Mechanisms for the Resolution of Organ Fibrosis

Abstract

Fibrosis is a dynamic process with the potential for reversibility and restoration of near-normal tissue architecture and organ function. Herein, we review mechanisms for resolution of organ fibrosis, in particular that involving the lung, with an emphasis on the critical roles of myofibroblast apoptosis and clearance of deposited matrix.

Introduction

Organ fibrosis is characterized by loss of cellular homeostasis and disruption of normal tissue architecture. In most organs, this tissue response develops when there is persistent or recurrent injury to the epithelium or endothelium. The etiological agent that induces such injury may range from infectious agents to toxic/metabolic exposures to autoimmunity, and, in some cases, the cause of the injury is never identified. When regeneration of the epithelium/endothelium is insufficient or incomplete, the subtending mesenchyme becomes activated, resulting in exuberant deposition of extracellular matrix (ECM). Although transient activation of the mesenchyme in response to tissue injury may represent an evolutionarily conserved, adaptive response to facilitate wound-repair and to contain microbial pathogens, maladaptive repair responses can culminate in organ fibrosis (165). Restoration of tissue homeostasis following injury requires tightly orchestrated temporal and spatial responses by multiple resident and recruited cells that communicate through an array of soluble factors under the influence of a dynamic ECM. In some cases, non-resolving fibrosis may result from persistent or recurrent extrinsic injuries, whereas, in others, such maladaptive fibrotic reactions may develop from an autonomous and self-propagating process. Thus, even when the inciting cause of injury is identified and eliminated, the resultant fibrosis may persist and progress (79, 175).

Recent estimates suggest that fibrotic disease is responsible for almost half of all deaths in Western developed countries (182). The mechanisms that resolve established fibrosis and restore tissue function are incompletely understood. The capacity for fibrosis resolution may differ depending on the organ involved, the nature and chronicity of the injurious stimulus, and host-specific factors, including age and genetic predisposition (13, 49, 165). Nevertheless, accumulating studies from humans and from animal models suggest that the potential to reverse fibrotic changes exists in most organs/tissues studied, including the liver, lung, kidney, skeletal muscle, heart, and bone marrow. In this review, we discuss the mechanisms that are currently known to regulate the resolution of normal wound repair and how these mechanisms are perturbed in fibrotic diseases. We broadly explore potential therapeutic strategies to facilitate the resolution of fibrosis and restore tissue homeostasis. In consideration of the topic, we submit that, although fibrosis is a stereotypical response with common mechanisms across various organs, there are also notable differences that determine both the capacity for, and the mechanisms of, fibrosis resolution. Thus our goal for this review is to address general themes that may apply to all organs, although we focus on lung fibrosis as an exemplar of fibrotic repair with the recognition that the capacity for resolution of fibrosis may be organ-specific (13). Additionally, the time required for the resolution of fibrosis may vary depending on the organ involved, the cause of injury, and host-specific factors such as age, genetic and epigenetic background, and immune competence. Furthermore, each organ or tissue may be subject to a “point of no return” at which any ability to resolve a fibrotic wound becomes markedly diminished, as has been shown in the carbon tetrachloride model of murine liver fibrosis (70). Only two pharmacological agents (pirfenidone and nintedanib) have been shown to alter the natural history of idiopathic pulmonary fibrosis (IPF), the most common and severe form of lung fibrosis that is associated with the worst outcomes. In IPF, although each drug slows the rate of decline in lung function, neither has been shown to reverse existing fibrosis based on physiological, histological, or radiological assessments. Interestingly, a recent analysis revealed that a subpopulation of patients with IPF treated with nintedanib had an improvement in lung physiology, challenging the notion that IPF is irreversible (35). Further studies are warranted to define the extent to which fibrosis is reversible in different human fibrotic disorders.

Wound Repair

The host response to tissue injury is a complex but evolutionarily conserved stereotypic process that involves the precise temporal and spatial orchestration of resident and recruited inflammatory cells (FIGURE 1). In addition to a number of soluble mediators, the changing biochemical composition and mechanical properties of the ECM determine the eventual outcome of the repair process. Our understanding of normal wound repair comes largely from studies of skin wounds, although the core cellular and molecular mechanisms are fundamentally similar in other organs (34, 46, 149, 167).

FIGURE 1.

Homeostatic and fibrotic wound repair

The host response to injury is a complex interplay of epithelial cells, mesenchymal cells, vascular endothelium, and inflammatory cells that are regulated by plasma proteins, coagulation factors, other soluble mediators synthesized from cells and/or liberated from protein-bound niches in the extracellular matrix, and mechanical cues transmitted to cells from the environment. Resolution of wound repair requires the highly orchestrated spatial and temporal regulation of these processes, which, when successful, culminate in the reestablishment of an intact epithelium coupled with degradation of extracellular matrix components and clearance of activated myofibroblasts. Intrinsic and extrinsic factors, including genetic/epigenetic background, the nature of the stimulus for injury, and the extent of the initial injury are additional factors that determine the outcome of wound repair.

Briefly, in response to tissue injury, there are several overlapping “phases” of repair. The first phase is characterized by initiation of the clotting cascade and the influx of platelets leading to the formation of a fibrin clot that is also rich in fibronectin. Platelet aggregation assists in hemostasis, and platelet degranulation serves as a rich source of cytokines and growth factors, including TGF-β. This is rapidly followed by recruitment of neutrophils and monocytes/macrophages with amplification of the acute inflammatory response, efferocytosis of dead/dying cells, phagocytosis of damaged tissue, and protection from microbial invasion (34, 46, 149). The second phase of repair is characterized by proliferation and effector cell activation. Epithelial cells proliferate and migrate on the provisional matrix to reestablish barrier function. Endothelial cells proliferate, and angiogenesis is evident. Fibroblasts derived from different progenitor populations are recruited. Next comes an effector phase of ECM deposition and remodeling in which the accumulated fibroblasts predominate. These cells differentiate into “myofibroblasts,” which are smooth muscle-like contractile cells characterized by the neo-expression of organized “stress fibers” composed of alpha-smooth muscle actin and myosin filaments; these myofibroblasts are primarily responsible for the deposition, remodeling, organization, and maturation of scar tissue (167). The deposition and remodeling of ECM leads to formation of a physiological scar that is rich in cross-linked type-1 collagen (46). With the conclusion of the normal wound-repair process, myofibroblasts are cleared via apoptosis, although the in vivo trigger(s) have not been defined (29). Studies also demonstrate the ability of myofibroblasts to de-differentiate, and one study of liver repair indicates that the de-differentiated cells can persist in a quiescent state and remain primed for activation when confronted with a subsequent injury stimulus (39, 52, 88, 188).

Fibrosis: Failed Resolution of the Repair Response

Fibrosis represents the consequence of a dysregulated repair response characterized by persistent epithelial injury, death, and failed regeneration coupled with the accumulation of activated myofibroblasts. In some cases, such as chronic viral hepatitis, connective tissue disease, or environmental/occupational exposures to inhaled particulates or antigens, there is an identifiable stimulus for ongoing epithelial injury. In other instances, such as IPF or fibrostenotic Crohn’s disease that progress despite anti-inflammatory therapy, the repair response is seen in the absence of a clearly persistent exogenous stimulus (44, 175). In these cases, interactions between the myofibroblasts and the juxtaposed epithelium are thought to maintain a vicious cycle of continued epithelial damage and fibroblast activation (65, 187). Interactions between each of these cell types and a biomechanically stiffened ECM can perpetuate a positive feedback loop by directly contributing to fibroblast survival and epithelial cell apoptosis (31, 64, 77, 173, 177, 178).

Myofibroblast Apoptosis

Myofibroblast apoptosis heralds the resolution phase of normal wound repair, although the temporal and mechanistic triggers have not been determined (42, 46). In contrast, fibrosis is characterized by a distinct absence of myofibroblast apoptosis despite persistent epithelial cell injury and apoptosis (29, 56, 57, 72, 162, 170). There is growing evidence that this is related to the acquisition of an apoptosis-resistant phenotype of myofibroblasts (42, 80, 162). It is likely that these factors function synergistically in a manner that involves both cell autonomous and non-autonomous mechanisms. Apoptosis of any cell is influenced by a number of factors, including the nature of the stimulus, which can be “extrinsic” (death receptor/ligand-mediated) or “intrinsic” (cellular response to stress without a specific receptor-ligand interaction) (162). Additionally, apoptosis is regulated by the ability of a cell to sense an external stimulus, the ability to propagate a receptor-mediated signal intracellularly, the balance of pro- and anti-apoptotic BCL-2 family proteins that regulate mitochondrial release of cytochrome-c and the apoptosome, and the levels of intracellular anti-apoptotic proteins that can inhibit the caspase-mediated execution phase of the apoptosis program. Each of these mechanisms can, in turn, be controlled by genetic, epigenetic, transcriptional, and posttranslational regulation. Thus the apoptotic susceptibility of a cell, or a population of cells, represents a tightly regulated balance of pro- and anti-apoptotic factors, which can be skewed to favor survival or programmed death in response to environmental cues (33, 162). Despite the complexity of the interacting and somewhat redundant mechanisms regulating fibroblast apoptosis and survival in the context of wound repair and fibrosis, several consistent themes have emerged.

The Resolution of Fibrosis is Associated with Myofibroblast Apoptosis

Consistent with the critical role of myofibroblast persistence in the maintenance and progression of fibrosis, in vivo studies demonstrate that fibrosis resolution is coupled with myofibroblast apoptosis. This association has been most firmly established in murine models of liver fibrosis (18, 70, 73, 74, 88, 116, 136). In the carbon tetrachloride model of liver fibrosis, withdrawal of the stimulus (carbon tetrachloride) within 4 wk of the onset of exposure is associated with spontaneous myofibroblast apoptosis and ECM degradation with restoration of normal tissue architecture within 4 wk of the withdrawal (70). Similar evidence has emerged to support the association between myofibroblast apoptosis and the resolution of established fibrosis in murine models of lung fibrosis (4, 53, 77, 144, 195). For example, in young mice exposed to a single dose of intratracheal bleomycin, lung fibrosis reached a peak between 3 and 4 wk, and then significantly diminished between 2 and 4 mo post-injury (53). Aged mice had significantly impaired fibrosis resolution in association with increased markers of myofibroblast senescence and decreased evidence of apoptosis in the lungs at 3 wk post-injury (53). Similar findings have been reported in animal models of skin fibrosis, with one study showing enhanced myofibroblast apoptosis and diminished fibrosis within 2 wk of treatment with a pan-BCL2 inhibitor, and another study showing resolution of hypertrophic scars associated with myofibroblast apoptosis within 1–2 wk of TGF-β inhibition (95, 192). In 2-yr-old transgenic mice with skeletal muscle fibrosis due to a dystrophin deficiency, inhibition of myostatin led to myofibroblast apoptosis and a reduction in fibrosis over 6 wk (15).

Soluble Pro-Fibrotic Mediators Induce Myofibroblast Resistance to Apoptosis

A number of soluble mediators, including growth factors, cytokines, and lipid mediators, in addition to matrix alterations have been implicated in the pathobiology of fibrosis through activation of a growing number of signaling mechanisms (FIGURE 2). Most of the soluble mediators implicated in fibrogenesis have been found to promote myofibroblast activation and resistance to apoptosis, and, in many cases, common signaling mechanisms have been identified. TGF-β1 has been strongly and broadly implicated in the pathophysiology of fibrosis (14, 56, 149). Beyond its role in myofibroblast differentiation and activation, TGF-β1 diminishes fibroblast susceptibility to apoptosis by activation of pro-survival protein kinase pathways involving PI3K/AKT and focal adhesion kinase (FAK) (60, 61, 63, 124), upregulation of NADPH oxidase 4 (Nox4) (53, 54), upregulation of the inhibitor of apoptosis-family proteins (IAPs) survivin and XIAP (X-linked IAP) (2, 6, 155), suppression of Fas (CD-95) expression (33, 58, 119), induction of Rho-kinase mediated nuclear localization of myocardin-related transcription factor-A (MRTF-A) (139, 154, 195), and regulation of BCL2 family protein expression (60, 95, 130, 190) (FIGURE 2). Many of these same signaling pathways are activated by other soluble mediators implicated in lung fibrosis, including endothelin-1 (59, 63) and lysophasphatidic acid (135, 147).

FIGURE 2.

Mechanisms of non-resolving fibrosis

Resolution of fibrosis requires the orchestrated apoptosis of myofibroblasts, and the degradation and clearance of deposited matrix molecules. Persistent TGF-β1 activation and/or altered matrix biochemistry/biomechanics can lead to the activation of signaling pathways that promote both differentiation and inhibit apoptosis of myofibroblasts.

Biochemical and Biomechanical Properties of the Extracellular Matrix Regulate Fibroblast Susceptibility to Apoptosis

Cell-matrix interactions and signals generated in response to cell adhesion are well recognized to regulate survival/apoptosis of many cell types; a form of apoptosis, termed “anoikis,” results from the loss of cell adhesion or disruption of adhesion-mediated signals (36, 37). Focal adhesion kinase (FAK) activity is a critical mediator of adhesion-mediated survival signaling and EMT (epithelial-mesenchymal transition) responses in epithelial cells (31), and is required for TGF-β1-induced myofibroblast differentiation and survival (63, 163). Consistently, interruption of fibroblast-matrix interactions using soluble peptides to competitively inhibit cell interactions with RGD motifs on ECM proteins was sufficient to reduce FAK phosphorylation and induce fibroblast apoptosis (48, 60, 63, 186). In a similar manner, plasmin-mediated fibronectin proteolysis (64) and inhibition of the collagen binding discoidin domain receptor-2 (DDR2) are also associated with fibroblast apoptosis (77).

Over the last decade, it has become increasingly evident that matrix-mediated regulation of fibroblast phenotypes extends beyond the biochemical composition of the matrix and that biomechanical cues are critical to the maintenance and persistence of myofibroblasts (55, 105, 168). A series of studies showed that polymerized, but not monomeric, collagen functions to suppress proliferation in normal human lung fibroblasts and that fibroblasts isolated from IPF lung tissue elude suppressive signals from polymerized collagen and maintain their proliferative phenotype (128, 183–185). Additionally, in vitro model systems employing polyacrylamide hydrogels with varying stiffness and decellularized lung matrices from patients with fibrotic lung disease have shown that fibroblasts exposed to “stiff” substrates have decreased basal rates of apoptosis and decreased susceptibility to extrinsic apoptotic stimuli compared with cells cultured on more compliant substrates (16, 33, 104, 105). This relationship is also consistent with evidence demonstrating that increased substrate stiffness promotes Rho-kinase-mediated nuclear translocation of MRTF-A (also known as MKL1) (195). Importantly, matrix stiffness was found to be a key mechanism for the activation of latent TGF-β, suggesting that increased matrix stiffness can, by itself, perpetuate a feed-forward cycle of fibroblast activation, survival, and ECM production (179). Increased matrix-stiffness was found to be associated with suppression of PGE2, which generally antagonizes TGF-β in the regulation of fibroblast phenotypes (66, 105, 115, 166). Thus matrix stiffness-mediated suppression of PGE2 could functionally amplify the impact of active TGF-β in the cellular microenvironment. However, some studies have also shown that the matrix-mediated effects on fibroblast phenotype are independent of TGF-β activation (16). Although our understanding continues to grow and evolve, the importance of biomechanical signals supporting fibroblast survival in the context of fibrosis is well substantiated. Interestingly, increased tissue stiffness has been shown to precede substantial increases in collagen production in murine models of liver fibrosis (40), suggesting that these biomechanical signals contribute not only to fibrosis progression but to its initiation.

Interruption of Pro-Survival Signaling Pathways Enhances Myofibroblast Susceptibility to Apoptosis and Facilitates the Resolution of Fibrosis

There is now substantial evidence from in vivo models of lung fibrosis indicating that interruption of the signaling mechanisms that mediate fibroblast resistance to apoptosis diminishes the severity of lung fibrosis and may even reverse established fibrosis. In lung fibrosis, for example, administration of a pharmacological inhibitor that suppresses AKT and FAK activation 8 days after administration of intratracheal bleomycin (i.e., during the late-inflammatory, early fibrotic phase of the model) was associated with a significant reduction in lung collagen accumulation by day 15 (171). Similarly, pharmacological and siRNA-mediated inhibition of FAK was shown to diminish lung fibrosis induced by bleomycin administration and by endothelin overexpression (86, 93, 94). Conversely, deficiency of FAK-related non-kinase (FRNK), which functions as an endogenous inhibitor of FAK and is decreased in the lungs of patients with IPF, exacerbates lung fibrosis, whereas overexpression of FRNK diminishes lung fibrosis in mice (30). Inhibition of AKT signaling alone or in combination with inhibition of MAP-kinase signaling through MEK was shown to diminish lung fibrosis induced by TGF-α overexpression (91, 98, 112), bleomycin (113), and TGF-β overexpression (81). Notably, FAK and/or AKT activation also have been implicated in hypertrophic scar formation (138), liver fibrosis (78, 137), and kidney fibrosis (51, 83). In addition to FAK, inhibition of downstream mechanotransduction-regulated signaling intermediates, including Rho-kinase and MRTF-A, have been shown to reduce fibrosis in the lung (9, 90, 154, 195) and skin (47, 68).

The resolution of fibrosis that is enhanced by targeting fibroblast survival signaling extends to pathways beyond mechanotransduction. Recently, targeted inhibition of pro-survival BCL2 family proteins was shown to promote myofibroblast apoptosis and to diminish bleomycin-induced skin fibrosis (95). Increased expression of pro-fibrotic BCL2 family proteins was also identified in apoptosis-resistant fibroblasts isolated from patients with non-resolving acute respiratory distress syndrome (60). Consistently, inhibition of pro-survival IAP family proteins reduces bleomycin-induced lung fibrosis (4), as does upregulation of Fas expression induced by quercetin (58) or administration of TNF-α (145). Outside of the lung, inhibition of myostatin was shown to induce myofibroblast apoptosis and to reverse established skeletal muscle fibrosis in a murine model (15). In the kidney, treatment of mice with an ALK3 agonist led to the resolution of established fibrosis in complementary murine models of nephrotoxin-induced fibrosis and diabetic glomerulosclerosis (158).

Senescence and Aging in the Regulation of Fibroblast Survival/Apoptosis

IPF is a disease of aging (143, 164); this has motivated investigators to identify age-related factors that predispose to lung fibrosis or contribute to diminished capacity for fibrosis resolution (53, 144, 159). Cellular senescence is a hallmark of aging (111). The induction of a senescence program in cells is intricately linked to co-activation of survival signaling and the acquisition of an anti-apoptotic phenotype (87). Indeed, explanted fibroblasts from patients with IPF have been shown to impair proliferative capacity compared with fibroblasts from normal lungs, which was associated with increased AKT activation and decreased Fas expression. In vitro, the senolytic drug quercetin reduced AKT activation, increased Fas expression, and sensitized fibroblasts to undergo apoptosis. Moreover, treatment of aged mice with quercetin led to diminished bleomycin-induced lung fibrosis and decreased lung levels of senescence-related proteins (58). Additionally, survival signaling mediated by BCL2 family proteins has been used to target senescent cells, including fibroblasts, to undergo apoptosis (22, 174).

The accumulation of apoptosis-resistant senescent myofibroblasts contributes to a non-resolving fibrosis in an aging model of lung fibrosis in mice, and inhibition of the senescence-inducing, oxidant-generating enzyme Nox4 reverses established lung fibrosis in aged mice (53). Nox4 is induced in replication-induced senescent fibroblasts, and gene silencing of Nox4 mitigates this senescence phenotype (151). Similar to Nox4 inhibition, strategies to target fibroblast and epithelial cell senescence using “senolytic” cocktails are being developed as potential anti-fibrotic strategies for recalcitrant fibrotic disorders such as IPF (100, 152).

Extracellular Matrix Degradation

In addition to myofibroblast apoptosis, the degradation and clearance of ECM from tissues is essential for resolution of fibrosis. ECM deposition and degradation are dynamic processes, with the balance shifting during the development vs. recovery phases (12, 25, 84, 92, 142, 169, 193). Fibrosis is characterized by the persistence of increased levels of ECM proteins, particularly type 1 collagen; biochemical measurements of collagen are a standard experimental endpoint of fibrosis, whereas collagen mRNA and protein are commonly reported as surrogates for fibroblast activation (76). In vivo studies have estimated that, under homeostatic conditions, new collagen is synthesized at rates between 1 and 2% per week and as high as 10% per day in the lung, and between 3 and 5% per day in the skin, heart, and skeletal muscle (5, 12, 97). Non-collagen ECM proteins were estimated to have an even higher turnover rate ranging from 10 to 30% per day, depending on the organ (97). However, the relationship between synthesis and deposition is not linear, since there is also a high rate of intracellular degradation before secretion (144). Nevertheless, secreted collagen can increase up to 100% in the context of an acute injury, and, to prevent progressive accumulation, any increase in collagen deposition must be matched by degradation of extracellular collagen fibers (5, 10, 11, 120, 121, 125, 129, 193). The importance of this equilibrium is evidenced by the demonstration that transgenic mice expressing a collagenase-resistant mutant collagen develop skin fibrosis (107) and have impaired resolution of liver fibrosis that is associated with decreased stellate cell apoptosis (75). Although the mechanisms that promote collagen synthesis by fibroblasts have been extensively studied, the mechanisms regulating collagen degradation, and how these mechanisms are impaired in the context of fibrosis, have received relatively little attention (5, 13, 161, 194).

Proteases and Their Inhibitors in Matrix Degradation and Fibrosis

Degradation of collagen that is incorporated into ECM fibers is largely accomplished by sequential proteolysis mediated by matrix metalloproteinases (MMPS) and antagonized by tissue-inhibitors of MMPs (TIMPS) (121). Additionally, cathepsins, mepins, and plasmin have been implicated in degradation of collagen, fibronectin, and other matrix components (146). There is an extensive literature on MMPs, TIMPs, and their relative expression in the context of fibrosis and several studies support impaired collagenolysis in fibrotic diseases (121, 131). For example, samples taken from sites of skin involvement in patients with scleroderma had reduced collagenase activity compared with samples from clinically uninvolved skin (17). However, MMPs have diverse effects in addition to ECM proteolysis; MMPs can activate latent growth factors, including TGF-β1, that reside in the matrix, thereby increasing the availability of trophic factors during repair and remodeling (1). In line with this concept, studies variably show that, in some cases, MMPs can contribute to fibrosis, and several MMPs have been reported to promote lung fibrosis in murine models (121, 123, 146). Consistent with a pro-fibrotic role for some MMPs, a broad-spectrum MMP inhibitor prevented lung fibrosis by reducing MMP-2 and MMP-9 activity (1, 27, 123). Similarly, mice lacking TIMP3 develop increased renal fibrosis (82). Moreover, the physiological roles of MMPs and TIMPs in wound repair extend beyond catalytic effects on matrix and matrix-bound growth factors since they may also regulate inflammatory responses to injury (41).

Collectively, these studies indicate that, despite some evidence supporting a paradigm of skewed MMP/TIMP balance favoring an anti-proteolytic environment in fibrotic disease, the conceptualization of fibrosis as the consequence of an imbalance between collagen proteases and their inhibitors is an over-simplification (1, 3, 38, 71, 103, 126, 153, 189). The complexity and diversity cells that secrete and respond to MMPs, the overlapping specificity and redundancy of MMPs and their substrates, and the biological activities of cleaved matrix peptides (matrikines) highlight the temporal and contextual actions of MMPs in lung injury repair. Thus a more nuanced mechanistic understanding is required if MMPs and/or TIMPS are to be targeted for the development of precision therapeutics to treat fibrotic disease (1, 146).

In addition to MMPs/TIMPs, fibrosis has been strongly associated with a dysregulated balance in serine proteases and their inhibitors, although the role of these in matrix proteolysis is less clear. Among the serine proteases, activation of urokinase plasminogen activator (uPA) in the lung has been shown to protect mice from bleomycin-induced lung fibrosis, whereas excessive levels of plasminogen activator-inhibitor 1 (PAI-1) promote fibrosis in the lung (157). Surprisingly, however, the pro-fibrotic effects of PAI-1 have been found to be largely independent of its anti-protease activity (28). Nevertheless, uPA-mediated plasminogen activation itself can diminish fibrosis, and several mechanisms have been identified (67, 123, 156). Plasmin liberates hepatocyte growth factor (HGF) from the ECM and promotes HGF-associated anti-fibrotic actions, including increased PGE2 synthesis (7, 118, 132). Additionally, plasminogen activation by fibroblasts promotes pericellular fibronectin degradation in association with induction of fibroblast apoptosis (23, 64). Signaling from an intact ECM impacts this plasminogen activation system, since matrix stiffness-regulated activation of Hippo-pathway transcription factors and nuclear localization of myocardin-related transcription factor A (MRTF-A) have both been shown to increase production of PAI-1 and to promote lung fibrosis in vivo (104, 154, 195). Although the precise role of the plasminogen-activation system in ECM proteolysis remains unclear, evidence indicates that signaling from a rigid matrix supports a pro-fibrotic perturbation of the plasminogen activation and that ECM proteolysis could enhance the restoration of a homeostatic balance between plasminogen activators and inhibitors.

Extracellular Matrix Cross-Linking

Extracellular matrix cross-linking, primarily mediated by transglutaminase (Tg) enzymes and lysyl oxidase (LOX) or its homologs, lysyl oxidase-like (LOXL) enzymes, is increasingly recognized as a critical regulatory checkpoint in fibrosis development and resolution (80). First, cross-linking stabilizes the matrix and promotes resistance to proteolysis (42, 121, 141, 146). Second, cross-linking promotes fibroblast adhesion and proliferation, possibly by increasing matrix stiffness and engaging integrin-mediated signaling (101). As with MMPs, there is a broad recognition that tissue transglutaminase and LOX/LOXL proteins are critical to lung, liver, cardiac, biliary, and dermal fibrosis (146). In the liver, transglutaminase-mediated cross-linking stabilizes matrix and promotes liver fibrosis (45), and Tg-mediated cross-linking accounts, at least in part, for incomplete resolution of fibrosis in the carbon tetrachloride model (74). In the lung, tissue transglutaminase-2 expression and activity were increased in patients with IPF, and mice lacking Tg2 or treated with Tg inhibitors had decreased fibrosis following bleomycin administration (133, 134).

Lysyl oxidase (LOX) and its homologous LOX-like (LOXL) proteins have been strongly implicated in fibroblast activity and fibrosis (69, 106, 150), and LOX enzymes have been evaluated as a biomarker for fibrotic disease in patients with scleroderma and IPF (26, 148). Pro-fibrotic stimuli increase cardiac and kidney fibroblast LOX expression (43, 172), whereas deficiency in, or inhibition of, these enzymes reduces fibrosis in the lung, heart, and peritoneum (8, 50, 117). Other types of ECM cross-linking reactions, such as those involving advanced glycation end-product protein adducts (180) and dityrosine cross-linking (96, 140), require further study in the context of progressive, non-resolving fibrosis.

Intracellular Processing of Cleaved Collagen

Matrix degradation does not conclude with extracellular proteolysis, and the cleaved products generated, including collagen peptides, can function as biologically active “matrikines” (176). Such matrikines have been associated with a wide range of biological processes, including wound repair and inflammation, although they have not been extensively studied in the pathobiology of fibrosis (21, 176). Collagen peptides are taken up by macrophages, fibrocytes, and fibroblasts, which further degrade those peptides via the lysosomal pathway (5, 42, 89, 99, 121). The specific contribution of intracellular collagen processing by each of these cell types during normal tissue homeostasis, physiological wound repair, and fibrosis is unclear. The importance of intracellular collagen degradation is established by studies demonstrating an anti-fibrotic role for uPARP/endo180, one of the receptors that mediates collagen uptake and delivery to lysosomes, in models of lung, kidney, and liver models (20, 110, 114). Similarly, milk fat globule epidermal growth factor 8 (Mfge8) expression on macrophages was shown to mediate cellular uptake of collagen, and mice lacking Mfge8 developed increased lung fibrosis following bleomycin administration (114). In another study, an unbiased assessment of mechanisms regulating collagen internalization in phagocytic cells identified a critical role for beclin-1, the mammalian homolog of the autophagy protein ATG6 (99). Given the emerging role of autophagy in fibrosis, this finding suggests that insufficient autophagy may promote dysregulated intracellular collagen processing and cellular toxicity (32, 102, 108, 109, 191, 194). A mechanistic association between autophagy, collagen processing, and fibrosis is supported by a study demonstrating increased collagen accumulation in the kidneys of transgenic mice with reduced levels of beclin-1 and increased intracellular collagen in mesangial cells in which beclin-1 levels have been suppressed by siRNA (85). Notably, however, there are also reports indicating that autophagy can promote the development of fibrosis, further highlighting the need to understand the cell-specific, temporal, and spatial regulation of this process during the evolution of fibrosis development and resolution (24, 122, 181) (FIGURE 3).

FIGURE 3.

Therapeutic strategies to promote fibrosis resolution

Strategies that may stimulate or accelerate the resolution of fibrosis may include the restoration of myofibroblast sensitivity to apoptosis, killing/elimination of senescent cells, disruption of collagen-matrix cross-links, augmentation of matrix proteolytic activity, and the clearance of degraded matrix molecules by cellular uptake and autophagy.

Conclusions

Disruption of tissue architecture and reduced organ function due to fibrosis across diverse organ systems are major causes of morbidity and mortality. Historically, fibrosis has been viewed as an irreversible process, such that attempts to identify and eradicate the inciting stimulus was the only hope in halting progression. More recently, evidence has emerged to suggest that fibrosis may be, at least partly, reversible (42, 80, 149). However, the extent to which reversibility can be achieved may be organ-/tissue- and stimulus-dependent. As outlined in this review, strategies to promote fibrosis resolution may include restoration of myofibroblast susceptibility to apoptosis, enhancing mechanisms of ECM degradation/clearance, inhibition of collagen cross-linking, and elimination of senescent cells (FIGURE 3).

To date, clinical trials of drugs targeting specific mechanisms in human fibrotic diseases such as IPF have been disappointing, and none have been shown to “reverse” fibrosis. The two drugs that have received FDA approval for treatment of IPF slow disease progression. Most compounds tested in clinical trials have had no clear impact on primary end points in clinical trials, and some interventions that were based on studies suggesting potential benefits were subsequently found to be detrimental (127). Collectively, these studies highlight not only the complexity and heterogeneity of wound repair and fibrosis in humans but also the substantial challenges in translating basic and pre-clinical studies into therapies in humans. In doing so, these studies accentuate the necessity of continued investment in efforts to understand the relevant mechanisms of persistent fibrosis within individual patients during the evolution of aberrant wound repair (19, 62, 160). Along with this improved understanding, the translational application of precision-based interventions necessitates the identification of mechanistic biomarkers and surrogates of fibrosis resolution that will facilitate the assessment of fibrosis and its resolution.