Increased Airway Smooth Muscle Cell Research Articles

Kisspeptin/KISS1R Signaling Modulates Human Airway Smooth Muscle Cell Migration.

Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle (ASM) cell mass and upregulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of Kp (kisspeptin) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF [platelet-derived growth factor])-induced human ASM cell proliferation invitro and airway remodeling invivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of promigration proteins like CDC42 (cell division control protein 42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The antimigratory effect of KISS1R was abolished by PKA (protein kinase A)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of CREB (cAMP-response element binding protein) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.

Vascular endothelial growth factor induces the migration of human airway smooth muscle cells by activating the RhoA/ROCK pathway

BackgroundAirway remodeling due to increased airway smooth muscle cell (ASMC) mass, likely due to enhanced proliferation, hypertrophy, and migration, has been proven to be highly correlated with decreased lung function in asthma patients. Vascular endothelial growth factor (VEGF) mediates vascular and extravascular remodeling and inflammation and has been proven to be involved in the progression of asthma. Previous studies have focused on the effects of VEGF on ASMC proliferation, but few researchers have focused on the effects of VEGF on human ASMC migration. The purpose of this study was to explore the effect of VEGF on the migration of ASMCs and its related signaling pathway mechanism to provide evidence for the treatment of airway remodeling.MethodsWe examined the effects of VEGF induction on ASMC migration and explored the mechanisms involved in ASMC migration.ResultsWe found by wound healing and Transwell assays that VEGF promoted ASMC migration. Through the Cell Counting Kit-8 (CCK-8) experiment, we found that VEGF had no significant effect on the proliferation of ASMCs, which excluded the involvement of cell proliferation in the process of wound healing. Moreover, a cellular immunofluorescence assay showed that VEGF promoted F-actin reorganization, and Western blotting showed that VEGF improved RhoA activation and myosin phosphatase targeting subunit-1 (MYPT1) and myosin light chain (MLC) phosphorylation in ASMCs. Treatment with the ROCK inhibitor Y27632 significantly attenuated the effects of VEGF on MYPT1/MLC activation and cell migration.ConclusionIn conclusion, the results suggest that the promigratory function of VEGF activates the RhoA/ROCK pathway, induces F-actin reorganization, improves the migration of ASMCs, and provides a better rationale for targeting the RhoA/ROCK pathway for therapeutic approaches in airway remodeling.

Asthmatic Bronchial Matrices Determine the Gene Expression and Behavior of Smooth Muscle Cells in a 3D Culture Model.

Asthma is associated with increased deposition and altered phenotype of airway smooth muscle (ASM) cells. However, little is known about the processes responsible for these changes. It has been suggested that alterations of the extracellular matrix (ECM) contribute to the remodeling of ASM cells in asthma. Three-dimensional matrices allow the in vitro study of complex cellular responses to different stimuli in a close-to-natural environment. Thus, we investigated the ultrastructural and genic variations of ASM cells cultured on acellular asthmatic and control bronchial matrices. We studied horses, as they spontaneously develop a human asthma-like condition (heaves) with similarities to chronic pulmonary changes observed in human asthma. Primary bronchial ASM cells from asthmatic (n = 3) and control (n = 3) horses were cultured on decellularized bronchi from control (n = 3) and asthmatic (n = 3) horses. Each cell lineage was used to recellularize six different bronchi for 41 days. Histomorphometry on HEPS-stained-recellularized matrices revealed an increased ASM cell number in the control cell/control matrix (p = 0.02) and asthmatic cell/control matrix group (p = 0.04) compared with the asthmatic cell/asthmatic matrix group. Scan electron microscopy revealed a cell invasion of the ECM. While ASM cells showed high adhesion and proliferation processes on the control ECM, the presence of senescent cells and cellular debris in the asthmatic ECM with control or asthmatic ASM cells suggested cell death. When comparing asthmatic with control cell/matrix combinations by targeted next generation sequencing, only AGC1 (p = 0.04), MYO10 (p = 0.009), JAM3 (p = 0.02), and TAGLN (p = 0.001) were differentially expressed out of a 70-gene pool previously associated with smooth muscle remodeling. To our knowledge, this is the first attempt to evaluate the effects of asthmatic ECM on an ASM cell phenotype using a biological bronchial matrix. Our results indicate that bronchial ECM health status contributes to ASM cell gene expression and, possibly, its survival.

Cross-Talk between Transforming Growth Factor–β1 and Muscarinic M2 Receptors Augments Airway Smooth Muscle Proliferation

Transforming growth factor-β₁ (TGF-β₁) is a central mediator in tissue remodeling processes, including fibrosis and airway smooth muscle (ASM) hyperplasia, as observed in asthma. The mechanisms underlying this response, however, remain unclear because TGF-β₁ exerts only weak mitogenic effects on ASM cells. In this study, we hypothesized that the mitogenic effect of TGF-β₁ on ASM is indirect and requires prolonged exposure to allow for extracellular matrix (ECM) deposition. To address this hypothesis, we investigated the effects of acute and prolonged treatment with TGF-β₁, alone and in combination with the muscarinic receptor agonist methacholine, on human ASM cell proliferation. Acutely, TGF-β₁ exerted no mitogenic effect. However, prolonged treatment (for 7 d) with TGF-β₁ increased ASM cell proliferation and potentiated the platelet-derived growth factor-induced mitogenic response. Muscarinic receptor stimulation with methacholine synergistically enhanced the effect of TGF-β₁. Interestingly, the integrin-blocking peptide Arg-Gly-Asp-Ser, as well as integrin α5β1 function-blocking antibodies, inhibited the effects of TGF-β₁ and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-β₁ increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-β₁-induced proliferation were reduced by the long-acting muscarinic receptor antagonist tiotropium and the M₂ receptor subtype-selective antagonist gallamine, but not the M₃-selective antagonist DAU5884. In line with these findings, the irreversible Gi protein inhibitor pertussis toxin also prevented the potentiation of TGF-β₁-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-β₁ enhances ASM cell proliferation, which is mediated by extracellular matrix-integrin interactions, and which can be enhanced by muscarinic M₂ receptor stimulation.

The Role of IgE-Receptors in IgE-Dependent Airway Smooth Muscle Cell Remodelling

BackgroundIn allergic asthma, IgE increases airway remodelling but the mechanism is incompletely understood. Airway remodelling consists of two independent events increased cell numbers and enhanced extracellular matrix deposition, and the mechanism by which IgE up-regulates cell proliferation and extracellular matrix deposition by human airway smooth muscle cells in asthma is unclear.ObjectiveCharacterise the role of the two IgE receptors and associated signalling cascades in airway smooth muscle cell remodelling.MethodsPrimary human airway smooth muscle cells (8 asthmatics, 8 non-asthmatics) were stimulated with human purified antibody-activated IgE. Proliferation was determined by direct cell counts. Total collagen deposition was determined by Sircol; collagen species deposition by ELISA. IgE receptors were silenced by siRNA and mitogen activated protein kinase (MAPK) signalling was blocked by chemical inhibitors.ResultsIgE dose-dependently increased extracellular matrix and collagen deposition by airway smooth muscle cells as well as their proliferation. Specifically in cells of asthma patients IgE increased the deposition of collagen-type-I, -III, –VII and fibronectin, but did not affect the deposition of collagens type-IV. IgE stimulated collagen type-I and type-VII deposition through IgE receptor-I and Erk1/2 MAPK. Proliferation and deposition of collagens type-III and fibronectin involved both IgE receptors as well as Erk1/2 and p38 MAPK. Pre-incubation (30 minutes) with Omalizumab prevented all remodelling effects completely. We observed no changes in gelatinase activity or their inhibitors.Conclusion & Clincal RelevanceOur study provides the molecular biological mechanism by which IgE increases airway remodelling in asthma through increased airway smooth muscle cell proliferation and deposition of pro-inflammatory collagens and fibronectin. Blocking IgE action prevents several aspects of airway smooth muscle cell remodelling. Our findings may explain the recently described reduction of airway wall thickness in severe asthma patients treated with humanised anti-IgE antibodies.

Claudin-1 expression in airway smooth muscle exacerbates airway remodeling in asthmatic subjects

Increased airway smooth muscle (ASM) mass is an essential component of airway remodeling and asthma development, and there is no medication specifically against it. Tight junction (TJ) proteins, which are expressed in endothelial and epithelial cells and affect tissue integrity, might exist in other types of cells and display additional functions in the asthmatic lung. The aim of this study was to investigate the existence, regulation, and function of TJ proteins in ASM in asthmatic patients. The expression and function of TJ proteins in primary ASM cell lines, human bronchial biopsy specimens, and a murine model of asthma were analyzed by means of RT-PCR, multispectral imaging flow cytometry, immunohistochemistry, Western blotting, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester staining, tritiated thymidine incorporation, wound-healing assay, and luminometric bead array. Increased claudin-1 expression was observed in ASM of asthmatic patients, as well as in a murine model of asthma-like airway inflammation. Whereas IL-1β and TNF-α upregulated claudin-1 expression, it was downregulated by the T(H)2 cytokines IL-4 and IL-13 in primary human ASM cells. Claudin-1 was localized to the nucleus and cytoplasm but not to the cell surface in ASM cells. Claudin-1 played a central role in ASM cell proliferation, as demonstrated by increased ASM cell proliferation seen with overexpression and decreased proliferation seen with small interfering RNA knockdown of claudin-1. Overexpression of claudin-1 induced vascular endothelial growth factor and downregulated IL-6, IL-8, and IFN-γ-induced protein 10 production by ASM cells. Claudin-1 upregulation by IL-1β or TNF-α was suppressed by dexamethasone but not by rapamycin, FK506, or salbutamol. These results demonstrate that claudin-1 might play a role in airway remodeling in asthmatic patients by means of regulation of ASM cell proliferation, angiogenesis, and inflammation.

In VivoRole of Platelet-Derived Growth Factor–BB in Airway Smooth Muscle Proliferation in Mouse Lung

Airway smooth muscle (ASM) hyperplasia in asthma likely contributes considerably to functional changes. Investigating the mechanisms behind proliferation of these cells may lead to therapeutic benefit. Platelet-derived growth factor (PDGF)-BB is a well known ASM mitogen in vitro but has yet to be directly explored using in vivo mouse models in the context of ASM proliferation and airway responsiveness. To determine the in vivo influence of PDGF-BB on gene transcripts encoding contractile proteins, ASM proliferation, and airway physiology, we used an adenovirus overexpression system and a model of chronic allergen exposure. We used adenovirus technology to selectively overexpress PDGF-BB in the airway epithelium of mice. Outcome measurements, including airway physiology, real time RT-PCR measurements, proliferating cell nuclear antigen staining, and airway smooth muscle quantification, were performed 7 days after exposure. The same outcome measurements were performed 24 hours and 4 weeks after a chronic allergen exposure model. PDGF-BB overexpression resulted in airway hyperresponsiveness, decreased lung compliance, increased airway smooth muscle cell numbers, positive proliferating cell nuclear antigen-stained airway smooth muscle cells, and a reduction in genes encoding contractile proteins. Chronic allergen exposure resulted in elevations in lung lavage PDGF-BB, which were observed in conjunction with changes in gene transcript expression encoding contractile proteins and ASM proliferation. We demonstrate for the first time in vivo that PDGF-BB induces ASM hyperplasia and changes in lung mechanics in mice and that, during periods of allergen exposure changes in lung, PDGF-BB are associated with changes in airway structure and function.

Interferons modulate mitogen-induced protein synthesis in airway smooth muscle.

Severe asthma is characterized by increased airway smooth muscle (ASM) mass due, in part, to ASM cell growth and contractile protein expression associated with increased protein synthesis. Little is known regarding the combined effects of mitogens and interferons on ASM cytosolic protein synthesis. We demonstrate that human ASM mitogens including PDGF, EGF, and thrombin stimulate protein synthesis. Surprisingly, pleiotropic cytokines IFN-beta and IFN-gamma, which inhibit ASM proliferation, also increased cytosolic protein content in ASM cells. Thus IFN-beta alone significantly increased protein synthesis by 1.62 +/- 0.09-fold that was further enhanced by EGF to 2.52 +/- 0.17-fold. IFN-gamma alone also stimulated protein synthesis by 1.91 +/- 0.15-fold; treatment of cells with PDGF, EGF, and thrombin in the presence of IFN-gamma stimulated protein synthesis by 2.24 +/- 0.3-, 1.25 +/- 0.17-, and 2.67 +/- 0.34-fold, respectively, compared with growth factors alone. The mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) inhibition with rapamycin inhibited IFN- and EGF-induced protein synthesis, suggesting that IFN-induced protein synthesis is modulated by mTOR/S6K1 activation. Furthermore, overexpression of tumor suppressor protein tuberous sclerosis complex 2 (TSC2), which is an upstream negative regulator of mTOR/S6K1 signaling, also inhibited mitogen-induced protein synthesis in ASM cells. IFN-beta and IFN-gamma stimulated miR143/145 microRNA expression and increased SM alpha-actin accumulation but had little effect on ASM cell size. In contrast, EGF increased ASM cell size but had little effect on miR143/145 expression. Our data demonstrate that both IFNs and mitogens stimulate protein synthesis but have differential effects on cell size and contractile protein expression and suggest that combined effects of IFNs and mitogens may contribute to ASM cell growth, contractile protein expression, and ASM remodeling in asthma.

Epithelium‐derived chemokines induce airway smooth muscle cell migration

The remodelling of airway smooth muscle (ASM) associated with asthma severity may involve the migration of ASM cells towards the epithelium. However, little is known about the mechanisms of cell migration and the effect of epithelial-derived mediators on this process. The main objective of the current study is to assess the effects of epithelial-derived chemokines on ASM cell migration. Normal human ASM cells were incubated with supernatants from cells of the bronchial epithelial cell line BEAS-2B and normal human bronchial epithelial (NHBE) cells. To induce chemokine production, epithelial cells were treated with TNF-alpha. Chemokine expression by epithelial cells was evaluated by quantitative real-time PCR, ELISA and membrane antibody array. To identify the role of individual chemokines in ASM cell migration, we performed migration assays with a modified Boyden chamber using specific neutralizing antibodies to block chemokine effects. Supernatants from BEAS-2B cells treated with TNF-alpha increased ASM cell migration; migration was increased 1.6 and 2.5-fold by supernatant from BEAS-2B cells treated with 10 and 100 ng/mL TNF-alpha, respectively. Protein levels in supernatants and mRNA expression by BEAS-2B cells of regulated on activation, normal T cell expressed and secreted (RANTES) and IL-8 were significantly increased by 100 ng/mL TNF-alpha treatment. The incubation of supernatant with antibodies to RANTES or IL-8 significantly reduced ASM cell migration, and the combined antibodies further inhibited the cell migration. The migratory effects of supernatants and inhibiting effects of RANTES and/or IL-8 were confirmed also using NHBE cells. The results show that chemokines from airway epithelial cells cause ASM cell migration and might potentially play a role in the process of airway remodelling in asthma.

4E-Binding Protein Phosphorylation and Eukaryotic Initiation Factor-4E Release Are Required for Airway Smooth Muscle Hypertrophy

The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.

Protective Effects of Tiotropium Bromide in the Progression of Airway Smooth Muscle Remodeling

Recent findings have demonstrated that muscarinic M(3) receptor stimulation enhances airway smooth muscle proliferation to peptide growth factors in vitro. Because both peptide growth factor expression and acetylcholine release are known to be augmented in allergic airway inflammation, it is possible that anticholinergics protect against allergen-induced airway smooth muscle remodeling in vivo. We investigated the effects of treatment with the long-acting muscarinic receptor antagonist tiotropium on airway smooth muscle changes in a guinea pig model of ongoing allergic asthma. Twelve weekly repeated allergen challenges induced an increase in airway smooth muscle mass in the noncartilaginous airways. This increase was not accompanied by alterations in cell size, indicating that the allergen-induced changes were entirely from increased airway smooth muscle cell number. Morphometric analysis showed no allergen-induced changes in airway smooth muscle area in the cartilaginous airways. However, repeated ovalbumin challenge enhanced maximal contraction of open tracheal ring preparations ex vivo. This was associated with an increase in smooth muscle-specific myosin expression in the lung. Treatment with inhaled tiotropium considerably inhibited the increase in airway smooth muscle mass, myosin expression, and contractility. These results indicate a prominent role for acetylcholine in allergen-induced airway smooth muscle remodeling in vivo, a process that has been thus far considered to be primarily caused by growth factors and other mediators of inflammation. Therefore, muscarinic receptor antagonists, like the long-acting anticholinergic tiotropium bromide, could be beneficial in preventing chronic airway hyperresponsiveness and decline in lung function in allergic asthma.

Potential role of c-Jun NH 2-terminal kinase in allergic airway inflammation and remodelling: effects of SP600125

Asthma is a chronic inflammatory disease of the airways associated with structural changes such as increased airway smooth muscle mass, which may contribute to impairment of lung function. To determine whether c-Jun NH 2-terminal kinase (JNK) of the mitogen-activated protein kinase signalling pathway participated in these changes, the effects of an inhibitor, SP600125 (anthra [1, 9-cd] pyrazole-6 (2H)-one), were examined in a murine model of chronic airway inflammation and remodelling. Mice sensitised to ovalbumin were exposed to ovalbumin aerosol and were treated with SP600125 [30 mg kg −1 intraperitoneal (i.p.)] on days of exposure. SP600125 significantly reduced eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid, suppressed eosinophilic inflammation within the bronchial submucosa, inhibited goblet cell hyperplasia, and increased airway smooth muscle cell number in allergen-exposed mice. SP600125 also inhibited allergen-induced increase in bronchial responsiveness. SP600125 inhibited JNK activity in the challenged lungs. Although SP 600125 may also have other effects, we conclude that c-Jun NH 2-terminal kinase may play a role in allergen-induced inflammation and remodelling associated with bronchial hyperresponsiveness.

Human neutrophil-derived elastase induces airway smooth muscle cell proliferation

Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 μg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 μM), p38 kinase inhibitor (SB203580, 10 μM) or elastase inhibitor II (100 μg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D 1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.