Human Airway Fibroblasts Research Articles

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.

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

Mast cell chymase affects the functional properties of primary human airway fibroblasts: Implications for asthma

Mast cells (MCs) have a profound impact on allergic asthma. Under such conditions, MCs undergo degranulation, resulting in the release of exceptionally large amounts of MC-restricted proteases. However, the role of these proteases in asthma is only partially understood. We sought to test our hypothesis that MC proteases can influence the functionality of human lung fibroblasts (HLFs). Primary HLFs were treated with MC chymase or tryptase, followed by assessment of parameters related to fibroblast function. HLFs underwent major morphologic changes in response to chymase, showing signs of cellular contraction, but were refractory to tryptase. However, no effects of chymase on HLF viability or proliferation were seen. Chymase, but not tryptase, had a major impact on the output of extracellular matrix-associated compounds from the HLFs, including degradation of fibronectin and collagen-1, and activation of pro-matrix metalloprotease 2. Further, chymase induced the release of various chemotactic factors from HLFs. In line with this, conditioned medium from chymase-treated HLFs showed chemotactic activity on neutrophils. Transcriptome analysis revealed that chymase induced a proinflammatory gene transcription profile in HLFs, whereas tryptase had minimal effects. Chymase, but not tryptase, has a major impact on the phenotype of primary airway fibroblasts by modifying their output of extracellular matrix components and by inducing a proinflammatory phenotype.

Abnormal ADAM17 expression causes airway fibrosis in chronic obstructive asthma

Patients with chronic obstructive asthma (COA) develop airflow obstruction caused by subepithelial fibrosis. Although a disintegrin and metalloproteinase 17 (ADAM17) has been implicated in lung inflammation and tissue fibrosis, its role in airway fibrosis in COA has not been explored. Here, we found marked overexpression of ADAM17, phosphorylated ADAM17, and connective tissue growth factor (CTGF) in human airway fibroblasts from COA patients, compared with those of normal subjects. Similarly, levels of ADAM17, CTGF, α-smooth muscle actin (α-SMA), and collagen were increased in endobronchial biopsies from COA patients, but not in controls. In an ovalbumin-challenge asthma model, airway fibrosis was inhibited in ADAM17f/f/Cre+ mice compared to control mice. TGF-β- and thrombin-induced fibrotic protein expression was reduced by ADAM17 small interfering (si)RNA, TAPI-0 (an ADAM17 inhibitor), and EGFR siRNA. In addition, exogenous HB-EGF reversed fibrotic response in ADAM17 knockdown human lung fibroblasts. ADAM17 causes subepithelial fibrosis through regulation of enhanced extracellular matrix production and fibroblast differentiation and is the common pathway for airway fibrosis mediated by TGF-β and thrombin through an aberrant ADAM17/EGFR signalling pathway.

Effect of Prostaglandin D2 on mRNA Expression of Three Isoforms of Hyaluronic Acid Synthase in Nasal Polyp Fibroblasts.

Hyaluronan is one of the major extracellular matrixes in chronic rhinosinusitis (CRS) associated with tissue remodeling. Prostaglandin D2 (PGD2) is also associated with the pathogenesis of CRS. However, little is known about whether PGD2 regulates hyaluronan production by human airway fibroblasts. We sought to determine the effect of PGD2 on the mRNA expression of three isoforms of membrane-bound hyaluronic acid synthase (HAS1, HAS2 and HAS3) in fibroblasts, the major source of hyaluronan production, derived from CRS patients. Nasal polyp-derived fibroblasts (NPDF) and uncinate tissue-derived fibroblasts (UTDF) were established from CRS patients with nasal polyps and those without, respectively. These fibroblasts were stimulated with PGD2 or PGD2 receptor (DP/CRTH2)-selective agonists in the presence or absence of receptor-selective antagonists. mRNA levels for HAS1, HAS2 and HAS3 were determined by real-time quantitative PCR. PGD2 (1 µM) significantly enhanced HAS1 but not HAS2 or HAS3 mRNA expression by NPDF. Enhanced HAS1 mRNA expression was also obtained by stimulation with a DP receptor-selective agonist, but not with a CRTH2 receptor-selective agonist. In addition, PGD2-induced HAS1 mRNA expression was significantly inhibited by pre-treatment with DP receptor-selective antagonists. Similar induction of PGD2-induced HAS1 mRNA expression was seen in UTDF. PGD2 selectively stimulates HAS1 mRNA expression in local fibroblasts in CRS via DP, but not CRTH2, receptors.

β-Elemene suppresses the proliferation of human airway granulation fibroblasts via attenuation of TGF-β/Smad signaling pathway.

Benign airway stenosis is easily to recur as a result of hyperplastic airway granulation tissues. Our previous study proved that a Chinese drug, β-elemene, could inhibit the proliferation of fibroblasts cultured from these tissues, which could decrease the recurrence rate of this disease. To find out the mechanism for this effect, we first cultured normal human airway fibroblasts and human airway granulation fibroblasts with different concentrations of β-elemene for the same time or the same dose of β-elemene for different times to explorethe dose/time-effect relationship between the drug and these cells. Then we used gene microarray to screen out the most important pathway by which the drug influenced human airway granulation fibroblasts. At last, we assessed the condition of this pathway in human airway granulation fibroblasts and verified the effect of this drug on this pathway. β-Elemene inhibited the proliferation of human airway granulation fibroblasts in a dose but no time-dependent manner and did not affect normal human airway fibroblasts. Affecting the expression of transforming growth factor β (TGF-β)/Smad pathway may be the key mechanism for this effect. This pathway was activated in human airway granulation fibroblasts and β-elemene inhibited it in a dose-dependent manner. This effect was similar to the pathway inhibitor, SB431542, and exogenous TGF-β could attenuate this effect. These results indicated that suppressing TGF-β/Smad pathway was possibly the key mechanism by which β-elemene inhibits the proliferation of human airway granulation fibroblasts. This pathway may be a promising target to treat benign airway stenosis.

β-Elemene inhibits the proliferation of primary human airway granulation fibroblasts by down-regulating canonical Wnt/β-catenin pathway.

Benign airway stenosis is a clinical challenge because of recurrent granulation tissues. Our previous study proved that a Chinese drug, β-elemene, could effectively inhibit the growth of fibroblasts cultured from hyperplastic human airway granulation tissues, which could slow down the progression of this disease. The purpose of the present study is to find out the mechanism for this effect. We cultured fibroblasts from normal human airway tissues and human airway granulation tissues. These cells were cultured with 160 μg/ml normal saline (NS), different doses of β-elemene, or 10 ng/ml canonical Wnt/β-catenin pathway inhibitor (Dickkopf-1, DKK-1). The proliferation rate of cells and the expression of six molecules involved in canonical Wnt/β-catenin pathway, Wnt3a, glycogen synthase kinase-3β (GSK-3β), β-catenin, α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and Collagen I (Col-I), were measured. At last, we used canonical Wnt/β-catenin pathway activator (LiCl) to further ascertain the mechanism of β-elemene. Canonical Wnt/β-catenin pathway is activated in human airway granulation fibroblasts. β-Elemene didn’t affect normal human airway fibroblasts; however, it had a dose–responsive inhibitive effect on the proliferation and expression of Wnt3a, non-active GSK-3β, β-catenin, α-SMA, TGF-β, and Col-I of human airway granulation fibroblasts. More importantly, it had the same effect on the expression and nuclear translocation of active β-catenin. All these effects were similar to 10 ng/ml DKK-1 and could be attenuated by 10 mM LiCl. Thus, β-elemene inhibits the proliferation of primary human airway granulation fibroblasts by down-regulating canonical Wnt/β-catenin pathway. This pathway is possibly a promising target to treat benign tracheobronchial stenosis.

Helicase proteins DHX29 and RIG-I cosense cytosolic nucleic acids in the human airway system.

The recognition of cytoplasmic nucleic acid is critical for innate immune responses against microbial infection and is responsible for autoimmunity induced by dead cells. Here, we report the identification of a unique cytosolic nucleic acid cosensor in human airway epithelial cells and fibroblasts: DEAH (Asp-Glu-Ala-His) box polypeptide 29 (DHX29), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family. Knocking down DHX29 by siRNA attenuated the ability of cells to mount type I IFN and IL-6 in response to cytosolic nucleic acids and various viruses by blocking the activation of interferon regulatory factor 3 and NF-κB-p65. The cytosolic nucleic acid sensing by DHX29 in human epithelial cells and fibroblasts is independent of stimulator of interferon genes but is dependent on retinoic acid-inducible gene 1 (RIG-I) and mitochondrial antiviral signaling protein (MAVS). DHX29 binds directly to nucleic acids and interacts with RIG-I and MAVS through its helicase 1 domain, activating the RIG-I-MAVS-dependent cytosolic nucleic acid response. These results suggest that DHX29 is a cytosolic nucleic acid cosensor that triggers RIG-I/MAVS-dependent signaling pathways. This study will have important implications in drug and vaccine design for control of viral infections and viral-induced pathology in the airway.

Airway mesenchymal cell death by mevalonate cascade inhibition: Integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins

HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1α) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX−/−/BAK−/− murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease.

Expression of Myoferlin in Human Airway Epithelium and Its Role in Cell Adhesion and Zonula Occludens-1 Expression

BackgroundNormal airway epithelial barrier function is maintained by cell-cell contacts which require the translocation of adhesion proteins at the cell surface, through membrane vesicle trafficking and fusion events. Myoferlin and dysferlin, members of the multiple-C2-domain Ferlin superfamily, have been implicated in membrane fusion processes through the induction of membrane curvature. The objectives of this study were to examine the expression of dysferlin and myoferlin within the human airway and determine the roles of these proteins in airway epithelial homeostasis.MethodsThe expression of dysferlin and myoferlin were evaluated in normal human airway sections by immunohistochemistry, and primary human airway epithelial cells and fibroblasts by immuno blot. Localization of dysferlin and myoferlin in epithelial cells were determined using confocal microscopy. Functional outcomes analyzed included cell adhesion, protein expression, and cell detachment following dysferlin and myoferlin siRNA knock-down, using the human bronchial epithelial cell line, 16HBE.ResultsPrimary human airway epithelial cells express both dysferlin and myoferlin whereas fibroblasts isolated from bronchi and the parenchyma only express myoferlin. Expression of dysferlin and myoferlin was further localized within the Golgi, cell cytoplasm and plasma membrane of 16HBE cells using confocal micrscopy. Treatment of 16HBE cells with myoferlin siRNA, but not dysferlin siRNA, resulted in a rounded cell morphology and loss of cell adhesion. This cell shedding following myoferlin knockdown was associated with decreased expression of tight junction molecule, zonula occludens-1 (ZO-1) and increased number of cells positive for apoptotic markers Annexin V and propidium iodide. Cell shedding was not associated with release of the innate inflammatory cytokines IL-6 and IL-8.Conclusions/SignificanceThis study demonstrates the heterogeneous expression of myoferlin within epithelial cells and fibroblasts of the respiratory airway. The effect of myoferlin on the expression of ZO-1 in airway epithelial cells indicates its role in membrane fusion events that regulate cell detachment and apoptosis within the airway epithelium.

MAPK Regulation of IL-4/IL-13 Receptors Contributes to the Synergistic Increase in CCL11/Eotaxin-1 in Response to TGF-β1 and IL-13 in Human Airway Fibroblasts

CCL11/eotaxin-1 is a potent eosinophilic CC chemokine expressed by primary human fibroblasts. The combination of TGF-β1 and IL-13 synergistically increases CCL11 expression, but the mechanisms behind the synergy are unclear. To address this, human airway fibroblast cultures from normal and asthmatic subjects were exposed to IL-13 alone or TGF-β1 plus IL-13. Transcriptional (nuclear run-on) and posttranscriptional (mRNA stability) assays confirmed that transcriptional regulation is critical for synergistic expression of CCL11. TGF-β1 plus IL-13 synergistically increased STAT-6 phosphorylation, nuclear translocation, and binding to the CCL11 promoter as compared with IL-13 alone. STAT-6 small interfering RNA significantly knocked down both STAT-6 mRNA expression and phosphorylation and inhibited CCL11 mRNA and protein expression. Regulation of the IL-4Rα complex by TGF-β1 augmented IL-13 signaling by dampening IL-13Rα2 expression, overcoming IL-13's autoregulation of its pathway and enhancing the expression of CCL11. Our data suggest that TGF-β1 induced activation of the MEK/ERK pathway reduces IL-13Rα2 expression induced by IL-13. Thus, TGF-β1, a pleiotropic cytokine upregulated in asthmatic airways, can augment eosinophilic inflammation by interfering with IL-13's negative feedback autoregulatory loop under MEK/ERK-dependent conditions.

Similarities And Differences Between Lung Ligands For Mouse Siglec-f And Human Siglec-8

S A T U R D A Y 185 Pro-fibrotic Effect Of Dexamethasone In Human Airway Fibroblasts W. Manuyakorn, D. Davies, P. Howarth; Faculty of Medicine, University of Southampton, Southampton, UNITED KINGDOM, Faculty of Medicine, Ramathobodi Hospital, Mahidol University, Bangkok, THAILAND. RATIONALE: Airway remodelling in asthma is characterized by deposition of extracellular matrix proteins (ECMs). Collagen is the most abundant ECMprotein in the airways and its cross-linking by lysyl oxidase (LOX) influences its strength and stability. Inhaled corticosteroids are the standard treatment for chronic asthma. We hypothesized that corticosteroids may have a role in regulating the production of LOX by fibroblasts from asthmatic airways. METHODS: Bronchial fibroblasts fromnormal or asthmatic subjectswere treated with dexamethasone in the presence and absence of TGFb2. LOX and collagen I mRNA expression was measured using RT-qPCR. The level of LOX in culture supernatants was measured using western blot analysis. RESULTS: TGFb2 significantly up-regulated collagen I mRNA expression. In contrast, dexamethasone had no effect on collagen I mRNA expression. TGFb2 also up-regulated LOX mRNA expression in fibroblasts from both healthy and asthmatic subjects (p50.002). In fibroblasts from asthmatic donors only, dexamethasone up-regulated LOX mRNA expression and had an additive effect with TGFb2 (p50.02). Both TGFb2 and dexamethasone enhanced the production of pro-LOX and active LOX in fibroblasts from normal or asthmatic donors (p50.03). CONCLUSIONS: TGFb2 and dexamethasone up-regulated LOX expression by bronchial fibroblasts with the potential for increased cross-linking of collagen in the airways. These findings suggest that apart from their immediate anti-inflammatory effects, corticosteroids may have a long term detrimental effect on collagencross-linking, affecting airwaywallmechanics. Should suchanactionbeevident invivo this couldhave adetrimental effect on decline in lung function and disease persistence in asthma.

Simvastatin inhibits TGFβ1-induced fibronectin in human airway fibroblasts

BackgroundBronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.MethodsWe used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).ResultsImmunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.ConclusionsWe conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

MMP-2 Inhibition Attenuates IL-13 Induced Collagen Type-1 mRNA Expression in Human Airway Fibroblasts in Asthma

RATIONALE: Collagen is an extracellular matrix protein involved in airway fibrosis and has been implicated in the pathogenesis of airway remodeling in asthma. We have previously demonstrated that IL-13 increased collagen type 1 alpha 2 (COL1A2) mRNA and protein expression in human airway fibroblasts from asthmatic patients compared to normal controls. Our data has shown that this IL-13 induced expression may be attenuated using a non-specific Matrix Metalloproteinase (MMP) inhibitor suggesting that MMPs mediate IL-13 induced collagen expression.

Mevalonate Cascade Regulation of Airway Mesenchymal Cell Autophagy and Apoptosis: A Dual Role for p53

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.

IL-13 Induces Procollagen 1 mRNA Expression in Human Airway Fibroblasts in Asthma

IL-13 Induces Procollagen 1 mRNA Expression in Human Airway Fibroblasts in Asthma

Poly(I:C) induces BLyS-expression of airway fibroblasts through phosphatidylinositol 3-kinase

B lymphocyte stimulator (BLyS), B cell activating factor (BAFF), a member of the tumor necrosis factor ligand superfamily has potent co-stimulatory activity on B cells, and BLyS-production in the airway mucosa is of potential importance as it triggers innate and adaptive immune responses. To investigate whether airway fibroblast could express BLyS, we examined BLyS-expression in human nasal airway fibroblasts and compared to its expression in tonsillar and skin fibroblasts as well as the effect of the Toll-like receptor (TLR) ligands on that in human nasal airway fibroblasts. The expression of BLyS by nasal fibroblasts in the presence of polyinocinic-polycytidykic acid (poly(I:C)) was markedly induced, to a level of more than 100 times higher than that observed in the absence of poly(I:C). In order to demonstrate the intracellular pathways involved in poly(I:C)-induced BLyS-expression, we used specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), spleen tyrosine kinase (Syk), p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and extracellular-signal related kinase (ERK)-signaling in these events. Pre-incubation with the PI3-kinase inhibitor LY294002 or Wortmanin reversed the poly(I:C)-induced production and expression of BLyS. Syk kinase inhibitor Piceatannol partially reduced its production and expression. Thus, we were able to show that PI3-kinase signaling is directly involved in poly(I:C)-induced BLyS-expression in nasal airway fibroblasts. These results indicate that human nasal airway fibroblasts strongly induce BLyS-expression and production by poly(I:C) through PI3-K signaling during airway immune responses.

Prostaglandin E2 is activated by airway injury and regulates fibroblast cytoskeletal dynamics

To characterize the activation of cyclooxygenase (COX)-2/prostaglandin (PG) E2 signaling during airway mucosal repair and its subsequent role during the wound healing process. Prospective animal study. The subglottis was approached via cricothyroidotomy. Sham airways were closed, and wounded airways were subjected to laser injury and closed. Subglottic tissue was harvested at 12 hours, 24 hours, 48 hours, and 72 hours postinjury. Secretions were collected preoperatively and at time of sacrifice. Inflammatory gene expression was analyzed using quantitative reverse transcriptase polymerase chain reaction. Subglottic/tracheal explants were exposed to exogenous IL-1beta in the presence or absence of COX inhibitors. Explant-produced PGE2 levels were assayed using enzyme linked immunoassays. Human airway fibroblast migration and collagen contraction were assayed in the presence or absence of prostaglandin E2. Laser injury triggers a rapid, dose-dependent increase in mucosal IL-1beta and COX-2 gene expression, with an anatomical distribution proportional to the distance from the site of injury. Gene upregulation correlates with dose-dependent increases in PGE2 mucosal secretion levels. Ex vivo analysis indicates IL-1beta is responsible for the activation of the COX-2 / PGE2 pathway. Prostaglandin E2 differentially inhibits airway fibroblast migration and contraction in a specific, dose-dependent manner. PGE2 is activated during mucosal inflammation and acts to decrease fibroplastic activity in the mucosal wound bed. During subglottic stenosis (SGS) development, the levels of PGE2 generated in response to injury may be insufficient to blunt the intrinsically fibroplastic phenotype of SGS fibroblasts, resulting in excessive scarring.