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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.

Background

Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.

Methods

The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.

Results

TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.

Conclusion

We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

The histopathology of idiopathic pulmonary fibrosis (IPF) includes the presence of myofibroblasts within so-called fibroblastic foci, and studies suggest that lung myofibroblasts may be derived from epithelial cells through epithelial–mesenchymal transition (EMT). Transforming growth factor (TGF)–β1 is expressed and/or activated in fibrogenesis, and induces EMT in lung epithelial cells in a dose-dependent manner. A higher occurrence of Epstein-Barr virus (EBV) has been reported in the lung tissue of patients with IPF. EBV expresses latent membrane protein (LMP) 1 during the latent phase of infection, and may play a role in the pathogenesis of pulmonary fibrosis inasmuch as LMP-1 may act as a constitutively active TNF-α receptor. Our data show a remarkable increase in mesenchymal cell markers, along with a concurrent reduction in the expression of epithelial cell markers in lung epithelial cells cotreated with LMP-1, and very low doses of TGF-β1. This effect was mirrored in lung epithelial cells infected with EBV expressing LMP1 and cotreated with TGF-β1. LMP1 pro-EMT signaling was identified, and occurs primarily through the nuclear factor–κB pathway and secondarily through the extracellular signal–regulated kinase (ERK) pathway. Activation of the ERK pathway was shown to be critical for aspects of TGF-β1–induced EMT. LMP1 accentuates the TGF-β1 activation of ERK. Together, these data demonstrate that the presence of EBV-LMP1 in lung epithelial cells synergizes with TGF-β1 to induce EMT. Our in vitro data may help to explain the observation that patients with IPF demonstrating positive staining for LMP1 in lung epithelial cells have a more rapid demise than patients in whom LMP1 is not detected.

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.

Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.

Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.

Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin.

Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis.

Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.

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.

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.

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.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease that is unresponsive to current therapies and characterized by excessive collagen deposition and subsequent fibrosis. While inflammatory cytokines, including interleukin (IL)-6, are elevated in IPF, the molecular mechanisms that underlie this disease are incompletely understood, although the development of fibrosis is believed to depend on canonical transforming growth factor (TGF)-β signalling. We examined bleomycin-induced inflammation and fibrosis in mice carrying a mutation in the shared IL-6 family receptor gp130. Using genetic complementation, we directly correlate the extent of IL-6-mediated, excessive Stat3 activity with inflammatory infiltrates in the lung and the severity of fibrosis in corresponding gp130757F mice. The extent of fibrosis was attenuated in B lymphocyte-deficient gp130757F;µMT−/− compound mutant mice, but fibrosis still occurred in their Smad3−/− counterparts consistent with the capacity of excessive Stat3 activity to induce collagen 1α1 gene transcription independently of canonical TGF-β/Smad3 signalling. These findings are of therapeutic relevance, since we confirmed abundant STAT3 activation in fibrotic lungs from IPF patients and showed that genetic reduction of Stat3 protected mice from bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in most adult tissues except the lung. In this study, we evaluated the interaction of ligand advanced glycation end products (AGE) with RAGE during the epithelial to myofibroblast transition in rat AECs. Our results indicate that AGE inhibited the TGF-β-dependent alveolar EMT by increasing Smad7 expression, and that the effect was abolished by RAGE siRNA treatment. Thus, the induction of Smad7 by the AGE-RAGE interaction limits the development of pulmonary fibrosis by inhibiting TGF-β-dependent signaling in AECs.

Aim

This study explored the cellular and biological interrelationships involved in Idiopathic Pulmonary Fibrosis (IPF) lung tissue remodelling using immunohistochemical analysis.

Methods and results

IPF and control lung tissues were examined for localisation of Epithelial Mesenchymal Transition (EMT), proliferation and growth factor markers assessing their relationship to key histological aberrations. E-cadherin was expressed in IPF and control (Alveolar type II) ATII cells (>75%). In IPF, mean expression of N-cadherin was scanty (<10%): however 4 cases demonstrated augmented expression in ATII cells correlating to histological disease status (Pearson correlation score 0.557). Twist was expressed within fibroblastic foci but not in ATII cells. Transforming Growth Factor- β (TGF-β) protein expression was significantly increased in IPF ATII cells with variable expression within fibroblastic foci. Antigen Ki-67 was observed within hyperplastic ATII cells but not in cells overlying foci. Collagen I and α-smooth muscle actin (α-SMA) were strongly expressed within fibroblastic foci (>75%); cytoplasmic collagen I in ATII cells was present in 3 IPF cases. IPF ATII cells demonstrated variable Surfactant Protein-C (SP-C).

Conclusions

The pathogenesis of IPF is complex and involves multiple factors, possibly including EMT. Histological analysis suggests TGF-β-stimulated myofib rob lasts initiate a contractile response within established fibroblastic foci while proliferating ATII cells attempt to instigate alveolar epithelium repair. Marker expression (N-cadherin and Ki-67) correlation with histological disease activity (as reflected by fibroblastic foci extent) may emerge as future prognostic indicators for IPF.

The mechanisms underlying the pathogenesis of idiopathic pulmonary fibrosis (IPF) involve multiple pathways, such as inflammation, epithelial mesenchymal transition, coagulation, oxidative stress, and developmental processes. The small GTPase, RhoA, and its target protein, Rho-kinase (ROCK), may interact with other signaling pathways known to contribute to pulmonary fibrosis. This study aimed to determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice. Our results showed that the Aschcroft score and hydroxyproline content of the bleomycin-treated mouse lung decreased in response to fasudil treatment. The number of infiltrated inflammatory cells in the bronchoalveolar lavage fluid (BALF) was attenuated by fasudil. In addition, fasudil reduced the production of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), and plasminogen activator inhibitor-1 (PAI-1) mRNA and protein expression in bleomycin-induced pulmonary fibrosis. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease of the lung parenchyma, without curative treatment. Gremlin is a bone morphogenic protein (BMP) antagonist, its expression being increased in IPF lungs. It has been implicated in promoting myofibroblast accumulation, likely through inhibited fibroblast apoptosis and epithelial-to-mesenchymal transition. In the current study, we examined the effects of selective adenovirus-mediated overexpression of Gremlin in rat lungs. We show that transient Gremlin overexpression results in activation of alveolar epithelial cells with proliferation and apoptosis, as well as partly reversible lung fibrosis. We found myofibroblasts arranged in fibroblastic foci. Fibroblast proliferation occurred delayed as compared with epithelial changes. Fibrotic pathology significantly declined after Day 14, the reversal being associated with an increase of the epithelium-protective element, fibroblast growth factor (FGF)–10. Our data indicate that Gremlin-mediated BMP inhibition results in activation of epithelial cells and transient fibrosis, but also induction of epithelium-protective FGF10. A Gremlin–BMP–FGF10 loop may explain these results, and demonstrate that the interactions between different factors are quite complex in fibrotic lung disease. Increased Gremlin expression in human IPF tissue may be an expression of continuing epithelial injury, and Gremlin may be part of activated repair mechanisms.

Idiopathic pulmonary fibrosis (IPF) is a chronic, relentlessly progressive fibrosing disease of the lung of unknown etiology. Significant progress has been made in recent years in elucidating key aspects of the pathobiology of IPF. Insights into disease pathogenesis have come from studies of cell biology, growth factor/cytokine signaling, animal models of pulmonary fibrosis, and human IPF cells and tissue. A consistent finding in the ultrastructural pathology of IPF is alveolar epithelial cell injury and apoptosis. Another consistent finding in the histopathology of human IPF, described as usual interstitial pneumonia, is the accumulation of aggregates of myofibroblasts in fibroblastic foci. The extent or profusion of fibroblastic foci in lung biopsies is strongly correlated with increased mortality in patients with IPF. There is emerging evidence that myofibroblasts in IPF/usual interstitial pneumonia, both in the in vivo microenvironment and during the process of differentiation in vitro, acquire resistance to apoptosis. Here, we review the current evidence and mechanisms for this apparent “apoptosis paradox” in the pathogenesis of IPF.

Idiopathic pulmonary fibrosis (IPF) is a chronic, relentlessly progressive fibrosing disease of the lung of unknown etiology. Significant progress has been made in recent years in elucidating key aspects of the pathobiology of IPF. Insights into disease pathogenesis have come from studies of cell biology, growth factor/cytokine signaling, animal models of pulmonary fibrosis, and human IPF cells and tissue. A consistent finding in the ultrastructural pathology of IPF is alveolar epithelial cell injury and apoptosis. Another consistent finding in the histopathology of human IPF, described as usual interstitial pneumonia, is the accumulation of aggregates of myofibroblasts in fibroblastic foci. The extent or profusion of fibroblastic foci in lung biopsies is strongly correlated with increased mortality in patients with IPF. There is emerging evidence that myofibroblasts in IPF/usual interstitial pneumonia, both in the in vivo microenvironment and during the process of differentiation in vitro, acquire resistance to apoptosis. Here, we review the current evidence and mechanisms for this apparent “apoptosis paradox” in the pathogenesis of IPF.

Aims

Recent studies suggest the importance of oxidant stress in the progression of pulmonary fibrosis. The aim of this study was to investigate extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme of the extracellular matrix of human lung, in biopsy-proven idiopathic pulmonary fibrosis (IPF) related to usual interstitial pneumonia (UIP).

Methods and results

Fibrotic areas and fibroblastic foci in UIP lungs were notable for absence of ECSOD by immunohistochemistry. Western blotting showed significantly lowered immunoreactivity of ECSOD in fibrotic compared with non-fibrotic areas of the diseased lung. The only cell type that showed intense ECSOD positivity in UIP was the interstitial mast cell. In order to investigate the mechanism for ECSOD depletion in fibrotic areas, alveolar epithelial cells were exposed to tumour necrosis factor-α and transforming growth factor (TGF)-β1; TGF-β suggested a trend towards decreased synthesis. Patients with UIP were also assessed to determine whether this disease is associated with a naturally occurring mutation in ECSOD (Arg213Gly) which leads to a loss of tissue binding of ECSOD. No significant differences could be found in the allele or genotype frequencies of this polymorphism between 63 UIP patients and 61 control subjects.

Conclusion

Overall, consistent with several other antioxidant enzymes, ECSOD is very low in fibrotic areas of UIP, which may further increase the oxidant burden in this disease.

Hyaluronan synthase 2 and CD44 are required for severe lung fibrosis in response to bleomycin.

Tissue fibrosis is a major cause of morbidity, and idiopathic pulmonary fibrosis (IPF) is a terminal illness characterized by unremitting matrix deposition in the lung. The mechanisms that control progressive fibrosis are unknown. Myofibroblasts accumulate at sites of tissue remodeling and produce extracellular matrix components such as collagen and hyaluronan (HA) that ultimately compromise organ function. We found that targeted overexpression of HAS2 (HA synthase 2) by myofibroblasts produced an aggressive phenotype leading to severe lung fibrosis and death after bleomycin-induced injury. Fibroblasts isolated from transgenic mice overexpressing HAS2 showed a greater capacity to invade matrix. Conditional deletion of HAS2 in mesenchymal cells abrogated the invasive fibroblast phenotype, impeded myofibroblast accumulation, and inhibited the development of lung fibrosis. Both the invasive phenotype and the progressive fibrosis were inhibited in the absence of CD44. Treatment with a blocking antibody to CD44 reduced lung fibrosis in mice in vivo. Finally, fibroblasts isolated from patients with IPF exhibited an invasive phenotype that was also dependent on HAS2 and CD44. Understanding the mechanisms leading to an invasive fibroblast phenotype could lead to novel approaches to the treatment of disorders characterized by severe tissue fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β–mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.

Rationale: Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis.

Objectives: To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E2, accounts for this imbalance.

Methods: Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE2 on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed.

Measurements and Main Results: IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE2 almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE2 protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE2 inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE2 modulates cellular apoptotic responses.

Conclusions: The observation that PGE2 deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive, usually fatal, form of interstitial lung disease characterized by failure of alveolar re-epithelialization, persistence of fibroblasts/myofibroblasts, deposition of extracellular matrix, and distortion of lung architecture which ultimately results in respiratory failure. Clinical IPF is associated with a histopathological pattern of usual interstitial pneumonia (UIP) on surgical lung biopsy. Therapy for this disease with glucocorticoids and other immunomodulatory agents is largely ineffective and recent trials of newer anti-fibrotic agents have been disappointing. While the inciting event(s) leading to the initiation of scar formation in UIP remain unknown, recent advances in our understanding of the mechanisms underlying both normal and aberrant wound healing have shed some light on pathogenetic mechanisms that may play significant roles in this disease. Unlike other fibrotic diseases of the lung, such as those associated with collagen vascular disease, occupational exposure, or chemotherapeutic agents, UIP is not associated with a significant inflammatory response; rather, dysregulated epithelial–mesenchymal interactions predominate. Identification of pathways crucial to fibrogenesis might offer potentially novel therapeutic targets to slow or halt the progression of IPF. This review focuses on evolving concepts of cellular and molecular mechanisms in the pathogenesis of UIP/IPF.

Purpose

To review the scientific literature supporting the participation of caveolin-1 in the pathogenesis of tissue fibrosis and that modulation of the caveolin-1 pathway may represent a novel treatment for systemic sclerosis (SSc) and other fibrotic diseases.

Recent Findings

Caveolin-1 plays an important role in the regulation of transforming growth factor β (TGF-β) signaling owing to its participation in TGF-β receptor (TβR) internalization. TβR internalized through caveolin-1 lipid rafts undergoes rapid degradation, effectively decreasing TGF-β signaling. Studies have shown that caveolin-1 knockdown in vitro markedly increased collagen gene expression in normal human lung fibroblasts. Caveolin-1 was reduced in affected SSc lungs and skin and in idiopathic pulmonary fibrosis (IPF) lung tissues and fibroblasts. Increasing caveolin-1 expression markedly improved bleomycin-induced pulmonary fibrosis. Restoration of caveolin bioavailability employing penetratin, a cell-permeable peptide carrier for a bioactive caveolin-1 fragment abrogated TGF-β activation of cultured human dermal fibroblasts. Systemic administration of penetratin-caveolin-1 peptide to mice with bleomycin-induced lung fibrosis reduced fibrosis.

Summary

Caveolin-1 plays an important role in the regulation of TGF-β signaling and participates in the pathogenesis of SSc and IPF. Restoration of caveolin function employing active caveolin-1 fragments coupled to cell-permeable carrier peptides may represent a novel approach for their treatment.

Background

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.

Methods

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.

Results

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.

Conclusion

NOX4 is critical for modulation of pulmonary myofibroblast phenotype in IPF, probably by modulating the response to TGF-β1 and PDGF.

The endothelin system participates in a number of critical biologic pathways, including normal wound healing. In addition, emerging basic science, animal and human data all suggest that endothelin-1 (ET-1) is a potentially important contributor in the pathobiology of fibrosing disorders, including those that affect the lung. For example, ET-1 drives fibroblast activation, proliferation, as well as differentiation into myofibroblasts—processes that lead to excessive collagen deposition. Patients with idiopathic pulmonary fibrosis (IPF) have increased levels of ET-1 in both their bronchoalveolar lavage fluid and lung tissue. Beyond this, rodent models suggest that endothelin receptor antagonists can limit bleomycin-induced lung fibrosis. This suggests a biologic rationale for the blockade of ET-1 to limit the evolution of lung fibrosis in humans. Initial results from a trial examining the efficacy of a dual endothelin receptor antagonist suggest that this approach may delay disease progression in a subset of patients with IPF.

Idiopathic pulmonary fibrosis (IPF) comprises an aggregate of mesenchymal cells. However, the cellular origin of these mesenchymal phenotypes remains unclear. Transforming growth factor β1 (TGF-β1) has been known as the main cytokine involved in the pathogenesis of IPF. We examined whether the potent fibrogenic cytokine TGF-β1 could induce the epithelial-to-mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and determined whether snail expression is associated with the phenotypic changes observed in the A549 cells. EMT was investigated with cells morphology changes under phase-contrast microscopy, western blotting, and indirect immunofluorescence stains. E-cadherin and transcription factor, snail, were also evaluated by measuring mRNA levels using reverse transcriptase-polymerase chain rection (RT-PCR) analysis. The data showed that TGF-β1 induced A549 cells with epithelial cell characteristics to undergo EMT in a concentration-dependent manner. Following TGF-β1 treatment, A549 cells induced EMT characterized by cells morphological changes, loss of epithelial markers E-caherin and cytokeratin, increased stress fiber reorganization by F-actin, and cytokeratin replacement by vimentin. Although IL-1β failed to induce A549 cells to undergo EMT, the combination of TGF-β1 and IL-1β showed synergy effects in cells morphology changes and the expression of mesenchymal markers. The snail expression study using RT-PCR analysis provided that loss of E-cadherin expression was associated with snail expression. Stimulation of A54 cells with TGF-β1 plus IL-1β revealed a higher level of snail expression. Our data showed that EMT of A549 cells might be closely associated with snail expression.

Rationale: Studies in patients and experimental animals provide compelling evidence of the involvement of the major thrombin receptor, proteinase-activated receptor-1 (PAR1), and the potent chemokine, chemokine (CC motif) ligand-2 (CCL2)/monocyte chemotactic protein-1, in the pathogenesis of idiopathic pulmonary fibrosis (IPF). PAR1 knockout mice are protected from bleomycin-induced lung inflammation and fibrosis and this protection is associated with marked attenuation in CCL2 induction.

Objectives: The aim of this study was to determine which cell types represent the major source of PAR1-inducible CCL2 in the fibrotic lung.

Methods: Using immunohistochemistry and dual immunofluorescence, we examined PAR1 and CCL2 expression in the bleomycin model and human IPF lung. PAR1 and CCL2 gene expression was also assessed in laser-captured alveolar septae from patients with IPF. The ability of PAR1 to induce CCL2 production by lung epithelial cells was also examined in vitro.

Measurements and Main Results: We report for the first time that PAR1 and CCL2 are coexpressed and co–up-regulated on the activated epithelium in fibrotic areas in IPF. Similar observations were found in bleomycin-induced lung injury. Furthermore, we show that thrombin is a potent inducer of CCL2 gene expression and protein release by cultured lung epithelial cells via a PAR1-dependent mechanism.

Conclusions: These data support the notion that PAR1 activation on lung epithelial cells may represent an important mechanism leading to increased local CCL2 release in pulmonary fibrosis. Targeting PAR1 on the pulmonary epithelium may offer a unique opportunity for therapeutic intervention in pulmonary fibrosis and other inflammatory and fibroproliferative conditions associated with excessive local generation of thrombin and CCL2 release.