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Meixuan Liu,1,2 Mengtian Shan,1 Yunxuan Zhang,3 Zhongliang Guo1,2 1Department of Respiratory Medicine, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, 200123, People’s Republic of China; 2Department of Respiratory Medicine, Shanghai East Clinical Medical College, Nanjing Medical University, Shanghai, 200123, People’s Republic of China; 3Department of Pharmacy, Huadong Hospital, Fudan University, Shanghai, 200040, People’s Republic of ChinaCorrespondence: Zhongliang Guo Tel +86 18901681968Email drguozhl@163.comPurpose: Airway remodeling is an important feature of chronic asthma, and yet there are few effective therapeutic strategies. Progranulin (PGRN) has been shown to have lung protective functions, but the role of PGRN in asthmatic airway remodeling is unclear. We aim to explore the protective potential of PGRN on house dust mite (HDM)-induced airway remodeling and the underlying mechanisms.Methods: In this study, a murine model of chronic asthma was established by HDM sensitization and challenge. Recombinant PGRN was intranasally administrated to mice during the phase of HDM challenge. TGF-β 1-treated human airway epithelial BEAS-2B cells were utilized to explore the effect of PGRN on airway epithelia exposed to profibrotic conditions and molecular mechanisms.Results: We found that PGRN treatment attenuated HDM-induced airway remodeling, as evidenced by the suppression of collagen accumulation, mucus overproduction and airway smooth muscle synthesis in HDM-challenged asthmatic mice lungs. Meanwhile, PGRN also reversed the increased levels of autophagy markers and autophagosomes in airway epithelia under mimic asthmatic conditions, thereby controlling the fibrotic process in vivo and in vitro. Specifically, overexpressed HMGB1 and the subsequent RAGE/MAPKs signaling activation due to HDM exposure were abrogated in PGRN-treated asthmatic mice. Furthermore, knockdown of HMGB1 expression significantly restrained the fibrosis formation in TGF-β 1-induced airway epithelia accompanied by the downregulation of autophagic activity. However, enhancement of extracellular HMGB1 levels blunted the inhibition of autophagic flux by PGRN in airway epithelia, thereby resulting in the augmentation of collagen synthesis and fibrosis.Conclusion: Taken together, our data revealed that PGRN protected against asthmatic airway remodeling by negatively regulating autophagy via HMGB1 suppression, which might provide new insights into the therapeutic options for HDM-induced chronic asthma.Keywords: house dust mite-induced asthma, airway remodeling, progranulin, autophagy, HMGB1

Purpose Recent studies have demonstrated that macrophage migration inhibitory factor (MIF) is of importance in asthmatic inflammation. The role of MIF in modulating airway remodeling has not yet been thoroughly elucidated to date. In the present study, we hypothesized that MIF promoted airway remodeling by intensifying airway smooth muscle cell (ASMC) autophagy and explored the specific mechanisms. Methods MIF knockdown in the lung tissues of C57BL/6 mice was conducted by instilling intratracheally adeno-associated virus (AAV) vectors (MIF-mutant AAV9) into mouse lung tissues. Mice genetically deficient in the autophagy marker ATG5 (ATG5+/-) was used to detect the role of autophagy in ovalbumin (OVA)-asthmatic murine models. Moreover, to block the expression of MIF and CD74 in vitro models, inhibitors, antibodies and lentivirus transfection techniques were employed. Results First, MIF knockdown in the lung tissues of mice showed markedly reduced airway remodeling in OVA murine mice models. Secondly, ASMC autophagy was increased in the OVA-challenged models. Mice genetically deficient in the autophagy marker ATG5 (ATG5+/-) that were primed and challenged with OVA showed lower airway remodeling than genetically wild-type asthmatic mice. Thirdly, MIF can induce ASMC autophagy in vitro. Moreover, the cellular source of MIF which promoted ASMC autophagy was macrophages. Finally, MIF promoted ASMC autophagy in a CD74-dependent manner. Conclusions MIF can increase asthmatic airway remodeling by enhancing ASMC autophagy. Macrophage-derived MIF can promote ASMC autophagy by targeting CD74.

Asthma is a chronic disease of the airways that has long been viewed predominately as an inflammatory condition. Accordingly, current therapeutic interventions focus primarily on resolving inflammation. However, the mainstay of asthma therapy neither fully improves lung function nor prevents disease exacerbations, suggesting involvement of other factors. An emerging concept now holds that airway remodeling, another major pathological feature of asthma, is as important as inflammation in asthma pathogenesis. Structural changes associated with asthma include disrupted epithelial integrity, subepithelial fibrosis, goblet cell hyperplasia/metaplasia, smooth muscle hypertrophy/hyperplasia, and enhanced vascularity. These alterations are hypothesized to contribute to airway hyperresponsiveness, airway obstruction, airflow limitation, and progressive decline of lung function in asthmatic individuals. Consequently, targeting inflammation alone does not suffice to provide optimal clinical benefits. Here we review asthmatic airway remodeling, focusing on airway epithelium, which is critical to maintaining a healthy respiratory system, and is the primary defense against inhaled irritants. In asthma, airway epithelium is both a mediator and target of inflammation, manifesting remodeling and resulting obstruction among its downstream effects. We also highlight the potential benefits of therapeutically targeting airway structural alterations. Since pathological tissue remodeling is likewise observed in other injury- and inflammation-prone tissues and organs, our discussion may have implications beyond asthma and lung disease.

Lung development is mediated by assorted signaling proteins and orchestrated by complex mesenchymal-epithelial interactions. Notch signaling is an evolutionarily conserved cell-cell communication mechanism that exhibits a pivotal role in lung development. Notably, both aberrant expression and loss of regulation of Notch signaling are critically linked to the pathogenesis of various lung diseases, in particular, pulmonary fibrosis, lung cancer, pulmonary arterial hypertension, and asthmatic airway remodeling; implying that precise regulation of intensity and duration of Notch signaling is imperative for appropriate lung development. Moreover, evidence suggests that Notch signaling links embryonic lung development and asthmatic airway remodeling. Herein, we summarized all-recent advances associated with the mechanistic role of Notch signaling in lung development, consequences of aberrant expression or deletion of Notch signaling in linking early-impaired lung development and asthmatic airway remodeling, and all recently investigated potential therapeutic strategies to treat asthmatic airway remodeling.

There was no effective measures can be obtained at present to reverse or prevent airway remodeling. We investigated the therapeutic effect of Erythropoietin (EPO) gene modified mesenchymal stem cells (MSCs) on asthmatic airway remodeling and the possible underlied molecular mechanisms. EPO gene was transfected into MSCs via lentivirus vector. The transfected cells (EPO-MSCs) were identified by flow cytometry and the EPO secreting function was detected by PCR and Western blot. MSCs or EPO-MSCs were administrated to albumin (OVA)-induced chronic asthmatic mouse model via tail veins. The asthmatic phenotype was analyzed. Number of cells in bronchoalveolar lavage fluid (BALF) was counted using a hemocytometer. Histological findings of airways were evaluated by microscopic examination. The concentrations of interleukin 4(IL-4), interleukin 5(IL-5), and interleukin 13(IL-13) in lung homogenate were determined by ELISA. The activation state of transforming growth factor-β 1 (TGF-β1), Transforming growth factor beta-activated kinase 1 (TAK1), and p38 Mitogen Activated Protein Kinase (p38MAPK) signaling was detected by Real-Time PCR and Western blotting. EPO-MSCs were successfully constructed. EPO-MSCs showed a more potently suppressive effect on local asthmatic airway inflammation and the level of IL-4, IL-5, and IL-13 in lung tissue than MSCs. Moreover, the numbers of goblet cells, the thicknesses of smooth muscle layer, collagen density, percentage of proliferating cell nuclear antigen positive (PCNA+ ) mesenchymal cells, and von Willebrand factor positive(vWF+ ) vessels were also significantly inhibited by EPO-MSCs. Furthermore, EPO-MSCs could downregulate the expression of TGF-β1, TAK1, and p38MAPK in lung tissue both in mRNA level and in protein level. EPO gene modified MSCs may more efficiently attenuate asthmatic airway remodeling, which maybe related with the downregulation of TGF-β1-TAK1-p38MAPK pathway activity.

Background: The tumor necrosis factor superfamily member LIGHT (the official gene symbol approved by NCBI Gene Database), an inflammatory factor secreted by T cells after allergen exposure, recently discovered to play crucial roles in asthmatic airway remodeling. However, it is unclear whether LIGHT could be controlled by inhaled corticosteroids, a key component of asthma management. This study was to investigate the effects and potential mechanisms of inhaled budesonide on the expressions of LIGHT and its receptors (LTβR and HVEM) of lung tissues in ovalbumin-sensitized mice. Materials and Methods: Thirty-three BALB/c mice were randomly divided into the control, asthma model, and budesonide treatment groups (11 in each group). Mice were sensitized and challenged by OVA to develop mouse model of chronic asthma, and treated with aerosolized budesonide before OVA challenge. Bronchoalveolar lavage fluid (BALF) and lungs were obtained after the final OVA challenge. Protein and mRNA Levels of LIGHT, LTβR, and HVEM in the lungs were investigated by immunohistochemistry, image analysis, and real-time PCR. Expressions of IL-6 and IFN-γ in BALF were measured by ELISA. Results: Inhaled budesonide significantly reduced protein and mRNA levels of lung LIGHT, LTβR, and HVEM in asthmatic mice. Correspondingly, the number of eosinophils and neutrophils and IL-6 levels in BALF after budesonide treatment were found to be decreased, whereas the IFN-γ levels in BALF were increased. Moreover, the expressions of LIGHT and HVEM mRNA showed positive correlation with IL-6 levels in the treatment group. Conclusions: Inhaled budesonide can down-regulate the expressions of LIGHT, LTβR, and HVEM in the lungs of asthmatic mice, and LIGHT/LTβR/HVEM interactions may be a potentially key target for asthma treatment.

Embryonic lung development requires reciprocal endodermal-mesodermal interactions; mediated by various signaling proteins. Wnt/β-catenin is a signaling protein that exhibits the pivotal role in lung development, injury and repair while aberrant expression of Wnt/β-catenin signaling leads to asthmatic airway remodeling: characterized by hyperplasia and hypertrophy of airway smooth muscle cells, alveolar and vascular damage goblet cells metaplasia, and deposition of extracellular matrix; resulting in decreased lung compliance and increased airway resistance. The substantial evidence suggests that Wnt/β-catenin signaling links embryonic lung development and asthmatic airway remodeling. Here, we summarized the recent advances related to the mechanistic role of Wnt/β-catenin signaling in lung development, consequences of aberrant expression or deletion of Wnt/β-catenin signaling in expansion and progression of asthmatic airway remodeling, and linking early-impaired pulmonary development and airway remodeling later in life. Finally, we emphasized all possible recent potential therapeutic significance and future prospectives, that are adaptable for therapeutic intervention to treat asthmatic airway remodeling.

Shp2 played an important role in cigarette-smoke-mediated inflammation, surfactant homeostasis and asthmatic airway remodeling. However, whether shp2 plays a key role in epithelium-associated allergic reaction is still unknown. In this study, LPS and OVA were observed to induce the production of IL-25 in bronchial epithelial cells in vitro via the activation of MAPK p38 and JNK. Furthermore, blockage of Shp2 by its specific inhibitor PHPS1 or by siRNA-mediated depletion was found to reduce the production of IL-25 in epithelial cells as well as the up-regulated LPS-triggered activation of JNK but not p38. To confirm the role of intra-bronchial epithelial Shp2 in OVA-induced allergic reaction, we generated CC10-rtTA/(tetO)7-Cre/Shp2f/f mice, where Shp2 was conditionally knocked out in bronchial epithelial cells. Surprisingly, specific deletion of Shp2 in bronchial epithelial cells showed a mild but insignificant effect on the expressions of epithelium-derived cytokines as well as TH2 and TH17 polarization following allergen-induced murine airway inflammation. Collectively, our data suggested that deletion of Shp2 impaired IL-25 production in bronchial epithelial cells in vitro, but might yet have minor influence on OVA-induced allergic reaction in vivo.

Introduction: Pathological alteration in the airway structure, termed as airway remodeling, is a hallmark feature of individuals with asthma and has been described to negatively impact lung function in asthmatics. Recent studies have raised considerable interest in the regulatory role of beta-catenin in remodeling asthmatic airways. The WNT/beta-catenin signaling pathway is the key to normal lung development and tightly coordinates the maintenance of tissue homeostasis under steady-state conditions. Several studies indicate the crucial role of beta-catenin signaling in airway remodeling in asthma and suggest that this pathway may be activated by both the growth factors and mechanical stimuli such as bronchoconstriction. Areas covered: In this review, we discuss recent literature regarding the mechanisms of beta-catenin signaling activation and its mechanistic role in asthmatic airway remodeling. Further, we discuss the possibilities of therapeutic targeting of beta-catenin. Expert opinion: The aberrant activation of beta-catenin signaling by both WNT-dependent and -independent mechanisms in asthmatic airways plays a key role in remodeling the airways, including cell proliferation, differentiation, tissue repair and extracellular matrix production. These findings are interesting from both a mechanistic and therapeutic perspective, as several drug classes have now been described that target beta-catenin signaling directly.

Subepithelial fibrosis is one of the common pathological features of asthmatic airway remodeling. During subepithelial fibrosis, type I collagen becomes the most abundant extracellular protein component. Studies have shown that Notch signaling participates in the progression of fibrosis; however, whether Notch signaling is involved in regulating type I collagen expression in airway fibroblasts remains unclear. The aim of the present study was to examine whether Notch signaling can regulate type I collagen expression in airway fibroblasts and to explore the underlying molecular mechanisms. Here, the expression of Notch signaling components was examined in mouse L929 cells and human MRC-5 cells. After upregulating or downregulating Notch signaling in these cell lines, col1α1 and col1α2 expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the role of Hes1 binding sites in both the mouse and human COL1A1 and COL1A2 promoters was investigated. This study revealed that Notch signaling-related molecules (including Notch1, Hes1, and others) are expressed in L929 and MRC-5 cells and that Notch signaling regulates the expression of col1α1 and col1α2 in both cell lines. Additionally, over-expression of the Notch intracellular domain resulted in activation of the COL1A1 and COL1A2 promoters, and site-directed mutagenesis reporter assays revealed that Hes1 proteins might augment both mouse and human COL1A1 and COL1A2 promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the COL1A1 and COL1A2 promoters in both L929 and MRC-5 cells. Therefore, it is reasonable to assume that Notch signaling can directly upregulate COL1A1 and COL1A2 promoter activity through a Hes1-dependent mechanism, which could serve as a possible target for pharmacotherapy of airway subepithelial fibrosis.

Allergic asthma can cause airway structural remodeling, involving the accumulation of extracellular matrix and thickening of smooth muscle. Tumor necrosis factor (TNF) family ligand LIGHT (TNFSF14) is a cytokine that binds herpesvirus entry mediator (HVEM)/TNFRSF14 and lymphotoxinβreceptor (LTβR). LIGHT induces asthmatic cytokine IL-13 and fibrogenic cytokine transforming growth factor-βrelease from allergic asthma-related eosinophils expressing HVEM and alveolar macrophages expressing LTβR, respectively, thereby playing crucial roles in asthmatic airway remodeling. In this study, we investigated the effects of LIGHT on the coculture of human basophils/eosinophils and bronchial epithelial BEAS-2B cells. The expression of adhesion molecules, cytokines/chemokines, and matrix metalloproteinases (MMP) was measured by flow cytometry, multiplex, assay or ELISA. Results showed that LIGHT could significantly promote intercellular adhesion, cell surface expression of intercellular adhesion molecule-1, release of airway remodeling-related IL-6, CXCL8, and MMP-9 from BEAS-2B cells upon interaction with basophils/eosinophils, probably via the intercellular interaction, cell surface receptors HVEM and LTβR on BEAS-2B cells, and extracellular signal-regulated kinase, p38 mitogen activated protein kinase, and NF-κB signaling pathways. The above results, therefore, enhance our understanding of the immunopathological roles of LIGHT in allergic asthma and shed light on the potential therapeutic targets for airway remodeling.

Over-expression of WISP1 has been described in multi-organ fibrosis and tissue remodeling. Moreover, it has recently been found that polymorphism of WISP1 gene is related with the change of lung function in asthmatic subjects. Therefore, we hypothesized that WISP1 might be closely linked to occurrence and development of asthmatic airway remodeling. Aim of this study was to examine the roles of WISP1 in an asthmatic model with airway remodeling and assess the specific effects of WISP1 on human lung fibroblast in vitro. Animal models were developed by challenged with ovalbumin. The levels of WISP1 expression in animal models were assessed by real-time PCR and immunohistochemistry. To examine the specific effects of WISP1 on airway remodeling, WISP1 was depleted by neutralizing α-WISP1 antibodies in vivo. Moreover, human lung fibroblast (HFL-1) was challenged with WISP1 in the presence and absence of SH-5 in vitro. Our study showed that OVA exposure increased the levels of WISP1 expression in a rat asthma model. WISP1 depletion could partially inhibit OVA-induced airway remodeling. In vitro, WISP1-treated HFL-1 cells presented abnormal proliferation and over-expression of Col1a1 and Fn1. However, WISP1-induced collagen release from HFL-1 cells could be attenuated by pretreatment with an Akt inhibitor. Moreover, the levels of p-Akt and p-GSK-3β in WISP1-treated HFL-1 cells were also significantly elevated. In summary, WISP1 might initiate and perpetuate the pathological remodeling of asthma by inducing fibroblast proliferation and ECM deposition. The specific effects of WISP1 were likely due to activation of pulmonary Akt/GSK-3β signaling.

In asthma, inflammation results in bronchial constriction, mucus hypersecretion, and airway remodeling. This pathology is thought to stem from a collusion of genetic predisposition and environmental factors, such as viruses or allergens, but specific airway molecules that choreograph the interaction between genes and environment remain unidentified. Genetic polymorphisms in ADAM33, a membrane-anchored protease expressed in multiple airway cell types, have been associated in population studies with asthma susceptibility and bronchial hyperresponsiveness. Davies et al. explored the pathobiology of this protein and have discovered a link between ADAM33 and allergic inflammation. The authors first found that the expression of enzymatically active ADAM33 was increased in bronchoalveolar lavage fluid from patients with asthma and in mice sensitized and challenged with house dust mite extract, a model of allergic airway inflammation. Interestingly, inducible overexpression of soluble ADAM33 in the airway of both embryonic and adult mice increased lung expression of genes associated with asthmatic airway remodeling. Human fetal lung explants treated with recombinant ADAM33 had similar gene expression changes but not when treated with a recombinant mutant lacking protease activity. Interestingly, ADAM33 overexpression alone did not increase inflammatory genes or cause bronchial hyperresponsiveness. However, the authors found an interaction between ADAM33 and the allergic stimulus: induction of soluble ADAM33 overexpression in the house dust mite model increased bronchial hyperresponsiveness, bronchoalveolar lavage eosinophilia, and lung expression of genes associated with T helper 2 (TH2) inflammation compared with the house dust mite model alone. Furthermore, ADAM33–/– mice in the house dust mite model were protected from airway remodeling and also had decreased bronchial hyperresponsivness, bronchoalveolar lavage eosinophil count, and lung expression of TH2 genes. Of note, airway remodeling could be reversed with cessation of transgene induction, which suggests that inhibition of ADAM33 could reverse airway pathology related to allergic inflammation. Taken together, these studies establish that ADAM33 mediates the airway inflammatory and remodeling effects of allergen and is a potential therapeutic target for asthma. Future studies aiming for clinical translation should devise strategies for inhibition of ADAM33 in vivo and test its role in other models of asthmatic airway inflammation. E. R. Davies et al. , Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life. JCI Insight 1 , e87632 (2016). [[Full Text]][1] [1]: https://insight.jci.org/articles/view/87632

Background Allergic asthma is characterized by inflammation and airway remodeling. Airway remodeling with excessive deposition of extracellular matrix (ECM) and larger smooth muscle mass are correlated with increased airway responsiveness and asthma severity. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in airway smooth muscle remodeling remains unknown. Objective To investigate the role of calpain in asthmatic airway remodeling as well as the underling mechanism. Methods The mouse asthma model was made by ovalbumin sensitization and challenge. Calpain conditional knockout mice were studied in the model. Airway smooth muscle cells (ASMCs) were isolated from smooth muscle bundles in airway of rats. Cytokines IL-4, IL-5, TNF-α and TGF-β1, and serum from asthmatic patients were selected to treated ASMCs. Collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs were analyzed. Results Inhibition of calpain using calpain knockout mice attenuated airway smooth muscle remodeling in mouse asthma models. Cytokines IL-4, IL-5, TNF-α and TGF-β1, and serum from asthmatic patients increased collagen-I synthesis, cell proliferation, and phosphorylation of Akt in ASMCs, which were blocked by the calpain inhibitor MDL28170. Moreover, MDL28170 reduced cytokine-induced increases in Rictor protein which is the most important component of mammalian target of rapamycin complex 2 (mTORC2). Blockage of the mTORC2 signal pathway prevented cytokine-induced phosphorylation of Akt, collagen-I synthesis and cell proliferation of ASMCs, and attenuated airway smooth muscle remodeling in mouse asthma models. Conclusions Our results indicate that calpain mediates cytokine-induced collagen-I synthesis and proliferation of ASMCs via the mTORC2/Akt signaling pathway, thereby regulating airway smooth muscle remodeling in asthma. This article is protected by copyright. All rights reserved.

Severe asthma is associated with airway remodeling, characterized by structural changes including increased smooth muscle mass and matrix deposition in the airway, leading to deteriorating lung function. TGF-β is a pleiotropic cytokine leading to increased synthesis of matrix molecules by human airway smooth muscle (HASM) cells and is implicated in asthmatic airway remodeling. TGF-β is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-β is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-β activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-β activation by HASM cells via the integrin αvβ5. Our findings highlight the importance of the β5 cytoplasmic domain because a polymorphism in the β5 subunit rendered the integrin unable to activate TGF-β. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-β. These data demonstrate that murine airway smooth muscle cells express αvβ5 integrins and activate TGF-β. Finally, these data show that inhibition, or genetic loss, of αvβ5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-β activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-β activation.

Background: Asthmatic airway remodeling is an abnormal injury/repair process of the small airways caused by chronic inflammation in which the quantities of multiple cells increase dramatically. However, the origin of these proliferative cells is still undetermined. Objective: The aim of this study was to examine whether bone marrow (BM)-derived adult stem cells are responsible for the proliferative cells in asthmatic airway remodeling. Methods: Adult mice were durably engrafted with BM isolated from green fluorescent protein (GFP) transgenic mice. Using GFP BM chimera mice, an ovalbumin (OVA)-induced chronic asthma mouse model was established. The distribution of BM-derived GFP+ cells in the lungs of chronic asthma mice was detected by fluorescence microscopy. The phenotype of BM-derived GFP+ cells in the lung tissues of chronic asthma mice was analyzed by flow cytometry. Results: BM chimera mice were successfully generated, with no detectable radioactive inflammation observed. Using BM chimera mice, we established a mouse model of chronic asthma characterized by a significant increase in the thickness of the airway subepithelial basement membrane and smooth muscle layers. OVA treatment caused many GFP+ cells to appear at sites of small airway inflammation. The extravascular localization of some GFP+ cells and their morphology were not consistent with leukocytes. Flow cytometric analysis of lung cells revealed a significant increase in type I collagen (Col I)+GFP+ cells and α-smooth muscle actin (α-SMA)+GFP+ cells in OVA-treated GFP BM chimera mice. Conclusions: Considerable numbers of Col I- and α-SMA-producing cells originated from BM in the lung tissues of mice with OVA-induced chronic asthma.

Asthmatic airway remodeling is the pathophysiological modifications of the normal airway wall structure which include changes in the composition and organization of its cellular and molecular constituents. These modifications are the major cause of the symptoms associated with decreased pulmonary function. Airway remodeling is partially reversible in mild asthma but mostly irreversible in chronic severe asthma. It is initiated as a repair process in response to airway wall injuries caused by inflammation; however, dysregulation of this process leads to airway remodeling. In this review, we will summarize the most recent findings about the different structural changes in airways of asthmatics as well as mediators involved in this process.

Background Eosinophilic inflammation is closely related to angiogenesis in asthmatic airway remodeling. In ovalbumin (OVA)–sensitized mice bone marrow–derived, proangiogenic endothelial progenitor cells (EPCs) are rapidly recruited into the lungs after OVA aerosol challenge and promptly followed by mobilization and recruitment of eosinophils. Objective We hypothesized that bone marrow–derived EPCs initiate the recruitment of eosinophils through expression of the eosinophil chemoattractant eotaxin-1. Methods EPCs were isolated from an OVA murine model of allergic airway inflammation and from asthmatic patients. Endothelial and smooth muscle cells were isolated from mice. Eotaxin-1 expression was analyzed by means of immunofluorescence, real-time PCR, or ELISA. In vivo recruitment of eosinophils by EPCs was analyzed in mice. Results Circulating EPCs of asthmatic patients had higher levels of eotaxin-1 compared with those seen in control subjects. In the murine model OVA allergen exposure augmented eotaxin-1 mRNA and protein levels in EPCs. The EPCs from OVA-sensitized and OVA-challenged mice released high levels of eotaxin-1 on contact with lung endothelial cells from sensitized and challenged mice but not from control animals and not on contact with cardiac or hepatic endothelial cells from sensitized and challenged mice. Intranasal administration of the eotaxin-rich media overlying cultures of EPCs caused recruitment into the lungs, confirming functional chemoattractant activity. Conclusions Bone marrow–derived EPCs are early responders to environmental allergen exposures and initiate a parallel switch to a proangiogenic and proeosinophilic environment in the lungs of asthmatic patients.

Asthmatic airway remodeling is characterized by goblet cell hyperplasia, angiogenesis, smooth muscle hypertrophy, and subepithelial fibrosis. This study evaluated whether acquired changes in fibroblast phenotype could contribute to this remodeling. Airway and parenchymal fibroblasts from control or chronically ovalbumin (OVA)-sensitized and challenged "asthmatic" mice were assessed for several functions related to repair and remodeling +/- exogenous transforming growth factor (TGF)-beta. All OVA-challenged mouse fibroblasts demonstrated augmented gel contraction (P < 0.05) and chemotaxis (P < 0.05); increased TGF-beta(1) (P < 0.05), fibronectin (P < 0.05), and vascular endothelial growth factor (P < 0.05) release; and expressed more alpha-smooth muscle actin (P < 0.05). TGF-beta(1) stimulated both control and asthmatic fibroblasts, which retained all differences from control fibroblasts for all features(P < 0.05, all comparisons). Parenchymal fibroblasts proliferated more rapidly (P < 0.05), while airway fibroblasts proliferated similarly compared with control fibroblasts (P = 0.25). Thus, in this animal model, OVA-challenged mouse fibroblasts acquire a distinct phenotype that differs from control fibroblasts. The augmented profibrotic activity and mediator release of asthmatic fibroblasts could contribute to airway remodeling in asthma.

Chronic asthma is associated with airway remodeling and decline in lung function. In this article, we show that follistatin-like 1 (Fstl1), a mediator not previously associated with asthma, is highly expressed by macrophages in the lungs of humans with severe asthma. Chronic allergen-challenged Lys-Cretg /Fstl1Δ/Δ mice in whom Fstl1 is inactivated in macrophages/myeloid cells had significantly reduced airway remodeling and reduced levels of oncostatin M (OSM), a cytokine previously not known to be regulated by Fstl1. The importance of the Fstl1 induction of OSM to airway remodeling was demonstrated in murine studies in which administration of Fstl1 induced airway remodeling and increased OSM, whereas administration of an anti-OSM Ab blocked the effect of Fstl1 on inducing airway remodeling, eosinophilic airway inflammation, and airway hyperresponsiveness, all cardinal features of asthma. Overall, these studies demonstrate that the Fstl1/OSM pathway may be a novel pathway to inhibit airway remodeling in severe human asthma.

Previous studies have demonstrated that interleukin-20 (IL-20) is a pro-inflammatory cytokine, and it has been implicated in psoriasis, lupus nephritis, rheumatoid arthritis, atherosclerosis, and ulcerative colitis. Little is known about the effects of IL-20 in airway remodeling in asthma. The aim of our study was to demonstrate the function of IL-20 in airway remodeling in asthma. To identify the expression of IL-20 and its receptor, IL-20R1/IL-20R2, in the airway epithelium in bronchial tissues, bronchial biopsy specimens were collected from patients and mice with asthma and healthy subjects and stained with specific antibodies. To characterize the effects of IL-20 in asthmatic airway remodeling, we silenced and stimulated IL-20 in cell lines isolated from mice by shRNA and recombinant protein approaches, respectively, and detected the expression of α-SMA and FN-1 by Western blot analysis. First, overexpression of IL-20 and its receptor, IL-20R1/IL-20R2, was detected in the airway epithelium collected from patients and mice with asthma. Second, IL-20 increased the expression of fibronectin-1 and α-SMA, and silencing of IL-20 in mouse lung epithelial (MLE)-12 cells decreased the expression of fibronectin-1 and α-SMA. IL-20 may be a critical cytokine in airway remodeling in asthma. This study indicates that targeting IL-20 and/or its receptors may be a new therapeutic strategy for asthma.

To investigate the function of interleukin-33 (IL-33) in the asthmatic airway remodeling and the relationship between IL-33 and asthma severity.IL-33 levels, sputum eosinophils percentage (EOS%), pulmonary function and total immunoglobulin (IgE) were measured for 45 patients with asthma and 40 non-allergic controls. Asthma severity was assessed. The expressions of IL-33 and reticular basement membrane (RBM) on bronchial biopsy specimens from eight asthma patients and eight non-allergic controls were observed after hematoxylin-eosin staining (HE) and immunohistochemical staining. In vitro experiments, real-time polymerase chain reactions and western blotting analysis were used to identify the specific effects of IL-33 administration.Serum IL-33 levels in patients with asthma were higher than those in non-allergic controls. Moreover, in asthmatic patients, serum IL-33 levels were negatively correlated to forced expiratory volume in one second (FEV1, % predicted), and positively correlated to asthma severity. Increased expression of IL-33 and RBM thickening were observed on bronchial biopsy specimens obtained from patients with asthma. Serum IL-33 levels were positively correlated to basement membrane thickness. The production of fibronectin1 and type I collagen in human lung fibroblasts (HLF-1) increased at 24 h after IL-33 treatment in vitro. Pre-treatment with anti-ST2 antibody or fluticasone propionate (FP) suppressed the production of fibronectin1 and types I collagen induced by IL-33.IL-33 is a marker of asthma severity, and may contribute to airway remodeling in asthma by acting on human lung fibroblasts.

Asthma remains a common chronic inflammatory disease whose incidence has markedly increased over the past two decades. In addition, the chronic inflammation associated with a given patient is often linked with the remodeling of airway structure that may impair lung function [1, 2]. These changes include peribronchial fibrosis, fibroblast proliferation and conversion to myofibroblasts, and smooth muscle hypertrophy [3]. Although these features are well recognized, the mechanisms leading to remodeling and the effect of therapy on preventing or reversing these changes are largely unknown. A variety of cells, cytokines, chemokines, and growth factors that are released from inflammatory and structural cells in the airway have been implicated in promoting airway inflammation and, in turn, remodeling events. Furthermore, the cross-talk between epithelium and the underlying mesenchyme appears to be critical for driving remodeling responses in asthma. Findings from long-term patient studies, which have been ongoing for more than a decade, have indicated that transforming growth factor-β1 (TGF-β1) may be an important mediator of allergic inflammation and airway remodeling. TGF-β1 is produced by various cell types in the lung, such as airway epithelial cells and leukocytes comprising the pulmonary pro-inflammatory cell infiltrate [4, 5], and more recent studies suggested that this pulmonary TGF-β1 contributes to subepithelial fibrosis and airway smooth muscle remodeling linked with many asthma patients [6, 7]. An important positive regulator of many growth factor signaling pathways, including TGF-β, is the protein tyrosine phosphatase SHP2. SHP2 is a cytoplasmic phosphotyrosine phosphatase with two SH2 domains (N-SHP2 and C-SHP2) that is widely expressed in the airway epithelium [8]. Studies of allergic respiratory inflammation have also identified SHP2 as both a positive and negative regulator of key pulmonary immune signaling pathways. These SHP2 functions include fine-tuning of cellular response to cytokines [9–12], effects on inhibitory receptor signaling [13–15], and mediating T-cell development and function [16–18]. In the present study, the role of SHP2 expression in airway epithelial cells was defined further using a mouse model of chronic allergen exposure that is accompanied by characteristic airway remodeling events. These studies avoid the early embryonic lethality associated with homozygous shp2 knockout mice [19, 20] and instead exploit an inducible shp2 knockout strategy to specifically delete the expression in the mouse airway epithelia. Airway epithelial cell shp2 expression was linked with the airway remodeling events following chronic allergen exposure. Subsequent cell culture studies with BEAS-2B airway epithelial cells showed that the shp2-mediated airway remodeling occurring in mice may be mediated by the regulation of airway epithelial TGF-β1 production. These data suggest that shp2 may be a key regulator of airway remodeling events and thus a previously underappreciated therapeutic target.

Evaluation of: Doherty TA, Soroosh P, Khorram N et al. The tumor necrosis factor family member LIGHT is a target for asthmatic airway remodeling. Nat. Med. 17, 596–603 (2011).Individuals with chronic asthma demonstrate a progressive decline in lung function that is thought to be due to structural remodeling of the airways, characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we report on a recent publication showing that the tumor necrosis factor family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin-β-receptor reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung TGF-β and IL-13, cytokines implicated...

The concept of eosinophil-associated tissue remodeling and fibrosis has its roots in the hypereosinophilic syndrome and asthmatic airways. In asthmatic patients TH2-associated eotaxins and interleukins activate eosinophils to release granule products and growth factors that propagate inflammation and cause tissue damage. Continual and recurrent dysregulated repair leads to tissue remodeling with subsequent fibrosis and end-organ dysfunction. The reversibility, natural history, and clinical consequences of eosinophil-associated remodeling have been largely debated, especially in human disease, in which the paucity of tissue for repeated analysis has been a rate-limiting factor in our understanding. The availability of adequate surrogate markers in patients with disorders such as asthma has obviated any clinical requirement for direct tissue assessment. In contrast, eosinophilic esophagitis (EoE), a relatively recently described antigen-driven disease of increasing worldwide prevalence, by its definition requires microscopic esophageal tissue evaluation for its diagnosis and management. This necessity for repeated histologic analysis allows EoE to function as a new model for studying allergic, eosinophil-associated remodeling over time. Although asthmatic airway remodeling has been hypothesized to begin in early childhood, the ability to study airway tissue in children remains elusive. EoE, on the other hand, affords the potential to monitor the genesis and evolution of remodeling in young children. However, despite readily available esophageal tissue, remodeling analysis in patients with EoE still has its challenges. Esophageal biopsy specimens are small, approximately 3 mm. Although the esophageal epithelium is uniformly available from all biopsy specimens, only the minority of biopsy specimens contain adequate subepithelial lamina propria (LP) and muscularis mucosa for analysis. Because a large number of remodeling events, including fibrosis and angiogenesis, occur in the LP, this can generally only be studied in a subset of patients with EoE. Whether this required selection of biopsy

Individuals with chronic asthma show a progressive decline in lung function that is thought to be due to structural remodeling of the airways characterized by subepithelial fibrosis and smooth muscle hyperplasia. Here we show that the tumor necrosis factor (TNF) family member LIGHT is expressed on lung inflammatory cells after allergen exposure. Pharmacological inhibition of LIGHT using a fusion protein between the IgG Fc domain and lymphotoxin β receptor (LTβR) reduces lung fibrosis, smooth muscle hyperplasia and airway hyperresponsiveness in mouse models of chronic asthma, despite having little effect on airway eosinophilia. LIGHT-deficient mice also show a similar impairment in fibrosis and smooth muscle accumulation. Blockade of LIGHT suppresses expression of lung transforming growth factor-β (TGF-β) and interleukin-13 (IL-13), cytokines implicated in remodeling in humans, whereas exogenous administration of LIGHT to the airways induces fibrosis and smooth muscle hyperplasia, Thus, LIGHT may be targeted to prevent asthma-related airway remodeling.

Our primary objective was to investigate the possible contribution of controller medications to asthmatic airway remodeling, by (1) comparing the apoptotic and necrotic effects of several corticosteroids and montelukast on cultured airway human bronchial surface epithelial (16HBE) and submucosal (Calu3) cells; (2) measuring epithelial shedding potential and desmosome length in response to a cytokine challenge, with or without co-administered corticosteroids; and (3) studying corticosteroids and montelukast effects on inter-cellular adhesion molecule (ICAM) expression in both 16HBE and human umbilical vein endothelial cells (HUVEC). For this purpose, apoptosis, necrosis, and ICAM expression were quantified by flow cytometry, with 16HBE cells sensitive to both the apoptotic and necrotic effects of dexamethasone and montelukast; Calu3 cells sensitive only to budesonide. Transmission electron microscopy revealed decreased desmosome length in the presence of cytokines (TNF-alpha and INF-gamma), with corticosteroids counteracting this reduction. Dexamethasone, beclomethasone, and montelukast decreased versus increased ICAM-1 expression in airway epithelial cells and HUVEC, respectively. For conclusions, bronchial surface epithelial and submucosal cells exhibit a different sensitivity profile toward dexamethasone, budesonide, and montelukast, with corticosteroids preventing cytokineinduced desmosomal damage in 16HBE cells. The studied drugs led to increased ICAM-1 expression in endothelium, potentially facilitating inflammatory cell migration into lung tissue.

Increased numbers of submucosal vessels are a consistent pathologic component of asthmatic airway remodeling. However, the relationship between new vessel formation and asthmatic inflammatory response is unknown. We hypothesized that angiogenesis is a primary event during the initiation of airway inflammation and is linked to the recruitment of bone marrow-derived endothelial progenitor cells (EPC). To test this hypothesis, circulating EPC and EPC-derived endothelial cell colony formation of individuals with asthma or allergic rhinitis and health controls was evaluated. Circulating EPC were increased in asthma, highly proliferative, and exhibited enhanced incorporation into endothelial cell tubes as compared with controls. In an acute allergen challenge murine asthma model, EPC mobilization occurred within hours of challenge and mobilized EPC were selectively recruited into the challenged lungs of sensitized animals, but not into other organs. EPC recruitment was Th1 and Th2 dependent and was temporally associated with an increased microvessel density that was noted within 48 h of allergen challenge, indicating an early switch to an angiogenic lung environment. A chronic allergen challenge model provided evidence that EPC recruitment to the lung persisted and was associated with increasing microvessel density over time. Thus, a Th1- and Th2-dependent angiogenic switch with EPC mobilization, recruitment, and increased lung vessel formation occurs early but becomes a sustained and cumulative component of the allergen-induced asthmatic response.

Background Increased airway smooth muscle mass is a prominent feature of asthmatic airway remodeling. Airway smooth muscle hyperplasia occurs in rodent models of experimental asthma, but the relevance of such finding to spontaneously occurring disease in large mammals is unknown. Objective We examined horses with heaves, a naturally occurring equine asthma related to sensitization and exposure to moldy hay. We hypothesized that airway remodeling occurs in heaves and shares disease mechanisms with asthma. Methods We quantified the airway smooth muscle mass and the numbers of proliferating and apoptotic airway smooth muscle cells in 5 horses with heaves and 5 control horses using morphometric techniques. Cell proliferation was detected in tissue sections by immunostaining for proliferating cell nuclear antigen, and apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of fragmented DNA. Both signals were colocalized with smooth muscle specific α-actin. Results Horses with heaves had a significant increase in the amount of smooth muscle in the airways (nearly triple that of the controls) associated with increased myocyte proliferation (7-fold proliferating cell nuclear antigen–positive airway myocytes) and apoptosis (6-fold). Conclusion Heaves involves airway smooth muscle growth associated with myocyte hyperplasia, which may contribute to the growth, and increased myocyte apoptosis that may reflect a compensatory mechanism serving to limit the abnormal smooth muscle growth. Clinical implications Airway smooth muscle remodeling in heaves may be involved in the mechanism of airway hyperresponsiveness and chronic lung function impairment in a way comparable to human asthma.

Airway remodeling is a major change responsible for irreversible asthmatic airflow restriction. The Th-2 cytokines-dominant eosinophilic inflammatory mechanism cannot fully explain the progressive subepithelial fibrosis and structural changes in the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are the key enzymes responsible for ECM degradation. MMPs are normally produced and secreted under the tight regulation of, at least, 3 different levels: the gene transcriptional level, the activation of the latent form of enzyme, and the inactivation by specific endogenous inhibitors. In asthmatic condition, as shown by the large amount of accumulated evidence in this review, MMP-9 is the most relevant among the 23 kinds of human MMPs at present detected. Although the mechanism is still under investigation and not accurately known, the imbalance between MMP-9 and tissue inhibitor of metalloproteinase-1 is considered a major theory to explain the progression of asthmatic airway remodeling. Various inflammatory cytokines including TGF beta and growth factors play a pivotal role in MMP-9 production and secretion. This review mainly focuses upon the pivotal role of MMP-9 in airway remodeling, and also upon major cellular source of MMP-9 in asthma such as eosinophils, neutrophils, epithelial cells and alveolar macrophages. This review also refers to the partial contribution of nitric oxide to MMP-9 in asthma.

Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (SOD) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -3 and poly (ADP-ribose) polymerase were cleaved. On the basis that SOD inactivation triggers cell death and low SOD levels occur in asthma, we tested whether SOD inactivation plays a role in airway epithelial cell death. SOD inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Furthermore, oxidation and nitration of MnSOD were identified in the asthmatic airway, correlating with physiological parameters of asthma severity. These findings link oxidative and nitrative stress to loss of SOD activity and downstream events that typify asthma, including apoptosis and shedding of the airway epithelium and hyperresponsiveness.

Asthma continues to challenge physicians and scientists. After declining for years, the prevalence, morbidity and mortality of asthma have increased over the last two decades. In keeping with the importance of this “epidemic,” significant attention has been focused on the pathogenesis of this disorder. These investigations have revised our concepts of the abnormalities that underlie the asthmatic diathesis and our understanding of crucial aspects of its natural history. They have also provided exciting insights into the cellular and molecular events that regulate airway inflammation and inflammation in other tissues. It is clear from these studies that a genetic predisposition plus an appropriate life event(s) conspire to generate the asthmatic diathesis and that often early life (and possibly in utero) events play a crucial role in disease pathogenesis. It is also clear that inflammation is the cornerstone of asthma and that, in the majority of cases, Th2 dominated tissue responses are responsible for this disorder. Recently, the non-inflammatory structural alterations in the asthmatic airway have received renewed attention and the roles these structural changes may play in generating symptomatology and altering natural history have begun to be elucidated. The first Perspective in this series, by Gern, Lemanske and Busse, deals with the early life origins of the asthmatic diathesis, and the second article, by Oettgen and Geha, highlights recent insights into the importance of IgE and new anti-IgE-based therapies in atopy and the asthmatic response. The third article by Ray and Cohn highlights the large body of data that has now been generated regarding the cellular and molecular events that regulate and the effector functions of Th2 inflammatory responses, and the fourth article by Lukacs and colleagues describes the organized chemokine network that is involved in the generation of airway inflammation. The last article in the series by Elias et al. deals with the structural changes and the concept of asthmatic airway remodeling, highlighting both our level of knowledge and the challenges for the future. When the information presented in this series is looked at in composite, one can easily be impressed by the depth and breadth of our knowledge. It is also easy to see how these studies will serve as a springboard for future investigations and the foundation upon which new and powerful therapeutic interventions for asthma can be developed. It is important to keep in mind, however, that the relationship between atopy and asthma and the cause of the asthmatic epidemic are still poorly understood despite significant investigation. Perspective is also provided by the fact that William Osler stated in 1892 that asthma “in many cases is a special form of inflammation of the smaller bronchioles.” It has taken us almost a century to fully embrace this concept and begin to carefully dissect this airway response. Although we have made impressive progress, we still have a long way to go.

Background Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs. Methods BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFβ2 in BEC-HLF co-cultures using monoclonal antibody inhibition. Results Expression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFβ2 led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone. Conclusion These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFβ2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling.