Search

Purpose of review: Interstitial lung disease (ILD) in children (chILD) is an umbrella term for a heterogeneous group of rare respiratory disorders that are mostly chronic and associated with high morbidity and mortality. The pathogenesis of the various chILD is complex and implicates genetic contributors. The purpose of this review is to provide updated information on the molecular defects associated with the development of chILD.Recent findings: Currently, the main mutations are identified in the surfactant genes SFTPA1, SFTPA2, SFTPB, SFTPC, ABCA3, and NKX2-1. In addition, pulmonary alveolar proteinosis is associated with mutations in CSF2RA, CSF2RB, and MARS, and specific auto-inflammatory forms of chILD implicate STING and COPA disorders. The relationships between the genetic defects and the disease expression remain poorly understood, with no genotype-phenotype correlations identified so far. Although targeted therapies are emerging, the management strategies are still largely empirical, relying mostly on corticosteroids.Summary: Genetic factors play an important role in chILD, and the ongoing development of novel technologies will rapidly broaden the genetic landscape of chILD. Therefore, in the coming years, it is expected that newly identified molecular defects and markers will help predicting disease courses and tailoring individual therapies.

International audience; Introduction: Children interstitial lung disease (chILD) is a heterogeneous group of rare respiratory disorders characterized by inflammatory and fibrotic changes of the lung parenchyma. They include ILD related to exposure/environment insults, ILD related to systemic diseases processes, ILD related to primary lung parenchyma dysfunctions and ILD specific to infancy. Areas covered: This review provides an update on chILD pathophysiology and diagnosis approaches in immunocompetent children. It includes current information on genetic causes. Expert commentary: ChILD covers a large spectrum of entities with heterogeneous disease expression. Various classifications have been reported, but none of them seems completely satisfactory. Recently, progress in molecular genetics has allowed identifying some genetic contributors, with, so far, a lack of correlations between gene disorders and disease expression. Despite improvements in patient management, chILD prognosis is still burdened by significant morbidity and mortality. Ongoing international collaborations will allow gathering larger longitudinal cohorts of patients to improve disease knowledge and personalized care. The overall goal is to help the children with ILD to reach the adulthood transition in a better condition, and to structure genetic counseling for their family.

Background: Heterozygous mutations in the SFTPA1 and SFTPA2 genes, encoding the surfactant protein SP-A1 and SP-A2 have been associated with rare forms of familial IIP and lung adenocarcinoma. We previously described 11 new heterozygous SFTPA1 and SFTPA2 variations in 13 unrelated patients including one newborn. The study aims to analyze the pathogenicity of those variations. Methods: We first analyzed the intrafamilial segregation of the identified variations with IIP and lung cancer. The production and the secretion of the mutant proteins were subsequently studied in HEK293T cells transiently expressing the SP-A proteins carrying the variations. Finally, SP-A expression was assessed on lung tissues of the patients. Results: In the studied families (38 adults, 3 children), the variations segregated with either IIP and/or lung cancer (37%) with an incomplete penetrance of the disease phenotype. The mean age at onset of the IIP was 45 years (0-68). Nine of the variations were located in the highly conserved carbohydrate recognition domain of the protein. In vitro, the mutant proteins were produced but their secretion was absent (n=9) or altered (n=2). The lung expression of SP-A studied in 3 unrelated patients showed an increased expression of SP-A in the cytoplasm of hypertrophic type 2 alveolar cells. Discussion and conclusion: The 11 identified SFTPA1 and SFTPA2 variations are pathogenic. They are associated with IIP and with an increased risk of lung cancer. They were identified mainly in adults but also in children. The pathophysiological consequences of their abnormal secretion/expression remain to be elucidated.

Coagulation activation accompanied by reduced anticoagulant activity is a key characteristic of patients with idiopathic pulmonary fibrosis (IPF). Although the importance of coagulation activation in IPF is well studied, the potential relevance of endogenous anticoagulant activity in IPF progression remains elusive. We assess the importance of the endogenous anticoagulant protein C pathway on disease progression during bleomycin‐induced pulmonary fibrosis. Wild‐type mice and mice with high endogenous activated protein C APC levels (APC high) were subjected to bleomycin‐induced pulmonary fibrosis. Fibrosis was assesses by hydroxyproline and histochemical analysis. Macrophage recruitment was assessed immunohistochemically. In vitro, macrophage migration was analysed by transwell migration assays. Fourteen days after bleomycin instillation, APC high mice developed pulmonary fibrosis to a similar degree as wild‐type mice. Interestingly, Aschcroft scores as well as lung hydroxyproline levels were significantly lower in APC high mice than in wild‐type mice on day 28. The reduction in fibrosis in APC high mice was accompanied by reduced macrophage numbers in their lungs and subsequent in vitro experiments showed that APC inhibits thrombin‐dependent macrophage migration. Our data suggest that high endogenous APC levels inhibit the progression of bleomycin‐induced pulmonary fibrosis and that APC modifies pulmonary fibrosis by limiting thrombin‐dependent macrophage recruitment.

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2.

Idiopathic pulmonary fibrosis (IPF) is a destructive disease in part resulting from premature or mature cellular aging. Protease-activated receptor-1 (PAR-1) recently emerged as a critical component in the context of fibrotic lung diseases. Therefore, we aimed to study the role of macrophages in PAR-1-mediated idiopathic pulmonary fibrosis. The number of macrophages were significantly reduced in lungs of PAR-1 antagonist (P1pal-12) treated animals upon bleomycin instillation. In line with these data, PAR-1 stimulation increased monocyte / macrophage recruitment in response to epithelium injury in in vitro trans-well assays. Moreover, macrophages induced fibroblasts migration, differentiation and secretion of collagen, which were inhibited in the presence of TGF-beta receptor inhibitors. Interestingly, these profibrotic effects were partially inhibited by treatment with the PAR-1 inhibitor P1pal-12. Using shRNA mediated PAR-1 knock down in fibroblasts, we demonstrate that fibroblast PAR-1 contributes to TGF-beta activation and production. Finally, we show that the macrophage-dependent induction of PAR-1 driven TGF-beta activation was mediated by FXa. Our data identify novel mechanisms by which PAR-1 stimulation on different cell types can contribute to IPF and identify macrophages as key players in PAR-1 dependent development of this devastating disease. IPF may result from cellular senescence mediated by macrophages in the lung.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease, characterized by accumulation of extracellular matrix (ECM) proteins. Matrix metalloprotease 1 (MMP-1) is an endopeptidase capable of degrading ECM proteins like fibrillar collagens, although pleiotropic actions have been described. We investigated MMP-1 expression in a cohort of IPF patients and determined the effect of genetic ablation of MMP-1 in the development of lung fibrosis in mice. We confirmed that MMP-1 levels are significantly increased in IPF lungs as compared to control lungs at both the mRNA and protein level. Interestingly, MMP-1 protein levels negatively correlated with Forced Vital Capacity (Spearman r = -0.45, p = 0.009). Next, wildtype and MMP-1a null (MMP-1a-/-) mice were subjected to bleomycin-induced lung fibrosis. MMP-1a-/- mice were protected from bleomycin-induced lung fibrosis as assessed by lung hydroxyproline content at d14, compared to wildtype mice (196±16 μg vs 146±11 μg/lung). Inflammatory cell influx in the BALF was significantly lower in MMP-1a-/- mice at d14 compared to wildtype mice (1.18x10^6±2.7x10^5 vs 0.54x10^6±0.67x10^5 cells/mL), whereas there was no difference at d7. We conclude that MMP-1 plays a detrimental role in the pathogenesis of lung fibrosis in mice and human. These data support our efforts in targeting MMP-1 as a new option against lung fibrosis progression.

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor 2 (PAR2) is expressed by different cell types in the lungs and can mediate inflammatory responses. We sought to determine the role of PAR2 during pneumococcal pneumonia. Pneumococcal pneumonia or sepsis was induced in wild-type and PAR2 knock-out (Par2−/−) mice by infection with viable S. pneumoniae. Par2−/− mice demonstrated improved host defense, a largely preserved lung barrier integrity, and reduced mortality during pneumococcal pneumonia. PAR2 deficiency did not influence bacterial growth after intravenous infection. Inhibition of the endogenous PAR2 activating proteases tissue factor/factor VIIa or tryptase did not impact on bacterial burdens during pneumonia. In a PAR2 reporter cell line it was demonstrated that S. pneumoniae-derived proteases are able to cleave PAR2. These results show that S. pneumoniae is able to cleave and exploit PAR2 to disseminate systemically from the airways.

Findings from recently published placebo-controlled trials in idiopathic pulmonary fibrosis have established that pirfenidone and nintedanib prevent about 50% of the decline in forced vital capacity typically seen in this disease; future trials are therefore unlikely to use placebo as a control group for ethical reasons. Future clinical assessment will probably include add-on trials in which a new drug is combined with an intervention with established efficacy; this development is in turn likely to herald the use of combination regimens in clinical practice. Personalised medicine (the selection of monotherapies on the basis of individualised biomarker signal) is an intrinsically attractive alternative approach, but is unlikely to be useful in routine management of idiopathic pulmonary fibrosis in the medium-term future because of the complex nature of the disease's pathogenesis. In this Personal View, we review the pleiotropic nature of disease pathogenesis in idiopathic pulmonary disease, the use of combination regimens in other selected chronic lung diseases, and the conceptual basis for combination therapies in interstitial lung disorders other than idiopathic pulmonary fibrosis. On the basis of these considerations, and the emergence of data from add-on trials, we believe that the future of management for idiopathic pulmonary fibrosis lies in the development of combination regimens.

Background Protease activated receptor (PAR)-1 expression is increased in a variety of tumor cells. In preclinical models, tumor cell PAR-1 appeared to be involved in the regulation of lung tumor growth and metastasis; however the role of PAR-1 in the lung tumor microenvironment, which is emerging as a key compartment in driving cancer progression, remained to be explored. Methods In the present study, PAR-1 gene expression was determined in lung tissue from patients with non-small-cell lung cancer (NSCLC) using a combination of publicly available RNA microarray datasets and in house-made tissue microarrays including tumor biopsies of 94 patients with NSCLC (40 cases of adenocarcinoma, 42 cases of squamous cell carcinoma and 12 cases of other type of NSCLC at different stages). Results PAR-1 gene expression strongly correlated with tumor stromal markers (i.e. macrophage, endothelial cells and (myo) fibroblast markers) but not with epithelial cell markers. Immunohistochemical analysis confirmed the presence of PAR-1 in the tumor stroma and showed that PAR-1 expression was significantly upregulated in malignant tissue compared with normal lung tissue. The overexpression of PAR-1 in tumor stroma of NSCLC appeared to be independent from tumor type, tumor stage, histopathological differentiation status, disease progression and patient survival. Conclusion Overall, our data provide evidence that PAR-1 in NSCLC is mainly expressed on cells that constitute the pulmonary tumor microenvironment, including vascular endothelial cells, macrophages and stromal fibroblasts. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3081-3) contains supplementary material, which is available to authorized users.

Idiopathic pulmonary fibrosis (IPF) is the most frequent fibrotic diffuse parenchymal lung disease. Its prognosis is devastating: >50% of the patients die within 3 years after diagnosis. Options for the treatment of IPF are limited and lung transplantation is the only ‘curative' therapy. Currently, in the absence of validated indicators of disease progression/activity and diagnostic tools, the clinical management of IPF remains a major challenge. A better understanding of the pathogenesis of IPF is critical for the identification of new therapeutic targets as well as molecules that may serve as surrogate markers for clinically significant endpoints. The current paradigm on the mechanisms leading from a normal to a fibrotic lung postulates that chronic epithelial lesion leads to aberrant wound healing activation, which is characterized by deregulated fibroblast proliferation and activation together with an uncontrolled extracellular matrix synthesis. In this review, we shed light on the role of epithelial cell damage in the pathogenesis of fibrosis. Finally, we examine the markers of epithelial damage and their potential use as biomarkers and the future of this continuously expanding field.

Introduction: Human Airway Trypsin-like protease (HAT) belongs to the membrane-anchored serine proteases family and is expressed in the lung. While its pathophysiological functions remain unknown, HAT is involved in pulmonary remodeling processes such as asthma. In bronchial airways HAT stimulates fibroblast proliferation through Protease-Activated Receptor-2, which is instrumental in IPF. So far, the role of HAT in IPF has never been investigated. Methods: In controls or patients with IPF, HAT expression was analyzed in lung biopsies by immunohistochemistry, and by Western-blot (WB) in lung homogenates, Broncho-Alveolar Lavages (BAL) and primary pulmonary fibroblasts. In lung homogenates and BAL, HAT activity was measured, by the cleavage of a specific chromogenic substrate. In vitro, the effects of HAT on fibrotic markers expression, migration and proliferation were assessed in human primary pulmonary fibroblasts using WB, WST-1 and Boyden chamber assays. Results: HAT is increased in IPF lungs in epithelial cells, (myo)fibroblasts and macrophages. Accordingly, HAT expression and activity are enhanced in lung and BAL from IPF patients. In fibroblasts, HAT induces phosphorylation of kinases such as ERK, decreases fibronectine and collagen expression, strongly impairs migration. Moreover, HAT does not increase pulmonary fibroblasts proliferation. Conclusions: Unlike its deleterious role in chronic inflammatory disease, in IPF, HAT induces anti-fibrotic responses in vitro. Its deregulated expression in patients with IPF may constitute an insufficient mechanism of protection. Although further studies are warranted in HAT-deficient mice, our results suggest that an induction of HAT in IPF could be beneficial.

Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention.

Idiopathic pulmonary fibrosis is currently believed to be driven by alveolar epithelial cells, with abnormally activated alveolar epithelial cells accumulating in an attempt to repair injured alveolar epithelium (1). Thus, targeting the alveolar epithelium to prevent or inhibit the development of pulmonary fibrosis might be an interesting therapeutic option in this disease. Hepatocyte growth factor (HGF) is a growth factor for epithelial and endothelial cells, which is secreted by different cell types, especially fibroblasts and neutrophils. HGF has mitogenic, motogenic, and morphogenic properties and exerts an antiapoptotic action on epithelial and endothelial cells. HGF has also proangiogenic effect. In vitro, HGF inhibits epithelial-to-mesenchymal cell transition and promotes myofibroblast apoptosis. In vivo, HGF has antifibrotic properties demonstrated in experimental models of lung, kidney, heart, skin, and liver fibrosis. Hence, the modulation of HGF may be an attractive target for the treatment of lung fibrosis.

CCAAT/enhancer-binding protein δ (C/EBPδ) recently emerged as an essential player in the inflammatory response to bacterial infections. C/EBPδ levels increase rapidly after a proinflammatory stimulus, and increasing C/EBPδ levels seem to be indispensable for amplification of the inflammatory response. Here we aimed to elucidate the role of C/EBPδ in host defense in community-acquired pneumococcal pneumonia. We show that C/EBPδ −/− mice are relatively resistant to pneumococcal pneumonia, as indicated by delayed and reduced mortality, diminished outgrowth of pneumococci in lungs, and reduced dissemination of the infection. Moreover, expression of platelet-activating factor receptor (PAFR), which is known to potentiate bacterial translocation of Gram-positive bacteria, was significantly reduced during infection in C/EBPδ −/− mice compared with WT controls. Importantly, cell stimulation experiments revealed that C/EBPδ potentiates PAFR expression induced by lipoteichoic acid and pneumococci. Thus, C/EBPδ exaggerates bacterial dissemination during Streptococcus pneumoniae -induced pulmonary infection, suggesting an important role for PAFR-dependent bacterial translocation.

Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of a-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype. (Am J Pathol 2011, 178:924-934; DOI: 10.1016/j.ajpath.2010.10.007)

Chronic inflammatory disorders constitute a heterogeneous group of complex and multifactorial diseases, which are often associated with increased cardiovascular morbidity and mortality, independent of the established cardiovascular risk factors. In keeping with this observation, hypercoagulability is frequently observed in patients suffering from atherosclerosis, chronic obstructive pulmonary disease and rheumatoid arthritis although the physiological significance of activated coagulation remained elusive. However, the identification of protease activated receptors (PAR) seem to provide a link between coagulation activation and disease progression as their activation by coagulation factors triggers a broad range of signaling pathways relevant for chronic inflammatory disorders. In experimental animal models, anticoagulation and/or genetic ablation of PAR signaling affords protection against the perpetuation of atherosclerosis, chronic obstructive pulmonary disease and rheumatoid arthritis. It is thus tempting to speculate that targeting the coagulation-PAR axis might have clinical relevance in the setting of chronic inflammatory disorders. In the current review, we discuss the current knowledge on coagulation activation in inflammatory disorders, we discuss the relationship between atherosclerosis, chronic obstructive pulmonary disease and rheumatoid arthritis and we review the current knowledge on PAR signaling in these disorders.

Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2−/− mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2−/− mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease.

Introduction Activation of the coagulation cascade during liver fibrosis: a puzzling paradox Protease-activated receptors: the link between coagulation cascade activation and liver fibrosis Expression and distribution of human PAR-2 in normal and pathological liver tissue FXa signalling on PAR-2 expressing cells, and the relevance for liver fibrosis FXa triggers fibroproliferative and pro-inflammatory responses in mesenchymal cells via PAR-2 activation FXa triggers pro-inflammatory responses and modulates the survival of epithelial cells via PAR-2 activation FXa and PAR-2 signalling in inflammatory cells FX-induced PAR-2 activation modulates endothelial barrier permeability Targeting FXa in animal models of liver fibrosis Summary and conclusions Hepatic fibrosis is a common response to virtually all forms of chronic liver injury independent of the etiologic agent. Despite the relatively large population of patients suffering from hepatic fibrosis and cirrhosis, no efficient and well-tolerated drugs are available for the treatment of this disorder. The lack of efficient treatment options is at least partly because the underlying cellular mechanisms leading to hepatic fibrosis are only partly understood. It is thus of pivotal importance to better understand the cellular processes contributing to the progression of hepatic fibrosis. Interestingly in this perspective, a common feature of fibrotic disease of various organs is the activation of the coagulation cascade and hepatic fibrosis is also accompanied by a local hypercoagulable state. Activated blood coagulation factors directly target liver cells by activating protease-activated receptors (PAR) thereby inducing a plethora of cellular responses like (among others) proliferation, migration and extracellular matrix production. Coagulation factor driven PAR activation thus establishes a potential link between activation of the coagulation cascade and the progression of fibrosis. The current review focuses on blood coagulation factor Xa and summarizes the variety of cellular functions induced by factor Xa-driven PAR-2 activation and the subsequent consequences for tissue repair and hepatic fibrosis.

Inflammatory bowel disease (IBD) is characterized by activation of the coagulation cascade and it has long been suspected that coagulation is an essential component of this still largely idiopathic group of diseases. The realization that coagulation factors are not only passive mediators in the propagation of coagulation, but also actively engage different cell types by activating proteinase-activated receptors (PARs) has provided mechanistic insight into how coagulation cascade propagation might participate in the progression of IBD. The emergence of PAR(2) as a key player in the progression of IBD has focused attention on its agonist, coagulation factor Xa (FXa). Recent findings on FXa and PAR(2) link the coagulation cascade to the progression of IBD. Here, we propose that FXa-induced PAR(2) activation has an important role in orchestrating intestinal inflammatory and fibroproliferative responses, and that PAR(2) might provide a novel therapeutic target for the management of IBD.

Activated factor Xa (FXa) is traditionally known as an important player in the coagulation cascade responsible for thrombin generation. Long considered a passive bystander, it is now evident that FXa exerts direct effects on a wide variety of cell types via activation of its two main receptors, protease-activated receptor-1 (PAR-1) and PAR-2. Recent findings suggest that PAR-2 plays a crucial role in fibro-proliferative diseases such as fibrosis, tissue remodeling and cancer and point towards FXa as the important mediator coordinating the interface between coagulation and disease progression. Here, we provide an overview of the FXa signaling pathways that mediate its effects in pathophysiology and explore the potential therapeutic implications of targeting FXa; in terms of arresting disease progression, the modulation of FXa activity might be more important than the modulation of FVIIa or thrombin. Non-hemostatic functions of FXa in health and disease: old thoughts and new developments

Coagulation proteases have been suggested to play a role in the pathogenesis of tissue remodeling and fibrosis. We therefore assessed the proinflammatory and fibroproliferative effects of coagulation protease factor (F)Xa. We show that FXa elicits a signaling response in C2C12 and NIH3T3 fibroblasts. FXa-induced ERK1/2 phosphorylation was dependent on protease-activated receptor (PAR)-2 cleavage because desensitization with a PAR-2 agonist (trypsin) but not a PAR-1 agonist (thrombin) abolished Fxa-induced signal transduction and PAR-2 siRNA abolished FXa-induced ERK1/2 phosphorylation. The PAR-2-dependent cellular effects of FXa led to fibroblast proliferation, migration, and differentiation into myofibroblasts, as demonstrated by the expression of a-smooth muscle actin and desmin, followed by the secretion of the cytokines monocyte chemotactic protein-1 and interleukin-6 as well as the expression of the fibrogenic proteins transforming growth factor-beta and fibronectin. To assess the relevance of FXa-induced proliferation and cell migration, we examined the effect of FXa in a wound scratch assay. indeed, FXa facilitated wound healing in a PAR-2- and ERK1/2-dependent manner. Taken together, these results support the notion that, beyond its role in coagulation, FXa-dependent PAR-2 cleavage might play a role in the progression of tissue fibrosis and remodeling.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease that remains refractory to current therapies. Objectives: To characterize the expression and activity of the membrane-anchored serine protease matriptase in IPF in humans and unravel its potential role in human and experimental pulmonary fibrogenesis. Methods: Matriptase expression was assessed in tissue specimens from patients with IPF versus control subjects using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blotting, while matriptase activity was monitored by fluorogenic substrate cleavage. Matriptase-induced fibroproliferative responses and the receptor involved were characterized in human primary pulmonary fibroblasts by Western blot, viability, and migration assays. In the murine model of bleomycin-induced pulmonary fibrosis, the consequences of matriptase depletion, either by using the pharmacological inhibitor camostat mesilate (CM), or by genetic down-regulation using matriptase hypomorphic mice, were characterized by quantification of secreted collagen and immunostainings. Measurements and Main Results: Matriptase expression and activity were up-regulated in IPF and bleomycin-induced pulmonary fibrosis. In cultured human pulmonary fibroblasts, matriptase expression was significantly induced by transforming growth factor-beta. Furthermore, matriptase elicited signaling via protease-activated receptor-2 (PAR-2), and promoted fibroblast activation, proliferation, and migration. In the experimental bleomycin model, matriptase depletion, by the pharmacological inhibitor CM or by genetic down-regulation, diminished lung injury, collagen production, and transforming growth factor-b expression and signaling. Conclusions: These results implicate increased matriptase expression and activity in the pathogenesis of pulmonary fibrosis in human IPF and in an experimental mouse model. Overall, targeting matriptase, or treatment by CM, which is already in clinical use for other diseases, may represent potential therapies for IPF.

Protease-activated receptors (PARs) are G-protein-coupled receptors (GPCRs) that are activated by a unique proteolytic mechanism. Besides the important role of blood coagulation factors in preventing bleeding after vascular injury, these serine proteinases actively engage target cells thereby fulfilling critical functions in cell biology. Cellular responses triggered by coagulation factor-induced PAR activation suggest that PARs play an important role in proliferation, survival and/or malignant transformation of tumor cells. Indeed, PAR expression correlates with cancer malignancy and clinical studies show that anticoagulant treatment is beneficial in cancer patients. In this review, we provide an overview on the PAR family, their mode of activation and mechanisms by which PAR signaling is terminated. In addition, we discuss the relationship between blood coagulation and cancer biology focusing on the potential role of PAR-induced modulation of cell survival, apoptosis and tumor growth.

Accumulating evidence shows that protease-activated receptor-1 (PAR-1) plays an important role in the development of fibrosis, including lung fibrosis. However, whether PAR-1 also plays a role in the development of skin fibrosis remains elusive. The aim of this study was to determine the role of PAR-1 in the development of skin fibrosis. To explore possible mechanisms by which PAR-1 could play a role, human dermal fibroblasts and keratinocytes were stimulated with specific PAR-1 agonists or antagonists. To investigate the role of PAR-1 in skin fibrosis, we subjected wild-type and PAR-1-deficient mice to a model of bleomycin-induced skin fibrosis. PAR-1 activation leads to increased proliferation and extra cellular matrix (ECM) production, but not migration of human dermal fibroblasts (HDF) in vitro. Moreover, transforming growth factor (TGF)-beta production was increased in keratinocytes upon PAR-1 activation, but not in HDF. The loss of PAR-1 in vivo significantly attenuated bleomycin-induced skin fibrosis. The bleomycin-induced increase in dermal thickness and ECM production was reduced significantly in PAR-1-deficient mice compared with wild-type mice. Moreover, TGF-beta expression and the number of proliferating fibroblasts were reduced in PAR-1-deficient mice although the difference did not reach statistical significance. This study demonstrates that PAR-1 contributes to the development of skin fibrosis and we suggest that PAR-1 potentiates the fibrotic response mainly by inducing fibroblast proliferation and ECM production.

Background Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical signalling pathways in pathology and physiology. Bleomycin-induced lung fibrosis has been shown to be diminished in PAR-1-deficient mice. The purpose of this study is to investigate whether pharmacological PAR-1 inhibition is a potential therapeutic option to combat pulmonary fibrosis. Methods Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type mice with or without a specific PAR-1 antagonist (ie, P1pal-12, a pepducin that blocks the PAR-1/G-protein interaction). Fibrosis was assessed by hydroxyproline analysis, immunohistochemistry, quantitative PCR and western blot for fibrotic markers expression. Results We first show that P1pal-12 effectively inhibits PAR-1-induced profibrotic responses in fibroblasts. Next, we show that once daily treatment with 0.5, 2.5 or 10 mg/kg P1pal-12 reduced the severity and extent of fibrotic lesions in a dose-dependent manner. These findings correlated with significant decreases in fibronectin, collagen and α smooth muscle actin expression at the mRNA and protein level in treated mice. To further establish the potential clinical applicability of PAR-1 inhibition, we analysed fibrosis in mice treated with P1pal-12 1 or 7 days after bleomycin instillation. Interestingly, when administered 7 days after the induction of fibrosis, P1pal-12 was as effective in limiting the development of pulmonary fibrosis as when administration was started before bleomycin instillation. Conclusions Overall, targeting PAR-1 may be a promising treatment for pulmonary fibrosis.

CCAAT-enhancer-binding protein delta (C/EBPδ) is a transcription factor mainly known for its role in inflammation and apoptosis/proliferation. Considering that these are key processes in renal fibrosis, we hypothesized that C/EBPδ would potentiate renal fibrosis. In line with this hypothesis, C/EBPδ has recently been suggested to regulate the fibrotic response during glomerulonephritis. Here we determined the importance of C/EBPδ in the development of renal tubulointerstitial fibrosis by subjecting 8- to 12-week-old C/EBPδ-deficient mice and age- and sex-matched wild-type controls to the unilateral ureteral obstruction model. Mice were killed at 1, 3, or 7 days post surgery, and renal tissues were obtained for RNA, protein, and immunohistochemical analysis. We show that C/EBPδ deficiency resulted in a more profound fibrotic response as evident from enhanced tubular injury, collagen deposition in the interstitial area, and higher expression of transforming growth factor-β. Moreover, we show that the increase in renal fibrosis in C/EBPδ-deficient mice does not depend on an altered proliferation/apoptosis balance or on a differential inflammatory response in the obstructed kidney. In conclusion, our study provides direct evidence that C/EBPδ is a novel mediator of renal fibrosis. Modulating C/EBPδ expression could consequently be a potential antifibrotic strategy in patients with chronic kidney disease.

Fibrotic lung disorders, either idiopathic, or associated with a specific etiology or a specific condition such as scleroderma, are increasingly recognized. As a whole they constitute a group of diseases characterized by the progressive destruction of the lung which ultimately leads to chronic respiratory failure and death. Improving the prognosis of these disorders requires the identification of drugs capable of inhibiting partially or totally the progression of lung fibrosis, and perhaps to reversing established fibrosis. This has been the focus of huge efforts from academic groups and pharma companies, and more than 20 different molecules are being investigated in clinical trials in idiopathic pulmonary fibrosis (IPF) a well defined and relatively frequent fibrotic lung disease of unknown etiology. However, until now, only one drug has been approved for lung fibrosis treatment. This drug, pirfenidone, has been shown to slow the decline of lung function in IPF, but no drug has demonstrated on the survival of patients with lung fibrosis (1). The effort must be prolonged and intensified.

Background Asthma is a complex disease with heterogeneous features of airway inflammation and remodeling. The increase in airway smooth muscle (ASM) mass is an essential component of airway remodeling in patients with severe asthma, yet the pathobiological mechanisms and clinical outcomes associated with ASM enlargement remain elusive. Objective We sought to compare ASM area in control subjects and patients with mild-to-moderate or severe asthma and to identify specific clinical and pathobiological characteristics associated with ASM enlargement. Methods Bronchial biopsy specimens from 12 control subjects, 24 patients with mild-to-moderate asthma, and 105 patients with severe asthma were analyzed for ASM area, basement membrane thickness, vessels, eosinophils, neutrophils, T lymphocytes, mast cells, and protease-activated receptor 2 (PAR-2). In parallel, the levels of several ASM mitogenic factors, including the PAR-2 ligands, mast cell tryptase, trypsin, tissue factor, and kallikrein (KLK) 5 and KLK14, were assessed in bronchoalveolar lavage fluid. Data were correlated with asthma severity and control both at inclusion and after 12 to 18 months of optimal management and therapy. Results Analyses across ASM quartiles in patients with severe asthma demonstrated that patients with the highest ASM quartile (median value of ASM area, 26.3%) were younger (42.5 vs ≥50 years old in the other groups, P ≤ .04) and had lower asthma control after 1 year of optimal management ( P ≤ .006). ASM enlargement occurred independently of features of airway inflammation and remodeling, whereas it was associated with PAR-2 overexpression and higher alveolar tryptase ( P ≤ .02) and KLK14 ( P ≤ .03) levels. Conclusion Increase in ASM mass, possibly involving aberrant expression and activation of PAR-2–mediated pathways, characterizes younger patients with severe asthma with poor asthma control.

Cell migration is critically important in (patho) physiological processes. The metastatic potential of cancer cells partly depends on activation of the coagulation cascade. The aim of the present study was to determine whether coagulation factor X (FXa) can regulate the migration and invasion of cancer cells. Quite unexpectedly, we found that FXa markedly diminished the migration of different cancer cell lines of various origins (breast, lung and colon cancer cells). We showed that FXa mediated inhibition of cancer cell migration was specific, as it was inhibited by TAP (a specific FXa inhibitor) but not by Hirudin (a specific thrombin inhibitor). Moreover, the FXa effect was dose dependent, with a maximal inhibitory effect reached at 0.75 U/ml FXa (corresponding to 130.5 nM). Next, we determined that FXa acted via protease-activated receptor (PAR)-1-dependent signaling, and PAR-1 desensitization, as well as knocking-down PAR-1 expression, abolished the FXa effects. Finally, we showed that Gi alpha was not involved in FXa mediated inhibition of cell migration as its effects were not reverted by pertussis toxin. These results suggest that, beyond its role in blood coagulation, FXa plays a key role in cancer cell migration. They also shed light on an unexpected role of PAR-1, which appears to be a Janus-like receptor in cancer cell biology. (C) 2009 Elsevier Ltd. All rights reserved.

We describe here five F7 mutations found in four patients without bleeding history, despite constitutional coagulation Factor VII (FVII) deficiency. All five mutations are missense and affect the catalytic domain of FVII (A191T, A191V, T239P, R224Q and M298I). The A191V and T239P mutations are novel and were found in homozygous patients with no clinical bleeding tendency. The patient diagnosed with the A191V mutation had a phenotype corresponding to a moderate type 1 FVII deficiency (FVII:C 4%, FVII:Ag 5%). The T239P mutation was found in a patient with mild type 2 FVII deficiency (FVII:C 25%, FVII:Ag 95%). Novel mutations are both in close vicinity to the charge-stabilizing system of FVII. Modeling studies allow understanding in part the molecular basis for the loss of function.

Mucociliary clearance (MC) is a key defense mechanism in airways. Smokers and patients with chronic obstructive pulmonary disease (COPD) exhibit modifications in MC, which predisposes these populations to recurrent infections. It is known that ex-smokers with normal lung function may present MC reversing after smoking cessation, but there are no studies that evaluate COPD ex-smokers' MC. Aim: To evaluate and to compare the MC and exhaled carbon monoxide (eCO) in smokers and COPD ex-smokers. Methods: We evaluated 83 subjects, divided in four groups: severe COPD (n=22), moderate COPD (n=19), current smokers (n=20) and nonsmokers (n=22). Severe and moderate COPD patients were ex-smokers (FEV1% = 38[34-43] and 60[53-63], 48[11-100] and 50[40-75] pack/years, respectively). Current smokers presented normal lung function and 40[22-44] pack/years. Nonsmokers were matched for age and sex. Were evaluated eCO levels and MC by saccharin transit time (STT) test. Tests were conducted between 8 and 9 AM with air temperature and relative humidity controlled. Statistical analyses were performed using Kruskal-Wallis test followed by Dunn's test. Results: STT was higher in smokers compared to control group (p=0,006). There was no difference in STT between smokers and COPD groups, but in both groups of COPD STT values were similar to control group. Also, there was no difference in STT between severe and moderate COPD. eCO levels was higher in smokers compared to other three groups (p

We read with interest the paper by Calvaruso and colleagues discussing the role of coagulation in fibrosis in chronic liver disease.1 Fibrotic diseases, and especially liver fibrosis, are a major public health issue, and remain too often refractory to therapy. Novel treatment options are therefore eagerly awaited. Overall, we agree with the author’s conclusions that compelling evidence supports a close relationship between thrombin and hepatic fibrogenesis and that targeting the coagulation cascade might be an attractive therapeutic avenue for the management of liver fibrosis. This notion is underscored by the fact that anticoagulant therapy already showed promise in idiopathic pulmonary fibrosis where it improved overall survival.2 Although we would …

Fibrosis represents the end-stage of a broad range of disorders affecting organ function. These disorders are often associated with aberrant angiogenesis, but whether vascular abnormalities during fibrosis are characterized by excessive or diminished neo-vascularization remains questionable. Strikingly, activation of the coagulation cascade is frequently observed in association with the progression of fibroproliferative disorders. As we recently showed that coagulation factor (F)Xa induced fibrotic responses in fibroblasts, we hypothesized that FXa might indirectly induce angiogenesis by triggering fibroblasts to secrete proangiogenic factors. In the present study, we show that although FXa induces p42/44 MAP Kinase phosphorylation in endothelial cells, it has no direct effect on endothelial cell proliferation, protein synthesis and tube formation. In contrast, conditioned medium of fibroblasts stimulated with FXa enhanced endothelial cell proliferation, extra cellular matrix synthesis, wound healing and endothelial tube formation. FXa induced VEGF production by fibroblasts and a VEGF neutralizing antibody blocked the indirect effect of FXa on proliferation and realignment of endothelial cells identifying VEGF as a crucial player in angiogenesis during coagulation factor-induced fibrosis. Overall, our results establish a link between the coagulation cascade and angiogenesis during fibrosis. (C) 2009 Elsevier B.V. All rights reserved.