Bidirectional epithelial–mesenchymal crosstalk provides self-sustaining profibrotic signals in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1–mediated epithelial–mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β–induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial–mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial–mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1–tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β–activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor–like repeats. Together, these data identify that aberrant bidirectional epithelial–mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.     

Transglutaminase 2: a novel therapeutic target for idiopathic pulmonary fibrosis using selective small molecule inhibitors

This study investigates the effects of a site-directed TG2-selective inhibitor on the lung myofibroblast phenotype and ECM deposition to elucidate TG2 as a novel therapeutic target in idiopathic pulmonary fibrosis (IPF)—an incurable progressive fibrotic disease. IPF fibroblasts showed increased expression of TG2, α smooth muscle actin (αSMA) and fibronectin (FN) with increased extracellular TG2 and transforming growth factor β1 (TGFβ1) compared to normal human lung fibroblasts (NHLFs) which do not express αSMA and express lower levels of FN. The myofibroblast phenotype shown by IPF fibroblasts could be reversed by selective TG2 inhibition with a reduction in matrix FN and TGFβ1 deposition. TG2 transduction or TGFβ1 treatment of NHLFs led to a comparable phenotype to that of IPF fibroblasts which was reversible following selective TG2 inhibition. Addition of exogenous TG2 to NHLFs also induced the myofibroblast phenotype by a mechanism involving TGFβ1 activation which could be ameliorated by selective TG2 inhibition. SMAD3-deleted IPF fibroblasts via CRISPR-cas9 genome editing, showed reduced TG2 protein levels following TGFβ1 stimulation. This study demonstrates a key role for TG2 in the induction of the myofibroblast phenotype and shows the potential for TG2-selective inhibitors as therapeutic agents for the treatment of fibrotic lung diseases like IPF.

     

Fibroblast to myofibroblast transition is enhanced by increased cell density

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of the fibroblast-to-myofibroblast transition (FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. We found that human lung fibroblasts (HLFs) express more α-SMA and deposit more collagen matrix, which are both characteristics of myofibroblasts, in response to TGF-β1 when cells are seeded at a high density compared with a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-β1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.     

Molecular Mechanism Responsible for Fibronectin-controlled Alterations in Matrix Stiffness in Advanced Chronic Liver Fibrogenesis

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to noncancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver matrix stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-β activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-β1 through canonical and noncanonical Smad such as PI3 kinase-mediated pathways. TGF-β1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared with wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrhosis in negatively regulating TGF-β bioavailability, which in turn modulates ECM remodeling and stiffening and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in a broader variety of chronic fibrotic diseases.     

Mechanosensitivity of the 2nd Kind: TGF-β Mechanism of Cell Sensing the Substrate Stiffness

Cells can sense forces applied to them, but also the stiffness of their environment. These are two different phenomena, and here we investigate the mechanosensitivity of the 2^nd kind: how the cell can measure an elastic modulus at a single point of adhesion—and how the cell can receive and interpret the chemical signal released from the sensor. Our model uses the example of large latent complex of TGF-β as a sensor. Stochastic theory gives the rate of breaking of latent complex, which initiates the signaling feedback loop after the active TGF-β release and leads to a change of cell phenotype driven by the α-smooth muscle actin. We investigate the dynamic and steady-state behaviors of the model, comparing them with experiments. In particular, we analyse the timescale of approach to the steady state, the stability of the non-linear dynamical system, and how the steady-state concentrations of the key markers vary depending on the elasticity of the substrate. We discover a crossover region for values of substrate elasticity closely corresponding to that of the fibroblast to myofibroblast transition. We suggest that the cell could actively vary the parameters of its dynamic feedback loop to ‘choose’ the position of the transition region and the range of substrate elasticity that it can detect. In this way, the theory offers the unifying mechanism for a variety of phenomena, such as the myofibroblast conversion in fibrosis of wounds and lungs and smooth muscle cell dysfunction in cardiac disease.     

Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.     

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation.

Measurements and Main Results

In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture.

Conclusions

We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.     

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.     

A Critical Role for the mTORC2 Pathway in Lung Fibrosis

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.     

Urokinase-type Plasminogen Activator Receptor (uPAR) Ligation Induces a Raft-localized Integrin Signaling Switch That Mediates the Hypermotile Phenotype of Fibrotic Fibroblasts

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.     

Duvelisib attenuates bleomycin-induced pulmonary fibrosis via inhibiting the PI3K/Akt/mTOR signalling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3K-δ and PI3K-γ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL). In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose-dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.     

Hyper-responsiveness of IPF/UIP fibroblasts: interplay between TGFbeta1, IL-13 and CCL2

One of the hallmarks of idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is excess collagen deposition, due to enhanced fibroblast extracellular matrix synthetic activity. Studies using murine models of lung fibrosis have elucidated a pro-fibrotic pathway involving IL-13 driving CCL2, which in turn drives TGFbeta1 in lung fibroblasts. Therefore, we sought to determine whether this pathway exists in the human fibrotic setting by evaluating human IPF/UIP fibroblasts. IPF/UIP fibroblasts have an increased baseline fibrotic phenotype compared to non-fibrotic fibroblasts. Interestingly, non-fibrotic fibroblasts responded in a pro-fibrotic manner to TGFbeta1 but were relatively non-responsive to IL-13 or CCL2, whereas, IPF/UIP cells were hyper-responsive to TGFbeta1, IL-13 and CCL2. Interestingly, TGFbeta1, CCL2 and IL-13 all upregulated TGFbeta receptor and IL-13 receptor expression, suggesting an ability of the mediators to modulate the function of each other. Furthermore, in vivo, neutralization of both JE and MCP5, the two functional orthologs of CCL2, during bleomycin-induced pulmonary fibrosis significantly reduced collagen deposition as well as JE and CCR2 expression. Also in the bleomycin model, CTGF, which is highly induced following TGFbeta stimulation, was attenuated with anti-JE/anti-MCP5 treatment. Overall this study demonstrates an interplay between TGFbeta1, IL-13 and CCL2 in IPF/UIP, where these three mediators feedback on each other, promoting the fibrotic response.     

Approaching the degradome in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating, lethal and currently untreatable lung disorder of unknown etiology. It is characterized by epithelial injury and activation, fibroblastic foci formation, and exaggerated accumulation of extracellular matrix (ECM) with the destruction of the lung parenchyma. Despite important progress in our understanding of the general mechanisms involved in lung fibrogenesis, the pathogenesis of the IPF remains unclear. Although the irreversible and progressive fibrosis in the lung suggests a decrease in lung degradative machinery, an increasing body of evidence, primarily obtained by global gene expression studies, demonstrates a significant upregulation of degrading enzymes in IPF. In this context, this review will focus on some families of the degradome, a term proposed for the complete set of proteases that are expressed at a specific time by a cell, tissue or an organism. In particular, we will approach recent progress in our understanding of the behavior of two families of metalloproteases M10 and M12 which are significantly changed in the IPF lungs. In general, evidence highlights the increasing diversity in both substrates and functions of these enzymes and the complexity of the processes in which they are involved, and indicate a critical role in the abnormal remodeling of IPF.     

A Highlights from MBoC Selection: Soft matrix is a natural stimulator for cellular invasiveness

Directional mesenchymal cell invasion in vivo is understood to be a stimulated event and to be regulated by cytokines, chemokines, and types of extracellular matrix (ECM). Instead, by focusing on the cellular response to ECM stiffness, we found that soft ECM (low stiffness) itself is sufficient to prevent stable cell-to-cell adherens junction formation, up-regulate matrix metalloproteinase (MMP) secretion, promote MMP activity, and induce invadosome-like protrusion (ILP) formation. Consistently, similar ILP formation was also detected in a three-dimensional directional invasion assay in soft matrix. Primary human fibroblasts spontaneously form ILPs in a very narrow range of ECM stiffness (0.1–0.4 kPa), and such ILP formation is Src family kinase dependent. In contrast, spontaneous ILP formation in malignant cancer cells and fibrosarcoma cells occurs across a much wider range of ECM stiffness, and these tumor cell ILPs are also more prominent at lower stiffness. These findings suggest that ECM softness is a natural stimulator for cellular invasiveness.     

Chemokine (C-X-C motif) ligand 14 contributes to lipopolysaccharide-induced fibrogenesis in mouse L929 fibroblasts via modulating PPM1A

Herein, we found that serum chemokine ligand 14 (CXCL14) was significantly enhanced in patients with idiopathic pulmonary fibrosis (IPF). In our current study, mouse L929 fibroblasts were stimulated with lipopolysaccharide (LPS) (100 ng/mL). Cell proliferation, the levels of matrix metalloproteinase 2 (MMP2) and MMP9, as well as extracellular matrix (ECM) content were assessed to evaluate the fibrogenesis of L929 cells. Proliferating cell nuclear antigen and cell viability were assessed to evaluate cell proliferation. Hydroxyproline (Hyp), collagen I/III, connective tissue growth factor (CTGF), and phosphorylated Smad2/3 (p-Smad2/3) were assessed to evaluate ECM secretion and deposition. α-Smooth muscle actin (α-SMA) was used to measure the occurrence of differentiation from fibroblast toward myofibroblast. Our data suggested that knockdown of CXCL14 prevented LPS-induced fibrogenesis of L929 cells through inhibiting cell proliferation and decreasing the expression of MMP2/9, Hyp, collagen I/III, CTGF, p-Smad2/3, and α-SMA. Notably, upregulation of protein phosphatase magnesium-dependent 1A (PPM1A) was involved in this process. On the contrary, recombinant CXCL14 protein led to an opposite effect. We first suggested that overexpression of PPM1A ameliorated LPS-induced fibrogenesis. Furthermore, we substantiated that knockdown of CXCL14 exerted an antifibrotic effect in IPF in vitro probably via the upregulation of PPM1A. Besides, evidently enhanced CXCL14, yet reduced PPM1A, was found in bleomycin-induced rat pulmonary fibrosis, confirming the roles of CXCL14 and its potential association with PPM1A in IPF in vivo. In conclusion, CXCL14 could be considered as a therapeutic target for preventing fibrogenesis of mouse L929 fibroblasts.     

Reactive oxygen species are required for maintenance and differentiation of primary lung fibroblasts in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms.

Methodology/Principal Findings

We first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger.

Conclusions/Significance

Robust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

Cartilage Oligomeric Matrix Protein in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5–3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded.