Proliferation Of Human Airway Smooth Muscle Cells Research Articles

Long non-coding RNA MEG3 knockdown represses airway smooth muscle cells proliferation and migration via sponging miR-143-3p/FGF9 in asthma

BackgroundAsthma is a respiratory disease characterized by airway remodeling. We aimed to find out the role and mechanism of lncRNA MEG3 in asthma.MethodsWe established a cellular model of asthma by inducing human airway smooth muscle cells (HASMCs) with PDGF-BB, and detected levels of lncRNA MEG3, miR-143-3p and FGF9 in HASMCs through qRT-PCR. The functions of lncRNA MEG3 or miR-143-3p on HASMCs were explored by cell transfection. The binding sites of miR-143-3p and FGF9 were subsequently analyzed with bioinformatics software, and validated with dual-luciferase reporter assay. MTT, 5-Ethynyl-2’-deoxyuridine (EdU) assay, and Transwell were used to detect the effects of lncRNA MEG3 or miR-143-3p on proliferation and migration of HASMCs. QRT-PCR and western blot assay were used to evaluate the level of proliferation-related marker PCNA in HASMCs.ResultsThe study found that lncRNA MEG3 negatively correlated with miR-143-3p, and miR-143-3p could directly target with FGF9. Silence of lncRNA MEG3 can suppress migration and proliferation of PDGF-BB-induced HASMCs via increasing miR-143-3p. Further mechanistic studies revealed that miR-143-3p negatively regulated FGF9 expression in HASMCs. MiR-143-3p could inhibit PDGF-BB-induced HASMCs migration and proliferation through downregulating FGF9.ConclusionLncRNA MEG3 silencing could inhibit the migration and proliferation of HASMCs through regulating miR-143-3p/FGF9 signaling axis. These results imply that lncRNA MEG3 plays a protective role against asthma.

β-Tocotrienol Decreases PDGF-BB-Induced Proliferation and Migration of Human Airway Smooth Muscle Cells by Inhibiting RhoA and Reducing ROS Production.

Tocotrienols exhibit antioxidant and anti-inflammatory activities. RhoA, a small GTPase protein, plays a crucial role in regulating contractility in airway smooth muscle (ASM). Previous studies have demonstrated that γ-tocotrienols reduce ASM proliferation and migration by inhibiting the activation of RhoA. In this present study, we investigate the effect of another vitamin E isoform, β-tocotrienols, on human ASM cell proliferation and migration stimulated by platelet-derived growth factor-BB (PDGF-BB). Human ASM cells were pre-treated with β-tocotrienol prior to being stimulated with PDGF-BB to induce ASM cell proliferation and migration. The proliferation and migration of PDGF-BB-induced human ASM cells were assessed using colorimetric and transwell migration assays. The intracellular ROS assay kit was employed to quantify reactive oxygen species (ROS) in human ASM cells. Additionally, we explored the effect of β-tocotrienols on the signaling pathways involved in PDGF-BB-induced ASM proliferation and migration. β-tocotrienol inhibited PDGF-BB-induced ASM cell proliferation and migration by reducing RhoA activation and ROS production. However, in this present study, β-tocotrienol did not affect the signaling pathways associated with cyclin D1, phosphorylated Akt1, and ERK1/2. In conclusion, the inhibition of RhoA activation and ROS production by β-tocotrienol, resulting in the reduction in human ASM proliferation and migration, suggests its potential as a treatment for asthma airway remodeling.

CST1 promotes the proliferation and migration of PDGF-BB-treated airway smooth muscle cells via the PI3K/AKT signaling pathway.

Typically, airway remodeling caused by migration and proliferation of airway smooth muscle cells (ASMCs) plays a crucial role in the pathophysiological characteristics of asthma development. Cystatin 1 (CST1), a protein-coding gene referred to as Cystatin SN, is highly expressed in asthma patients. However, the role of CST1 and related molecular mechanisms in the development of asthma remains to be explored. This study aims to investigate the role of CST1 in asthma progression and present related molecular mechanisms. To explore these aspects, human ASMCs with platelet-derived growth factor BB (PDGF-BB) are initially stimulated and applied as a cellular model of asthma. Further, CST1 is knocked down with small interfering ribose nucleic acid (siRNA) overexpressed with plasmids. Then, 5-ethynyl-2'-deoxyuridine (EdU) and Cell Count Kit (CCK)-8 assays are applied to assess the cell proliferation rates. Further, Transwell and Western blot analyses for migration of cells and expression of MMP1 and MMP9 proteins are assessed, respectively. Under PDGF-BB stimulation, human ASMCs showed an increased CST1 expression, enhanced proliferation, and migration abilities, as well as up-regulated PI3K/AKT signaling pathway. Further, knockdown or overexpression of CST1 presented the declined or enhanced proliferation, migration, and up-regulation of the PI3K/AKT signaling pathway of human ASMCs. Inhibiting PI3K/AKT signaling pathway displayed the reduced migration and proliferation of human ASMCs. In summary, these findings indicated that CST1 played an essential role in the progression of asthma by activating the PI3K/AKT signaling pathway and promoting the migration and proliferation abilities of human ASMCs treated with PDGF-BB.

Circular RNA circ_0002594 regulates PDGF-BB-induced proliferation and migration of human airway smooth muscle cells via sponging miR-139-5p/TRIM8 in asthma

Background Asthma is a chronic respiratory disease characterised by the contraction of smooth muscle and remodelling of the airway wall, which is correlated with increased airway smooth muscle mass. Circular RNA (circRNA) circ_0002594 has been reported as a pro-inflammatory factor in allergic asthma. Therefore, this study is designed to explore the role and mechanism of circ_0002594 in human airway smooth muscle cells (HASMC) proliferation and metastasis. Methods Cell proliferative ability, invasion, and migration were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2’-deoxyuridine (EdU), Transwell, and Wound healing assays. The protein levels of E-cadherin, N-cadherin, and tripartite motif 8 (TRIM8) were detected by western blot assay. The levels of interleukin-6 (IL-6) and IL-13 were detected using Enzyme-linked immunosorbent assays (ELISA). Levels of circ_0002594, microRNA-139-5p (miR-139-5p), TRIM8 were determined by real-time quantitative polymerase chain reaction (RT-qPCR). The binding between miR-139-5p and circ_0002594 or TRIM8 was predicted by Circinteractome or Starbase v2.0, and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. Results Platelet-derived growth factor-BB (PDGF-BB) could trigger HASMC proliferation, metastasis, and inflammation. Circ_0002594 and TRIM8 were elevated in asthma patients and PDGF-BB-treated HASMC, and the miR-139-5p level was decreased. Furthermore, circ_0002594 knockdown could suppress PDGF-BB- stimulated HASMC damage. Mechanism analysis exhibited that circ_0002594 could regulate TRIM8 expression through sponging miR-139-5p. Conclusion Our findings revealed that circ_0002594 could act as a regulator in the airway remodelling during asthma development partly by the miR-139-5p/TRIM8 axis, hinting at an underlying therapeutic strategy for asthma.

MiR-21 modulates proliferation and apoptosis of human airway smooth muscle cells by regulating autophagy via PARP-1/AMPK/mTOR signalling pathway

Superfluous human airway smooth muscle (HASM) cell proliferation is an important pathological feature of airway remodelling in asthma. This study aimed to determine whether miR-21 is involved in the regulation of HASM cell survival. Overexpressed miR-21 inhibited HASM cell apoptosis and autophagy and promoted proliferation, whereas a miR-21 inhibitor exerted the opposite effects (P < 0.05). Overexpressed poly (ADP-ribose) polymerase-1 (PARP-1) promoted apoptosis and inhibited proliferation of HASM cells (P < 0.05). Dual-luciferase assays confirmed that miR-21 directly targeted poly (ADP-ribose) polymerase-1 (PARP-1) mRNA (P < 0.05). Silencing PARP-1 based on miR-21 downregulation mimicked the role of 3-methyladenine (3-MA), an autophagy inhibitor (P < 0.05). Overexpressed PARP-1 reversed the effects of miR-21 on HASM cells, somewhat dependently on PARP-1-induced enhanced autophagy, which we elucidated by 3-MA block (P < 0.05). MicroRNA-21 mimics reduced AMPK and increased mTOR signalling by downregulating PARP-1, and a miR-21 inhibitor exerted the opposite effects (P < 0.05). Collectively, miR-21 inhibitor could upregulate PARP-1 in HASM cells to promote autophagy and thus inhibit proliferation and promote apoptosis that might be mediated by the AMPK/mTOR signalling pathway.

Silencing of miR-10b-5p alleviates the mechanical stretch-induced proliferation of HASMCs

MicroRNAs (miRNAs) are important mediators to human airway smooth muscle cells (HASMCs) phenotype remodeling and airway diseases. MicroRNA-10b-5p (miR-10b-5p) has been extensively studied in different fields. This study set out to probe into the effect of miR-10b-5p in cyclic mechanical stretch-induced apoptosis in HASMCs. The results showed that after 15 % deformation, 0.5 s stretching and 0.5 s cyclic mechanical stretching relaxation (0.5 Hz) occurred to HASMCs, miR-10b-5p showed up-regulation without inducing significant apoptosis. Moreover, the mRNA and protein expressions of FLT1 were reduced. Then, dual-luciferase reporter assay verified that FLT1 was targeted by miR-10b-5p, and miR-10b-5p silencing increased FLT1 expression, leading to a prolonged arrest of stretch-treated HASMCs at the G1/S stage, and increased cell apoptosis compared with control group. Furthermore, the activity of Caspase-3 was reinforced, and the ratio of Bcl-2 to Bax was markedly reduced after miR-10b-5p silencing. The current study proved that expression levels of p-PI3K and p-Akt in stretch-treated HASMCs of the inhibition group were significantly inhibited in comparison to those of the controls. The effects of miR-10b-5p overexpression are opposite to that of inhibition of miR-10b-5p in stretched HASMCs. In conclusion, this study showed that miR-10b-5p silencing could weaken the hypertrophy of HASMCs. MiR-10b-5p negatively regulated FLT1 expression, but positively regulated the PI3K/Akt pathway in HASMCs. By referring to other previous studies, we concluded that miR-10b-5p might be a potent target in the treatment of airway diseases.

Long non-coding RNA TUG1 promotes proliferation and migration in PDGF-BB-stimulated HASMCs by regulating miR-216a-3p/SMURF2 axis

BackgroundAbnormal proliferation and migration of human airway smooth muscle cells (HASMCs) play an important role in the development of childhood asthma. Long non-coding RNAs (lncRNAs) have been demonstrated to participate in HASMC proliferation and migration. We aimed to explore more effects and molecular mechanism of taurine upregulated gene 1 (TUG1) in childhood asthma.ResultsTUG1 and SMURF2 were overexpressed and miR-216a-3p was downregulated in childhood asthma patients and PDGF-BB-stimulated HASMCs. TUG1 knockdown attenuated PDGF-BB-triggered proliferation and migration of HASMCs. MiR-216a-3p was targeted by TUG1, and miR-216a-3p suppression counteracted the repressive effects of TUG1 interference on proliferation and migration in PDGF-BB-treated HASMCs. SMURF2 was a downstream target of miR-216a-3p, and SMURF2 upregulation abated the inhibiting effects of miR-216a-3p on migration and proliferation in PDGF-BB-exposed HASMCs. TUG1 sponged miR-216a-3p to positively regulate SMURF2 expression.ConclusionTUG1 downregulation inhibited PDGF-BB-induced HASMC proliferation and migration by regulating miR-216a-3p/SMURF2 axis, offering novel insight into the potential application of TUG1 for childhood asthma treatment.

Role of NEAT1/MiR-9-5p/SLC26A2 Pathway on Human Airway Smooth Muscle Cell.

PurposeAsthma is a serious inflammatory disease of the respiratory system in which airway smooth muscle cells (ASMCs) play a key role. This study aimed to investigate the expression of SLC26A2 in human ASMCs (HASMCs) and the regulatory mechanism of SLC26A2 in the proliferation and inflammatory factor production of HASMCs.Materials and MethodsWe obtained the asthma-associated differential mRNA SLC26A2 by bioinformatics analysis in childhood acute asthma samples. To investigate its role in airway inflammation and airway remodeling, we treated HASMCs with platelet-derived growth factor (PDGF) in an in vitro model and determined SLC26A2 expression in cells using western blotting. Cell proliferation was detected by MTT and EdU assays, and cell contractile phenotype marker proteins were measured. Cell migration and production of inflammatory factors were determined by Transwell and ELISA assays. Additionally, the upstream regulatory miRNA and LncRNA of SLC26A2 were identified by bioinformatics, luciferase reporter gene, and RIP analyses.ResultsSLC26A2 was significantly upregulated in bioinformatics analysis of pediatric asthma-related sample. PDGF treatment up-regulated SLC26A2 expression in HASMCs, whereas the knockdown of SLC26A2 inhibited PDGF-stimulated proliferation, migration, and production of inflammatory factors, and enhanced the expression of cell contractile phenotype marker proteins in HASMCs. Luciferase reporter and RIP experiments validated that NEAT1 targeted miR-9-5p to regulate SLC26A2, thereby influencing the biological function of PDGF-induced HASMCs.ConclusionThese findings indicate that NEAT1-mediated miR-9-5p targeting of SLC26A2 inhibits the PDGF-induced proliferation and production of inflammatory factors in HASMCs. These findings highlight potential therapeutic targets for asthma and airway inflammation.

Comparisons of ATP‐competitive (Type I) versus function‐selective (Type IV) ERK Inhibitors to Prevent Airway Smooth Muscle Cell Proliferation

Hyperproliferation of airway smooth muscle (ASM) cells is a characteristic of airway remodeling associated with inflammatory diseases, such as asthma. Currently, there are no effective therapies to stop ASM cell proliferation that contributes to debilitating bronchoconstriction. Previous studies have shown that stimuli such as growth factors, cytokines, and cellular stress induce signaling through the extracellular signal‐regulated kinases (ERK1/2) in ASM cells making them a potential therapeutic target. Although several Type I selective ATP‐competitive inhibitors of ERK1/2 have been developed, these compounds block all enzymatic activity, including ERK1/2 functions in normal cells not associated with a disease. Given the ubiquitous expression and multiple functions of ERK1/2, the use of ATP‐competitive or catalytic site inhibitors may lead to unwanted off‐target effects. To mitigate off‐target toxicity, we previously identified a novel function‐selective Type IV ERK2 inhibitor, referred to as SF‐3‐030. In the current studies, SF‐3‐030 was compared to a known Type I inhibitor, ulixertinib, in regulating platelet‐derived growth factor (PDGF) induced ASM cell proliferation. We show that both SF‐3‐030 and ulixertinib can prevent PDGF‐mediated proliferation of human ASM cells through the disruption of activator protein‐1 (AP‐1) activity along with the inhibition of several key signaling pathways associated with asthma pathogenesis. SF‐3‐030 inhibited ASM cell proliferation and significantly affected the expression of Fos family and c‐Jun proteins to disrupt AP‐1 downstream activity. Proteomic analysis of PDGF‐stimulated ASM cells showed that SF‐3‐030 regulated a subset of proteins that were affected by ulixertinib. Approximately 40% of all proteins that increased, and 20% of all proteins that decreased after 8 and 24 h were common to both treatments indicating SF‐3‐030 and ulixertinib share overlapping mechanisms of action. Both SF‐3‐030 and ulixertinib inhibited PDGF induced TGF‐β signaling, which plays a role in airway remodeling associated with asthma. Unique to SF‐3‐030 was the inhibition of proinflammatory interleukin‐6, collagens, nuclear factor kappa‐light‐chain‐enhancer of activated B cell (NF‐kB), and the production of nitric oxide and reactive oxygen species that are involved in the pathogenesis of asthma. Overall, ulixertinib inhibited the expression of more proteins than SF‐3‐030 but the total number of proteins whose expression increased with SF‐3‐030 or ulixertinib was similar. These data suggest that Type IV function‐selective inhibition of ERK2 is sufficient to mitigate ASM cell proliferation in asthma while reducing possible off‐target effects seen with Type I ATP‐competitive inhibitors.Support or Funding InformationR21AI126492 from the National Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Saponins of Dioscorea Nipponicae Inhibits IL-17A-Induced Changes in Biomechanical Behaviors of In Vitro Cultured Human Airway Smooth Muscle Cells.

Airway hyperresponsiveness (AHR) is one of the main pathologic features of bronchial asthma, which is largely attributable to enhanced contractile response of asthmatic airway smooth muscle. Although β2 adrenergic receptor agonists are commonly used to relax airway smooth muscle for treating AHR, there are side effects such as desensitization of long-term use. Therefore, it is desirable to develop alternative relaxant for airway smooth muscle, preferably based on natural products. One potential candidate is the inexpensive and widely available natural herb saponins of Dioscorea nipponicae (SDN), which has recently been reported to suppress the level of inflammatory factor IL-17A in ovalbumin-induced mice, thereby alleviating the inflammation symptoms of asthma. Here, we evaluated the biomechanical effect of SDN on IL-17A-mediated changes of cultured human airway smooth muscle cells (HASMCs) in vitro. The stiffness and traction force of the cells were measured by optical magnetic twisting cytometry (OMTC), and Fourier transform traction microscopy (FTTM), respectively. The cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry, the cell migration was measured by cell scratch test, and the changes of cell cytoskeleton were assessed by laser confocal microscopy. We found that the stiffness and traction force of HASMCs were enhanced along with the increases of IL-17A concentration and exposure time, and SDN treatment dose-dependently reduced these IL-17A-induced changes in cell mechanical properties. Furthermore, SDN alleviated IL-17A-mediated effects on HASMCs proliferation, migration, and cytoskeleton remodeling. These results demonstrate that SDN could potentially be a novel drug candidate as bronchodilator for treating asthma-associated AHR.

TRPC channels mediated calcium entry is required for proliferation of human airway smooth muscle cells induced by nicotine-nAChR

The present study was designed to explore the role of transient receptor potential canonical 3 (TRPC3) in nicotine-induced chronic obstructive pulmonary disease (COPD) and its underlying mechanism. In this study, the expression and localization of α5 nicotinic acetylcholine receptor (α5-nAchR) in lung tissues were determined by western blotting and immunohistochemistry. The quantitative real-time PCR (qRT-PCR) analysis was performed to examine the mRNA expression levels of α5-nAchR and TRPC3 in human airway smooth muscle cells (HASMCs). Cell viability was assessed by CCK-8 assay. Proliferation was detected by cell counting and EdU immunofluorescent staining. Fluorescence calcium imaging was carried out to measure cytosolic Ca2+ ([Ca2+]cyt) concentration. The results showed that the α5-nAchR and TRPC3 expressions were significantly up-regulated in lung tissues of COPD smokers. Nicotine promoted HASMC proliferation, which was accompanied by elevated α5-nAchR and TRPC3 expressions, basal [Ca2+]cyt, store-operated calcium entry (SOCE) and the rate of Mn2+ quenching in HASMCs. Further investigation indicated that nicotine-induced Ca2+ response and TRPC3 up-regulation was reversibly blocked by small interfering RNA (siRNA) suppression of α5-nAChR. The knockdown of TRPC3 blunted Ca2+ response and HASMC proliferation induced by nicotine. In conclusion, nicotine-induced HASMC proliferation was mediated by TRPC3-dependent calcium entry via α5-nAchR, which provided a potential target for treatment of COPD.

PO187 Smoking as An Independent Risk Factor For Severe Coronary Disease In Japanese Population With Zero of Coronary Artery Calcium Score

The present study was designed to explore the role of transient receptor potential canonical 3 (TRPC3) in nicotine-induced chronic obstructive pulmonary disease (COPD) and its underlying mechanism. In this study, the expression and localization of α5 nicotinic acetylcholine receptor (α5-nAchR) in lung tissues were determined by western blotting and immunohistochemistry. The quantitative real-time PCR (qRT-PCR) analysis was performed to examine the mRNA expression levels of α5-nAchR and TRPC3 in human airway smooth muscle cells (HASMCs). Cell viability was assessed by CCK-8 assay. Proliferation was detected by cell counting and EdU immunofluorescent staining. Fluorescence calcium imaging was carried out to measure cytosolic Ca2+ ([Ca2+]cyt) concentration. The results showed that the α5-nAchR and TRPC3 expressions were significantly up-regulated in lung tissues of COPD smokers. Nicotine promoted HASMC proliferation, which was accompanied by elevated α5-nAchR and TRPC3 expressions, basal [Ca2+]cyt, store-operated calcium entry (SOCE) and the rate of Mn2+ quenching in HASMCs. Further investigation indicated that nicotine-induced Ca2+ response and TRPC3 up-regulation was reversibly blocked by small interfering RNA (siRNA) suppression of α5-nAChR. The knockdown of TRPC3 blunted Ca2+ response and HASMC proliferation induced by nicotine. In conclusion, nicotine-induced HASMC proliferation was mediated by TRPC3-dependent calcium entry via α5-nAchR, which provided a potential target for treatment of COPD.

Airway epithelial compression promotes airway smooth muscle proliferation and contraction.

During acute bronchoconstriction, the airway epithelium becomes mechanically compressed, as airway smooth muscle contracts and the airway narrows. This mechanical compression activates airway epithelium to promote asthmatic airway remodeling. However, whether compressed airway epithelium can feed back on the cause of bronchoconstriction has remained an open question. Here we examine the potential for epithelial compression to augment proliferation and contraction of airway smooth muscle, and thus potentiate further bronchoconstriction and epithelial compression. Well-differentiated primary human bronchial epithelial (HBE) cells maintained in air-liquid interface culture were mechanically compressed to mimic the effect of bronchoconstriction. Primary human airway smooth muscle (HASM) cells were incubated with conditioned media collected from mechanically compressed HBE cells to examine the effect of epithelial-derived mediators on HASM cell proliferation using an EdU assay and HASM cell contraction using traction microscopy. An endothelin receptor antagonist, PD-145065, was employed to probe the role of HBE cell-derived endothelin-1 on the proliferation and contraction of HASM cells. Conditioned media from compressed HBE cells increased HASM cell proliferation, independent of the endothelin-1 signaling pathway. However, conditioned media from compressed HBE cells significantly increased HASM cell basal contraction and histamine-induced contraction, both of which depended on the endothelin-1 signaling pathway. Our data demonstrate that mechanical compression of bronchial epithelial cells contributes to proliferation and basal contraction of airway smooth muscle cells and that augmented contraction depends on epithelial cell-derived endothelin-1. By means of both airway smooth muscle remodeling and contractility, our findings suggest a causal role of epithelial compression on asthma pathogenesis.

Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation.

Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways, and may point to a novel perception for blunting airway remodeling.

Contraction-dependent TGF-β1 activation is required for thrombin-induced remodeling in human airway smooth muscle cells

AimsThrombin is a serine proteinase that is not only involved in coagulation cascade, but also mediates a number of biological responses relevant to tissues repair, and induces bronchoconstriction. TGF-β plays a pivotal role in airway remodeling due to its effects on airway smooth muscle proliferation and extracellular matrix (ECM) deposition. Recently, bronchoconstriction itself is found to constitute a form of strain and is highly relevant to asthmatic airway remodeling. However, the underlying mechanisms remain unknown. Here, we investigated the role of contraction- dependent TGF-β activation in thrombin-induced remodeling in human airway smooth muscle (HASM) cells. Materials and methodsPrimary HASM cells were treated with or without thrombin in the absence or presence of anti-TGF-β antibody, cytochalasin D and formoterol. CFSE labeling index or CCK-8 assay were performed to test cell proliferation. RT-PCR and Western blotting were used to examined ECM mRNA level and collagen Iα1, α-actin protein expression, respectively. Immunofluorescence was also used to confirm contraction induced by thrombin in HASM cells. Key findingThrombin stimulation enhanced HASM cells proliferation and activated TGF-β signaling. Thrombin induced ECM mRNA and collagen Iα1 protein expression, and these effects are mediated by TGF-β. Abrogation of TGF-β activation by contraction inhibitors cytochalasin D and formoterol prevents the thrombin-induced effects. SignificanceThese findings suggest that contraction-dependent TGF-β activation could be a mechanism by which thrombin leads to the development of asthmatic airway remodeling. Blocking physical forces with bronchodilator would be an intriguing way in reducing airway remodeling in asthma.

The ProS Peptide of Porcine Cathelicidins Is an Active Component of Swine Concentrated Animal Feeding Operations (CAFOs) Dust that Mobilizes Calcium and Stimulates Proliferation in Human Airway Smooth Muscle Cells

Components in the dust from swine concentrated animal feeding operations (CAFOs) can induce inflammatory lung disease with features of chronic obstructive lung disease. An aqueous hogbarn dust extract (HDE) induces pro‐inflammatory responses in both airway cells in culture and in mouse airways in vivo, thus providing a starting point for identifying the dust molecules that are involved. Proteomics analysis of an EGF receptor‐activating peak from gel filtration of HDE revealed multiple fragments from the ProS peptide precursor of a subset of porcine anti‐bacterial cathelicidins. Antibodies against ProS confirmed its presence in HDE. The full‐length ProS peptide, without the N‐terminal signal peptide and the C‐terminal anti‐bacterial peptide, stimulated proliferation of human airway smooth muscle (HASM) cells assessed as [3H]thymidine incorporation. Stimulation was concentration‐dependent, with a maximal stimulation of 2‐ to 3‐fold, greater than that with EGF and somewhat less than with the G protein‐coupled receptor (GPCR) mitogen lysophosphatidic acid (LPA). ProS stimulation was blocked by the EGF receptor inhibitor AG1478 and the phospholipase C inhibitor U73122, but not by the Gq inhibitor YM‐254890 or the matrix metalloprotease inhibitor GM6001. ProS also stimulated a concentration‐dependent increase in intracellular calcium that was not blocked by AG1478 but was blocked by U73122 and by the Gq inhibitor YM‐254890. These data indicate that ProS is likely to be a novel Gq‐coupled GPCR agonist that mobilizes calcium in HASM cells via Gq activation but stimulates HASM cell proliferation through a Gq‐independent pathway involving EGF receptor activation. Whether and how the phospholipase C‐dependent calcium response may be related to the phospholipase C‐dependent proliferation response remains to be established. Further studies are in progress to identify the specific receptor and signaling pathways involved. The simultaneous formation of both an anti‐bacterial peptide to fight infection and the ProS peptide to activate EGF receptors to initiate cell proliferation and repair suggests a unique dual role for the cathelicidin precursor protein.Support or Funding InformationFunded by NIOSH 1RO1‐0H008539‐09 to DJR and UNMC Institutional Funds to MLT

Different effects of simvastatin on human airway smooth muscle cell proliferation induced by platelet activating factor

Determine whether and how simvastatin (Sim) may influence human airway smooth muscle cells (HASMCs) proliferation induced by platelet activating factor (PAF) through NADPH oxidase-ROS signaling pathway. Cell proliferation was analyzed according to the protocol of CCK-8 assay. Intracellular reactive oxygen species was measured by the fluorescence using an oxidation sensitive fluorescent probe, 2′7′-dichlorofluorescin diacetate (DCFHDA). The mRNA of Nox4, MnSOD, and Catalase were determined by real-time PCR. Protein levels of Nox4, phospho-ERK, total ERK were determined by western blotting. Inhibitor of MEK1/2was used to confirm the involved signal pathway. Our data demonstrated that PAF induced HASMCs proliferation with the maximal effect seen at 10-6 M and 24 h, the proliferation rate was (1.77 ± 0.51)as fold as the control group. Simvastatin (at the concentration of 10-5 M) was (0.67 ± 0.18), inhibited HASMCs proliferation induced by PAF (P<0.01).Compared with the negative control group (76.79 ± 6.05), PAF-treated group (98.89 ± 1.28) led to a significant increase in DCF fluorescence, PAF combined with simvastatin group (66.40 ± 2.87) decreased ROS level (P<0.01).PAF could increase Nox4 mRNA expression, reduced MnSOD, Catalase levels, simvastatin played opposite role. Treating HASMCs with simvastatin abrogated the phosphorylation of ERK induced by PAF. When added N-acetyl-cysteine (NAC) or PD98059 (ERK inhibitor) to PAF group separately, phosphorylation of ERK reduced at different degree. PAF induced the proliferation of HASMCs through NADPH oxidase-ROS signaling pathway, which was reversed by Simvastatin. Pretreatment of Antioxidant (NAC) significantly reduced ERK phosphorylation. The present study demonstrates ROS dependent activation of ERK/MAPK signaling way.

Defining an olfactory receptor function in airway smooth muscle cells.

Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma.

Human airway smooth muscle cell proliferation from asthmatics is negatively regulated by semaphorin3A.

Airway smooth muscle (ASM) hyperplasia is a key feature of airway remodeling in development of lung diseases such as asthma. Anomalous proliferation of ASM cells directly contributes to ASM hyperplasia. However, the molecular mechanisms controlling ASM cell proliferation are not completely understood. Semaphorins are versatile regulators of various cellular processes including cell growth and proliferation. The role of semaphorins in ASM cell proliferation has remained to be addressed. Here, we report that semaphorin 3A (Sema3A) receptor, neuropilin 1 (Nrp1), is expressed on human ASM cells (HASMC) isolated from healthy and asthmatic donors and treatment of these cells with exogenous Sema3A inhibits growth factor-induced proliferation. Sema3A inhibitory effect on HASMC proliferation is associated with decreased tyrosine phosphorylation of PDGFR, downregulation of Rac1 activation, STAT3 and GSK-3β phosphorylation. Bronchial sections from severe asthmatics displayed immunoreactivity of Nrp1, suggestive of functional contribution of Sema3A-Nrp1 axis in airway remodeling. Together, our data suggest Sema3A-Nrp1 signaling as a novel regulatory pathway of ASM hyperplasia.

TNFα-induced airway smooth muscle cell proliferation depends on endothelin receptor signaling, GM-CSF and IL-6

Pathological proliferation of human airway smooth muscle cells (HASMCs) causes hyperplasia in chronic lung diseases. Signaling pathways that link airway inflammation to HASMC proliferation might provide therapeutic targets for the prevention of airway remodeling and chronic lung diseases. Endothelin-1 (ET-1) signals via endothelin-A- and B-receptors (ETAR, ETBR) to perpetuate HASMC-associated and TNFα-dependent inflammatory processes. Hypothesis: endothelin receptor antagonists (ERAs) suppress HASMC proliferation induced by inflammatory cytokines. HASMCs were stimulated ex vivo with cytokines in the presence or absence of ERAs (ETAR-specific/selective: BQ123, ambrisentan; ETBR-specific: BQ788; non-selective: bosentan, macitentan, ACT-132577) or cytokine-blocking antibodies. Cell counts, DNA-synthesis (BrdU-incorporation assay), cytokine production (ELISA) and ETBR expression (whole-genome microarray data, western blot) were analyzed. ET-1-induced HASMC proliferation and DNA-synthesis were reduced by protein kinase inhibitors and ETAR-specific/selective ERAs but not by BQ788. TNFα-induced HASMC proliferation and DNA-synthesis were reduced by all ERAs. TNFα induced ET-1 and ETBR expression. TNFα- and ET-1-induced GM-CSF releases were both reduced by BQ123 and BQ788. TNFα- and ET-1-induced IL-6 releases were both reduced by BQ123 but not by BQ788. Combined but not single blockade of GM-CSF-receptor-α-chain and IL-6 reduced TNFα- and ET-1-induced HASMC proliferation and DNA-synthesis. Combined but not single treatment with GM-CSF and IL-6 induced HASMC proliferation and DNA-synthesis in the presence of ET-1. In conclusion, TNFα induces HASMC proliferation via ET-1/GM-CSF/IL-6. ETBR requires up-regulation by TNFα to mediate ET-1 effects on HASMC proliferation. This signaling cascade links airway inflammation to HASMC-associated remodeling processes and is sensitive to ERAs. Therefore, ERAs could prevent inflammation-induced airway smooth muscle hyperplasia.