Investigating the role of platelets and platelet-derived transforming growth factor-ß in idiopathic pulmonary fibrosis.

Transforming growth factor-ß1 (TGFß1) is the key profibrotic cytokine in idiopathic pulmonary fibrosis (IPF), but the primary source of this cytokine in this disease is unknown. Platelets have abundant stores of TGFß1, although the role of these cells in IPF is ill-defined. In this study, we investigated whether platelets, and specifically platelet-derived TGFß1, mediate IPF disease progression. Patients with IPF and non-IPF patients were recruited to determine platelet reactivity, and separate cohorts of patients with IPF were followed for mortality. To study whether platelet-derived TGFß1 modulates pulmonary fibrosis (PF), mice with a targeted deletion of TGFß1 in megakaryocytes and platelets (TGFß1fl/fl.PF4-Cre) were used in the well-characterized bleomycin-induced pulmonary fibrosis (PF) animal model. In a discovery cohort, we found significantly higher mortality in patients with IPF who had elevated platelet counts within the normal range. However, our validation cohort did not confirm this observation, despite significantly increased platelets, neutrophils, active TGFß1, and CCL5, a chemokine produced by inflammatory cells, in the blood, lung, and bronchoalveolar lavage (BAL) of patients with IPF. In vivo, we showed that despite platelets being readily detected within the lungs of bleomycin-treated mice, neither the degree of pulmonary inflammation nor fibrosis was significantly different between TGFß1fl/fl.PF4-Cre and control mice. Our results demonstrate for the first time that platelet-derived TGFß1 does not significantly mediate inflammation or fibrosis in a PF animal model. Furthermore, our human studies revealed blood platelet counts do not consistently predict mortality in IPF but other platelet-derived mediators, such as C-C chemokine ligand 5 (CCL5), may promote neutrophil recruitment and human IPF.NEW & NOTEWORTHY Platelets are a rich source of profibrotic TGFß; however, the role of platelets in idiopathic pulmonary fibrosis (IPF) is unclear. We identified that patients with IPF have significantly more platelets, neutrophils, and active TGFß in their airways than control patients. Using an animal model of IPF, we demonstrated that platelet-derived TGFß does not significantly drive lung fibrosis or inflammation. Our findings offer a better understanding of platelets in both human and animal studies of IPF.

ICAM-1 and ICAM-2 Are Differentially Expressed and Up-Regulated on Inflamed Pulmonary Epithelium, but Neither ICAM-2 nor LFA-1: ICAM-1 Are Required for Neutrophil Migration Into the Airways In Vivo.

Neutrophil migration into the airways is an important process to fight infection and is mediated by cell adhesion molecules. The intercellular adhesion molecules, ICAM-1 (CD54) and ICAM-2 (CD102) are known ligands for the neutrophil integrins, lymphocyte function associated antigen (LFA)-1 (αLß2; CD11a/CD18), and macrophage-1 antigen (Mac-1;αMß2;CD11b/CD18) and are implicated in leukocyte migration into the lung. However, it is ill-defined how neutrophils exit the lung and the role for ICAMs in trans-epithelial migration (TEpM) across the bronchial or alveolar epithelium. We found that human and murine alveolar epithelium expressed ICAM-1, whilst the bronchial epithelium expressed ICAM-2, and both were up-regulated during inflammatory stimulation in vitro and in inflammatory lung diseases such as cystic fibrosis. Although ß2 integrins interacting with ICAM-1 and -2 mediated neutrophil migration across human bronchial epithelium in vitro, neither ICAM-2 nor LFA-1 binding of ICAM-1 mediated murine neutrophil migration into the lung or broncho-alveolar space during LPS-induced inflammation in vivo. Furthermore, TEpM of neutrophils themselves resulted in increased epithelial junctional permeability and reduced barrier function in vitro. This suggests that although ß2 integrins interacting with ICAMs may regulate low levels of neutrophil traffic in healthy lung or early in inflammation when the epithelial barrier is intact; these interactions may be redundant later in inflammation when epithelial junctions are disrupted and no longer limit TEpM.

Gasping for Sulfide: A Critical Appraisal of Hydrogen Sulfide in Lung Disease and Accelerated Aging.

Hydrogen sulfide (H2S) is a gaseous signaling molecule involved in a plethora of physiological and pathological processes. It is primarily synthesized by cystathionine-ß-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase as a metabolite of the transsulfuration pathway. H2S has been shown to exert beneficial roles in lung disease acting as an anti-inflammatory and antiviral and to ameliorate cell metabolism and protect from oxidative stress. H2S interacts with transcription factors, ion channels, and a multitude of proteins via post-translational modifications through S-persulfidation ("sulfhydration"). Perturbation of endogenous H2S synthesis and/or levels have been implicated in the development of accelerated lung aging and diseases, including asthma, chronic obstructive pulmonary disease, and fibrosis. Furthermore, evidence indicates that persulfidation is decreased with aging. Here, we review the use of H2S as a biomarker of lung pathologies and discuss the potential of using H2S-generating molecules and synthesis inhibitors to treat respiratory diseases. Furthermore, we provide a critical appraisal of methods of detection used to quantify H2S concentration in biological samples and discuss the challenges of characterizing physiological and pathological levels. Considerations and caveats of using H2S delivery molecules, the choice of generating molecules, and concentrations are also reviewed. Antioxid. Redox Signal. 35, 551-579.

Culture-independent multilocus sequence typing of Pseudomonas aeruginosa for cross-infection screening.

The genotyping of pathogens within cystic fibrosis cohorts is an important process, enabling the detection of transmissible and clinically-important strains. Traditionally this has been via culture-dependent processes. However, culture-independent investigation of respiratory samples is becoming more common, with such approaches highlighting the limitations of culture-based methods. In this study we describe the culture-independent application of multilocus sequence typing (MLST) for Pseudomonas aeruginosa, performed on DNA extracted from the sputa of cystic fibrosis patients. We compare the output to conventional culture-dependent MLST applied to the same samples and demonstrate high concordance. Culture-independent MLST enabled genotyping of culture-negative samples in patients from whom P. aeruginosa was intermittently isolated, and revealed the hidden presence of transmissible strains. Culture-independent MLST is also capable of highlighting samples containing multiple strains, albeit inconsistently. We conclude that culture-independent MLST can be a useful genotyping tool for screening cohorts and identifying patients that warrant further detailed investigation.

Telomere length and risk of idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease: a mendelian randomisation study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease accounting for 1% of UK deaths. In the familial form of pulmonary fibrosis, causal genes have been identified in about 30% of cases, and a majority of these causal genes are associated with telomere maintenance. Prematurely shortened leukocyte telomere length is associated with IPF and chronic obstructive pulmonary disease (COPD), a disease with similar demographics and shared risk factors. Using mendelian randomisation, we investigated evidence supporting a causal role for short telomeres in IPF and COPD. METHODS: Mendelian randomisation inference of telomere length causality was done for IPF (up to 1369 cases) and COPD (13 538 cases) against 435 866 controls of European ancestry in UK Biobank. Polygenic risk scores were calculated and two-sample mendelian randomisation analyses were done using seven genetic variants previously associated with telomere length, with replication analysis in an IPF cohort (2668 cases vs 8591 controls) and COPD cohort (15 256 cases vs 47 936 controls). FINDINGS: In the UK Biobank, a genetically instrumented one-SD shorter telomere length was associated with higher odds of IPF (odds ratio [OR] 4·19, 95% CI 2·33-7·55; p=0·0031) but not COPD (1·07, 0·88-1·30; p=0·51). Similarly, an association was found in the IPF replication cohort (12·3, 5·05-30·1; p=0·0015) and not in the COPD replication cohort (1·04, 0·71-1·53; p=0·83). Meta-analysis of the two-sample mendelian randomisation results provided evidence inferring that shorter telomeres cause IPF (5·81 higher odds of IPF, 95% CI 3·56-9·50; p=2·19 × 10-12). There was no evidence to infer that telomere length caused COPD (OR 1·07, 95% CI 0·90-1·27; p=0·46). INTERPRETATION: Cellular senescence is hypothesised as a major driving force in IPF and COPD; telomere shortening might be a contributory factor in IPF, suggesting divergent mechanisms in COPD. Defining a key role for telomere shortening enables greater focus in telomere-related diagnostics, treatments, and the search for a cure in IPF. Investigation of therapies that improve telomere length is warranted. FUNDING: Medical Research Council.

Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort.

While Pseudomonas aeruginosa (PA) cross-infection is well documented among patients with cystic fibrosis (CF), the equivalent risk among patients with non-CF bronchiectasis (NCFB) is unclear, particularly those managed alongside patients with CF. We performed analysis of PA within a single centre that manages an unsegregated NCFB cohort alongside a segregated CF cohort. We found no evidence of cross-infection between the two cohorts or within the segregated CF cohort. However, within the unsegregated NCFB cohort, evidence of cross-infection was found between three (of 46) patients. While we do not presently advocate any change in the management of our NCFB cohort, longitudinal surveillance is clearly warranted.

Differential Expression of VEGF-Axxx Isoforms Is Critical for Development of Pulmonary Fibrosis.

RATIONALE: Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-A165a and VEGF-A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. OBJECTIVES: To establish VEGF-A isoform expression and functional effects in IPF. METHODS: We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTA+/-TetoCre+/-LoxP-VEGF-A+/+ to conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. MEASUREMENTS AND MAIN RESULTS: IPF and normal lung fibroblasts differentially expressed and responded to VEGF-A165a and VEGF-A165b in terms of proliferation and matrix expression. Increased VEGF-A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-A165b to wild-type mice. CONCLUSIONS: These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-Axxxa to VEGF-Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.

TGFß upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells.

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFß in response to tissue injury via an αvß6 integrin-mediated mechanism. TGFß is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFß is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2- and Sp1-dependent. We also show that TGFß-mediated PAR-1 upregulation is accompanied by increased expression of integrin αv and ß6 subunits. Finally, TGFß pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFß signalling responses in lung cancer.

The anti-fibrotic effect of inhibition of TGFß-ALK5 signalling in experimental pulmonary fibrosis in mice is attenuated in the presence of concurrent γ-herpesvirus infection.

TGFß-ALK5 pro-fibrotic signalling and herpesvirus infections have been implicated in the pathogenesis and exacerbation of pulmonary fibrosis. In this study we addressed the role of TGFß-ALK5 signalling during the progression of fibrosis in a two-hit mouse model of murine γ-herpesvirus 68 (MHV-68) infection on the background of pre-existing bleomycin-induced pulmonary fibrosis. Assessment of total lung collagen levels in combination with ex vivo micro-computed tomography (µCT) analysis of whole lungs demonstrated that MHV-68 infection did not enhance lung collagen deposition in this two-hit model but led to a persistent and exacerbated inflammatory response. Moreover, µCT reconstruction and analysis of the two-hit model revealed distinguishing features of diffuse ground-glass opacities and consolidation superimposed on pre-existing fibrosis that were reminiscent of those observed in acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). Virally-infected murine fibrotic lungs further displayed evidence of extensive inflammatory cell infiltration and increased levels of CCL2, TNFα, IL-1ß and IL-10. Blockade of TGFß-ALK5 signalling attenuated lung collagen accumulation in bleomycin-alone injured mice, but this anti-fibrotic effect was reduced in the presence of concomitant viral infection. In contrast, inhibition of TGFß-ALK5 signalling in virally-infected fibrotic lungs was associated with reduced inflammatory cell aggregates and increased levels of the antiviral cytokine IFNγ. These data reveal newly identified intricacies for the TGFß-ALK5 signalling axis in experimental lung fibrosis, with different outcomes in response to ALK5 inhibition depending on the presence of viral infection. These findings raise important considerations for the targeting of TGFß signalling responses in the context of pulmonary fibrosis.

Evidence for a functional thymic stromal lymphopoietin signaling axis in fibrotic lung disease.

Thymic stromal lymphopoietin (TSLP) recently has emerged as a key cytokine in the development of type 2 immune responses. Although traditionally associated with allergic inflammation, type 2 responses are also recognized to contribute to the pathogenesis of tissue fibrosis. However, the role of TSLP in the development of non-allergen-driven diseases, characterized by profibrotic type 2 immune phenotypes and excessive fibroblast activation, remains underexplored. Fibroblasts represent the key effector cells responsible for extracellular matrix production but additionally play important immunoregulatory roles, including choreographing immune cell recruitment through chemokine regulation. The aim of this study was to examine whether TSLP may be involved in the pathogenesis of a proto-typical fibrotic disease, idiopathic pulmonary fibrosis (IPF). We combined the immunohistochemical analysis of human IPF biopsy material with signaling studies by using cultured primary human lung fibroblasts and report for the first time, to our knowledge, that TSLP and its receptor (TSLPR) are highly upregulated in IPF. We further show that lung fibroblasts represent both a novel cellular source and target of TSLP and that TSLP induces fibroblast CCL2 release (via STAT3) and subsequent monocyte chemotaxis. These studies extend our understanding of TSLP as a master regulator of type 2 immune responses beyond that of allergic inflammatory conditions and suggest a novel role for TSLP in the context of chronic fibrotic lung disease.

Coagulation cascade proteinases in lung injury and fibrosis.

The primary function of the coagulation cascade is to promote hemostasis and limit blood loss in response to tissue injury. In addition, there is now considerable evidence that coagulation plays pivotal roles in orchestrating inflammatory and tissue repair responses via both the generation of fibrin and activation of the family of proteinase-activated receptors (PARs). Consequently, uncontrolled coagulation and PAR signaling responses have been shown to contribute to excessive inflammatory and fibroproliferative responses in the context of a broad range of conditions, including acute lung injury and fibrotic lung disease. In terms of the cellular origin of excessive coagulation activity in the context of lung injury, coagulation zymogens are principally thought to be derived from the circulation and locally activated via the extrinsic tissue factor-dependent coagulation pathway within the intraalveolar compartment. More recently, we have provided compelling evidence that several key coagulation zymogens are locally synthesized by the hyperplastic alveolar epithelium in pulmonary fibrosis. In terms of signaling receptors activated in response to the coagulation cascade, current evidence suggests a major role for PAR1 in influencing endothelial-epithelial barrier disruption, inflammatory cell recruitment, and collagen deposition in response to lung injury, whereas PAR2 signaling has been implicated mainly in mediating lung inflammatory responses. This article reviews current understanding of coagulation pathways in acute and fibrotic lung injury and expands on the scientific rationale for strategies that specifically target intraalveolar coagulation or PAR signaling responses.

Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes.

BACKGROUND: Large production volumes of zinc oxide nanoparticles (ZnONP) might be anticipated to pose risks, of accidental inhalation in occupational and even in consumer settings. Herein, we further investigated the pathological changes induced by ZnONP and their possible mechanism of action. METHODS: Two doses of ZnONP (50 and 150 cm2/rat) were intratracheally instilled into the lungs of rats with assessments made at 24 h, 1 wk, and 4 wks after instillation to evaluate dose- and time-course responses. Assessments included bronchoalveolar lavage (BAL) fluid analysis, histological analysis, transmission electron microscopy, and IgE and IgA measurement in the serum and BAL fluid. To evaluate the mechanism, alternative ZnONP, ZnONP-free bronchoalveolar lavage exudate, and dissolved Zn2+ (92.5 µg/rat) were also instilled to rats. Acridine orange staining was utilized in macrophages in culture to evaluate the lysosomal membrane destabilization by NP. RESULTS: ZnONP induced eosinophilia, proliferation of airway epithelial cells, goblet cell hyperplasia, and pulmonary fibrosis. Bronchocentric interstitial pulmonary fibrosis at the chronic phase was associated with increased myofibroblast accumulation and transforming growth factor-ß positivity. Serum IgE levels were up-regulated by ZnONP along with the eosinophilia whilst serum IgA levels were down-regulated by ZnONP. ZnONP are rapidly dissolved under acidic conditions (pH 4.5) whilst they remained intact around neutrality (pH 7.4). The instillation of dissolved Zn2+ into rat lungs showed similar pathologies (eg., eosinophilia, bronchocentric interstitial fibrosis) as were elicited by ZnONP. Lysosomal stability was decreased and cell death resulted following treatment of macrophages with ZnONP in vitro. CONCLUSIONS: We hypothesise that rapid, pH-dependent dissolution of ZnONP inside of phagosomes is the main cause of ZnONP-induced diverse progressive severe lung injuries.

Novel therapeutic approaches for pulmonary fibrosis.

Pulmonary fibrosis represents the end stage of a number of heterogeneous conditions and is, to a greater or lesser degree, the hallmark of the interstitial lung diseases. It is characterized by the excessive deposition of extracellular matrix proteins within the pulmonary interstitium leading to the obliteration of functional alveolar units and in many cases, respiratory failure. While a small number of interstitial lung diseases have known aetiologies, most are idiopathic in nature, and of these, idiopathic pulmonary fibrosis is the most common and carries with it an appalling prognosis - median survival from the time of diagnosis is less than 3 years. This reflects the lack of any effective therapy to modify the course of the disease, which in turn is indicative of our incomplete understanding of the pathogenesis of this condition. Current prevailing hypotheses focus on dysregulated epithelial-mesenchymal interactions promoting a cycle of continued epithelial cell injury and fibroblast activation leading to progressive fibrosis. However, it is likely that multiple abnormalities in a myriad of biological pathways affecting inflammation and wound repair - including matrix regulation, epithelial reconstitution, the coagulation cascade, neovascularization and antioxidant pathways - modulate this defective crosstalk and promote fibrogenesis. This review aims to offer a pathogenetic rationale behind current therapies, briefly outlining previous and ongoing clinical trials, but will focus on recent and exciting advancements in our understanding of the pathogenesis of idiopathic pulmonary fibrosis, which may ultimately lead to the development of novel and effective therapeutic interventions for this devastating condition.

Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis.

Many common diseases of the gas exchange surface of the lung have no specific treatment but cause serious morbidity and mortality. Idiopathic Pulmonary Fibrosis (IPF) is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma. Keratinocyte Growth Factor (KGF, also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. During repair, the lung not only uses resident cells after injury but also recruits circulating bone marrow-derived cells (BMDC). Several groups have used Mesenchymal Stromal Cells (MSCs) as therapeutic vectors, but little is known about the potential of Hematopoietic Stem cells (HSCs). Using an inducible lentiviral vector (Tet-On) expressing KGF, we were able to efficiently transduce both MSCs and HSCs, and demonstrated that KGF expression is induced in a regulated manner both in vitro and in vivo. We used the in vivo bleomycin-induced lung fibrosis model to assess the potential therapeutic effect of MSCs and HSCs. While both populations reduced the collagen accumulation associated with bleomycin-induced lung fibrosis, only transplantation of transduced HSCs greatly attenuated the histological damage. Using double immunohistochemistry, we show that the reduced lung damage likely occurs through endogenous type II pneumocyte proliferation induced by KGF. Taken together, our data indicates that bone marrow transplantation of lentivirus-transduced HSCs can attenuate lung damage, and shows for the first time the potential of using an inducible Tet-On system for cell based gene therapy in the lung.

Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury.

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-beta activation that was mediated by proteinase-activated receptor-1 (PAR1) and integrin alphavbeta5. PAR1, alphavbeta5, and alpha-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.

Absence of proteinase-activated receptor-1 signaling in mice confers protection from fMLP-induced goblet cell metaplasia.

The morphological features of chronic obstructive pulmonary disease in man include emphysema and chronic bronchitis associated with mucus hypersecretion. These alterations can be induced in mice by a single intratracheal instillation of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), a chemoattractant and degranulating agent for neutrophils. The mechanisms underlying excessive mucus production and, in particular, goblet cell hyperplasia/metaplasia in chronic obstructive pulmonary disease remain poorly understood. The proteinase-activated receptors (PARs) are widely recognized for their modulatory properties during inflammation. In this study, we examined whether PAR-1 contributes to inflammation and lung damage induced by fMLP by comparing the response of PAR-1-deficient (PAR-1(-/-)) mice with that of wild-type (WT) mice. Mice were killed at various time points after fMLP instillation (200 microg/50 microl). WT mice developed emphysema and goblet cell metaplasia. The onset of pulmonary lesions was preceded by an increase in thrombin immunoreactivity in bronchial airways and alveolar tissue. This was followed by a decrease in PAR-1 immunoreactivity, and by an increase in IL-13 immunostaining on the luminal surface of airway epithelial cells. In PAR-1(-/-) mice, fMLP administration induced similar responses in terms of inflammation and emphysema, but these mice were protected from the development of goblet cell metaplasia. The involvement of PAR-1 in airway epithelial cell transdifferentiation was confirmed by demonstrating that intratracheal instillation of the selective PAR-1 agonist (TFLLR) induced goblet cell metaplasia in the airways of WT mice only. These data suggest that emphysema and goblet cell metaplasia occur independently, and that PAR-1 signaling through IL-13 stimulation may play an important role in inducing goblet cell metaplasia.

Pulmonary epithelium is a prominent source of proteinase-activated receptor-1-inducible CCL2 in pulmonary fibrosis.

RATIONALE: Studies in patients and experimental animals provide compelling evidence of the involvement of the major thrombin receptor, proteinase-activated receptor-1 (PAR(1)), and the potent chemokine, chemokine (CC motif) ligand-2 (CCL2)/monocyte chemotactic protein-1, in the pathogenesis of idiopathic pulmonary fibrosis (IPF). PAR(1) 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 PAR(1)-inducible CCL2 in the fibrotic lung. METHODS: Using immunohistochemistry and dual immunofluorescence, we examined PAR(1) and CCL2 expression in the bleomycin model and human IPF lung. PAR(1) and CCL2 gene expression was also assessed in laser-captured alveolar septae from patients with IPF. The ability of PAR(1) to induce CCL2 production by lung epithelial cells was also examined in vitro. MEASUREMENTS AND MAIN RESULTS: We report for the first time that PAR(1) 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 PAR(1)-dependent mechanism. CONCLUSIONS: These data support the notion that PAR(1) activation on lung epithelial cells may represent an important mechanism leading to increased local CCL2 release in pulmonary fibrosis. Targeting PAR(1) 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.

Coagulation signalling following tissue injury: focus on the role of factor Xa.

The primary function of the coagulation cascade is to promote haemostasis and limit blood loss in response to tissue injury. However, it is now recognized that the physiological functions of the coagulation cascade extend beyond blood coagulation and that this cascade plays a pivotal role in influencing inflammatory and tissue repair responses via the activation of their signalling responses, the proteinase-activated receptors (PARs). Consequently, uncontrolled coagulation activity and PAR signalling contributes to the pathophysiology of several conditions, including thrombosis, arthritis, cancer, kidney disease, and acute and chronic lung injury. Much of the work thus far has focused on the role of thrombin-mediated signalling in the pathophysiology of these conditions. However, recent evidence suggests that coagulation proteinases upstream of thrombin, including factor Xa (FXa), may also signal via PARs and thus induce cellular effects independent of thrombin generation. These studies have highlighted a novel and important role for FXa signalling in influencing proinflammatory and pro-fibrotic effects following tissue injury. This article will provide an overview of FXa as a central proteinase of the coagulation cascade and will review more recent evidence that FXa signalling may contribute to inflammation and tissue remodelling. The novel opportunities that this may present for therapeutic intervention will also be highlighted.