Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
fibrosis; epithelial cells; fibroblasts
Persistence of myofibroblasts is believed to contribute to the development of fibrosis in idiopathic pulmonary fibrosis (IPF). Transforming growth factor-β1 (TGF-β1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle α-actin (α-SMA) and produce extracellular matrix proteins, such as procollagen I (α1). Reactive oxygen species (ROS) produced by NADPH oxidases (NOX) have been shown to regulate cell differentiation. We hypothesized that NOX could be expressed in parenchymal pulmonary fibroblasts and could mediate TGF-β1–stimulated conversion of fibroblasts to myofibroblasts.
Fibroblasts were cultured from the lung of 9 Control and 8 IPF patients. We quantified NOX4, α-SMA and procollagen I (α1) mRNA and protein expression, ROS production and Smad2/3 phosphorylation, in the absence and in the presence of incubation with TGF-β1. PDGF-induced fibroblasts migration was also assessed.
We found that (1) NOX4 mRNA and protein expression was upregulated in pulmonary fibroblasts from patients with IPF and correlated with mRNA expression of α-SMA and procollagen I (α1) mRNA; (2) TGF-β1 upregulated NOX4, α-SMA and procollagen I (α1) expression in control and IPF fibroblasts; (3) the change in α-SMA and procollagen I (α1) expression in response to TGF-β1 was inhibited by antioxidants and by a NOX4 siRNA; (4) NOX4 modulated α-SMA and procollagen I (α1) expression by controlling activation of Smad 2/3, and (5) NOX4 modulated PDGF-induced fibroblasts migration.
NOX4 is critical for modulation of pulmonary myofibroblast phenotype in IPF, probably by modulating the response to TGF-β1 and PDGF.
Adult; Aged; Cell Differentiation; drug effects; physiology; Cells, Cultured; Female; Fibroblasts; drug effects; enzymology; pathology; Gene Expression Regulation, Enzymologic; Humans; Idiopathic Pulmonary Fibrosis; enzymology; pathology; Lung; enzymology; pathology; Male; Middle Aged; NADPH Oxidase; biosynthesis; genetics; physiology; Platelet-Derived Growth Factor; pharmacology; RNA, Messenger; genetics; Reactive Oxygen Species; metabolism; Reverse Transcriptase Polymerase Chain Reaction; methods; Smad2 Protein; metabolism; Smad3 Protein; metabolism; Transforming Growth Factor beta1; pharmacology; Up-Regulation
Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents.
Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs.
Methods: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and α–smooth muscle actin (α-SMA). We used cell culture of pten−/− and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits α-SMA, fibroblast proliferation, and collagen production.
Results: In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten−/− fibroblasts, and in normal fibroblasts in which PTEN was inhibited, α-SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor-β to wild-type cells, but not pten−/− cells, resulted in increased α-SMA expression in a time-dependent fashion. In pten−/− cells, reconstitution of PTEN decreased α-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-β–induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype.
Conclusions: The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.
fibrosis; myofibroblast; phosphatase; PTEN; smooth muscle actin
Prostaglandin E2 (PGE2), the main metabolite of cyclooxygenase (COX), is a well-known anti-fibrotic agent. Moreover, myofibroblasts expressing α-smooth muscle actin (α-SMA), fibroblast expansion and epithelial-mesenchymal transition (EMT) are critical to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our aim was to investigate the expression of COX-2 and PGE2 in human lung myofibroblasts and establish whether fibroblast-myofibroblast transition (FMT) and EMT are associated with COX-2 and PGE2 down-regulation.
Fibroblasts obtained from IPF patients (n = 6) and patients undergoing spontaneous pneumothorax (control, n = 6) and alveolar epithelial cell line A549 were incubated with TGF-β1 and FMT and EMT markers were evaluated. COX-2 and α-SMA expression, PGE2 secretion and cell proliferation were measured after IL-1β and PGE2 incubation.
Myofibroblasts from both control and IPF fibroblast cultures stimulated with IL-1β showed no COX-2 expression. IPF fibroblasts showed increased myofibroblast population and reduced COX-2 expression in response to IL-1β. TGF-β1 increased the number of myofibroblasts in a time-dependent manner. In contrast, TGF-β1 induced slight COX-2 expression at 4 h (without increase in myofibroblasts) and 24 h, but not at 72 h. Both IPF and control cultures incubated with TGF-β1 for 72 h showed diminished COX-2 induction, PGE2 secretion and α-SMA expression after IL-1β addition. The latter decreased proliferation in fibroblasts but not in myofibroblasts. A549 cells incubated with TGF-β1 for 72 h showed down-regulated COX-2 expression and low basal PGE2 secretion in response to IL-1β. Immuno-histochemical analysis of IPF lung tissue showed no COX-2 immuno-reactivity in myofibroblast foci.
Myofibroblasts are associated with COX-2 down-regulation and reduced PGE2 production, which could be crucial in IPF development and progression.
Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle–specific α-actin (α-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture. In this article, we demonstrate that MK2 also regulates myofibroblasts in vivo. Disruption of MK2 in mice prevented myofibroblast formation in a model of pulmonary fibrosis. However, MK2 disruption and consequent lack of myofibroblast formation exacerbated fibrosis rather than ameliorated it as previously postulated. When mice lacking MK2 (MK2−/−) were exposed to bleomycin, more collagen accumulated and more fibroblasts populated fibrotic regions in their lungs than in similarly treated wild-type mice. While there were many vimentin-positive cells in the bleomycin-treated MK2−/− mouse lungs, few α-SMA–positive cells were observed in these lungs compared with wild-type mouse lungs. siRNA against MK2 reduced α-SMA expression in wild-type mouse embryonic fibroblasts (MEF), consistent with its suppression in MK2−/− MEF. On the other hand expressing constitutively active MK2 in MK2−/− MEF significantly increased α-SMA expression. MK2−/−MEF proliferated at a faster rate and produced more collagen; however, they migrated at a slower rate than wild-type MEF. Overexpressing phosphomimicking HSP27, a target of MK2, did not reverse the effect of MK2 disruption on fibroblast migration. MK2 disruption did not affect Smad2 activation by transforming growth factor-β. Thus, MK2 appears to mediate myofibroblast differentiation, and inhibiting that differentiation might contribute to fibrosis rather than protect against it.
actin; cytoskeleton; fibroblast; p38; idiopathic pulmonary fibrosis
Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5–7 d after initiation of pulmonary injury. TGF-β1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-β1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.
Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-β1. As the ECM critically regulates AEC responses to TGF-β1, we explored the role of the prominent epithelial integrin α3β1 in experimental fibrosis by generating mice with lung epithelial cell–specific loss of α3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through β-catenin has been implicated in EMT; we found that in primary AECs, α3 integrin was required for β-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654–β-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654–β-catenin/pSmad2 complexes and showed accumulation of pY654–β-catenin in myofibroblasts. These findings demonstrate epithelial integrin–dependent profibrotic crosstalk between β-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.
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.
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.
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.
Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of α-smooth muscle actin (α-SMA)–expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-β–induced expression of myofibroblast markers such as α-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-β–induced α-SMA expression in primary murine alveolar epithelial cells. Since TGF-β is known to regulate α-SMA expression, the effect of HGF on components of TGF-β signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-β signaling, in a mitogen-activated protein kinase–dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in α-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells.
EMT; HGF; Smad7
Rationale: Tissue fibrosis is considered a dysregulated wound-healing response. Fibronectin containing extra type III domain A (EDA) is implicated in the regulation of wound healing. EDA-containing fibronectin is deposited during wound repair, and its presence precedes that of collagen.
Objectives: To investigate the role of EDA-containing fibronectin in lung fibrogenesis.
Methods: Primary lung fibroblasts from patients with idiopathic pulmonary fibrosis or from patients undergoing resection for lung cancer were assessed for EDA-containing fibronectin and α-smooth muscle actin (α-SMA) expression. Mice lacking the EDA domain of fibronectin and their wild-type littermates were challenged with the bleomycin model of lung fibrosis. Primary lung fibroblasts from these mice were assayed in vitro to determine the contribution of EDA-containing fibronectin to fibroblast phenotypes.
Measurements and Main Results: Idiopathic pulmonary fibrosis lung fibroblasts produced markedly more EDA-containing fibronectin and α-SMA than control fibroblasts. EDA-null mice failed to develop significant fibrosis 21 days after bleomycin challenge, whereas wild-type controls developed the expected increase in total lung collagen. Histologic analysis of EDA-null lungs after bleomycin showed less collagen and fewer α-SMA–expressing myofibroblasts compared with that observed in wild-type mice. Failure to develop lung fibrosis in EDA-null mice correlated with diminished activation of latent transforming growth factor (TGF)-β and decreased lung fibroblast responsiveness to active TGF-β in vitro.
Conclusions: The data show that EDA-containing fibronectin is essential for the fibrotic resolution of lung injury through TGF-β activation and responsiveness, and suggest that EDA-containing fibronectin plays a critical role in tissue fibrogenesis.
fibrosis; fibronectin; TGF-β; myofibroblast
Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.
Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis. Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents. Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity in vivo and to identify a potential mechanism by which this occurs. Methods: We utilized tissue sections of surgical lung biopsies from patients with idiopathic pulmonary fibrosis to localize expression of PTEN and α-SMA. We utilized cell culture of pten-/- and wild-type fibroblasts as well as adenoviral strategies and pharmacologic inhibitors to determine the mechanism by which PTEN inhibits α-SMA, fibroblast proliferation, and collagen production. Results: In human lung specimens of idiopathic pulmonary fibrosis, myofibroblasts within fibroblastic foci demonstrate diminished PTEN expression. Further, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten-/- fibroblasts, and in normal fibroblasts in which PTEN is inhibited, α-SMA, proliferation, and collagen production is upregulated. Addition of transforming growth factor-β to wild-type cells, but not pten-/- cells, results in increased α-SMA expression in a time-dependent fashion. In pten-/- cells, reconstitution of PTEN decreases α-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibits transforming growth factor-β-induced myofibroblast differentiation. Both the protein and lipid phosphatase actions of PTEN are capable of modulating the myofibroblast phenotype. Conclusions: The results indicate that in idiopathic pulmonary fibrosis, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.
myofibroblast; fibrosis; PTEN; phosphatase; smooth muscle actin
Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-β is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator–activated receptor (PPAR) γ is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARγ ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-β–stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of α–smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15 d-PGJ2), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARγ-independent pathway. CDDO and 15 d-PGJ2 contain an α/β unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A1 and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ2 lacking the electrophilic center is much less potent. CDDO does not alter TGF-β–induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-β–responsive genes. Overall, these data indicate that certain PPARγ ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-β–mediated profibrogenic activities through pathways independent of PPARγ. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.
myofibroblast; peroxisome proliferator–activated receptor γ; 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; 15-deoxy-Δ12,14-prostaglandin J2; α-smooth muscle actin
Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex disease for which the pathogenesis is poorly understood. In this study, we identified lactic acid as a metabolite that is elevated in the lung tissue of patients with IPF.
Objectives: This study examines the effect of lactic acid on myofibroblast differentiation and pulmonary fibrosis.
Methods: We used metabolomic analysis to examine cellular metabolism in lung tissue from patients with IPF and determined the effects of lactic acid and lactate dehydrogenase-5 (LDH5) overexpression on myofibroblast differentiation and transforming growth factor (TGF)-β activation in vitro.
Measurements and Main Results: Lactic acid concentrations from healthy and IPF lung tissue were determined by nuclear magnetic resonance spectroscopy; α-smooth muscle actin, calponin, and LDH5 expression were assessed by Western blot of cell culture lysates. Lactic acid and LDH5 were significantly elevated in IPF lung tissue compared with controls. Physiologic concentrations of lactic acid induced myofibroblast differentiation via activation of TGF-β. TGF-β induced expression of LDH5 via hypoxia-inducible factor 1α (HIF1α). Importantly, overexpression of both HIF1α and LDH5 in human lung fibroblasts induced myofibroblast differentiation and synergized with low-dose TGF-β to induce differentiation. Furthermore, inhibition of both HIF1α and LDH5 inhibited TGF-β–induced myofibroblast differentiation.
Conclusions: We have identified the metabolite lactic acid as an important mediator of myofibroblast differentiation via a pH-dependent activation of TGF-β. We propose that the metabolic milieu of the lung, and potentially other tissues, is an important driving force behind myofibroblast differentiation and potentially the initiation and progression of fibrotic disorders.
lactate; idiopathic pulmonary fibrosis; myofibroblast; lactate dehydrogenase; hypoxia-inducible factor 1α
Rationale: Idiopathic pulmonary fibrosis (IPF) is a deadly progressive disease with few treatment options. Transglutaminase 2 (TG2) is a multifunctional protein, but its function in pulmonary fibrosis is unknown.
Objectives: To determine the role of TG2 in pulmonary fibrosis.
Methods: The fibrotic response to bleomycin was compared between wild-type and TG2 knockout mice. Transglutaminase and transglutaminase-catalyzed isopeptide bond expression was examined in formalin-fixed human lung biopsy sections by immunohistochemistry from patients with IPF. In addition, primary human lung fibroblasts were used to study TG2 function in vitro.
Measurements and Main Results: TG2 knockout mice developed significantly reduced fibrosis compared with wild-type mice as determined by hydroxyproline content and histologic fibrosis score (P < 0.05). TG2 expression and activity are increased in lung biopsy sections in humans with IPF compared with normal control subjects. In vitro overexpression of TG2 led to increased fibronectin deposition, whereas transglutaminase knockdown led to defects in contraction and adhesion. The profibrotic cytokine transforming growth factor-β causes an increase in membrane-localized TG2, increasing its enzymatic activity.
Conclusions: TG2 is involved in pulmonary fibrosis in a mouse model and in human disease and is important in normal fibroblast function. With continued research on TG2, it may offer a new therapeutic target.
fibroblast; myofibroblast; animal model; collagen; fibronectin
Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-β (TGF-β)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-β signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-β: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-β-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and β. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-β-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.
Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle α-actin (smα) in response to profibrotic agents such as TGFβ is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF). MK2 is a kinase substrate of p38 MAP kinase that mediates some effects of p38 activation on the actin cytoskeleton. Using mouse embryonic fibroblasts (MEF) from MK2 knockout (MK2−/−) mice, we demonstrate that disrupting expression of MK2 expression reduces filamentous actin and stress fibers. It also causes MK2−/− MEF to express less smα than their corresponding wild-type (WT) MEF at baseline and in response to TGFβ. Furthermore, TGFβ causes downregulation of smα in MK2−/− MEF, instead of upregulation observed in WT MEF. Expression of other fibroblast markers, such as collagen, is not altered in MK2−/− MEF. Our results further suggest that downregulation of smα in MK2−/− MEF is not due to lack of activation of serum responsive promoter elements, but probably due to reduced smα message stability in these cells. These results indicate that MK2 plays a key role in regulation of smα expression, and that targeting MK2 might present a therapeutic approach in managing conditions such as pulmonary fibrosis.
fibrosis; smooth muscle actin; cytoskeleton; MAPKAPK2; fibroblast; stress fibers
Idiopathic pulmonary fibrosis (IPF) is a serious progressive and irreversible lung disease with unknown etiology and few treatment options. This disease was once thought to be a chronic inflammatory-driven process, but it is increasingly recognized that the epithelial–mesenchymal transition (EMT) contributes to the cellular origin of fibroblast accumulation in response to injury. During the pathogenesis of pulmonary fibrotic diseases, transforming growth factor-β (TGF-β) signaling is considered a pivotal inducer of EMT and fibroblast activation, and a number of therapeutic interventions that interfere with TGF-β signaling have been developed to reverse established fibrosis. However, efficient and well-tolerated antifibrotic agents are not currently available. Previously, we reported the identification of sorafenib to antagonize TGF-β signaling in mouse hepatocytes in vitro. In this manuscript, we continued to evaluate the antifibrotic effects of sorafenib on bleomycin (BLM)-induced pulmonary fibrosis in mice. We further demonstrated that sorafenib not only profoundly inhibited TGF-β1-induced EMT in alveolar epithelial cells, but also simultaneously reduced the proliferation and collagen synthesis in fibroblasts. Additionally, we presented in vivo evidence that sorafenib inhibited the symptoms of BLM-mediated EMT and fibroblast activation in mice, warranting the therapeutic potential of this drug for patients with IPF.
sorafenib; TGF-β signaling; pulmonary fibrosis; EMT; fibroblast activation
Rationale: Members of the transforming growth factor (TGF)-β superfamily, including TGF-βs and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF).
Objectives: To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis.
Methods: Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-β and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure.
Measurements and Main Results: Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-β receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice.
Conclusions: The TGF-β and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.
gremlin; pulmonary fibrosis; bone morphogenetic protein; transforming growth factor-β
Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the recurrent damage of alveolar epithelial cells as well as inappropriate expansion and activation of fibroblasts resulting in pronounced extracellular matrix (ECM) deposition. Although recent studies have indicated the involvement of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein regulating ECM deposition, in the pathogenesis of fibrosis, factors regulating SPARC expression or roles of SPARC in fibrosis have not been fully elucidated.
Among the profibrotic factors examined in cultured fibroblasts, we showed that SPARC expression was upregulated mainly by transforming growth factor (TGF)-β. We also showed that expression of SPARC in the lung was upregulated in the murine bleomycin-induced pulmonary fibrosis model, which was inhibited by TGF-β receptor I inhibitor. Knockdown of SPARC in fibroblasts using siRNA or treatment with the antioxidant N-acetylcysteine attenuated epithelial cell injury induced by TGF-β-activated fibroblasts in a coculture system. We also demonstrated that SPARC was required for hydrogen peroxide (H2O2) production in fibroblasts treated with TGF-β. Furthermore, TGF-β activated integrin-linked kinase (ILK), which was inhibited by SPARC siRNA. Knockdown of ILK attenuated extracellular H2O2 generation in TGF-β-stimulated fibroblasts. Our results indicated that SPARC is upregulated by TGF-β and is required for TGF-β-induced H2O2 production via activation of ILK, and this H2O2 production from fibroblasts is capable of causing epithelial cell injury.
The results presented in this study suggest that SPARC plays a role in epithelial damage in the IPF lung via enhanced H2O2 production from fibroblasts activated by TGF-β. Therefore, SPARC inhibition may prevent epithelial injury in IPF lung and represent a potential therapeutic approach for IPF.
SPARC; TGF-β; Hydrogen peroxide; Fibroblast; Pulmonary fibrosis
Mechanisms regulating myofibroblastic differentiation of fibroblasts within fibroblastic foci in idiopathic pulmonary fibrosis (IPF) remain unclear. Epigenetic processes, including DNA methylation, produce heritable but potentially reversible changes in DNA or its associated proteins and are prominent in development and oncogenesis. We have shown that Thy-1 suppresses myofibroblastic differentiation of lung fibroblasts and that fibroblasts in fibroblastic foci are Thy-1(−). Epigenetic down-regulation of Thy-1 has been demonstrated in cellular transformation and clinical cancer. We hypothesized that epigenetic regulation of Thy-1 affects the lung fibroblast fibrogenic phenotype. RT-PCR, methylation-specific PCR (MSP), and bisulfite genomic sequencing were used to determine the methylation status of the Thy-1 promoter in Thy-1(+) and Thy-1(−) lung fibroblasts, and MSP–in situ hybridization (MSPISH) was performed on fibrotic tissue. Thy-1 gene expression is absent in Thy-1(−) human and rat fibroblasts despite intact Thy-1 genomic DNA. Cytosine-guanine islands in the Thy-1 gene promoter are hypermethylated in Thy-1(−), but not Thy-1(+), fibroblasts. RT-PCR and MSP demonstrate that, in IPF samples in which Thy-1 expression is absent, the Thy-1 promoter region is methylated, whereas in lung samples retaining Thy-1 expression, the promoter region is unmethylated. MSPISH confirms methylation of the Thy-1 promoter in fibroblastic foci in IPF. Treatment with DNA methyltransferase inhibitors restores Thy-1 expression in Thy-1(−) fibroblasts. Epigenetic regulation of Thy-1 is a novel and potentially reversible mechanism in fibrosis that may offer the possibility of new therapeutic options.
pulmonary fibrosis; epigenetic processes; Thy-1 antigen; fibroblasts
Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease with an average survival of only 3 to 5 years. The mechanisms underlying the initiation and progression of IPF are poorly understood, and treatments available have only modest effect on disease progression. Interestingly, the incidence of IPF is approximately 60 times more common in individuals aged 75 years and older, but the mechanism by which aging promotes fibrosis is unclear. The authors hypothesized that aged lungs have a profibrotic phenotype that render it susceptible to disrepair after injury.
Young and old mice were treated with bleomycin to examine disrepair in the aged lung. In addition, uninjured young and old mouse lungs were analyzed for transforming growth factor-beta 1 (TGF-β1) production, extracellular matrix composition and lung fibroblast phenotype. Lung fibroblasts were treated with a DNA methyltransferase inhibitor to examine the potential epigenetic mechanisms involved in age-associated phenotypic alterations.
The lungs of old mice showed worse fibrosis after bleomycin-induced injury compared with the lungs from young mice. At baseline, aged lungs expressed a profibrotic phenotype characterized by increased mRNA expression for fibronectin extracellular domain A (Fn-EDA) and the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Old lungs also expressed higher levels of TGF-β receptor 1 and TGF-β1 mRNA, protein and activity as determined by increased Smad3 expression, protein phosphorylation and DNA binding. Lung fibroblasts harvested from aged lungs showed reduced expression of the surface molecule Thy-1, a finding also implicated in lung fibrosis; the latter did not seem related to Thy-1 gene methylation.
Altogether, aged lungs manifest a profibrotic phenotype characterized by enhanced fibronectin extracellular domain A and MMP expression and increased TGF-β1 expression and signaling and are populated by Thy-1–negative fibroblasts, all implicated in the pathogenesis of lung fibrosis.
Lung fibrosis; Aging; Injury; Extracellular matrix; Lung fibroblast
The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.
Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-β activation that was mediated by proteinase-activated receptor–1 (PAR1) and integrin αvβ5. PAR1, αvβ5, and α-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.
Rationale: An increase in the number of mononuclear phagocytes in lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) worsens prognosis. Chemokines that recruit mononuclear phagocytes, such as CC chemokine ligand 2 (CCL2), are elevated in bronchoalveolar lavage (BAL) fluid (BALF) from patients with IPF. However, little attention is given to the role of the mononuclear phagocyte survival and recruitment factor, macrophage colony-stimulating factor (M-CSF), in pulmonary fibrosis.
Objectives: To investigate the role of mononuclear phagocytes and M-CSF in pulmonary fibrosis.
Methods: Wild-type, M-CSF−/−, or CCL2−/− mice received intraperitoneal bleomycin. Lung inflammation and fibrosis were measured by immunohistochemistry, ELISA, collagen assay, BAL differentials, real-time polymerase chain reaction, and Western blot analysis. Human and mouse macrophages were stimulated with M-CSF for CCL2 expression. BALF from patients with IPF was examined for M-CSF and CCL2.
Measurements and Main Results: M-CSF−/− and CCL2−/− mice had less lung fibrosis, mononuclear phagocyte recruitment, collagen deposition, and connective tissue growth factor (CTGF) expression after bleomycin administration than wild-type littermates. Human and mouse macrophages stimulated with M-CSF had increased CCL2 production, and intratracheal administration of M-CSF in mice induced CCL2 production in BALF. Finally, BALF from patients with IPF contained significantly more M-CSF and CCL2 than BALF from normal volunteers. Elevated levels of M-CSF were associated with elevated CCL2 in BALF and the diagnosis of IPF.
Conclusions: These data suggest that M-CSF contributes to the pathogenesis of pulmonary fibrosis in mice and in patients with IPF through the involvement of mononuclear phagocytes and CCL2 production.
bleomycin; CC chemokine ligand 2; macrophage colony-stimulating factor; mononuclear phagocytes; pulmonary fibrosis