Protease‐activated receptor (PAR)‐2 is required for PAR‐1 signalling in pulmonary fibrosis

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.     

Psoralen attenuates bleomycin‐induced pulmonary fibrosis in mice through inhibiting myofibroblast activation and collagen deposition

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by excessive deposition of extracellular matrix (ECM) and chronic inflammation with limited therapeutic options. Psoralen, a major active component extracted from Psoralea corylifolia L. seed, has several biological effects. However, the role of psoralen in IPF is still unclear. Here, we hypothesized that psoralen played an essential role in IPF in the inhibition of fibroblast proliferation and inflammatory response. A murine model of IPF was established by injecting bleomycin (BLM) intratracheally, and psoralen was administered for 14 days from the 7^th to 21^st day after BLM injection. Our results demonstrated that psoralen treatment reduced body weight loss and improved the survival rate of mice with IPF. Histological and immunofluorescent examination showed that psoralen alleviated BLM‐induced lung parenchymal inflammatory and fibrotic alteration. Furthermore, psoralen inhibited proliferation and collagen synthesis of mouse fibroblasts and partially reversed BLM‐induced expression of α‐smooth muscle actin at both the tissue and cell level. Moreover, psoralen decreased the expression of transforming growth factor‐β1, interleukin‐1β, and tumor necrosis factor‐α in the lungs of BLM‐stimulated mice. Our results reveale for the first time that psoralen exerts therapeutic effects against IPF in a BLM‐induced murine model.     

In vivo gene transfer of an extracellular domain of platelet-derived growth factor beta receptor by the HVJ-liposome method ameliorates bleomycin-induced pulmonary fibrosis

A number of investigators have reported augmented expression of PDGF in lungs with idiopathic pulmonary fibrosis (IPF) or with other types of pulmonary fibrosis. To accomplish such a regulation of PDGF activity, we constructed an expression plasmid of the extracellular domain of PDGF receptor beta chain (XR), which lacks intracellular tyrosine kinase domain and transmembrane portions, and estimated the therapeutic effects of XR gene transfer through the trachea on bleomycin-induced lung fibrosis of C57BL/6 mice using the hemagglutinating virus of Japan(HVJ)-liposome method. The XR gene transfer ameliorated the increases in the wet weight and hydroxyproline content and the histopathologic changes of the lung induced by bleomycin. These findings suggest that PDGF plays a crucial role in the pathogenesis of pulmonary fibrosis, and that XR gene transfer using the HVJ-liposome method may limit the progression of pulmonary fibrosis.     

Deficiency of developmental endothelial locus-1 (Del-1) aggravates bleomycin-induced pulmonary fibrosis in mice

Pulmonary fibrosis is a lung disease wherein lung parenchyma is gradually and irreversibly replaced with collagen. The molecular pathogenesis of pulmonary fibrosis is not fully understood and the only effective treatment available is lung transplantation. To test if Del-1, an endogenous anti-inflammatory molecule, may be implicated in the development of pulmonary fibrosis, we induced pulmonary fibrosis in wild type (WT) and Del-1^−/− mice by intratracheal administration of bleomycin. Del-1 expression in the lung was decreased in the WT mice treated with bleomycin compared to control mice. In addition, bleomycin-induced pulmonary fibrosis increased collagen deposition and TGF-β production in the lung of Del-1^−/− mice. Finally, Del-1^−/− mice treated with bleomycin displayed higher weight loss and greater mortality than did WT mice identically treated. These findings suggest that Del-1 may negatively regulate development of pulmonary fibrosis. Further delineation of a role for Del-1 in the development of pulmonary fibrosis will broaden our understanding of the molecular pathogenesis of this disease and hopefully help develop potential therapeutics.     

Vascular dysfunction in aged mice contributes to persistent lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.     

Serpine 1 induces alveolar type II cell senescence through activating p53‐p21‐Rb pathway in fibrotic lung disease

Senescence of alveolar type 2 (ATII) cells, progenitors of the alveolar epithelium, is implicated in the pathogeneses of idiopathic pulmonary fibrosis (IPF), an aging‐related progressive fatal lung disorder with unknown etiology. The mechanism underlying ATII cell senescence in fibrotic lung diseases, however, remains poorly understood. In this study, we report that ATII cells in IPF lungs express higher levels of serpine 1, also known as plasminogen activator inhibitor 1 (PAI‐1), and cell senescence markers p21 and p16, compared to ATII cells in control lungs. Silencing PAI‐1 or inhibition of PAI‐1 activity in cultured rat ATII (L2) cells leads to decreases in p53 serine 18 phosphorylation (p53^S18P), p53 and p21 protein expressions; an increase in retinoblastoma protein phosphorylation (ppRb); and a reduction in the sensitivity to bleomycin‐ and doxorubicin‐induced senescence. Silencing p53, on the other hand, abrogates PAI‐1 protein‐stimulated p21 expression and cell senescence. In vivo studies, using ATII cell‐specific PAI‐1 conditional knockout mouse model generated recently in this laboratory, further support the role of PAI‐1 in the activation of p53‐p21‐Rb cell cycle repression pathway, ATII cell senescence, and lung fibrosis induced by bleomycin. This study reveals a novel function of PAI‐1 in regulation of cell cycle and suggests that elevation of PAI‐1 contributes importantly to ATII cell senescence in fibrotic lung diseases.     

Down-regulation of the inhibitor of growth family member 4 (ING4) in different forms of pulmonary fibrosis

Background

Recent evidence has underscored the role of hypoxia and angiogenesis in the pathogenesis of idiopathic fibrotic lung disease. Inhibitor of growth family member 4 (ING4) has recently attracted much attention as a tumor suppressor gene, due to its ability to inhibit cancer cell proliferation, migration and angiogenesis. The aim of our study was to investigate the role of ING4 in the pathogenesis of pulmonary fibrosis both in the bleomycin (BLM)-model and in two different types of human pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and cryptogenic organizing pneumonia (COP).

Methods

Experimental model of pulmonary fibrosis was induced by a single tail vein injection of bleomycin in 6- to 8-wk-old C57BL/6mice. Tissue microarrays coupled with qRT-PCR and immunohistochemistry were applied in whole lung samples and paraffin-embedded tissue sections of 30 patients with IPF, 20 with COP and 20 control subjects.

Results

A gradual decline of ING4 expression in both mRNA and protein levels was reported in the BLM-model. ING4 was also found down-regulated in IPF patients compared to COP and control subjects. Immunolocalization analyses revealed increased expression in areas of normal epithelium and in alveolar epithelium surrounding Masson bodies, in COP lung, whereas showed no expression within areas of active fibrosis within IPF and COP lung. In addition, ING4 expression levels were negatively correlated with pulmonary function parameters in IPF patients.

Conclusion

Our data suggest a potential role for ING4 in lung fibrogenesis. ING4 down-regulation may facilitate aberrant vascular remodelling and fibroblast proliferation and migration leading to progressive disease.     

Lysophosphatidic acid accelerates lung fibrosis by inducing differentiation of mesenchymal stem cells into myofibroblasts

Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type‐1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow‐derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA‐LPA1 signalling in this is yet unclear. Using a bleomycin‐induced mouse lung‐fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived‐mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin‐injured lung. The migrated BMSC contributed significantly to α‐smooth muscle actin (α‐SMA)‐positive myofibroblasts. By transplantation of GFP‐labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative‐RT‐PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA‐induced BMSC differentiation into myofibroblast and the secretion of ECM via LPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.     

Tetraspanin 1 as a mediator of fibrosis inhibits EMT process and Smad2/3 and beta‐catenin pathway in human pulmonary fibrosis

Tetraspanin 1(TSPAN1) as a clinically relevant gene target in cancer has been studied, but there is no direct in vivo or vitro evidence for pulmonary fibrosis (PF). Using reanalysing Gene Expression Omnibus data, here, we show for the first time that TSPAN1 was markedly down‐regulated in lung tissue of patient with idiopathic PF (IPF) and verified the reduced protein expression of TSPAN1 in lung tissue samples of patient with IPF and bleomycin‐induced PF mice. The expression of TSPAN1 was decreased and associated with transforming growth factor‐β1 (TGF‐β₁)‐induced molecular characteristics of epithelial‐to‐mesenchymal transition (EMT) in alveolar epithelial cells (AECs). Silencing TSPAN1 promoted cell migration, and the expression of alpha‐smooth muscle actin, vimentin and E‐cadherin in AECs with TGF‐β₁ treatment, while exogenous TSPAN1 has the converse effects. Moreover, silencing TSPAN1 promotes the phosphorylation of Smad2/3 and stabilizes beta‐catenin protein, however, overexpressed TSPAN1 impeded TGF‐β₁‐induced activation of Smad2/3 and beta‐catenin pathway in AECs. Together, our study implicates TSPAN1 as a key regulator in the process of EMT in AECs of IPF.     

Nintedanib reduces ventilation‐augmented bleomycin‐induced epithelial–mesenchymal transition and lung fibrosis through suppression of the Src pathway

Mechanical ventilation (MV) used in patients with acute respiratory distress syndrome (ARDS) can increase lung inflammation and pulmonary fibrogenesis. Src is crucial in mediating the transforming growth factor (TGF)‐β1‐induced epithelial–mesenchymal transition (EMT) during the fibroproliferative phase of ARDS. Nintedanib, a multitargeted tyrosine kinase inhibitor that directly blocks Src, has been approved for the treatment of idiopathic pulmonary fibrosis. The mechanisms regulating interactions among MV, EMT and Src remain unclear. In this study, we suggested hypothesized that nintedanib can suppress MV‐augmented bleomycin‐induced EMT and pulmonary fibrosis by inhibiting the Src pathway. Five days after administrating bleomycin to mimic acute lung injury (ALI), C57BL/6 mice, either wild‐type or Src‐deficient were exposed to low tidal volume (V_T) (6 ml/kg) or high V_T (30 ml/kg) MV with room air for 5 hrs. Oral nintedanib was administered once daily in doses of 30, 60 and 100 mg/kg for 5 days before MV. Non‐ventilated mice were used as control groups. Following bleomycin exposure in wild‐type mice, high V_T MV induced substantial increases in microvascular permeability, TGF‐β1, malondialdehyde, Masson's trichrome staining, collagen 1a1 gene expression, EMT (identified by colocalization of increased staining of α‐smooth muscle actin and decreased staining of E‐cadherin) and alveolar epithelial apoptosis (P < 0.05). Oral nintedanib, which simulated genetic downregulation of Src signalling using Src‐deficient mice, dampened the MV‐augmented profibrotic mediators, EMT profile, epithelial apoptotic cell death and pathologic fibrotic scores (P < 0.05). Our data indicate that nintedanib reduces high V_T MV‐augmented EMT and pulmonary fibrosis after bleomycin‐induced ALI, partly by inhibiting the Src pathway.     

Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis

Epithelial‐mesenchymal transition (EMT) plays an important role in idiopathic pulmonary fibrosis (IPF). Astragaloside IV (ASV), a natural saponin from astragalus membranaceus, has shown anti‐fibrotic property in bleomycin (BLM)‐induced pulmonary fibrosis. The current study was undertaken to determine whether EMT was involved in the beneficial of ASV against BLM‐induced pulmonary fibrosis and to elucidate its potential mechanism. As expected, in BLM‐induced IPF, ASV exerted protective effects on pulmonary fibrosis and ASV significantly reversed BLM‐induced EMT. Intriguing, transforming growth factor‐β1 (TGF‐β1) was found to be up‐regulated, whereas Forkhead box O3a (FOXO3a) was hyperphosphorylated and less expressed. However, ASV treatment inhibited increased TGF‐β1 and activated FOXO3a in lung tissues. TGF‐β1 was administered to alveolar epithelial cells A549 to induce EMT in vitro. Meanwhile, stimulation with TGF‐β1‐activated phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway and induced FOXO3a hyperphosphorylated and down‐regulated. It was found that overexpression of FOXO3a leading to the suppression of TGF‐β1‐induced EMT. Moreover, ASV treatment, similar with the TGF‐β1 or PI3K/Akt inhibitor, reverted these cellular changes and inhibited EMT in A549 cells. Collectively, the results suggested that ASV significantly inhibited TGF‐β1/PI3K/Akt‐induced FOXO3a hyperphosphorylation and down‐regulation to reverse EMT during the progression of fibrosis.     

Rac2 is involved in bleomycin-induced lung inflammation leading to pulmonary fibrosis

Background

Pulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available. Rac2, a ras-related guanosine triphosphatase expressed mainly in hematopoietic cells, is a crucial molecule regulating a diversity of mast cell, macrophage, and neutrophil functions. All these cell types have been implicated in the development of pulmonary fibrosis in a variety of animal models. For the studies described here we hypothesized that Rac2 deficiency protects mice from bleomycin-induced pulmonary fibrosis.

Methods

To determine the role of Rac2 in pulmonary fibrosis we used a bleomycin-induced mouse model. Anesthetized C57BL/6 wild type and rac2 ^-/- mice were instilled intratracheally with bleomycin sulphate (1.25 U/Kg) or saline as control. Bronchoalveolar lavage (BAL) samples were collected at days 3 and 7 of treatment and analyzed for matrix metalloproteinases (MMPs). On day 21 after bleomycin treatment, we measured airway resistance and elastance in tracheotomized animals. Lung sections were stained for histological analysis, while homogenates were analyzed for hydroxyproline and total collagen content.

Results

BLM-treated rac2 ^-/- mice had reduced MMP-9 levels in the BAL on day 3 and reduced neutrophilia and TNF and CCL3/MIP-1α levels in the BAL on day 7 compared to BLM-treated WT mice. We also showed that rac2 ^-/- mice had significantly lower mortality (30%) than WT mice (70%) at day 21 of bleomycin treatment. Lung function was diminished in bleomycin-treated WT mice, while it was unaffected in bleomycin-treated rac2 ^-/- mice. Histological analysis of inflammation and fibrosis as well as collagen and hydroxyproline content in the lungs did not show significant differences between BLM-treated rac2 ^-/- and WT and mice that survived to day 21.

Conclusion

Rac2 plays an important role in bleomycin-induced lung injury. It is an important signaling molecule leading to BLM-induced mortality and it also mediates the physiological changes seen in the airways after BLM-induced injury.     

CCR6, the Sole Receptor for the Chemokine CCL20, Promotes Spontaneous Intestinal Tumorigenesis

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APC^MIN/+ mice) to generate APC^MIN/+ mice with CCR6 knocked out (CCR6KO-APC^MIN/+ mice). CCR6KO-APC^MIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APC^MIN/+ also had normal sized spleens as compared to the enlarged spleens found in APC^MIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APC^MIN/+ as compared to APC^MIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.     

Targeting Cpt1a-Bcl-2 interaction modulates apoptosis resistance and fibrotic remodeling

The mitochondrial calcium uniporter (MCU) regulates metabolic reprogramming in lung macrophages and the progression of pulmonary fibrosis. Fibrosis progression is associated with apoptosis resistance in lung macrophages; however, the mechanism(s) by which apoptosis resistance occurs is poorly understood. Here, we found a marked increase in mitochondrial B-cell lymphoma-2 (Bcl-2) in lung macrophages from subjects with idiopathic pulmonary fibrosis (IPF). Similar findings were seen in bleomycin-injured wild-type (WT) mice, whereas Bcl-2 was markedly decreased in mice expressing a dominant-negative mitochondrial calcium uniporter (DN-MCU). Carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for fatty acid β-oxidation, directly interacted with Bcl-2 by binding to its BH3 domain, which anchored Bcl-2 in the mitochondria to attenuate apoptosis. This interaction was dependent on Cpt1a activity. Lung macrophages from IPF subjects had a direct correlation between CPT1A and Bcl-2, whereas the absence of binding induced apoptosis. The deletion of Bcl-2 in macrophages protected mice from developing pulmonary fibrosis. Moreover, mice had resolution when Bcl-2 was deleted or was inhibited with ABT-199 after fibrosis was established. These observations implicate an interplay between macrophage fatty acid β-oxidation, apoptosis resistance, and dysregulated fibrotic remodeling.Subject terms: Cell biology, Medical research     

Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2 deficiency persistently reduces bleomycin-induced pulmonary fibrosis or merely delays disease progression and whether pharmacological PAR-2 inhibition limits experimental pulmonary fibrosis. Bleomycin was instilled intranasally into wild-type or PAR-2–deficient mice in the presence/absence of a specific PAR-2 antagonist (P2pal-18S). Pulmonary fibrosis was consistently reduced in PAR-2–deficient mice throughout the fibrotic phase, as evident from reduced Ashcroft scores (29%) and hydroxyproline levels (26%) at d 28. Moreover, P2pal-18S inhibited PAR-2–induced profibrotic responses in both murine and primary human pulmonary fibroblasts (p < 0.05). Once daily treatment with P2pal-18S reduced the severity and extent of fibrotic lesions in lungs of bleomycin-treated wild-type mice but did not further reduce fibrosis in PAR-2–deficient mice. Importantly, P2pal-18S treatment starting even 7 d after the onset of fibrosis limits pulmonary fibrosis as effectively as when treatment was started together with bleomycin instillation. Overall, PAR-2 contributes to the progression of pulmonary fibrosis, and targeting PAR-2 may be a promising therapeutic strategy for treating pulmonary fibrosis.     

Asiaticoside might attenuate bleomycin‐induced pulmonary fibrosis by activating cAMP and Rap1 signalling pathway assisted by A2AR

Asiaticoside (AS) has been reported to have protective effect on pulmonary fibrosis (PF). In this study, we aimed to explore the potential mechanism of the therapeutic role of AS and its relationship with A2AR in PF. Adenosine 2A receptor gene knockout (A2AR^?/?) mice and wild‐type (WT) mice were used to establish bleomycin (BLM)‐induced PF models and were then treated with AS (50 mg/kg/d). Pulmonary inflammation and fibrosis were observed in the PF model with much higher severity in A2AR^?/?mice than that in WT mice and AS significantly alleviated lung inflammation and fibrosis; however, it was less effective in A2AR^?/? mice than in WT mice via histopathological analysis. Using RNA sequencing analysis, we found up‐regulated differentially expressed genes (DEGs) in BLM group were enriched in immune and inflammation‐associated pathways compared with control group. There were 242 common DEGs between down‐regulated in BLM vs control group and up‐regulated in BLM + AS vs BLM group, which were enriched in cAMP and Rap1 signalling pathways. Furthermore, the expression of five key factors of these two pathways including adenylate cyclase (ADCY1, ADCY5, ADCY8, cAMP and Rap1) were confirmed up‐regulated by AS with the presence of A2AR. Therefore, AS might attenuate BLM‐induced PF by activating cAMP and Rap1 signalling pathways which is assisted by A2AR, making it a promising therapeutic optional for PF.     

Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis

Background

Effective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFβ) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFβ superfamily of growth factors, modulate fibrogenesis driven by TGFβ. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis.

Methods

Lung fibrosis was induced in wild-type or noggin haploinsufficient (Nog^+/LacZ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis.

Results

Nog^+/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFβ and BMP signaling followed an inverse course, with dynamic activation of TGFβ signaling and repression of BMP signaling activity.

Conclusions

Upon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog^+/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFβ, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.