Airway remodeling in murine asthma correlates with a defect in PGE2 synthesis by lung fibroblasts

Asthma is a chronic lung disease characterized by local inflammation that can result in structural alterations termed airway remodeling. One component of airway remodeling involves fibroblast accumulation and activation, resulting in deposition of collagen I around small bronchi. Prostaglandin E(2) (PGE(2)) is the main eicosanoid lipid mediator produced by lung fibroblasts, and it exerts diverse anti-fibrotic actions. Dysregulation of the PGE(2) synthesis/response axis has been identified in human pulmonary fibrotic diseases and implicated in the pathogenesis of animal models of lung parenchymal fibrosis. Here we investigated the relationship between the fibroblast PGE(2) axis and airway fibrosis in an animal model of chronic allergic asthma. Airway fibrosis increased progressively as the number of airway challenges with antigen increased from 3 to 7 to 12. Compared with cells from control lungs, fibroblasts grown from the lungs of asthmatic animals, regardless of challenge number, exhibited no defect in the ability of PGE(2) or its analogs to inhibit cellular proliferation and collagen I expression. This correlated with intact expression of the EP(2) receptor, which is pivotal for PGE(2) responsiveness. However, cytokine-induced upregulation of PGE(2) biosynthesis as well as expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 declined with increasing numbers of antigen challenges. In addition, treatment with the COX-2-selective inhibitor nimesulide potentiated the degree of airway fibrosis following repeated allergen challenge. Because endogenous COX-2-derived PGE(2) acts as a brake on airway fibrosis, the inability of fibroblasts to upregulate PGE(2) generation in the inflammatory milieu presented by repeated allergen exposure could contribute to the airway remodeling and fibrosis observed in chronic asthma.     

Free radical generation induces epithelial-to-mesenchymal transition in lung epithelium via a TGF-β1-dependent mechanism

Fibrotic remodelling of lung parenchymal and airway compartments is the major contributor to life-threatening organ dysfunction in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD). Since transforming growth factor-β1 (TGF-β1) is believed to play a key role in disease pathogenesis and markers of oxidative stress are also commonly detected in bronchoalveolar lavage (BAL) from such patients we sought to investigate whether both factors might be interrelated. Here we investigated the hypothesis that oxidative stress to the lung epithelium promotes fibrotic repair by driving epithelial-to-mesenchymal transition (EMT) via the augmentation of TGF-β1. We show that in response to 400μM hydrogen peroxide (H(2)O(2)) A549 cells, used a model for alveolar epithelium, and human primary bronchial epithelial cells (PBECs) undergo EMT displaying morphology changes, decreased expression of epithelial markers (E-cadherin and ZO-1), increased expression of mesenchymal markers (vimentin and α-smooth muscle actin) as well as increased secretion of extracelluar matrix components. The same oxidative stress also promotes expression of TGF-β1. Inhibition of TGF-β1 signalling as well as treatment with antioxidants such as phenyl tert-butylnitrone (PBN) and superoxide dismutase 3 (SOD3) prevent the oxidative stress driven EMT-like changes described above. Interventions also inhibited EMT-like changes. This study identifies a link between oxidative stress, TGF-β1 and EMT in lung epithelium and highlights the potential for antioxidant therapies to limit EMT and its potential contribution to chronic lung disease.     

IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.     

Mechanotransduction determines the structure and function of lung and bone: a theoretical model for the pathophysiology of chronic disease

Multicellular organisms have evolved in adaptation to the Earth's gravitational and oxygen environment. This epigenetic process is dependent on the capacity of mesodermal cells to act as mechanosensors that can conform, deform, and reform in adaptation to the organism's physical environment. Mechanical forces, such as hydrostatic pressure and gravity, play important roles in the embryonic development, homeostasis, and repair of lung and bone. We discuss the role of parathyroid hormone-related protein (PTHrP) as a mechanotransducer for stretch in these organs during normal development, particularly as it lends itself to homeostasis; we further demonstrate that "uncoupling" of such mechanisms may play a central role in injury repair, particularly as it relates to chronic diseases of lung and bone. Endothermal PTHrP signaling through its G-protein coupled receptor promotes normal cell-cell signaling that maintains the homeostatic phenotypes of lung and bone. Molecular disruption of the PTHrP/PTHrP receptor pathway from endoderm to mesoderm, because of such factors as volutrauma, hyperoxia, inflammation, and microgravity, alters intracellular signaling, causing maladaptive cellular changes, resulting in myofibroblast proliferation and granulation. Examples of such pathologic changes specifically related to this cellular/molecular mechanism of maladaptation are chronic lung disease and osteoporosis. We suggest a new paradigm that may help in the future creation of diagnostic and therapeutic modalities for a wide range of developmental and chronic diseases ranging from bronchopulmonary dysplasia in newborns to idiopathic pulmonary fibrosis and osteoporosis as a result of aging or microgravity.     

Adenosine-dependent pulmonary fibrosis in adenosine deaminase-deficient mice

Pulmonary fibrosis is a common feature of numerous lung disorders, including interstitial lung diseases, asthma, and chronic obstructive pulmonary disease. Despite the prevalence of pulmonary fibrosis, the molecular mechanisms governing inflammatory and fibroproliferative aspects of the disorder are not clear. Adenosine is a purine-signaling nucleoside that is generated in excess during cellular stress and damage. This signaling molecule has been implicated in the regulation of features of chronic lung disease; however, the impact of adenosine on pulmonary fibrosis is not well understood. The goal of this study was to explore the impact of endogenous adenosine elevations on pulmonary fibrosis. To accomplish this, adenosine deaminase (ADA)-deficient mice were treated with various levels of ADA enzyme replacement therapy to regulate endogenous adenosine levels in the lung. Maintaining ADA-deficient mice on low dosages of ADA enzyme therapy led to chronic elevations in lung adenosine levels that were associated with pulmonary inflammation, expression of profibrotic molecules, collagen deposition, and extreme alteration in airway structure. These features could be blocked by preventing elevations in lung adenosine. Furthermore, lowering lung adenosine levels after the establishment of pulmonary fibrosis resulted in a resolution of fibrosis. These findings demonstrate that chronic adenosine elevations are associated with pulmonary fibrosis in ADA-deficient mice and suggest that the adenosine functions as a profibrotic signal in the lung.     

Genetically manipulated mouse models of lung disease: potential and pitfalls

Gene targeting in mice (transgenic and knockout) has provided investigators with an unparalleled armamentarium in recent decades to dissect the cellular and molecular basis of critical pathophysiological states. Fruitful information has been derived from studies using these genetically engineered mice with significant impact on our understanding, not only of specific biological processes spanning cell proliferation to cell death, but also of critical molecular events involved in the pathogenesis of human disease. This review will focus on the use of gene-targeted mice to study various models of lung disease including airways diseases such as asthma and chronic obstructive pulmonary disease, and parenchymal lung diseases including idiopathic pulmonary fibrosis, pulmonary hypertension, pneumonia, and acute lung injury. We will attempt to review the current technological approaches of generating gene-targeted mice and the enormous dataset derived from these studies, providing a template for lung investigators.     

High‐mobility group box‐1 induces vascular remodelling processes via c‐Jun activation

Extracellular high‐mobility group box‐1 (HMGB1) acts as a signalling molecule during inflammation, cell differentiation and angiogenesis. Increased abundance of HMGB1 is associated with several pathological disorders such as cancer, asthma and chronic obstructive pulmonary disease (COPD). In this study, we investigated the relevance of HMGB1 in the pathological remodelling present in patients with idiopathic pulmonary arterial hypertension (IPAH) and pulmonary hypertension (PH) associated with COPD. Remodelled vessels present in COPD with PH and IPAH lung samples were often surrounded by HMGB1‐positive cells. Increased HMGB1 serum levels were detected in both patient populations compared to control samples. The effects of physiological HMGB1 concentrations were then examined on cellular responses in vitro. HMGB1 enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC) and primary human arterial endothelial cells (PAEC). HMGB1 stimulated p38, extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) phosphorylation. Furthermore, activation of the downstream AP‐1 complex proteins c‐Fos and c‐Jun was observed. Silencing of c‐Jun ablated the HMGB1‐induced proliferation in PASMC. Thus, an inflammatory component such as HMGB1 can contribute to PASMC and PAEC proliferation and therefore potentially to vascular remodelling and PH pathogenesis.     

Mechanotransduction determines the structure and function of lung and bone

Multicellular organisms have evolved in adaptation to the Earth's gravitational and oxygen environment. This epigenetic process is dependent on the capacity of mesodermal cells to act as mechanosensors that can conform, deform, and reform in adaptation to the organism's physical environment. Mechanical forces, such as hydrostatic pressure and gravity, play important roles in the embryonic development, homeostasis, and repair of lung and bone. We discuss the role of parathyroid hormone-related protein (PTHrP) as a mechanotransducer for stretch in these organs during normal development, particularly as it lends itself to homeostasis; we further demonstrate that “uncoupling” of such mechanisms may play a central role in injury repair, particularly as it relates to chronic diseases of lung and bone. Endothermal PTHrP signaling through its G-protein coupled receptor promotes normal cell-cell signaling that maintains the homeostatic phenotypes of lung and bone. Molecular disruption of the PTHrP/PTHrP receptor pathway from endoderm to mesoderm, because of such factors as volutrauma, hyperoxia, inflammation, and microgravity, alters intracellular signaling, causing maladaptive cellular changes, resulting in myofibroblast proliferation and granulation. Examples of such pathologic changes specifically related to this cellular/molecular mechanism of maladaptation are chronic lung disease and osteoporosis. We suggest a new paradigm that may help in the future creation of diagnostic and therapeutic modalities for a wide range of developmental and chronic diseases ranging from bronchopulmonary dysplasia in newborns to idiopathic pulmonary fibrosis and osteoporosis as a result of aging or microgravity.     

Transforming growth factor-beta 1 induces angiogenesis in vitro via VEGF production in human airway smooth muscle cells

Increase in size and number of bronchial blood vessels as well as hyperaemia are factors that contribute to airway wall remodelling in patients with chronic airway diseases, such as asthma and chronic obstructive pulmonary diseases (COPD). Expression of transforming growth factor beta 1 (TGF-beta 1), a multifunctional cytokine as well as vascular endothelial growth factor (VEGF), a key angiogenic molecule, has been shown in the inflammed airways in patients with chronic airway diseases. TGF-beta 1 has been implicated in the regulation of extracellular matrix, leading to airway remodelling in patients with chronic airway diseases. However, the role of TGF-beta 1 in regulating VEGF expression in patients with chronic airway diseases, as well as the underlying mechanisms are not yet well established. We investigated whether TGF-beta 1 stimulates VEGF expression in vitro and hence could influence vascular remodelling. Cultured human airway smooth muscle cells (HASMC) were serum deprived for 60 h before incubation with 5ng/ml of TGF-beta 1 for different time points. Control cells received serum-free culture medium. TGF-beta 1 treatment resulted in time dependent HASMC cell proliferation with maximal values for DNA biosynthesis at 24 h and cell number at 48 h. Northern blot analysis of VEGF mRNA expression showed increased levels in cells treated with TGF-beta 1 for 4 to 8 h. TGF-beta 1 also induced a time-dependent release of VEGF proteins in the conditioned medium after 48 h of treatment. Furthermore, the ability of HASMC-released VEGF proteins to induce human umbilical vein endothelial cells proliferation was inhibited by VEGF receptor antagonist, confirming that TGF-beta 1 induced VEGF was biologically active. We conclude that TGF-beta 1 in addition to an extracellular matrix regulator also could play a key role in bronchial angiogenesis and vascular remodelling via VEGF pathway in asthma.     

HYDAMTIQ,a selective PARP‐1 inhibitor,improves bleomycin‐induced lung fibrosis by dampening the TGF‐β/SMAD signalling pathway

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP‐ribose) polymerases‐1 (PARP‐1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP‐1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP‐1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor‐β (TGF‐β) expression and TGF‐β/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF‐β/SMAD signalling pathway with alpha‐smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor‐α, interleukin‐1β, iNOS and COX‐2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF‐β levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above‐mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF‐β expression and the TGF‐β/SMAD transduction pathway.     

Epithelium-fibroblast interactions in response to airway inflammation

Dramatic changes to the architecture of the airway walls have been commonly described in the airways of patients with asthma, cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Much research has focused on how airway inflammation drives these structural changes, particularly in terms of the mechanisms/mediators that are involved, and a number of parallels are observed between the disease phenotypes. For example, the increased deposition of extracellular matrix (ECM) at focal sites in the airway wall is seen in asthma and all interstitial lung diseases that involve fibrosis. In addition, increased expression of a number of well characterized cytokines and growth factors, such as TGF-beta and epidermal growth factor (EGF) have been demonstrated in these diseases. However, the role of the lesser-known cytokines, including the leukaemia inhibitory factor (LIF) and other members of the IL-6 family of cytokines in the pathogenesis of airway remodelling and fibrosis is largely unknown. However, the use of genetic manipulation in vivo and more specific inhibitors/antibodies in vitro has now provided increasing evidence to support the hypothesis that a complex interaction exists between these cytokines, ECM and integrins in regulating the function of both epithelial cells and fibroblasts.     

Elevated CO(2) levels cause mitochondrial dysfunction and impair cell proliferation

Elevated CO(2) concentrations (hypercapnia) occur in patients with severe lung diseases. Here, we provide evidence that high CO(2) levels decrease O(2) consumption and ATP production and impair cell proliferation independently of acidosis and hypoxia in fibroblasts (N12) and alveolar epithelial cells (A549). Cells exposed to elevated CO(2) died in galactose medium as well as when glucose-6-phosphate isomerase was knocked down, suggesting mitochondrial dysfunction. High CO(2) levels led to increased levels of microRNA-183 (miR-183), which in turn decreased expression of IDH2 (isocitrate dehydrogenase 2). The high CO(2)-induced decrease in cell proliferation was rescued by α-ketoglutarate and overexpression of IDH2, whereas proliferation decreased in normocapnic cells transfected with siRNA for IDH2. Also, overexpression of miR-183 decreased IDH2 (mRNA and protein) as well as cell proliferation under normocapnic conditions, whereas inhibition of miR-183 rescued the normal proliferation phenotype in cells exposed to elevated levels of CO(2). Accordingly, we provide evidence that high CO(2) induces miR-183, which down-regulates IDH2, thus impairing mitochondrial function and cell proliferation. These results are of relevance to patients with hypercapnia such as those with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchopulmonary dysplasia, and muscular dystrophies.     

MicroRNAs in inflammatory lung disease - master regulators or target practice?

MicroRNAs (miRNAs) have emerged as a class of regulatory RNAs with immense significance in numerous biological processes. When aberrantly expressed miRNAs have been shown to play a role in the pathogenesis of several disease states. Extensive research has explored miRNA involvement in the development and fate of immune cells and in both the innate and adaptive immune responses whereby strong evidence links miRNA expression to signalling pathways and receptors with critical roles in the inflammatory response such as NF-κB and the toll-like receptors, respectively. Recent studies have revealed that unique miRNA expression profiles exist in inflammatory lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis and lung cancer. Evaluation of the global expression of miRNAs provides a unique opportunity to identify important target gene sets regulating susceptibility and response to infection and treatment, and control of inflammation in chronic airway disorders. Over 800 human miRNAs have been discovered to date, however the biological function of the majority remains to be uncovered. Understanding the role that miRNAs play in the modulation of gene expression leading to sustained chronic pulmonary inflammation is important for the development of new therapies which focus on prevention of disease progression rather than symptom relief. Here we discuss the current understanding of miRNA involvement in innate immunity, specifically in LPS/TLR4 signalling and in the progression of the chronic inflammatory lung diseases cystic fibrosis, COPD and asthma. miRNA in lung cancer and IPF are also reviewed.     

Whole lung proteome of an acute epithelial injury mouse model in comparison to spatially resolved proteomes

Epithelial injury is one of the major drivers of acute pulmonary diseases. Recurring injury followed by aberrant repair is considered as the primary cause of chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF). Preclinical in vivo models allow studying early disease-driving mechanisms like the recently established adeno-associated virus-diphtheria toxin receptor (AAV-DTR) mouse model of acute epithelial lung injury, which utilises AAV mediated expression of the human DTR. We performed quantitative proteomics of homogenised lung samples from this model and compared the results to spatially resolved proteomics data of epithelial cell regions from the same animals. In whole lung tissue proteins involved in cGAS-STING and interferon pathways, proliferation, DNA replication and the composition of the provisional extracellular matrix were upregulated upon injury. Besides epithelial cell markers SP-A, SP-C and Scgb1a1, proteins involved in cilium assembly, lipid metabolism and redox pathways were among downregulated proteins. Comparison of the bulk to spatially resolved proteomics data revealed a large overlap of protein changes and striking differences. Together our study underpins the broad usability of bulk proteomics and pinpoints to the benefit of sophisticated proteomic analyses of specific tissue regions or single cell types.     

In vivo potential effects of adenovirus type 5 E1A and E1B on lung carcinogenesis and lymphoproliferative inflammation

Triggering uncontrolled cellular proliferation, chronic inflammation, and/or disruption of p53 activity is critical for tumorigenesis initiated by latent viral oncogenes. The adenovirus type 5 (Ad5) early genes E1A and E1B can maintain lifelong latency in the lungs of patients with chronic pulmonary diseases. To determine the in vivo effects of the latent Ad5 E1A and E1B oncogenes, we have examined the influence of Ad5 E1A and E1B gene products on mouse lung carcinogenesis and inflammation by generation and characterization of lung-specific transgenic mouse models. Here, we show that either the Ad5 E1A 243-amino-acid (aa) protein or the E1B 58-kDa protein was dominantly expressed in the transgenic lung. Preferential expression of Ad5 E1A 243-aa protein alone was not sufficient to induce lung carcinogenesis but resulted in low-grade cellular proliferation and high-grade lymphoproliferative inflammation in the lung. The presence of Ad5 E1B dramatically enhanced the expression of the E1A 243-aa protein, in addition to impairing p53 and apoptosis response, resulting in uncontrolled cellular proliferation, lymphoproliferative inflammation, and metastatic carcinomas in the lung after a period of latency. Our studies may provide clues to understanding the potential in vivo biological effects of Ad5 E1A and E1B latent in the lung and a new scope for assessing in vivo functions of viral genes latent in the infection target tissue.