Airway Fibroblasts Research Articles

Leptin augments IL-13-induced airway eotaxins and submucosal eosinophilia in obesity-associated asthma

BackgroundAirway tissue eosinophilia can be an observed feature of obesity associated T2 asthma, but the processes mediating this inflammation are unknown. ObjectiveWe investigated a process whereby leptin, an adipokine elevated in obesity, potentiates pulmonary eosinophilia and eotaxin production by airway fibroblasts in T2 asthma. MethodsWe assessed associations between body mass index and airway eosinophilia, leptin and eotaxin production in 78 asthma participants, 36 of which exhibited obesity. Cultured human airway fibroblasts and mouse models of chronic allergic airway disease were used to evaluate leptin’s effect on eotaxin production and lung eosinophilia. The role of interleukin-13 receptor alpha 2 (IL-13Rα2) in mediating these processes was examined using specific neutralizing antibodies in vitro. ResultsIn T2 asthma participants with obesity, we observed that airway tissue eosinophilia did not associate with traditional T2 inflammation metrics such as peripheral and/or bronchoalveolar lavage fluid (BALF) eosinophil counts or with fractional concentration of exhaled nitric oxide (FeNO). Alternatively, we observed elevated BALF leptin and eotaxin-1 levels. In airway fibroblasts from asthma participants, leptin augmented IL-13-induced eotaxin-1 and -3 production and IL13RA2 expression. In mice, elevated leptin promoted airway IL-13Rα2 and eotaxin production by lung fibroblasts and lung tissue eosinophilia following chronic house dust mite allergen exposure. Inhibition of IL-13Rα2 reduced combined leptin and IL-13-stimulated eotaxin secretion by human airway fibroblasts. ConclusionWe identified a potential association explaining airway tissue eosinophil retention in obesity-associated T2 asthma through leptin-mediated enhancement of IL-13-induced eosinophil chemokine production by airway fibroblasts, a process requiring IL-13Rα2.

A 3D Epithelial-Mesenchymal Co-Culture Model of the Airway Wall Using Native Lung Extracellular Matrix.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by ongoing inflammation, impaired tissue repair, and aberrant interplay between airway epithelium and fibroblasts, resulting in an altered extracellular matrix (ECM) composition. The ECM is the three-dimensional (3D) scaffold that provides mechanical support and biochemical signals to cells, now recognized not only as a consequence but as a potential driver of disease progression. To elucidate how the ECM influences pathophysiological changes occurring in COPD, in vitro models are needed that incorporate the ECM. ECM hydrogels are a novel experimental tool for incorporating the ECM in experimental setups. We developed an airway wall model by combining lung-derived ECM hydrogels with a co-culture of primary human fibroblasts and epithelial cells at an air-liquid interface. Collagen IV and a mixture of collagen I, fibronectin, and bovine serum albumin were used as basement membrane-mimicking coatings. The model was initially assembled using porcine lung-derived ECM hydrogels and subsequently with COPD and non-COPD human lung-derived ECM hydrogels. The resulting 3D construct exhibited considerable contraction and supported co-culture, resulting in a differentiated epithelial layer. This multi-component 3D model allows the investigation of remodelling mechanisms, exploring ECM involvement in cellular crosstalk, and holds promise as a model for drug discovery studies exploring ECM involvement in cellular interactions.

Azithromycin Prevents Subglottic Stenosis in Mice.

Pediatric subglottic stenosis (SGS) is characterized by subglottic narrowing which occurs when pathological fibroblasts deposit extracellular matrix that reduces airway patency. Recent clinical observations have suggested that azithromycin may have favorable impacts on SGS reduction while treating airway infections; furthermore, our recent work in mice demonstrated that the airway microbiome influences SGS. In this work, we characterize the protective effect of azithromycin as an immunomodulatory and antibacterial therapeutic against subglottic stenosis. Immunomodulatory and antifibrotic effects of azithromycin were assessed on TGF-β1-stimulated airway fibroblasts at 10 μg/mL for 5 days. Changes in gene expression were quantified by RT-qPCR and myofibroblast differentiation by α-SMA immunostaining. Murine airways were pretreated (2-weeks) with intranasal azithromycin before SGS injury by a twisted wire brush. Disease severity and immune response were characterized by histology and immunostaining for immune cells. In vitro, azithromycin treatment of TGF-β1-stimulated fibroblasts exhibited strong reductions in extracellular matrix (COL1A1, LOX) and myofibroblast-related gene expression (ACTA2). Notably, there was a significant reduction in pro-fibrotic expression, which was observed with 10 μg/mL azithromycin. Immunostaining of fibroblasts for α-SMA revealed strong reductions in the number of positive-staining cells and the intensity of each positive cell. In vivo, azithromycin exhibited a significant decrease in lamina propria thickness indicative of reduced stenosis with associated changes in T-cell infiltration. Overall, we show azithromycin prevents pro-fibrotic gene expression and myofibroblast differentiation and can help protect mice from developing SGS. This introduces azithromycin as a potential treatment for SGS. NA Laryngoscope, 2024.

Staphylococcus aureus adapts to exploit collagen-derived proline during chronic infection

Staphylococcus aureus is a pulmonary pathogen associated with substantial human morbidity and mortality. As vaccines targeting virulence determinants have failed to be protective in humans, other factors are likely involved in pathogenesis. Here we analysed transcriptomic responses of human clinical isolates of S. aureus from initial and chronic infections. We observed upregulated collagenase and proline transporter gene expression in chronic infection isolates. Metabolomics of bronchiolar lavage fluid and fibroblast infection, growth assays and analysis of bacterial mutant strains showed that airway fibroblasts produce collagen during S. aureus infection. Host-adapted bacteria upregulate collagenase, which degrades collagen and releases proline. S. aureus then imports proline, which fuels oxidative metabolism via the tricarboxylic acid cycle. Proline metabolism provides host-adapted S. aureus with a metabolic benefit enabling out-competition of non-adapted strains. These data suggest that clinical settings characterized by airway repair processes and fibrosis provide a milieu that promotes S. aureus adaptation and supports infection.

Inhaled nanoparticles for treating idiopathic pulmonary fibrosis by inhibiting honeycomb cyst and alveoli interstitium remodeling

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with high mortality. The Food and Drug Administration-approved drugs, nintedanib and pirfenidone, could delay progressive fibrosis by inhibiting the overactivation of fibroblast, however, there was no significant improvement in patient survival due to low levels of drug accumulation and remodeling of honeycomb cyst and interstitium surrounding the alveoli. Herein, we constructed a dual drug (verteporfin and pirfenidone)-loaded nanoparticle (Lip@VP) with the function of inhibiting airway epithelium fluidization and fibroblast overactivation to prevent honeycomb cyst and interstitium remodeling. Specifically, Lip@VP extensively accumulated in lung tissues via atomized inhalation. Released verteporfin inhibited the fluidization of airway epithelium and the formation of honeycomb cyst, and pirfenidone inhibited fibroblast overactivation and reduced cytokine secretion that promoted the fluidization of airway epithelium. Our results indicated that Lip@VP successfully rescued lung function through inhibiting honeycomb cyst and interstitium remodeling. This study provided a promising strategy to improve the therapeutic efficacy for IPF.

Rhinovirus protease cleavage of nucleoporins: perspective on implications for airway remodeling.

Human Rhinoviruses (RV) are a major cause of common colds and infections in early childhood and can lead to subsequent development of asthma via an as yet unknown mechanism. Asthma is a chronic inflammatory pulmonary disease characterized by significant airway remodeling. A key component of airway remodeling is the transdifferentiation of airway epithelial and fibroblast cells into cells with a more contractile phenotype. Interestingly, transforming growth factor-beta (TGF-β), a well characterized inducer of transdifferentiation, is significantly higher in airways of asthmatics compared to non-asthmatics. RV infection induces TGF-β signaling, at the same time nucleoporins (Nups), including Nup153, are cleaved by RV proteases disrupting nucleocytoplasmic transport. As Nup153 regulates nuclear export of SMAD2, a key intermediate in the TGF-β transdifferentiation pathway, its loss of function would result in nuclear retention of SMAD2 and dysregulated TGF-β signaling. We hypothesize that RV infection leads to increased nuclear SMAD2, resulting in sustained TGF-β induced gene expression, priming the airway for subsequent development of asthma. Our hypothesis brings together disparate studies on RV, asthma and Nup153 with the aim to prompt new research into the role of RV infection in development of asthma.

Small airway fibroblasts from patients with chronic obstructive pulmonary disease exhibit cellular senescence.

Small airway disease (SAD) is a key early-stage pathology of chronic obstructive pulmonary disease (COPD). COPD is associated with cellular senescence whereby cells undergo growth arrest and express the senescence-associated secretory phenotype (SASP) leading to chronic inflammation and tissue remodeling. Parenchymal-derived fibroblasts have been shown to display senescent properties in COPD, however small airway fibroblasts (SAFs) have not been investigated. Therefore, this study investigated the role of these cells in COPD and their potential contribution to SAD. To investigate the senescent and fibrotic phenotype of SAF in COPD, SAFs were isolated from nonsmoker, smoker, and COPD lung resection tissue (n = 9-17 donors). Senescence and fibrotic marker expressions were determined using iCELLigence (proliferation), qPCR, Seahorse assay, and ELISAs. COPD SAFs were further enriched for senescent cells using FACSAria Fusion based on cell size and autofluorescence (10% largest/autofluorescent vs. 10% smallest/nonautofluorescent). The phenotype of the senescence-enriched population was investigated using RNA sequencing and pathway analysis. Markers of senescence were observed in COPD SAFs, including senescence-associated β-galactosidase, SASP release, and reduced proliferation. Because the pathways driving this phenotype were unclear, we used cell sorting to enrich senescent COPD SAFs. This population displayed increased p21CIP1 and p16INK4a expression and mitochondrial dysfunction. RNA sequencing suggested these senescent cells express genes involved in oxidative stress response, fibrosis, and mitochondrial dysfunction pathways. These data suggest COPD SAFs are senescent and may be associated with fibrotic properties and mitochondrial dysfunction. Further understanding of cellular senescence in SAFs may lead to potential therapies to limit SAD progression.NEW & NOTEWORTHY Fibroblasts and senescence are thought to play key roles in the pathogenesis of small airway disease and COPD; however, the characteristics of small airway-derived fibroblasts are not well explored. In this study we isolate and enrich the senescent small airway-derived fibroblast (SAF) population from COPD lungs and explore the pathways driving this phenotype using bulk RNA-seq.

Study on the effect and mechanism of verapamil on the growth of human airway granulosa fibroblasts

Objective: This study aimed to investigate the inhibitory effect of the calcium channel blocker verapamil on human airway sarcomeric fibroblasts through network pharmacology molecular docking combined with experimental validation, so as to provide a theoretical and experimental basis for clinical use of calcium channel blockers against benign airway stenosis. Methods: The relevant targets of verapamil, airway injury, sarcoidosis and fibroblast were collected and intersected by a public database, respectively. The protein-protein interaction network was constructed by combining STRING; the DAVID database was used for gene function GO and KEGG pathway enrichment analysis. Molecular docking validation of verapamil and key targets was performed using AutoDock. Meanwhile, human lung bronchial tissue and human airway granulation tissue were treated with different antibiotic regimens to prevent contamination, in which human normal airway fibroblasts were treated with 92.81 μg/ml verapamil, while airway granulosa fibroblasts were treated with 69.57 μg/ml verapamil. The flow cytometry test was performed to detect the apoptotic effect of verapamil on human airway granulosa fibroblasts. The relative expression of mRNA of relevant factors between human normal airway fibroblasts and human airway granulosa fibroblasts was detected using polymerase chain reaction (PCR). Meanwhile, the difference in mRNA expression between human airway fibroblasts in normal and disease conditions was measured by PCR, and the change in mRNA expression of related factors after treatment with verapamil was determined to verify the findings of network pharmacology analysis. Results: This study predicted that verapamil affects 162 targets in human airway granulosa fibroblasts, among which, the most concerned and core target proteins are TGF-β1, VEGFA and IL-6; mainly involved in biological processes such as apoptosis, angiogenesis, energy metabolism and inflammation, and AGE-RAGE, JAK-STAT, PI3K-AKT, HIF as well as IL-17 signaling pathways. Molecular docking results showed that verapamil can bind to key targets. The apoptosis rate of human airway granulomatous fibroblasts treated with verapamil was higher than that of the control group, and the difference was statistically significant (P < 0.05). PCR showed that the relative expression of TGF-β1, VEGFA, IL-6 and other mRNAs in human airway granulomatous fibroblasts was significantly higher than that in human normal airway fibroblasts (P < 0.05), whereas the relative expression of TGF-β1 mRNA was down-regulated (P < 0.05) and the mRNA expression of VEGFA, IL-6 did not change significantly (P > 0.05) in human airway granulosa fibroblasts after treatment with verapamil. Conclusions: Increasing the antibiotic concentration and combining multiple antibiotics to soak the tissue can effectively kill the tissue pathogens. Verapamil can inhibit the proliferation of airway granulomatous fibroblasts through multiple targets and pathways, and it may be a method to prevent and treat benign airway stenosis.

P14-21: Evaluation of pulmonary fibrosis of Humidifier Disinfectants (HD) in a 3D co-culture model composed of Primary Human Small airway epithelial and fibroblast

P14-21: Evaluation of pulmonary fibrosis of Humidifier Disinfectants (HD) in a 3D co-culture model composed of Primary Human Small airway epithelial and fibroblast

Inhibitory Effect of Verapamil on the Growth of Human Airway Granulation Fibroblasts.

To explore the inhibitory effect of verapamil, a calcium channel blocker, on the growth of human airway granulation fibroblasts to provide an experimental basis for the clinical use of calcium channel blockers in preventing and treating benign airway stenosis. Primary human airway normal fibroblasts and human airway granulation fibroblasts were cultured by tissue block attachment culture method, and the experimental studies were carried out using 3-8 generation cells. Cell Counting Kit-8 (CCK-8) was used to test the proliferation of human normal airway fibroblasts and human airway granulation fibroblasts and the semi-inhibitory concentration of verapamil on normal airway fibroblasts and airway granulation fibroblasts. A scratch test detected the migration effect of verapamil on human airway granulation fibroblasts. The mRNA relative expression levels of related factors were detected by PCR to compare the differences between normal airway fibroblasts and airway granulation fibroblasts. Western blot was used to detect the relative amount of related proteins and compare the differences between normal airway fibroblasts and granulation airway fibroblasts. After 48 hours of treatment with half of the inhibitory concentration of Vera Pammy for granulation airway fibroblasts, the relative expression levels of related factors on mRNA and protein were observed. Human normal airway fibroblasts and human airway granulation fibroblasts with a purity of more than 95% could be obtained from primary culture by tissue block adherence method. CCK8 results showed that the proliferation rate of human airway granulation fibroblasts was faster than that of the normal human airway fibroblasts. The semi-inhibitory concentration of verapamil on human normal airway fibroblasts was 92.81 ug/ml, while the semi-inhibitory concentration on human airway granulation fibroblasts was 69.57 ug/ml. The scratch test indicated that the cell migration rate of human airway granulation fibroblasts treated with verapamil decreased significantly (P < 0.05). PCR results showed that the mRNA relative expression levels of TGFβ1, COL1A1, Smad2/3, VEGFA, IL6, and IL8 in human airway granulation fibroblasts were significantly higher than those in normal human airway fibroblasts (P < 0.05). The mRNA relative expressions of TGFβ1, smad2/3, and COL1A1 in human airway granulation fibroblasts treated with semi-inhibited verapamil for 48h were down-regulated (P < 0.05), while the mRNA relative expressions of VEGFA, IL6 and IL8 had no significant changes (P > 0.05). WB test showed that the relative protein expressions of TGFβ1, Smad2, and VEGFC in human airway granulation fibroblasts were upregulated (P < 0.05) but downregulated after verapamil treatment compared with before treatment (P < 0.05). Calcium channel blockers can inhibit the proliferation of human airway granulation fibroblasts through TGFβ1/ Smad pathway, which may be a method to prevent and treat benign airway stenosis.

A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

The Rapid Development of Airway Organoids: A Direct Culture Strategy.

The lung is a complex organ composed of numerous cell types. Exposure to air pollutants, cigarette smoke, bacteria, viruses, and many others may cause injury to the epithelial cells that line the conducting airways and alveoli. Organoids are the 3D self-organising structures grown from stem cells and generated from adult stem and progenitor cells. Lung organoids are fascinating tools to investigate human lung development in vitro. The objective of this study was to establish a rapid method for generating lung organoids with a direct culture strategy. Trachea and lung organoids were derived from mixed cell populations of mice primary airway epithelial cells, fibroblasts, and lung microvascular endothelial cells and directly digested from the whole cell population in the distal lung. The formation of spheres appeared as early as 3days and continued to proliferate until day 5. The generation of trachea and lung organoids self-organised into discrete epithelial structures was formed within less than 10days. We conclude that researchers will be able to examine cellular involvement during organ formation and molecular networks because organoids come in a variety of morphologies and stages of development, and this organoid protocol may be used for modelling lung diseases as a platform for therapeutic purposes and suitable for personalised medicine for respiratory diseases.

P12-22 Transcriptome and proteome profiling of normal human bronchial epithelial cells cocultured with human airway fibroblasts after their exposure to air pollution-derived PM2.5 predicts a shift from healthy to COPD-phenotype

P12-22 Transcriptome and proteome profiling of normal human bronchial epithelial cells cocultured with human airway fibroblasts after their exposure to air pollution-derived PM2.5 predicts a shift from healthy to COPD-phenotype

IL-36 receptor agonist and antagonist imbalance drives neutrophilic inflammation in COPD.

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared with control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and is further induced by a viral mimetic, whereas IL-36Ra is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ, thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, which was inhibited by exogenous IL-36Ra. The use of a therapeutic antibody that inhibits binding to the IL-36R attenuated IL-36γ–driven inflammation and cellular crosstalk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and have shown that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising therapeutic strategy in the treatment of COPD.

Expression and significance of TGF-β1 in infant asthma model

This article aimed to study the expression and role of TGF-β1 in young rat asthma. The experimental method in this paper is to replicate the asthma model by aerosolization of egg protein. The young rats with asthma were sacrificed after 2, 4 and 8 weeks. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA of TGF-β1 and α-SMA. The expression level was detected by the immunohistochemical staining method to detect the protein expression of TGF-β1, α-SMA, Smad3, Smad4 and Smad7 in bronchopulmonary tissues. At the same time, the image analysis method was used to measure the airway morphological parameters of young rats. The experimental results showed that the expression of TGF-β1 in the lung tissue of young rats in the asthma group was increased after two weeks of the challenge. Compared with the control group, the difference was significant (P<0.05); the expression of TGF-β1, α-SMA and mRNA in the 4-week group was significantly higher, compared with the control group and the 2-week challenge group, the difference was significant (P<0.05). Therefore, the change of asthma airway remodeling is a dynamic process. The TGF-β1 pathway presents a dynamic process in the airway remodeling model of young asthmatic rats. Among them, TGF-β1 induces the division and proliferation of airway fibroblasts and promotes fibroblasts. The cells transform into myofibroblasts and increase the synthesis of fibronectin and collagen, suggesting that it plays an important role in asthma inflammation and pulmonary fibrosis.

Long noncoding RNA GAS5 attenuates cigarette smoke-induced airway remodeling by regulating miR-217-5p/PTEN axis.

Airway remodeling is a remarkable pathological characteristic of chronic obstructive pulmonary disease (COPD), and long noncoding RNAs have been demonstrated to participate in COPD development and pathogenesis. Here, we investigate the role of long noncoding RNA GAS5 in cigarette smoke (CS)-induced airway remodeling. GAS5 expression is significantly lower in lung tissues of CS-exposed mice than in tissues of control mice without exposure to CS. Forced GAS5 overexpression suppresses CS-induced airway inflammation and remodeling. GAS5 overexpression also inhibits CS extract-induced inflammatory-cytokine expression and fibroblast activation in vitro. Regarding the mechanism, GAS5 acts as a sponge of miR-217-5p, thereby increasing PTEN expression. MiR-217-5p overexpression and PTEN knockdown separately reverse the inhibitory effects of GAS5 overexpression on the inflammatory-cytokine expression and fibroblast activation. Collectively, these results suggest that GAS5 can suppress airway inflammation and fibroblast activation by regulating miR-217-5p/PTEN axis, which may help develop novel therapeutic strategies against COPD.

TARGETING THE HISTONE DEMETHYLASE JMJD2B/KDM4B AS A NOVEL THERAPEUTIC APPROACH IN IL-13-MEDIATED FIBROSIS IN AIRWAY FIBROBLASTS OF SEVERE ASTHMATICS

TYPE: Abstract TOPIC: Allergy and Airway PURPOSE: Asthma is a heterogeneous disorder characterized by chronic inflammation and remodeling of the airways. Asthma responses are driven mainly by type 2 immune responses, where IL-13 plays a key role in asthma pathogenesis. We previously identified JMJD2B as an IL-13-regulated epigenetic modifier in asthmatic airway fibroblasts. Therefore, this study aimed to target JMJD2B to potentially alleviate IL-13-mediated fibrosis in asthma. METHODS: Bronchial fibroblasts isolated from asthmatic and healthy individuals were stimulated with IL-13 and treated with JIB-04, a pan-selective inhibitor of histone demethylase(s). Gene and protein expression of extracellular matrix markers were assessed using qRT-PCR, western blotting and matrix metalloproteinases (MMPs) activity assay. Chromatin immunoprecipitation (ChIP) assay was used to determine the bindig of JMJD2B to promoter region of Tissue inhibitors of matrix metalloproteinases (TIMPs). RESULTS: Here we show that JIB-04 inhibited JMJD2B activity by reducing the demethylation of its downstream target, H3K36me3, in asthmatic fibroblasts. Inhibition of JMJD2B significantly affected the viability of the bronchial fibroblasts at 48 hours. JMJD2B inhibition was further associated with the downregulation of extracellular matrix (ECM) proteins such as MMP-2, MMP-9, collagen-1, and fibronectin, and upregulation of TIMP-2, at both the gene and protein levels. This was accompanied by inhibition of IL-13 mediated MMPs activity. JIB-04 further prevented the association of JMJD2B on TIMP-2 promoter region. CONCLUSIONS: Our results demonstrate the potential of JMJD2B inhibition in attenuating IL-13-mediated fibrosis in asthma. CLINICAL IMPLICATIONS: Therapeutic targeting of JMJD2B using JIB-04 is a promising candidate to alleviate IL-13-mediated responses in chronic disorders such as asthma. DISCLOSURE: Nothing to declare. KEYWORD: Asthma

Airway Fibroblast Secretory Products Enhance Cell Migration

Background: The nasal fibroblast secretome, which includes various cytokines, chemokines, and growth factors, promotes cell migration. Currently, the proteomics of Airway Fibroblast (AF) Conditioned Medium (AFCM) are being actively studied. Objective: This study was aimed at profiling and identifying the AF secreted proteins that can enhance wound healing of the airway epithelium and predict the potential pathway involved. Methods: Airway Epithelial Cells (AECs) and AFs were isolated from redundant human nasal turbinate and cultured. AFCM was collected by culturing the AFs either with serum-free airway epithelium basal medium (AECM) or with serum-free F12:DMEM (FDCM). For evaluating cell migration, the AECs were supplemented with airway epithelium medium and defined keratinocyte medium (1:1; AEDK; control), or with AEDK supplemented with 20% AECM or 20% FDCM. The mass spectrometry sample was prepared by protein precipitation, followed by gel electrophoresis and in-gel digestion. Results : AECM promoted better cell migration compared to the FDCM and the control medium. Bioinformatics analysis identified a total of 121, and 92 proteins from AECM and FDCM, respectively: 109 and 82 were identified as secreted proteins, respectively. STRING® analysis predicted that 23 proteins from the AECM and 16 proteins from the FDCM are involved in wound healing. Conclusion: Conditioned medium promotes wound healing by enhancing cell migration, and we successfully identified various secretory proteins in a conditioned medium that play important roles in wound healing.

MicroRNAs Associated with Chronic Mucus Hypersecretion in COPD Are Involved in Fibroblast-Epithelium Crosstalk.

We recently identified microRNAs (miRNAs) associated with chronic mucus hypersecretion (CMH) in chronic obstructive pulmonary disease (COPD), which were expressed in both airway epithelial cells and fibroblasts. We hypothesized that these miRNAs are involved in communication between fibroblasts and epithelium, contributing to airway remodeling and CMH in COPD. Primary bronchial epithelial cells (PBECs) differentiated at the air–liquid interface, and airway fibroblasts (PAFs) from severe COPD patients with CMH were cultured alone or together. RNA was isolated and miRNA expression assessed. miRNAs differentially expressed after co-culturing were studied functionally using overexpression with mimics in mucus-expressing human lung A549 epithelial cells or normal human lung fibroblasts. In PBECs, we observed higher miR-708-5pexpression upon co-culture with fibroblasts, and miR-708-5p expression decreased upon mucociliary differentiation. In PAFs, let-7a-5p, miR-31-5p and miR-146a-5p expression was significantly increased upon co-culture. miR-708-5p overexpression suppressed mucin 5AC (MUC5AC) secretion in A549, while let-7a-5poverexpression suppressed its target gene COL4A1 in lung fibroblasts. Our findings suggest that let-7a-5p, miR-31-5p and miR-146a-5p may be involved in CMH via fibroblasts–epithelium crosstalk, including extracellular matrix gene regulation, while airway epithelial expression of miR-708-5p may be involved directly, regulating mucin production. These findings shed light on miRNA-mediated mechanisms underlying CMH, an important symptom in COPD.

Syndecan-1 Amplifies Ovalbumin-Induced Airway Remodeling by Strengthening TGFβ1/Smad3 Action.

Syndecan-1 (SDC-1) is a transmembrane proteoglycan of heparin sulfate that can regulate various cell signal transduction pathways in the airway epithelial cells and fibroblasts. Airway epithelial cells and human bronchial fibroblasts are crucial in airway remodeling. However, the importance of SDC-1 in the remodeling of asthmatic airways has not been confirmed yet. The present study was the first to uncover SDC-1 overexpression in the airways of humans and mice with chronic asthma. This study also validated that an increase in SDC-1 expression was correlated with TGFβ1/Smad3-mediated airway remodeling in vivo and in vitro. A small interfering RNA targeting SDC-1 (SDC-1 siRNA) and homo-SDC-1 in pcDNA3.1 (pc-SDC-1) was designed to assess the effects of SDC-1 on TGFβ1/Smad3-mediated collagen I expression in Beas-2B (airway epithelial cells) and HLF-1 (fibroblasts) cells. Downregulation of the SDC-1 expression by SDC-1 siRNA remarkably attenuated TGFβ1-induced p-Smad3 levels and collagen I expression in Beas-2B and HLF-1 cells. In addition, SDC-1 overexpression with pc-SDC-1 enhanced TGFβ1-induced p-Smad3 level and collagen I expression in Beas-2B and HLF-1 cells. Furthermore, the levels of p-Smad3 and collagen I induced by TGFβ1 were slightly increased after the addition of the recombinant human SDC-1 protein to Beas-2B and HLF-1 cells. These findings in vitro were also confirmed in a mouse model. A short hairpin RNA targeting SDC-1 (SDC-1 shRNA) to interfere with SDC-1 expression considerably reduced the levels of p-Smad3 and remodeling protein (α-SMA, collagen I) in the airways induced by ovalbumin (OVA). Similarly, OVA-induced p-Smad3 and remodeling protein levels in airways increased after mice inhalation with the recombinant mouse SDC-1 protein. These results suggested that SDC-1 of airway epithelial cells and fibroblasts plays a key role in the development of airway remodeling in OVA-induced chronic asthma.