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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.

Both canonical and non-canonical Wnt signaling pathway alterations have been documented in pulmonary disease pathogenesis and progression; therefore, they can be an attractive target for pharmaceutical management of severe asthma. Wnt/β-catenin signaling was shown to link early embryonic lung development impairment to later in life asthmatic airway remodeling. Here we explored the changes in Wnt signaling associated with asthma initiation and progression in epithelial and fibroblasts using a comprehensive approach based onin silicoanalysis and followed byin vitrovalidation. In summary, thein silicoanalysis showed that the bronchial epithelium of severe asthmatic patients showed a deranged balance between Wnt enhancer and Wnt inhibitors. A Th2-high phenotype is associated with upregulated Wnt-negative regulators, while inflammatory and neutrophilic severe asthmatics showed higher canonical Wnt signaling member enrichment. Most of these genes are regulators of healthy lung development early in life and, if disturbed, can make people susceptible to developing asthma early in life and prone to developing a severe phenotype. Most of the Wnt members are secreted, and their effect can be in an autocrine fashion on the bronchial epithelium, paracrine on nearby adjacent structural cells like fibroblasts and smooth muscles, or systemic in blood. Our results showed that canonical Wnt signaling is needed for the proper response of cells to proliferative stimuli, which puts cells under stress. Cells in response to this proliferative stress will activate the senescence mechanism, which is also dependent on Wnt signaling. Inhibition of Wnt signaling using FH535 inhibits both proliferation and senescence markers in bronchial fibroblasts compared to DMSO-treated cells. In fibroblasts from asthmatic patients, inhibition of Wnt signaling did not show that effect as the Wnt signaling is deranged besides other pathways that might be non-functional.

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.

Purpose: Epithelial-mesenchymal transition (EMT) involved in asthmatic airway remodeling. Thymic stromal lymphopoietin (TSLP), an epithelial-derived cytokine, was a key component in airway immunological response in asthma. But the role of TSLP in the EMT process was unknown. We aimed to access whether TSLP could induce EMT in airway epithelia and its potential mechanism. Materials and Methods: Human bronchial epithelial (HBE) cells were incubated with TSLP or transforming growth factor beta 1 (TGF-β1) or both. SB431542 was used to block TGF-β1 signal while TSLP siRNA was used to performed TSLP knockdown. Changes in E-cadherin, vimentin, collagen I and fibronectin level were measured by real-time PCR, western blot and immunofluorescence staining. Expressions of TGF-β after TSLP administration were measured by real-time PCR, western blot and ELISA. Results: TSLP induced changes of EMT relevant markers alone and promoted TGF-β1-induced EMT in HBEs. Intracellular and extracellular expression of TGF-β1 were upregulated by TSLP. SB431542 blocked changes of EMT relevant markers induced by TSLP. Knockdown of TSLP not only reduced TSLP and TGF-β1 expression but also inhibited changes of EMT relevant markers induced by TGF-β1 in HBEs. Conclusions: TSLP could induce early stage of EMT in airway epithelial cells through upregulation of TGF-β1. This effect may act as a targeting point for suppression of asthma.

Background Bronchoconstriction and cough, a characteristic of the asthmatic response, leads to development of compressive stresses in the airway wall. We hypothesized that progressively pathological high mechanical stress could act on mechanosensitive cation channels, such as transient receptor potential channel 1 (TRPC1) and then contributes to airway remodeling. Methods We imitate the pathological airway pressure in vitro using cyclic stretch at 10 and 15% elongation. Ca2+ imaging was applied to measure the activity of TRPC1 after bronchial epithelial cells exposed to cyclic stretch for 0, 0.5, 1, 1.5, 2, 2.5 h. To further clarify the function of channnel TRPC1 in the process of mechano-transduction in airway remodeling, the experiment in vivo was implemented. The TRPC1 siRNA and budesonide were applied separately to asthmatic models. The morphological changes were measured by HE and Massion method. The expression levels of TRPC1 were evaluated by real-time PCR, western blot and immunohistochemistry. The protein expression level of IL-13, TGF-β1 and MMP-9 in BALF were measured by ELISA. Results The result showed that cyclic stretch for 15% elongation at 1.5 h could maximize the activity of TRPC1 channel. This influx in Ca2+ was blocked by TRPC1 siRNA. Higher TRPC1 expression was observed in the bronchial epithelial layer of ovalbumin induced asthmatic models. The knockdown of TRPC1 with TRPC1 siRNA was associated with a hampered airway remodeling process, such as decreased bronchial wall thickness and smooth muscle hypertrophy/hyperplasia, a decreased ECM deposition area and inflammation infiltration around airway wall. Meantime, expression of IL-13, TGF-β1 and MMP-9 in OVA+TRPC1 siRNA also showed reduced level. TRPC1 intervention treatment showed similar anti-remodeling therapeutic effect with budesonide. Conclusions These results demonstrate that most TRPC1 channels expressed in bronchial epithelial cells mediate the mechanotransduction mechanism. TRPC1 inducing abnormal Ca2+ signal mediates receptor-stimulated and mechanical stimulus-induced airway remodeling. The inhibition of TRPC1 channel could produce similar therapeutic effect as glucocortisteroid to curb the development of asthmatic airway remodeling.

Emerging evidences support that transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) participates in the pathogenesis of pulmonary fibrosis and asthmatic airway remodeling. Recent studies demonstrated that apolipoprotein A-I (Apo A-I) is the only known substance that can resolve established pulmonary fibrotic nodules, and Apo A-I mimetic D-4F (a synthetic polypeptide consisting of 18 amino acids) plays an inhibitory role in murine asthmatic model. However, cellular mechanisms for such therapeutic effects of Apo A-I and D-4F remain to be elucidated. This study evaluated the effects of D-4F on TGF-β1 induced EMT in human type II alveolar epithelial cell line A549. A549 cells treated with 10ng/ml of TGF-β1 manifested distinct EMT, including fibroblastic morphological changes, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. These EMT related changes were all inhibited by D-4F in a concentration dependent manner. Transcriptional investigation demonstrated clearly that D-4F dose-dependently compensated for the reduced E-cadherin mRNA level and the increased vimentin mRNA level in TGF-β1 treated A549 cells. Translational analysis revealed that D-4F significantly reversed the TGF-β1 induced changes of E-cadherin and vimentin levels. These results suggested that D-4F inhibits TGF-β1 induced EMT in human alveolar epithelial cell. Given the functional similarities between D-4F and Apo A-I, it is speculated that D-4F and Apo A-I are able to exert possible anti-fibrotic and anti-asthmatic effects via inhibiting alveolar EMT, and D-4F may possess beneficial clinical potential for patients suffering from pulmonary fibrosis and asthma.

Asthma is characterized by chronic inflammation, and long-term chronic inflammation leads to airway remodeling. But the potential regulatory mechanism of airway remodeling is not clearly understood, and there is still no effective way to prevent airway remodeling. Present studies have confirmed the role of microRNAs (miRNAs) in the development of disease, which is known as suppressing translation or degradation of messenger RNA (mRNA) at the posttranscriptional stage. In this study, we described the role of miRNA-133a in asthma and demonstrated it in regulating airway remodeling of asthma through the phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway by targeting IGF-1 receptor (IGF1R). IGF1R helps in mediating the intracellular signaling cascades. Asthmatic mice models were established by sensitization and Ovalbumin challenge. Adenovirus transfer vector carrying miR-133a or miR-133a sponge sequence was used to build the overexpression or downexpression of miR-133a modeling. Real-time polymerase chain reaction and Western blot were used to determine the alterations in the expression of miR-133a and mRNAs and their corresponding proteins. Results showed that miR-133a was downregulated in asthma. Upregulation of miR-133a expression in airway smooth muscle cells in vivo and in vitro could inhibit the activation of PI3K/AKT/mTOR pathway, and reduce the expression of α-smooth muscle actin (α-SMA), indicating that airway remodeling was inhibited. Functional studies based on luciferase reporter revealed miR-133a as a direct target of IGF1R mRNA. In conclusion, these data suggested that miR-133a regulated the expression of α-SMA through PI3K/AKT/mTOR signaling by targeting IGF1R. miR-133a plays a key role in airway remodeling of asthma and may serve as a potential therapeutic target for managing asthmatic airway remodeling.

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.

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.

1,25-Dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has immune- and inflammation-modulating properties in asthma, but its possible effects on asthmatic airway remodeling remain uncertain. In this study, we investigated the effects of 1,25-(OH)(2)D(3) on airway remodeling in a murine model of chronic asthma and investigated its role in regulating nuclear factor-κB (NF-κB) activation.BALB/c mice were sensitized to ovalbumin (OVA) and subsequently exposed to intranasal OVA challenges for 9 weeks. Some mice also received an intraperitoneal injection of 1,25-(OH)(2)D(3) at the time of challenge. At the end of the challenge period, mice were evaluated for chronic airway inflammation and airway remodeling. Nuclear translocation of NF-κB p65 in lung tissue was examined by Western blot. Inhibitor of NF-κB alpha (IκBα) expression was determined by real-time quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western blot. Phosphorylated IκBα protein expression was also determined by Western blot.1,25-(OH)(2)D(3) treatment reduced OVA-induced chronic inflammation in lung tissue and attenuated established structural changes of the airways, including subepithelial collagen deposition, goblet cell hyperplasia, and increased airway smooth muscle mass. 1,25-(OH)(2)D(3) also inhibited the nuclear translocation of NF-κB p65 in lung tissue. Concurrently, 1,25-(OH)(2)D(3) induced increased IκBα protein levels via inducing increased IκBα mRNA levels and decreased IκBα phosphorylation.1,25-(OH)(2)D(3) could attenuate asthmatic airway remodeling and its inhibition of NF-κB activation may underlie this protective effect.

In this research, compound Maqin decoction (CMD) has been shown to positively affect in airway inflammation of asthma models. We evaluated the effects of CMD on the expression of transforming growth factor (TGF)-β1/Smad proteins, interleukin (IL)-17, and IL-10 in lung tissue of asthmatic rats. Asthma was induced in a rat model using ovalbumin. After a 4-week treatment with CMD, rats were killed to evaluate the expression of TGF-β1 and Smad proteins in lung tissue. IL-10 and IL-17 levels in lung tissue homogenates were determined by ELISA. The expression of TGF-β1 and Smad3 protein increased, whereas expression of Smad7 protein decreased upon high-dose or low-dose treatment with CMD or by intervention with dexamethasone, compared to the control. There was a significant difference between treatment with a high dose CMD and the control treatment, but no significant difference was found between high-dose CMD treatment and dexamethasone intervention. The expression of TGF-β1 and Smad7 protein increased, whereas the expression of Smad3 protein decreased in the model group compared to other groups. In the CMD high-dose group, low-dose group, and dexamethasone intervention group, the IL-17 concentrations in lung tissue homogenates were decreased, while IL-10 levels were increased. Again, there was a significant difference between CMD high-dose and control treatment, but not between CMD high-dose treatment and dexamethasone intervention. Thus, positive effects of CMD against asthmatic airway remodeling may be due to its regulatory effect on TGF-β1, Smad3, and Smad7 protein levels and on cytokines such as IL-10 and IL-17.

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.

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.

In order to investigate the effect of nuclear factor-kappaB (NF-kappaB) on airway remodeling in asthmatic rats, 18 Wistar rats were divided into three groups: asthmatic group; pyrrolidine dithiocarbamate (PDTC) group, in which rats were injected intraperitoneally with NF-kappaB specific inhibitor PDTC (100 mg/kg) before ovalbumin (OVA) challenge; control group. The NF-kappaB activity and the expression of inhibitory protein kappaBalpha (I-kappaBalpha) in airway were detected by electrophoretic mobility shift assay (EMSA), Western blot and immunohistochemistry respectively. The infiltration of inflammatory cells, the number of Goblet cells, the area of collagen and smooth muscle in airway were measured by means of image analysis system. The results showed that with the up-regulation of airway NF-kappaB activity in asthmatic group, the number of goblet cells (3.084 +/- 0.86/100 microm basement membrane (BM)), the area of collagen (24.71 +/- 4.24 microm2/microm BM) and smooth muscle (13.81 +/- 2.11 microm2/microm BM) in airway were significantly increased (P0.05) as compared with control group (0.14 +/- 0.05/100 microm BM, 14.31 +/- 3.16 microm2/microm BM and 7.67 +/- 2.35 microm2/microm BM respectively) and PDTC group (0.33 +/- 0.14/100 microm BM, 18.16 +/- 2.85 microm/microm BM and 8.95 +/- 2.16 microm2/microm BM respectively). However, there was no significant difference between PDTC group and control group (P0.05). It was concluded that the activity of NF-kappaB is increased in airway of asthmatic rats. Inhibition of NF-kappaB activation can attenuate constructional changes in asthma airway, suggesting NF-kappaB may contribute to asthmatic airway remodeling.

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.

Airway remodeling is an important pathological feature of asthma and the basis of severe asthma. Proliferation of airway smooth muscle cells (ASMCs) is a major contributor to airway remodeling. As an important Ca(2+) channel, transient receptor potential vanilloid 1 (TRPV1) plays the key role in the cell pathological and physiological processes. This study investigated the expression and activity of TRPV1 channel, and further clarified the effect of TRPV1 channel on the ASMCs proliferation and apoptosis in order to provide the scientific basis to treat asthmatic airway remodeling in clinical practice. Immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of TRPV1 in rat ASMCs. Intracellular Ca(2+) was detected using the single cell confocal fluorescence microscopy measurement loaded with Fluo-4/AM. The cell cycles were observed by flow cytometry. MTT assay and Hoechst 33258 staining were used to detect the proliferation and apoptosis of ASMCs in rats respectively. The data showed that: (1) TRPV1 channel was present in rat ASMCs. (2) TRPV1 channel agonist, capsaicin, increased the Ca(2+) influx in a concentration-dependent manner (EC50=284.3±58 nmol/L). TRPV1 channel antagonist, capsazepine, inhibited Ca(2+) influx in rat ASMCs. (3) Capsaicin significantly increased the percentage of S+G2M ASMCs and the absorbance of MTT assay. Capsazepine had the opposite effect. (4) Capsaicin significantly inhibited the apoptosis, whereas capsazepine had the opposite effect. These results suggest that TRPV1 is present and mediates Ca(2+) influx in rat ASMCs. TRPV1 activity stimulates proliferation of ASMCs in rats.

Airway remodeling is an important characteristic of asthma, linking inflammation with airway hyperresponsiveness. Baicalin, a major active component, was isolated from Radix Scutellariae. Many studies show that baicalin has anti-inflammatory, anti-bacterial, and anti-allergic effects. Here we investigate the influence of baicalin on asthmatic airway remodeling and the mechanism underlining the anti-remodeling effect in vivo. Asthmatic airway remodeling mice model was established by ovalbumin exposure. Seventy female BALB/c mice were randomly assigned to seven experimental groups: blank, ovalbumin, hexadecadrol, control, and baicalin (25 mg/kg, 50 mg/kg, 100 mg/kg) groups. Pulmonary function was measured using a whole-body plethysmograph in conscious and unrestrained mice. The lung pathology was observed and measured. The production of cytokines in bronchoalveolar lavage fluid and serum was measured using enzyme-labeled immunosorbent assay kits, and the expression levels of transforming growth factor-β 1 and vascular endothelial growth factor were detected by immunohistochemistry. The protein expression levels of transforming growth factor-β 1, vascular endothelial growth factor, extracellular signal–regulated kinase, and p21ras were measured using Western blot. The results show that ovalbumin exposure significantly increased the expression of interleukin-13 in BALF and serum, and transforming growth factor-β 1, vascular endothelial growth factor, extracellular signal–regulated kinase and p21ras expressions in the lungs. Baicalin attenuated the effects of ovalbumin significantly. It can be concluded that baicalin has significant anti-remodeling effect on ovalbumin-induced asthmatic airway remodeling mice model by decreasing expression of transforming growth factor-β 1, interleukin-13, and vascular endothelial growth factor and inhibiting the activation of the extracellular signal–regulated kinase pathway.

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.

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.

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 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.

Airway remodeling is a main pathological characteristic of asthma, and strongly associated with migration and proliferation of airway smooth muscle cells. E-prostanoid (EP) receptor can regulate airway remodeling. This study established a rat model of asthma and evaluated EP changes in airway smooth muscle cells under the asthmatic state so as to provide theoretical evidence for developing EP drugs to treat airway remodeling in asthma. A total of 20 clean Sprague-Dawley rats were randomly assigned to asthma group and control group. 28 days later, they were sacrificed for histological examination. Airway smooth muscle cells were isolated, cultured and measured using quantitative fluorescent PCR. Histopathological examination revealed that rat models of asthma were in accordance with the manifestations of asthmatic airway remodeling. After reverse transcription, real-time quantitative fluorescent PCR was performed using miRNA Q-PCR diagnostic kit. GAPDH was considered the internal reference. Relative expressions of E-prostanoid 1–4 (EP1–4) (2-△△Ct) in the control and asthma groups were respectively as follows: EP1: 4.35±0.18, 6.55±1.21; EP2: 3.64±0.12, 1.35±1.06; EP3: 4.59±1.14, 5.89±1.74; EP4: 2.89±1.85, 1.69±0.44. EP2/4 significantly decreased, but EP1 significantly increased in the asthma group (P < 0.01). These results suggested that the reduced EP2/4 and increased EP1 expressions in airway smooth muscle cells of rat models of asthma were probably important factors for asthmatic airway remodeling.

Rationale Genistein is a dietary isoflavone and a broad-spectrum tyrosine kinase inhibitor contained in soy products. An epidemiologic study of asthma revealed that subjects with high consumption of dietary soy isoflavones had better lung function than those with lower intake. In a guinea pig model of allergic asthma, genistein reduced methacholine-induced bronchoconstriction. To explore the mechanisms underlying these observations, we tested genistein9s ability to block lung myofibroblast differentiation, a key phenotypic change in asthmatic airway remodeling. Methods Human fetal lung fibroblasts (IMR-90) were grown to subconfluence in DMEM containing 10% FBS, serum-deprived for 24 hours, and treated with genistein (10 μM) for an additional 24 hurs. Cells were then stimulated with TGF-b1 (2 ng/mL) for 24 hours, and a-smooth muscle actin (aSMA) expression, a marker of the myofibroblast phenotype, was assessed by both immunoblot and immunofluorescence microscopy. To determine the intracellular mechanism of inhibitory actions by genistein, we also assessed phosphorylation of Smad2 by immunoblot in the presence and absence of genistein after 30-minute stimulation with TGF-b1. Results Treatment with genistein resulted in 54.9 ± 17.1% reduction in TGF-b1 induced expression of aSMA. Immunofluorescence microscopy revealed a decrease in aSMA staining intensity and stress fiber formation in genistein-treated cells. In addition, Smad2 phosphorylation was inhibited by 71.1 ± 16.7% in genistein-treated cells. Conclusions These results demonstrate that genistein interferes with TGF-b1-stimulated myofibroblast differentiation and suggest that tyrosine kinase inhibition may have a role in modulation of asthmatic airway remodeling. Funded by NIH RO1HL072891, T32HL076139, The CHEST Foundation-GSK Clinical Research Trainee Award.