical distension during breathing.⁵⁵

Myofibroblasts are critical effector cells that function in the synthesis, secretion, deposition, and remodeling of the ECM in the wound repair response and fibrosis. Dysregulated myofibroblast function is closely associated with abnormal ECM accumulation and scar tissue formation. At the molecular level, collagen cross-linking accompanies tissue stiffness and fibrosis.⁶³ Crosslinking of collagen is driven by enzymatic and nonenzymatic mechanisms. Lysyl hydroxylases, lysyl oxidase (LOX), and transglutaminase 2 (TG2) can initiate the formation of both intramolecular and intermolecular cross-links, and each has been linked to lung fibrosis.⁶⁴ Collagen cross-linking can also be generated nonenzymatically, and the main nonenzymatic cross-links are those arising from advanced glycation end-products.⁶⁵

Among collagen cross-linking mechanisms, it is well established that LOX deregulation is linked to fibrosis. LOX initiates the process of covalent intramolecular and intermolecular cross-linking of collagen by oxidatively deaminating specific lysine and hydroxylysine residues located in the telopeptide domains.⁶⁶ Importantly, LOXL1 and LOXL2 gene expression and the corresponding protein levels are increased in IPF tissues compared with controls, and increased collagen fibril thickness in IPF versus non-IPF lung tissues is correlated with increased LOXL1 and LOXL2 protein expression.67, 68, 69 Moreover, TGF-β, AKT/mammalian target of rapamycin, and NF-κB–dependent pathways that are associated with lung fibrosis are known to regulate LOX genes.^27,28,70 Although the role of LOX enzymes in cross-linking and stiffness of collagen-rich tissues seems clear, and their contribution to fibrogenesis is plausible, a clinical trial targeting LOXL2 in patients with IPF failed to show a positive clinical outcome,⁷¹ highlighting the complexity of fibrotic disease and the difficulty in translation of basic mechanisms to clinical benefit.

As with the LOX enzymes, TG2 has been implicated in the development of pulmonary fibrosis and has received significant attention as a therapeutic target in fibrotic lung disease based on its matrix cross-linking properties.⁷² TG2 levels are increased in fibrotic lung tissues, and inhibition of TG2 attenuates lung fibrosis in murine models. The mechanistic role of TG2 in lung fibrosis may extend beyond its collagen cross-linking ability, as a recent study reported the identification of multiple additional TG2 substrates within fibrotic lungs. In contrast to LOXL2, inhibition of TG2 has not advanced to clinical trials for patients with fibrotic lung disease.^73,74

Rigidity of Fibrotic Lung Tissues Caused by Cellular Stiffness

Fibrotic disease is driven by dysregulation in mechanical forces at the organ, tissue, and cellular levels.⁷⁵ Cells sense and respond to the extracellular environment, and stiff matrices influence the cellular network of structural and signaling molecules, thereby affecting the mechanical properties of cells. Thus, as with ECM stiffness, cell stiffness should be considered as a mechanism contributing to fibrosis (Figure 2). Examples of the relationship between cell stiffness and human disorders have been previously described. In cancer cells, the measurements of cell stiffness have a strong correlation between cell deformability and malignancy.⁷⁶ In lung fibrosis, fibroblasts from patients with IPF were stiffer and had an augmented cytoskeletal response to TGF-β compared with fibroblasts from donors without IPF.⁷⁵ Atomic force microscopy analysis further supports the concept that the increased stiffness of lung fibroblasts from patients with IPF contributes to the increased rigidity of fibrotic lung tissue. The combination of a stiff mechanical environment (ECM stiffness) and increasing fibroblast cellular stiffness promotes the activation of TGF-β, whereas compliant matrices attenuate such activation.⁶⁰ One study documented that decellularized IPF lung tissues promote TGF-β–independent myofibroblast differentiation compared with normal matrices.²⁹ Collectively, these findings suggest that the formation of stiff fibrotic ECM and cellular stiffness cooperate in the development of lung fibrosis via TGF-β–dependent and TGF-β–independent mechanisms.

Figure 2.

Extracellular matrix (ECM) and fibroblast stiffness in lung fibrosis. Stiff ECM influences the cellular network of structural and signaling molecules while fibroblasts sense and respond to the extracellular environment. The dysregulated interplay causes the formation of scar tissues and the emergence of apoptosis-resistant fibroblasts, promoting lung fibrosis.

Several mechanisms are thought to augment cellular stiffness. First, myofibroblast contraction is a function of its cytoskeleton, and contractile forces are largely determined by α-smooth muscle actin organized into stress fibers.⁷⁷ Thus, enhanced expression and organization of α-smooth muscle actin by TGF-β is associated with the cellular stiffness.⁷⁵ Second, it has been suggested that the inactivation of the myosin light chain phosphatase through the RhoA-dependent Rho-(associated) kinase increases myofibroblast contraction.⁷⁸ Cytosolic Ca²⁺ levels are also thought to be associated with myofibroblast contraction. In addition, inhibition of the calcium-binding protein, calmodulin, impairs rat wound skin closure, and in vitro 3D models using colon myofibroblasts showed the link between cytosolic Ca²⁺ levels and contraction.⁷⁹ Together, these studies suggest that different mechanisms control myofibroblast contraction and cellular stiffness.

Mechanotransduction Pathways Engaged by Stiff ECM and Fibrotic Disease

Various cell surface protein receptors are involved in the matrix interactions of adherent cells. Mammalian collagen-binding receptors include discoidin receptors (DDR1 and DDR2), specific integrins, glycoprotein VI, leukocyte-associated immunoglobulin-like receptor 1, and mannose receptors. Among these, integrins are well-known mechanosensing receptors that mediate transduction of signals from the extracellular mechanical environment into intracellular biochemical signals. Structurally, integrins are heterodimeric transmembrane receptors composed of α and β subunits, various combinations of which provide a broad range of functional receptors with different matrix-binding specificities. Twenty-four distinct integrins have been identified in vertebrates, and individual integrins have ligand specificities. For example, α1β1, α2β1, α10β1, and α11β1 integrins are known to preferentially bind to type I collagen, whereas α5β1 and αvβ3 integrins are the major receptors for fibronectin, and αvβ3 integrins are the main receptor of vitronectin. Interestingly, integrins are signaling molecules that mediate bidirectional signaling: outside-in and inside-out signaling.⁸⁰ With outside-in signaling, engagement with ECM components initiates integrin clustering and assembly of cytoplasmic proteins such as FAK, c-Src, Paxillin, Ras, and Rho at the concentrated regions called focal adhesion complexes. Talin, a scaffold protein that directly binds to F-actin and vinculin, is also associated with the integrin β-subunit cytoplasmic domain. This event triggers various signal transduction pathways associated with proliferation, adhesion, survival, and migration. In the case of inside-out signaling, the presence of intracellular force stimulates molecules such as talin and kindlin to attach to the β-subunit, which promotes an extended and activated or open conformation, allowing interaction with the ECM.⁸⁰

In addition to direct mechanotransduction signaling, integrins are critical in the activation of TGF-β. Latency-associated peptide and latent TGF-ß–binding protein-1 contain the common recognition motif for integrin docking (arginine-glycine-aspartic acid), which allows for the cell-mediated activation of TGF-β stored in ECM.⁴ Several specific integrins, including αv integrins (αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8), α5β1, α8β1, and αIIbβ3, are known to activate latent TGF-β.^10,81 TGF-β is also activated by shear forces, plasmin, MMP-9, MMP-13, MT1-MMP, and reactive oxygen species.

The nonreceptor tyrosine kinase FAK is phosphorylated in response to a stiff ECM environment as well as by TGF-β. This mechanosensitive kinase is a critical mediator of profibrotic fibroblast functions, including actin polymerization and survival.²⁴ Downstream of FAK, Rho-associated kinase promotes actin polymerization.^82,83 Activated Rho-associated kinase and FAK induce the activation of the myosin light chain as well as inhibition of myosin light chain phosphatase, abruptly increasing myosin II activity and actomyosin contractility. In addition, actin polymerization facilitates the nuclear translocation of myocardin-related transcription factor-A, a transcriptional co-activator implicated in myofibroblast differentiation, survival, and fibrosis.⁸⁴ Similarly, nuclear translocation of the mechanosensitive transcription factors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) increase transcription of profibrotic genes such as collagen and other ECM proteins (Figure 3). These changes collectively translate into profibrotic phenotypic changes in fibroblasts.⁵⁵

Figure 3.

Mechanotransduction pathways engaged by stiff extracellular matrix (ECM). The external biomechanical forces promote the clustering of integrins to focal adhesions that integrate the extracellular inputs with the intracellular cytoskeleton. Focal adhesion kinase (FAK) can be activated by a stiff ECM as well as by transforming growth factor-β (TGF-β). Rho-associated protein kinase (ROCK), downstream of FAK, promotes actin polymerization, which facilitates the nuclear translocation of myocardin-related transcription factor A (MRTF-A) implicated in myofibroblast differentiation, survival, and fibrosis. Nuclear translocation of the mechanosensitive transcription factors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) also increases transcription of profibrotic genes. Extracellular enzymes such as lysyl oxidase (LOX) or transglutaminase 2 (TG2) initiate covalent intramolecular and intermolecular cross-linking of collagen, increasing ECM stiffness. These changes collectively translate into profibrotic phenotypic changes in fibroblasts. TGF-βR1/2, transforming growth factor-β receptor 1 and 2.

In Vitro and ex Vivo Models of Lung Fibrosis

Despite a number of advances in the understanding of the mechanisms underlying the pathogenesis of IPF, significant gaps remain. The absence of animal models that can faithfully recapitulate the complexity of disease highlights the potential value of in vitro and ex vivo approaches to integrate various components of disease pathogenesis.⁸⁵ Along with the lack of in vivo models, there are inherent limitations to reductionist approaches involving single cells cultured on a defined substrate (typically plastic). These in vitro studies can be helpful in addressing fundamental biologic questions, but the translational impact of these model systems must be viewed with caution. It is expected that ultimately, emerging in vitro and ex vivo models that increasingly mimic the complexity of cell–cell and cell–matrix interactions holds promise for the identification of critical mechanisms for therapeutic intervention. In the setting of fibrosis research, multidrug approaches that target different mechanisms of the fibrogenic pathways, ideally by inhaled delivery, and the identification of subsets of patients who may respond better to specific drugs, are likely to be more effective.⁸⁶

The remainder of this article reviews several in vitro and ex vivo model systems used in the study of lung fibrosis. It discusses the advantages and limitations of each and describes how each may recapitulate aspects of the lung biochemical and biomechanical microenvironment.

In Vitro Single Cell Models

A variety of in vitro models have been developed to examine cell–matrix interactions and their contribution to the development of pulmonary fibrosis. These systems range in complexity from simple two-dimensional systems of cultured fibroblasts on an ECM-coated plastic plate to complex hydrogels within a transwell system that measures functional properties of the cells.⁸⁷ A hydrogel is a 3D hydrophilic polymeric matrix that has physical properties similar to those of animal tissues. The advantage of using the in vitro hydrogel systems is that these models provide a platform to examine the role of ECM components and mechanical forces in regulating cellular activation and functional outcomes. For example, the contraction of collagen hydrogels as a result of fibroblast extension and interaction with the gel through integrin binding can be measured. Furthermore, the fibroblast cytoskeleton organization including α-smooth muscle actin stress fiber formation can also be visualized by immunostaining.⁸⁸ Hydrogels formed from type I collagen have particularly been a popular model for 3D fibroblast culture. Fibrin hydrogels have also been used for the measurement of proliferation and migration. Hyaluronic acid interacts with collagen and fibronectin to create a complex extracellular environment. Addition of hyaluronic acid alters the viscoelastic properties of collagen gels.⁸⁸ Thus, the incorporation of hyaluronic acid within collagen hydrogels may mimic the native multicomponent structure of ECM. Along with these components, elastic-like polypeptides, polyacrylamide, and polyethylene glycol have also been used to study the effects of biomechanical force of ECM. Thus, the components of a hydrogel system vary between models, with some using tunable substrates^87,89 whereas others use and incorporate different ECM components.⁹⁰

Polymeric source and composition denote whether the hydrogel is natural, synthetic, or hybrid.⁹¹ Natural hydrogels are polymers that have natural origins such as gelatin and collagen, whereas synthetic hydrogels are generated by using synthetic polymers such as polyamides and polyethylene glycol.⁹¹ Naturally obtained ECM hydrogels from the decellularized human and animal tissues,^92,93 cultured fibroblasts,⁹² or a combination of these components⁹⁴ have been used for their matrix mechanical properties. In vitro scaffolds have also been generated by using biodegradable foams and gels^95,96 or 3D printing of ECM components.⁹⁷ The physiological and nonphysiological effects of ECM stiffness on specific types of cells can provide insight into fundamental cell and molecular biology with potential relevance regarding disease processes.

In vitro models are also used to model the effect of stretch on cellular functions. Cells are placed on flexible membranes coated with ECM substrates and grown before being subjected to stretch. Stretch can be adjusted to be static or cyclical and can vary in amplitude and frequency. Recently, cyclic stretch has been incorporated into a “lung-on-a-chip” model⁹⁸ and has been combined with precision cut lung slices (PCLS), which are described in further detail in Ex Vivo Whole Lung Models).⁹⁹

Regardless of the in vitro system chosen, a challenge in using these models has been validating the extent to which the model truly reflects the in vivo naive lung microenvironment. Although these systems allow control of some ECM components, and model the stiffness present within the diseased and normal lung tissue, they frequently fail to incorporate multiple cell types and do not reflect the dynamic changes that can occur in ECM composition and biomechanics over time. In addition, they typically do not replicate the microscale heterogeneity of lung stiffness that has been described within fibrotic lesions. Finally, these models do not account for other types of mechanical forces such as pressure, stretch, and viscoelasticity.¹⁰⁰

In Vitro Multicellular Models

Growing understanding of the pathophysiology of IPF has supported the role of cell–cell interactions in the development of the disease, and it has become clear that IPF cannot be attributed to the activity on any single cell type. Data from single-cell RNA sequencing have shown the broad diversity of changes in cellular composition in the fibrotic lung. Acknowledging this diversity, prevailing theories place alveolar epithelial cell injury at the center of IPF pathogenesis and suggest that this injury leads to a reparative response by mesenchymal cells. More recent studies have broadened the scope of the study of cell–cell interactions to include macrophage–epithelial, endothelial–epithelial, and mesenchymal cell–macrophage interactions. In the simplest co-culture system, individual cells are cultured in a two-dimensional orientation in which a physical barrier separates the cell lines. This design is best suited for evaluation of cell–cell interactions mediated by soluble factors. However, the importance of direct cell–cell contact can also be essential in defining how one cell can influence neighboring cells. More recently, in vitro fibrosis model systems have evolved to encompass the multicellular microenvironment of the lung while also incorporating biomechanical variables. Organoids, for example, are 3D multicellular models that incorporate relevant cell types and matrix components to mimic intact tissues. Although early lung organoid studies were completed by using tissue progenitors or pluripotent stem cell groups in a 3D environment,101, 102, 103 recent attempts have used primary adult lung epithelial cells, fibroblasts, and microvascular endothelial cells.¹⁰⁴ Thus, lung organoids can be generated from stem cells or differentiated cells, taking advantage of advances in induced pluripotent stem technology.

Depending on the cells used, the lung organoids can recapitulate the 3D structures such as the airway or alveoli. Recent studies using lung organoids have been essential in defining the differentiation of epithelial cells by other cell types. For example, the applications of organoids using primary cells from patient lungs with IPF showed the role of matrix fibroblast in alveolar reseptation and alveolar epithelial differentiation via paracrine signaling.¹⁰⁵ Thus, organoid systems allow increased complexity regarding cell–cell interactions on defined ECM substrates. A drawback to date is that these models have been limited to the incorporation of structural cells. Further development to allow incorporation of immune cells, including macrophages, will be important to advance this field.¹⁰⁶

Another multicellular model system used in fibrosis research is the lung-on-a-chip. Originally focused on establishing a biomimetic microsystem to measure alveolar–capillary interfaces within the human lung,¹⁰⁷ these models are continuously evolving to include higher throughput systems¹⁰⁸ as well as 3D systems to investigate microfluidics and epithelial–endothelial interactions as well as mechanical forces.¹⁰⁹ Although organoids and lung-on-a-chip technologies provide tools to examine dynamic interactions between cells as well as the cell–matrix interactions, limitations remain regarding investigation of mechanical forces within the lung microenvironment.¹¹⁰ Additional studies incorporating specific cell types, accounting for age, disease status, and mechanical forces in the lungs, are needed to continue to advance our integrated understanding of how cell–cell and cell–matrix interactions drive disease complexity.

Ex Vivo Whole Lung Models

In vitro two-dimensional and 3D models do not recapitulate an organ- or tissue-specific ECM structure and/or the effects of heterogeneous composition on in vivo matrices, which hampers the characterization of disease-specific ECM factors. To overcome these limitations, investigators have developed new model systems that preserve the native ECM structure in both diseased and normal lung tissue. One example is the use of decellularized tissues that can be collected from both normal and diseased human and animal models. Previous work using IPF lung matrices revealed a marked increase in the ECM deposition in the lung interstitium with disorganized alignment, disrupted basement membranes, and increased but heterogeneous stiffness compared with decellularized matrices from normal lungs.²⁹ Although the potential benefits of studies incorporating ex vivo decellularized matrices are compelling, the methods and protocols used for obtaining this acellular matrix can be technically difficult and cumbersome, limiting their widespread adoption. In addition, this model does not incorporate the important cell–cell interactions that occur during pulmonary fibrosis, and it is unclear whether specific cell types establish the appropriate niches following the recellularization process.¹¹¹

PCLS, which have been gaining attention as an essential alternative to the two-dimensional and 3D cell culture, overcome the known limitations of these models. Since its initial description in the 1990s, multiple protocols have been developed with the goal of maintaining viability of all the different cell types, particularly alveolar epithelial cells, while retaining the intricate structure of the alveoli. Today, the use of PCLS is expanding from mechanistic studies on matrix–cell interaction to drug discovery. Arguably the most advanced technology that can be used for translational studies, PCLS offer the advantages of preservation of ECM as both a structural support and as a reservoir of soluble factors, while preserving all of the lung cell types.¹¹² These features, evident in PCLS derived from both human and murine tissues, make PCLS a unique model to study lung biology and to evaluate possible interventions.

In addition to preserving the 3D structure, PCLS also permit evaluation of regional differences within a tissue. This is important in the study of IPF, which is defined by spatial and temporal heterogeneity. For example, generating PCLS from explanted lungs from more fibrotic lower lobes and partially normal upper lobes allows identification of geographic differences in molecular signatures and the evaluation of therapeutic interventions. In PCLS derived from nondiseased lungs, a profibrotic combination of growth factors and inflammatory cytokines resulted in activation of intracellular profibrotic pathways, changes in multiple cell phenotypes, and increased matrix deposition mimicking changes identified in the IPF lung.¹¹³ The use of PCLS can be extended to up to 5 to 7 days, after which there is a decrease in viability of epithelial cells and a reduction in the number and proportion of immune cells. Although useful in answering a variety of different questions related to ECM and cell–cell interactions, systems of PCLS are limited in their ability to evaluate recruited immune cells and are typically used in static, nonmechanically induced models.¹¹⁴

Many of the concerns with PCLS are addressed in new models of human ex vivo lung perfusion. This system was originally designed to recondition donor lungs before transplantation, but more recently a group of investigators has been using it as a preclinical model for translational research on chronic lung disease.¹¹⁵ Explant lungs from patients with IPF were perfused on the human ex vivo lung perfusion circuit for 6 hours, and biological parameters, airway mechanics, pulmonary hemodynamics, and gas exchange were continuously measured. Although technically challenging, this new model provides a platform to examine ECM, cell–cell interactions, and mechanical forces imparted by respiration in an ex vivo system. The main limitation of human ex vivo lung perfusion is that after few hours, there is a rapid detriment of the quality of the lungs, limiting experiments to a maximum of 6 to 8 hours.

Summary

IPF is a progressive and fatal lung disease characterized by the excessive accumulation of collagen-rich ECM that disrupts lung architecture and function, leading to respiratory impairment. The abnormal cellular responses triggered by the fibrotic ECM are an important component of the pathologic process. We speculate that complex mechanisms involving multiple cell types must converge to result in a fibrotic outcome of wound repair. Thus, multimodal therapies that simultaneously target key pathogenic pathways may represent future therapeutic strategies of IPF. Improved understanding of how cell–cell interactions integrate with dynamic changes in the ECM stiffness to perpetuate the vicious cycle of lung injury and repair that underlies fibrosis will facilitate the development of novel strategies that target the development and maintenance of fibrosis. The integrated use of in vitro and ex vivo models can help address the multiple layers of complexity that have hampered progress to date.