Human Airway Smooth Muscle Research Articles

Asthma and inflammation transcriptionally up-regulate the aryl hydrocarbon receptor in airway smooth muscle via p38/JNK-AP1 signalling.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that maintains cellular homeostasis. AhR in airway fibroblasts, epithelial and immune cells inhibit inflammatory responses. Nevertheless, its expression and role in airway smooth muscle (ASM), an airway structural cell indispensable in asthma pathophysiology, are obscure. This study uncovers AhR expression, underlying mechanisms, and activity in ASM during inflammation and asthma. Cultured primary human nonasthmatic and asthmatic ASM cells were treated with TNFα/IL-13, with/without pharmacological inhibitors targeting PI3K, p38MAPK, JNK, NFkB, and AP1. AhR expression was analysed using RNA-sequencing, confocal microscopy, qPCR, and immunoblotting. AP1 specific role was confirmed using c-JUN (AP1) siRNA and ChIP-qPCR. AhR activity was determined by Nano luciferase in AhR agonist-treated cells. We found ubiquitous expression of AhR in ASM with up-regulation in asthmatic ASM. TNFα increased AhR expression, whereas IL-13 did not. Furthermore, p38 and JNK inhibition significantly reduced AhR expression with TNFα exposure, whereas PI3K inhibition had no effect. AP1 inhibition and c-JUN knockdown significantly down-regulated AhR expression, whereas NFkB inhibition showed no effect. TNFα promoted c-JUN binding to AhR promoter and increased AhR mRNA expression. Additionally, AhR agonists significantly increased the xenobiotic response element (XRE) activity, CYP1B1, and AhR nuclear expression. TNFα exposure reduced XRE activity and AhR nuclear expression. Our findings suggest inflammation and asthma transcriptionally up-regulates AhR through the p38/JNK-AP1 pathway in ASM, identifying a potential therapeutic target for modulating AhR and its downstream effects in asthma.

TFF-3 Modulates Responsiveness to Bronchodilators in Airways

Rhinovirus (RV) is the major cause of exacerbations, or worsening of symptoms, in asthmatic children and adults. This often reduces the efficacy of therapeutic interventions such as bronchodilators — a type of medication used to promote airflow and alleviate asthma symptoms. The exact mechanisms through which RV exposure decreases responsiveness to bronchodilators remain unclear. Previous data demonstrates that airway cells release a specific signature of inflammatory mediators following RV exposure. Other research has shown that Trefoil Factor 3 (TFF-3), one of the mediators identified by our screen, regulates cell motility in other cell types. We show that RV exposure attenuates relaxation in both the airway and human airway smooth muscle (HASM). Given our data, we aim to examine whether or not TFF-3 attenuates the relaxation of HASM and airways. Primary non-diseased human airway smooth muscle (HASM) was used to examine the consequences of TFF-3 in modulating HASM and airway relaxation. Following RV-C15 exposure, it was found that the airway and HASM relaxation was attenuated. TFF-3 exposure also attenuated both airway and HASM relaxation. Additionally, TFF-3 exposure partially weakened iso-induced reversal of carbachol-induced phosphorylation of the myosin light chain. Within the cADDis Live Cell Assays, which provide real-time kinetic measurements of cyclic Adenosine Monophosphate (cAMP) production, TFF-3 attenuated formoterol-induced cAMP production. Researching how bronchodilation pathways change following RV infection can lead to the development of effective treatments and pharmaceutical solutions to alleviate worsening asthma symptoms during a viral exacerbation of the disease.

Design and Evaluation of Novel Ginger 6-Shogaol-Inspired Phospholipase C Inhibitors to Enhance β-Agonist-Induced Relaxation in Human Airway Smooth Muscle.

Over 300 million people suffer from asthma, with many experiencing poor symptom control despite current medications. This highlights the need for novel therapeutics. Phospholipase Cβ (PLCβ), which contributes to bronchoconstriction, is a promising drug target. We previously identified 6-shogaol (6S) derivatives containing a dec-1,4-dien-3-one backbone as promising PLCβ inhibitors in cell-based assays. However, they failed to relax airways in mouse lung tissues. To improve efficacy, we synthesized 15 novel 6S derivatives. Most derivatives inhibited PLCβ activity and modulated downstream effectors, including inositol phosphates, diacylglycerol, intracellular calcium, and pMLC20. Notably, derivatives 8a and 8c exhibited enhanced inhibition in human airway smooth muscle cells without cytotoxicity. Structure-activity analysis revealed that a para-phenol-conjugated dec-1,4-dien-3-one core with additional meta-free hydroxyl groups in the phenol moiety of 6S derivatives significantly enhanced PLCβ inhibition. Importantly, these two derivatives also significantly potentiated β-agonist-induced relaxation in human tracheal tissue, highlighting their therapeutic potential as innovative asthma treatments.

TNFα-mediated subcellular heterogeneity of succinate dehydrogenase activity in human airway smooth muscle cells.

Tumor necrosis factor-α (TNFα) is a pro-inflammatory cytokine, which mediates acute inflammatory effects in response to allergens, pollutants, and respiratory infections. Previously, we reported that TNFα increased maximum O2 consumption rate (OCR) and mitochondrial volume density (MVD) in human airway smooth muscle (hASM) cells. However, TNFα decreased maximum OCR when normalized to mitochondrial volume. In addition, TNFα altered mitochondrial distribution and motility within hASM cells. Although high-resolution respirometry is valuable for assessing mitochondrial function, it overlooks mitochondrial structural and functional heterogeneity within cells. Therefore, a direct measurement of cellular mitochondrial function provides valuable information. Previously, we developed a confocal-based quantitative histochemical technique to determine the maximum velocity of the succinate dehydrogenase (SDH) reaction (SDHmax) in single cells and observed that cellular SDHmax corresponds with MVD. Therefore, we hypothesized that TNFα decreases SDHmax per mitochondrion in hASM cells. The hASM cells were treated with TNFα (20 ng/mL, 6 h, and 24 h) or untreated (time-matched control). Using three-dimensional (3-D) confocal imaging of labeled mitochondria and a concentric shell method for analysis, we quantified MVD, mitochondrial complexity index (MCI) and SDHmax relative to the nuclear membrane. Within each shell, SDHmax and MVD peaked in the perinuclear compartments and decreased toward the distal compartments of the cell. When normalized to mitochondrial volume, SDHmax decreased in the perinuclear compartments compared with distal compartments. TNFα caused a significant shift in mitochondrial morphometry and function compared to control. In conclusion, mitochondria within individual cells exhibit distinct morphological and functional heterogeneity, which is disrupted during acute inflammation.NEW & NOTEWORTHY Mitochondria show context-specific heterogeneity in their morphometry. Previously, we reported that acute TNFα exposure increased O2 consumption rate (OCR) and mitochondrial volume density, but decreased OCR per mitochondrion. TNFα also altered mitochondrial distribution and motility. To assess TNFα-mediated subcellular mitochondrial structural and functional heterogeneity, we used a confocal-based quantitative histochemical technique to determine the maximum velocity of succinate dehydrogenase reaction. Our findings highlight that mitochondria within cells exhibit functional heterogeneity, which is disrupted during inflammation.

Characterisation of the novel quinoline RCD405: Relaxant effects on cholinergic and histaminergic tone in human bronchi and small airways

Abstract Background and PurposeIncreased contractility of human airway smooth muscle (hASM) is a hallmark of asthma and chronic obstructive pulmonary disease (COPD). Developing new classes of bronchodilators has proved to be challenging because of efficacy and safety concerns. Quinolines hold potential therapeutic applications for the treatment of respiratory disorders.Experimental ApproachRelaxant effects of the novel quinoline RCD405 were investigated on contractile responses of hASM to carbachol, histamine and electrical field stimulation (EFS). The role of the non‐adrenergic non‐cholinergic (NANC) system was assessed using the inducible NO synthase inhibitor aminoguanidine and the TRPV1 agonist capsaicin.Key ResultsIn medium bronchi, RCD405 elicited a maximum relaxant effect (Emax) of 92 ± 4% with a half‐maximal effective concentration (EC50) of 45.71 μM for carbachol, and an Emax of 96 ± 1% with a EC50 of 12 μM for histamine. In small airways, RCD405 demonstrated significant relaxant responses, with an Emax of 54 ± 7% (EC50 17 μM) for carbachol and 90 ± 6% (EC50 20 μM) for histamine. RCD405 reduced contractility in response to EFS, with Emax values of 63 ± 10% at 25 Hz and 79 ± 9% at 50 Hz in medium bronchi. The NANC system did not affect the bronchorelaxation induced by RCD405.Conclusions and ImplicationsRCD405 showed significant potential as a novel bronchodilator drug for the treatment of asthma and COPD through its ability to induce relaxation of hASM. These findings suggest that further investigation of RCD405 is warranted as a possible novel treatment of chronic respiratory disorders.

Ovarian Cancer G protein-coupled receptor-1 signaling bias dictates anti-contractile effect of benzodiazepines on airway smooth muscle

BackgroundWe recently reported that the ovarian cancer G protein-coupled receptor-1 (OGR1) can be pharmacologically biased with specific benzodiazepines to couple with distinct heterotrimeric G proteins in human airway smooth muscle (ASM) cells. Lorazepam stimulated both Gs and Gq signaling via OGR1, whereas sulazepam only stimulated Gs signaling in ASM cells. The present study sought to determine the effects of sulazepam and lorazepam on contraction of human precision cut lung slices (hPCLS), and detail the biochemical mechanisms mediating these effects.MethodsModels of histamine (His) -stimulated contraction included imaging of ex vivo human precision cut lung slices (hPCLS) and Magnetic Twisting Cytometry (MTC) analysis of human ASM cell stiffness. To explore mechanisms of regulation, we examined effects on myosin light chain (pMLC) phosphorylation and PKA activity in primary human ASM cultures, as well as actin cytoskeleton integrity as defined by changes in the ratio of F to G actin assessed by immunofluorescence.ResultsIn a dose-dependent manner, sulazepam relaxed His-contracted hPCLS and reduced baseline cell stiffness. Lorazepam did not relax His-contracted hPCLS, and only at a maximal dose (100 μM) did lorazepam relax baseline cell stiffness. The Gs-biased ligand sulazepam stimulated PKA activity as evidenced by significant induction of VASP and HSP20 phosphorylation, which was associated with significant inhibition of His-induced pMLC phosphorylation. Conversely, the balanced ligand lorazepam did not significantly increase HSP20 phosphorylation or VASP phosphorylation and did not significantly inhibit His-induced MLC phosphorylation. Sulazepam was also able to inhibit histamine induced F-actin formation.ConclusionsThe Gs-biased OGR1 ligand sulazepam relaxed contracted ASM in both tissue- and cell- based models, via inhibition of MLC phosphorylation in a PKA-dependent manner and through inhibition of actin stress fiber formation. The relative inability of the balanced ligand lorazepam to influence ASM contractile state was likely due to competitive actions of concomitant Gq and Gs signaling.

Aryl Hydrocarbon Receptor Activation in Airway Smooth Muscle Maintains Mitochondrial Dynamics During Inflammation and Asthma

Rationale: Asthma is a chronic inflammatory disease of conducting airways characterized by airway hyperresponsiveness and remodeling. Airway smooth muscle (ASM) is a major structural cell in the airway that contributes to airway hyperresponsiveness and remodeling. Mitochondrial dynamics is a fission/fusion process crucial for optimal function and signal transduction. Inflammation alters the mitochondrial dynamics in ASM, thereby disrupt mitochondrial function and contributes to asthma pathophysiology. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates cellular homeostasis. Recently, we found upregulation of AhR in response to inflammation and asthma in ASM. Additionally, AhR activation inhibited inflammation-induced ASM proliferation and remodeling. However, the role of AhR in regulating mitochondrial dynamics in ASM during inflammation and asthma is obscure. Objective: The aim of this work is to unveil the role of AhR in regulating mitochondrial structure and function in human ASM during inflammation and asthma. Methods: Primary non-asthmatic and asthmatic human ASM cells isolated from surgical lung sections were cultured in DMEM-F12 medium supplemented with 10% FBS and 1% AbAm. Cells were serum deprived, treated with AhR agonist 6-Formylindolo[3,2-b]carbazole (FICZ: 10 nM) and antagonist CH223191 (10 μM), with/or without TNFα (20 ng/mL). Mitochondrial bioenergetics was determined by Seahorse XFp analyzer. Mitochondrial morphology was visualized using mitotracker dye. Expressions of fission and fusion mRNA and protein were determined by qPCR and immunoblotting. Additionally, AhR role in fusion and fission was confirmed in shRNA mediated AhR knockdown and AhR overexpressed ASM cells. Results: AhR activation by FICZ alleviated TNFα-induced disrupted mitochondrial bioenergetics in non-asthmatic ASM cells as well as in asthmatic ASM. Furthermore, TNFα-induced increased mitochondrial fission and reduced fusion was restored by AhR activation in both asthmatic and non-asthmatic ASM. AhR inhibition by CH223191 and knockdown promoted mitochondrial fission and blunted fusion in non-asthmatic ASM cells. Interestingly, AhR overexpression and activation by FICZ restored the mitochondrial dynamics altered by inflammation and asthma in ASM cells. Conclusion: Collectively, AhR activation promotes mitochondrial fusion and abolishes fission induced by inflammation and asthma in human ASM, thereby maintains mitochondrial structure and homeostasis. Therefore, AhR is a potential therapeutic target to mitigate altered mitochondrial dynamics in ASM during inflammation and asthma. Supported by NIH grants R01-HL171245 (Venkatachalem and Britt), R01-HL146705 (Venkatachalem), R01-HL155095 (Britt), R01-HL142061 (Pabelick, Prakash) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

The Acute Force Relaxation Effect of Pitavastatin Depends on Cofilin's Rapid Disassembly of F-actin in Human Airway Smooth Muscle

The Acute Force Relaxation Effect of Pitavastatin Depends on Cofilin's Rapid Disassembly of F-actin in Human Airway Smooth Muscle

Class C GPRC5A in Human Airway Smooth Muscle

Class C GPRC5A in Human Airway Smooth Muscle

Molecular Regulation of Mitochondrial Biogenesis by Mitophagy Mediated Inhibition of PARIS

Mitochondrial volume and quality are tightly regulated by two key mechanisms: removal of dysfunctional/damaged mitochondria by selective autophagy (mitophagy) and mitochondrial biogenesis. Disruption of either one or both processes results in the accumulation of dysfunctional mitochondria and increased oxidative stress. Emerging evidence suggest that mitophagy and mitochondrial biogenesis are tightly coordinated, working in concert to maintain mitochondrial homeostasis and quality control. Previously, we reported that tumor necrosis factor α (TNFα), a proinflammatory cytokine produced during inflammatory airway disease, promotes mitochondrial biogenesis by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling, the nodal regulator of mitochondrial biogenesis, thereby increasing mitochondrial volume density in human airway smooth muscle (hASM) cells. Additional evidence indicates that TNFα induces mitochondrial depolarization, which is known to trigger a mitophagy pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, an E3 ubiquitin ligase. Mitochondrial depolarization facilitates the mitochondrial accumulation of PINK1 and its activation by autophosphorylation at ser228 (pPINK1s228). pPINK1s228 promotes the recruitment and phosphorylation of Parkin at ser65 (pParkins65), which ubiquitylates several mitochondrial associated proteins promoting the removal of damaged mitochondria. The decrease in the number of functional mitochondria by mitophagy can be offset by increasing mitochondrial biogenesis through the upregulation of PGC-1α signaling. Therefore, we hypothesize that PARIS (ZNF746), a transcriptional repressor of PGC-1α, is a target of PINK1/Parkin-mediated ubiquitination and degradation in hASM cells. In the present study, primary hASM cells were isolated from bronchiolar tissue of 6 subjects with no history of chronic pulmonary disease or smoking and separated into the following treatment groups: 1) FCCP (1 µM, 6 h; positive control), a proton ionophore that induces mitochondrial depolarization; 2) TNFα (20 ng/mL, 6 h); and 3) untreated control. We observed that FCCP and TNFα-induced mitochondrial depolarization triggered mitochondrial translocation of PINK1 and increased phosphorylation of pPINK1s228. Additionally, phosphorylation of pParkins65 and ubiquitination of several mitochondrial associated proteins was increased. Immunoprecipitation of ubiquitinated proteins revealed increased ubiquitination of PARIS, while total expression of PARIS was decreased. Mitochondria and lysosomes were labelled with MitoTracker green (200 nM) and LysoTracker red (100 nM), respectively, and imaged in 3D (Z optical slice of 0.5 μm) to demonstrate the coefficient for colocalization of mitochondria with lysosomes. We observed increased formation of mitophagosomes, characteristic of mitophagy. Our findings demonstrate that TNFα-induces both mitophagy and mitochondrial biogenesis via the activation of PINK1/Parkin-mediated mitophagy pathway. Such coordination of mitophagy and mitochondrial biogenesis has significant ramifications for the maintenance of mitochondrial homeostasis, quality control and cellular energy metabolism. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Molecular Mechanism Underlying TNFα-Induced Proliferation of Human Airway Smooth Muscle Cells

Airway inflammation occurs as a defense mechanism in response to pathogens, allergens, toxins, or pollutants. The physiological responses of airway inflammation are mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), that causes airway remodeling, characterized by airway smooth muscle (ASM) hyperreactivity and proliferation. The molecular mechanism underlying TNFα-induced ASM cell proliferation is not fully understood. Previous studies from our lab demonstrated that TNFα exposure induces accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggering an ER stress response that involves autophosphorylation of inositol requiring enzyme 1α at Serine 724 (pIRE1αS724). pIRE1αS724 promotes alternative splicing of X-box binding protein 1 (XBP1s) mRNA. XBP1s acts as a ubiquitous transcription factor in ASM cells, targets a number of genes associated with different cellular responses. In the present study, we hypothesized that TNFα promotes XBP1s mediated transcriptional activation of the Cyclin B1, a protein essential for the control of the cell cycle, thus promoting ASM cell proliferation. Human ASM (hASM) cells were dissociated from bronchiolar tissue samples collected during lung surgery from female and male patients (49 to 61 years of age) with no history of chronic respiratory diseases or current smoking. Isolated hASM cells from the same patient were serum-deprived for 48 h and separated into two groups: 1) untreated controls, and 2) treated with TNFα (20 ng/ ml) for 6 h. To confirm the effect of TNFα on hASM cell proliferation, loss of function experiments were employed by: 1) transfecting hASM cells with a non-spliceable XBP1 mutant, and 2) treating the cells with 4μ8C, a pharmacological inhibitor for IRE1α mediated XBP1 splicing. hASM cell proliferation was measured using CyQuant cell proliferation assay. The binding site sequence of XBP1s to the Cyclin B1 promoter was identified by bioinformatic analysis and confirmed by chromatin immunoprecipitation (ChIP) assay. mRNA and protein expressions of Cyclin B1 were determined by qRT-PCR and Western blot respectively. Nuclear localization of Cyclin B1 was measured by nuclear fractionation, followed by western blot of nuclear and cytosolic fraction. Results were analyzed by paired t-tests. TNFα treatment for 6 h induced pIRE1αS724 phosphorylation and XBP1s splicing. Bioinformatic analysis revealed the presence of an XBP1s binding site on the Cyclin B1 promoter. The ChIP assay confirmed that XBP1s binds to the promoter region of the Cyclin B1 genes, and the binding affinity was increased in TNFα-treated hASM cells. Consistent with these results, we found that TNFα treatment increased both mRNA and protein expressions of Cyclin B1 in hASM cells. Nuclear localization of Cyclin B1 increased significantly in TNFα treated cells consistent with an increase in hASM cell proliferation. Inhibition of XBP1 splicing reduced the expression of Cyclin B1 in hASM cells and hASM cell proliferation. In summary, TNFα induces an ER stress response involving pIRE1αS724 phosphorylation and XBP1s splicing. In turn, XBP1s transcriptionally targets expression of Cyclin B1 that mediates hASM cell proliferation. Supported by NIH grant HL157984 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Data-independent acquisition (DIA) analysis shows specific regulation of TGF-β1 on airway smooth muscle cell extracellular matrix (ECM) protein interactions

Introduction: Throughout the lifetime of an asthmatic, the lung undergoes changes in the composition of its extracellular matrix (ECM) and becomes more fibrotic, a characteristic feature of airway remodeling. Due to this, lung functionality decreases as the lung becomes less elastic and airway resistance increases. The ECM proteins constantly network with their respective mechanosensory receptors and regulatory proteins on the cell membrane. These signaling interactions allow for continuous interactions and feedback between the ECM and airway smooth muscle cells (ASM). ASM cells are one of the major structural cells that regular airway remodeling and are the primary target for treating asthma. In this study, we investigated the specific alterations that occur in the ECM of ASM cells treated with TGF-β1. Methods: Human primary airway smooth muscle (ASM) cells were grown to confluency in DMEM/F-12 with 10% FBS and 1% AbAm in standard conditions. Cells were serum deprived for 48 hours prior to experiments. Cells were treated with TGF-β1 (2 ng/mL) in serum free media. Cells were harvested after 48 hours and 200 µg of protein lysate was prepared. Data-independent acquisition (DIA) mass spectrometry was performed at IDeA National Resource for Quantitative Proteomics. Fold changes for ECM class proteins were collected from DIA results. Pathway analysis was then done using Qiagen ingenuity pathway analysis. Furthermore, expression patterns of select proteins were confirmed via western blotting. Results: The effects of TGF-β1 on specific types of collagens, integrins, laminins, and additional ECM proteins were analyzed. Western blotting confirmed similar changes in expression as measured by DIA. TGF-β1 exposure upregulated LAMA4, LAMB2, and LAMC1 which is likely to increase the prevalence of Laminins 221 and 421. TGF-β1 augmented integrin signaling by upregulating ITGA7, ITGA11, ITGA1, ITGB1 and downregulating ITGA2 and ITGB8. TGF-β1 increased expression of TSP1, TSP2, TSP3, TNS-C, FB, Elastin and Versican while downregulating TNS-X. Several interactions between ECM proteins and their sensors and receptors were highlighted for their enrichment. Conclusion: The DIA analysis shows an upregulation of proteins involved in airway remodeling by TGF-β1 in ASM cells. These findings substantiate our previous results as well as provide insights into a more targeted approach in studying ECM-Cell interactions. NIH R01HL171245 (Venkatachalem), R01HL146705 (Venkatachalem), R01HL142061 (Pabelick, Prakash). and IDeA National Resource for Quantitative Proteomics (R24GM137786). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Kinase-mediated Phosphorylation of Glucocorticoid Receptors in Human Airway Smooth Muscle Cells

Kinase-mediated Phosphorylation of Glucocorticoid Receptors in Human Airway Smooth Muscle Cells

METTL3 regulates ASMCs proliferation and M2 macrophage polarization via mediating the m6A methylation of TIMMDC1 in asthma.

METTL3 regulates ASMCs proliferation and M2 macrophage polarization via mediating the m6A methylation of TIMMDC1 in asthma.

Role of Nestin in IL-17A Induced Human Airway Smooth Muscle Cell Proliferation

Role of Nestin in IL-17A Induced Human Airway Smooth Muscle Cell Proliferation

Mitochondrial Reactive Oxygen Species and Endoplasmic Reticulum Stress in Hyperoxia-induced Senescence in Developing Human Airway Smooth Muscle

Mitochondrial Reactive Oxygen Species and Endoplasmic Reticulum Stress in Hyperoxia-induced Senescence in Developing Human Airway Smooth Muscle

TRPC6 Channel Regulates Airway Remodeling in Chronic Obstructive Pulmonary Disease Causing Right Heart Failure.

The role of the canonical transient receptor potential 6 (TRPC6) channel in chronic obstructive pulmonary disease (COPD) remains poorly understood at the mechanistic level. Objects: This study aims to investigate the involvement of TRPC6 in COPD and its signaling mechanisms in human airway smooth muscle cells (HASMCs). Methods and Results: The study found that mRNA and protein expression of TRPC6 increased in cultured HASMCs that were incubated with nicotine, as measured by reverse transcription quantitative polymerase chain reaction and Western blot analysis. Nicotine treatment significantly enhanced TRPC6 transcriptional activity in HASMCs through nuclear factor (NF)-κB, as demonstrated by co-immunoprecipitation and electrophoretic mobility shift assays. Furthermore, miR-135a/b-5p was shown to downregulate TRPC6 expression in HASMCs at the mRNA and protein levels, as confirmed by luciferase reporter assays. Immunohistochemistry assays in a mouse model of cigarette-induced airway remodeling revealed a significant increase in smooth muscle (SM) cell proliferation and SM layer mass. Conclusion: These findings suggest that nicotine exposure increases HASMC proliferation and migration through NF-κB signaling, and that cigarette smoke inhalation causes airway SM layer remodeling via altered TRPC6-induced Ca2+ influx, which is abolished by miR-135a/b-5p both in vitro and invivo. Antioxid. Redox Signal. 42, 480-493.

PGAP3 is expressed at increased levels in asthmatic ASM and is associated with increased ASM proliferation, contractility and expression of GATA3 and ALOX5.

Post-GPI Attachment to Proteins phospholipase 3 (PGAP3) is a glycosylphosphatidylinositol (GPI) anchor-remodeling gene found on chromosome 17q12-21, which is a locus highly linked to asthma. Genetic association studies have linked PGAP3 SNPs to increased PGAP3 expression as well as asthma exacerbations, severity, and susceptibility. This study compared the levels of PGAP3 mRNA expression quantitated by RT-qPCR in human bronchial airway smooth muscle cells derived from postmortem lungs of asthmatics (ASM-A) to that derived from control non-asthmatics (ASM-NA). ASM-A expressed significantly higher levels of PGAP3 mRNA compared to ASM-NA. As ASM-A expressed higher levels of PGAP3 mRNA we performed functional studies of ASM-NA transfected with PGAP3 to determine if increased PGAP3 expression in ASM influenced ASM function including proliferation and contractility. Functional studies of ASM transfected with PGAP3 demonstrated that increased PGAP3 expression in ASM resulted in increased ASM proliferation and contractility. RNA-seq studies of ASM transfected with PGAP3 demonstrated significantly increased levels of genes linked to asthma including GATA3 and ALOX5. Fifteen genes upregulated by PGAP3 in ASM-NA were detected in asthmatic ASM data sets, underscoring the ability of PGAP3 to induce genes of importance to asthma in ASM. In summary, this study made the novel observation that ASM derived from the lungs of asthmatics express higher levels of PGAP3 compared to non-asthmatics. In addition, when ASM from non-asthmatics are transfected with PGAP3, the increased levels of PGAP3 increase ASM proliferation and contractility, and increase levels of genes previously linked to asthma including GATA3 and ALOX5. Overall, these studies suggest that increased PGAP3 expression in ASM plays a functional role in contributing to the pathogenesis of asthma.

Integrative Roles of Pro-Inflammatory Cytokines on Airway Smooth Muscle Structure and Function in Asthma.

Asthma has become more appreciated for its heterogeneity with studies identifying type 2 and non-type 2 phenotypes/endotypes that ultimately lead to airflow obstruction, airway hyperresponsiveness, and remodeling. The pro-inflammatory environment in asthma influences airway smooth muscle (ASM) structure and function. ASM has a vast repertoire of inflammatory receptors that, upon activation, contribute to prominent features in asthma, notably immune cell recruitment and activation, hypercontractility, proliferation, migration, and extracellular matrix protein deposition. These pro-inflammatory responses in ASM can be mediated by both type 2 (e.g., IL-4, IL-13, and TSLP) and non-type 2 (e.g., TNFα, IFNγ, IL-17A, and TGFβ) cytokines, highlighting roles for ASM in type 2 and non-type 2 asthma phenotypes/endotypes. In recent years, there has been considerable advances in understanding how pro-inflammatory cytokines promote ASM dysfunction and impair responsiveness to asthma therapy, corticosteroids and long-acting β2-adrenergic receptor agonists (LABAs). Transcriptomic analyses on human ASM cells and tissues have expanded our knowledge in this area but have also raised new questions regarding ASM and its role in asthma. In this review, we discuss how pro-inflammatory cytokines, corticosteroids, and LABAs affect ASM structure and function, with particular focus on changes in gene expression and transcriptional programs in type 2 and non-type 2 asthma.

Oxylipin Profiling of Airway Structural Cells Is Unique and Modified by Relevant Stimuli.

Oxylipins, diverse lipid mediators derived from fatty acids, play key roles in respiratory physiology, but the contribution of lung structural cells to this diverse profile is not well understood. This study aimed to characterize the oxylipin profiles of airway smooth muscle (ASM), lung fibroblasts (HLF), and epithelial (HBE) cells and define how they shift when they are exposed to stimuli related to contractility, fibrosis, and inflammation. Using HPLC-MS/MS, 162 oxylipins were measured in baseline media from cultured human ASM, HLF, and HBE cells as well as after stimulation with modulators of contractility and central regulators of fibrosis/inflammation. At the baseline, ASM and HLF cells had the most similar oxylipin profiles, dominated by oxylipins from cytochrome P450 (CYP450) epoxygenase metabolites. TGFβ stimulation of HLF suppressed CYP450-derived oxylipins, while ASM stimulation increased prostaglandin production. HBE showed the most distinct baseline profile enriched with cyclooxygenase (COX)-derived oxylipins. TGFβ stimulation of HBE increased the level of several oxylipins from CYP450 epoxygenases. These findings highlight the importance of CYP450 oxylipins, which are relatively unexplored in the context of respiratory physiology. By resolving these oxylipin profiles, we enable future respiratory research to understand the function of these oxylipins in regulating physiology, especially in the context of modifying contraction and inflammation.