Asthmatic Airway Smooth Muscle Research Articles

Downregulation of protein phosphatase 2Aα in asthmatic airway smooth muscle.

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca2+]i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca2+]i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca2+]i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

Unveiling the potent effect of vitamin D: harnessing Nrf2/HO-1 signaling pathways as molecular targets to alleviate urban particulate matter-induced asthma inflammation

BackgroundAsthma is the most common allergic disease characterized by an inflammatory response in the airways. Mechanismly, urban particulate matter (PM) is the most widely air pollutant associated with increased asthma morbidity and airway inflammation. Current research found that vitamin D is an essential vitamin with anti-inflammatory, antioxidant and other medical efficacy. Inadequate or deficient vitamin D often leads to the pathogenesis and stability of asthma. NGF exacerbates airway inflammation in asthma by promoting smooth muscle cell proliferation and inducing the Th2 immune response. Activation of the Nrf2/HO-1 signaling pathway can exert a protective effect on the inflammatory response in bronchial asthma. However, the specific mechanism of this pathway in PM-involved asthmatic airway smooth muscle cells remains unclear.MethodsMice were sensitized and challenged with Ovalbumin (OVA) to establish an asthma model. They were then exposed to either PM, vitamin D or a combination of both, and inflammatory responses were observed. Including, acetylcholine stimulation at different concentrations measured airway hyperresponsiveness in mice. Bronchoalveolar lavage fluid (BALF) and serum were collected for TNF-α, IL-1β, IL-6, and Nerve growth factor (NGF) analysis. Additionally, lung tissues underwent histopathological examination to observe alveolar structure and inflammatory cell infiltration. Specific ELISA kits were utilized to determine the levels of the inflammatory factors TNF-α, IL-1β, IL-6, and Nerve growth factor (NGF). Nrf2/HO-1 signaling pathways were examined by western blot analysis. Meanwhile, we constructed a cell system with low HO-1 expression by lentiviral transfection of airway smooth muscle cells. The changes of Nrf2, HO-1, and NGF were observed after the treatment of OVA, PM, and Vit D were given.ResultsThe in vivo results showed that vitamin D significantly alleviated pathological changes in lung tissue of PM-exposed mice models. Mechanismly, vitamin D decreased substantial inflammatory cell infiltration in lung tissue, as well as the number of inflammatory cells in BALF. Furthermore, vitamin D reduced the heightened inflammatory factors including of TNF-α, IL-1β, IL-6, and NGF caused by PM exposure, and triggered the activity of nucleus Nrf2 and HO-1 in PM-exposed asthmatic mice. Notably, knockdown HO-1 weakens the Vitamin D- mediated inhibition to pollution toxicity in asthma. Importantly, in vitro experiments on OVA-stimulated mice airway smooth muscle cells, the results showed that OVA and PM, respectively, reduced Nrf2/HO-1 and increased NGF’s expression, while vitamin D reversed the process. And in the HO-1 knockdown cell line of Lenti-si-HO-1 ASMCs, OVA and PM reduced Nrf2’s expression, while HO-1 and NGF’s expression were unchanged.ConclusionsThe above results demastrate that vitamin D downregulated the inflammatory response and the expression of NGF by regulating the Nrf2/HO-1 signaling pathways in airway smooth muscle cells, thereby showing potent anti-inflammatory activity in asthma.

NMDA Receptor Modulation in COVID-19-Associated Acute Respiratory Syndrome in both In Silico and In Vitro Approach.

The normal function of the N-methyl D-aspartate receptors (NMDAR) in human lungs depends on precisely regulated synaptic glutamate levels. Pathophysiology of the lungs is brought on by the changes in homeostasis of glutamate in the synapsis that leads to abnormal NMDAR activity. Severe acute respiratory syndrome (SARS) primarily results in lung infections, particularly lung muscle stiffening, and NMDA receptor potentiation may increase calcium ion influx and support downstream signaling mechanisms. Hence, NMDAR modulators that depend on glutamate levels could be therapeutically useful medications with fewer unintended side effects. A compound called THP (tetrahydropalmatine) that amplifies Ca2+ influx and potentiates NMDA receptors has been identified in the current study. In asthmatic human airway smooth muscle (HASM) cells, THP regulates the NMDA receptor and helps in asthmatic ASM contraction, and the pharmacological stimulation of ASM depends on both brain and respiratory NMDA receptors. Glutamate potency is altered by this substance without any voltage-dependent side effects. Additionally, a GGPP (geranylgeranyl pyrophosphate)-dependent mechanism of THP reduced the production of pro-inflammatory cytokines in ASM. THP is distinctive in terms of its chemical makeup, functioning, and agonist concentration-dependent and allosteric modulatory activity. To treat COVID-19-related SARS, THP, or any future-related compounds will make good drug-like molecule candidates.

Nicotine affects mitochondrial structure and function in human airway smooth muscle cells.

Exposure to cigarette smoke and e-cigarettes, with nicotine as the active constituent, contributes to increased health risks associated with asthma. Nicotine exerts its functional activity via nicotinic acetylcholine receptors (nAChRs), and the alpha7 subtype (α7nAChR) has recently been shown to adversely affect airway dynamics. The mechanisms of α7nAChR action in airways, particularly in the context of airway smooth muscle (ASM), a key cell type in asthma, are still under investigation. Mitochondria have garnered increasing interest for their role in regulating airway tone and adaptations to cellular stress. Here mitochondrial dynamics such as fusion versus fission, and mitochondrial Ca2+ ([Ca2+]m), play an important role in mitochondrial homeostasis. There is currently no information on effects and mechanisms by which nicotine regulates mitochondrial structure and function in ASM in the context of asthma. We hypothesized that nicotine disrupts mitochondrial morphology, fission-fusion balance, and [Ca2+]m regulation, with altered mitochondrial respiration and bioenergetics in the context of asthmatic ASM. Using human ASM (hASM) cells from nonasthmatics, asthmatics, and smokers, we examined the effects of nicotine on mitochondrial dynamics and [Ca2+]m. Fluorescence [Ca2+]m imaging of hASM cells with rhod-2 showed robust responses to 10 μM nicotine, particularly in asthmatics and smokers. In both asthmatics and smokers, nicotine increased the expression of fission proteins while decreasing fusion proteins. Seahorse analysis showed blunted oxidative phosphorylation parameters in response to nicotine in these groups. α7nAChR siRNA blunted nicotine effects, rescuing [Ca2+]m, changes in mitochondrial structural proteins, and mitochondrial dysfunction. These data highlight mitochondria as a target of nicotine effects on ASM, where mitochondrial disruption and impaired buffering could permit downstream effects of nicotine in the context of asthma.NEW & NOTEWORTHY Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

Percent Recovery Index Predicts Poor Asthma Control and Exacerbation in Adults.

Previous studies indicate that the percent recovery index (PRI: the percentage increase from the maximally reduced FEV1 after bronchodilator inhalation), one of the indexes of methacholine bronchial provocation, may predict acute asthma exacerbations in childhood and elderly asthmatics. It is known that childhood (<12) and elder (>60) asthmatics may be different to adult patients in many aspect including prognosis. However, in adults, a research for predicting value of PRI to exacerbation is still absence. Besides exacerbation, predicting value of PRI to poor asthma control is also unknown. We try to detect whether PRI can predict poor asthma control and exacerbation in adults in this research. Meanwhile, we try to detect whether treatment can influence PRI. In 61 adults with asthma, baseline PRI was measured during enrollment. And then baseline PRI was evaluated as a predictor of exacerbation or poor asthma control at an upcoming 3-month follow-up. The covariates included age, sex, BMI, previous exacerbation, smoking status and baseline lung function. After treatment for 3 months, PRI was measured again and compared with baseline PRI. After the 3-month follow-up, we found that baseline PRI was significantly related to asthma exacerbation (P = 0.023), poor asthma control (ACT at 3 months, P = 0.014), decreased quality of life (decrease of MiniAQLQ, P = 0.010) and cumulative number of EDHO at 3 months (P = 0.039). Meanwhile, no significant correlation was observed between baseline PRI and inflammation factors (FENO, CaNO, and EOS). Finally, PRI was dramatically reduced after standard treatment for 3 months. PRI is efficient in the prediction of poor asthma control and exacerbation in adults. The predictive value of PRI may rely on the inherent property of asthmatic airway smooth muscle (ASM) independent of inflammation factors. Effective treatment can alleviate PRI dramatically and that indicate PRI may also be valuable in evaluation of curative effect.

Noncoding RNAs in asthmatic airway smooth muscle cells.

Asthma is a complex and heterogeneous airway disease caused by genetic, environmental and epigenetic factors treated with hormones and biologics. Irreversible pathological changes to airway smooth muscle cells (ASMCs) such as hyperplasia and hypertrophy can occur in asthmatic patients. Determining the mechanisms responsible is vital for preventing such changes. In recent years, noncoding RNAs (ncRNAs), especially microRNAs, long noncoding RNAs and circular RNAs, have been found to be associated with abnormalities of the ASMCs. This review highlights recent ncRNA research into ASMC pathologies. We present a schematic that illustrates the role of ncRNAs in pathophysiological changes to ASMCs that may be useful in future research in diagnostic and treatment strategies for patients with asthma.

Purification of Myosin from Bovine Tracheal Smooth Muscle, Filament Formation and Endogenous Association of Its Regulatory Complex.

Dynamic regulation of myosin filaments is a crucial factor in the ability of airway smooth muscle (ASM) to adapt to a wide length range. Increased stability or robustness of myosin filaments may play a role in the pathophysiology of asthmatic airways. Biochemical techniques for the purification of myosin and associated regulatory proteins could help elucidate potential alterations in myosin filament properties of asthmatic ASM. An effective myosin purification approach was originally developed for chicken gizzard smooth muscle myosin. More recently, we successfully adapted the procedure to bovine tracheal smooth muscle. This method yields purified myosin with or without the endogenous regulatory complex of myosin light chain kinase and myosin light chain phosphatase. The tight association of the regulatory complex with the assembled myosin filaments can be valuable in functional experiments. The purification protocol discussed here allows for enzymatic comparisons of myosin regulatory proteins. Furthermore, we detail the methodology for quantification and removal of the co-purified regulatory enzymes as a tool for exploring potentially altered phenotypes of the contractile apparatus in diseases such as asthma.

Endothelin A (ETA) and Endothelin B (ETB) Receptor Subtypes Potentiate Epidermal Growth Factor (EGF)-Mediated Proliferation in Human Asthmatic Bronchial Airway Smooth Muscle.

BackgroundAsthma is a chronic disease characterized by chronic inflammation, reversible airway obstruction, airway hyperresponsiveness (AHR), and airway remodeling. One of the important features of asthma is airway remodeling, which plays a central role in airflow limitation. Airway remodeling involves numerous changes in the bronchial walls, including airway smooth muscle (ASM) cell hypertrophy and hyperplasia. Studies have shown that ASM hyperplasia in asthma is mediated by the increased production of mitogens. Endothelin-1 (ET-1) has been shown to induce proliferation and function as a co-mitogen in vascular and ASM. In patients with asthma, plasma and bronchoalveolar lavage fluid have been shown to have elevated ET-1 levels, which have been linked to airway remodeling and airflow obstruction in severe asthma. This study investigates the role of ET-1 in proliferation, the receptor subtype mediating its effect, and the signaling pathway.MethodologyNormal and asthmatic bronchial airway smooth muscle (BASM) cells were seeded into 5 × 103 cells/well. Cell proliferation was assayed using 5-bromo-2’-deoxyuridine (BrdU) incorporation. Confluent cells were treated with different concentrations of ET-1 in the presence or absence of the epidermal growth factor (EGF). Signaling pathways were explored using pretreatment of BASM with antagonists 15 minutes before ET-1/EGF stimulation.ResultsIn asthmatic BASM, ET-1 (0.1 nM) functions as a co-mitogen in the presence of EGF (10 nM), showing a significantly greater effect on asthmatic BASM proliferation compared with normal BASM. The ETA receptor antagonist BQ-123 (10-1,000 nM) significantly reduced the proliferative effect of ET-1/EGF on asthmatic BASM more than normal BASM. Moreover, the effect of ETB antagonist BQ-788 (1,000 nM) or pretreatment with the ETB agonist S6C (1-10 nM) followed by co-treatment with EGF in asthmatic BASM showed a small but significant decrease when pretreated with the inhibitor and increased with the agonist, thereby suggesting that the co-mitogenic effect of ET-1 is mainly via the activation of ETA receptors, with a small contribution by the ETB receptors in asthmatic BASM. Finally, pertussis toxin (PTX) pretreatment (25 and 50 ng/mL) showed that EGF and ET-1/EGF mitogenic and co-mitogenic signaling utilizes Gi/0-mediated transactivation by EGF and ET receptors, especially in asthmatic BASM, leading to the activation of Ras-ERK-PI3K pathways. Enhanced ERK and PI3K effects on proliferation suggested that these kinases modulate the co-mitogenic effect of ET-1 in asthmatic BASM. Enhanced cross-talk between ET and EGF receptors may be a potential mechanism contributing to airway remodeling in asthmatic BASM.ConclusionsET-1 enhances the mitogenic effect of EGF predominantly via the ETA receptor in asthmatic BASM with the activation of Ras, ERK, and PI3K. The cross-talk mechanism between ET and EGF receptors may be a potential therapeutic target to prevent the progression of airway remodeling in ASM in patients with asthma.

PaThe regulation mechanism of MUC5AC secretion in airway of obese asthma

The purpose of this study was to establish a rat asthma model and extract MUC5AC to explore the mechanism of mucin 5AC (MUC5AC) signaling pathway regulating the function of asthmatic airway smooth muscle cells (ASMC) and participating in asthmatic airway remodeling. Western blot was used to detect β-catenin (β-catenin), glycogen synthase kinase-3β (GSK-3β), proto-oncogene MUC5AC and cyclin D1 (cyclin D1) in MUC5AC of asthmatic and normal groups. After inhibiting the interaction between β-catenin and transcription cofactor p300 / CBP in ASMC of the asthma group and control group, the cell viability and cycle changes of ASMC were detected by the CCK-8 method and flow cytometry. After inhibiting the activity of P38 mitogen-activated protein kinase (MAPK), the protein expression changes of c-Myc and cyclin D1 were detected by Western blot. Results showed that comprehensive HE staining results of lung tissue sections indicate that the experimental rat model of asthma airway remodeling was successfully established. Compared with the control group, 100 fxmol and L1 Efaroxan promoted insulin secretion (P <0.01), and administration of the MUC5AC antagonist KU14R significantly inhibited the effect of MUC5AC.Western blot showed that the protein expression levels of β-catenin, c-Myc and cyclin D1 in ASMC of the obese asthma group were significantly higher than those of the control group (P <0.05), while the protein expression level of GSK-3β was lower than Control group (P <0.05). After inhibiting the interaction between β-catenin and p300 / CBP, the decrease in cell viability and the degree of cell cycle change of ASMC in the asthma group were more obvious than those in the control group (P <0.05). After inhibiting the activity of P38 MAPK, the expressions of the target proteins c-Myc and cyclin D1 in the MUC5AC signaling pathway in ASMC model rats and control rats were down-regulated, and the difference was statistically significant (P <0.05). The conclusion was that the Wnt/β-catenin signaling pathway can regulate the proliferation and differentiation of ASMC by up-regulating the expression level of cMyc. Cyclin D1 interacts with the MAPK signaling pathway, thereby affecting the function of ASMC and participating in asthma airway remodeling.

Abi1 mediates airway smooth muscle cell proliferation and airway remodeling via Jak2/STAT3 signaling

SummaryAsthma is a complex pulmonary disorder with multiple pathological mechanisms. A key pathological feature of chronic asthma is airway remodeling, which is largely attributed to airway smooth muscle (ASM) hyperplasia that contributes to thickening of the airway wall and further drives asthma pathology. The cellular processes that mediate ASM cell proliferation are not completely elucidated. Using multiple approaches, we demonstrate that the adapter protein Abi1 (Abelson interactor 1) is upregulated in ∼50% of ASM cell cultures derived from patients with asthma. Loss-of-function studies demonstrate that Abi1 regulates the activation of Jak2 (Janus kinase 2) and STAT3 (signal transducers and activators of transcription 3) as well as the proliferation of both nonasthmatic and asthmatic human ASM cell cultures. These findings identify Abi1 as a molecular switch that activates Jak2 kinase and STAT3 in ASM cells and demonstrate that a dysfunctional Abi1-associated pathway contributes to the progression of asthma.

Lysyl oxidase like 2 is increased in asthma and contributes to asthmatic airway remodelling.

BackgroundAirway smooth muscle (ASM) cells are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyperresponsiveness and airway remodelling. The extracellular matrix (ECM) can influence tissue remodelling pathways; however, to date no study has investigated the effect of ASM ECM stiffness and cross-linking on the development of asthmatic airway remodelling. We hypothesised that transforming growth factor-β (TGF-β) activation by ASM cells is influenced by ECM in asthma and sought to investigate the mechanisms involved.MethodsThis study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGF-β activation and expression of ECM cross-linking enzymes. Human bronchial biopsies from asthmatic and nonasthmatic donors were used to confirm lysyl oxidase like 2 (LOXL2) expression in ASM. A chronic ovalbumin (OVA) model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling.ResultsWe found that asthmatic ASM cells activated more TGF-β basally than nonasthmatic controls and that diseased cell-derived ECM influences levels of TGF-β activated. Our data demonstrate that the ECM cross-linking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGF-β activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an OVA mouse model of asthma.ConclusionThese data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further investigation as a potential therapy to reduce remodelling of the airways in severe asthma.

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.

Dysregulated retinoic acid signaling in airway smooth muscle cells in asthma.

Vitamin A deficiency has been shown to exacerbate allergic asthma. Previous studies have postulated that retinoic acid (RA), an active metabolite of vitamin A and high-affinity ligand for RA receptor (RAR), is reduced in airway inflammatory condition and contributes to multiple features of asthma including airway hyperresponsiveness and excessive accumulation of airway smooth muscle (ASM) cells. In this study, we directly quantified RA and examined the molecular basis for reduced RA levels and RA-mediated signaling in lungs and ASM cells obtained from asthmatic donors and in lungs from allergen-challenged mice. Levels of RA and retinol were significantly lower in lung tissues from asthmatic donors and house dust mite (HDM)-challenged mice compared to non-asthmatic human lungs and PBS-challenged mice, respectively. Quantification of mRNA and protein expression revealed dysregulation in the first step of RA biosynthesis consistent with reduced RA including decreased protein expression of retinol dehydrogenase (RDH)-10 and increased protein expression of RDH11 and dehydrogenase/reductase (DHRS)-4 in asthmatic lung. Proteomic profiling of non-asthmatic and asthmatic lungs also showed significant changes in the protein expression of AP-1 targets consistent with increased AP-1 activity. Further, basal RA levels and RA biosynthetic capabilities were decreased in asthmatic human ASM cells. Treatment of human ASM cells with all-trans RA (ATRA) or the RARγ-specific agonist (CD1530) resulted in the inhibition of mitogen-induced cell proliferation and AP-1-dependent transcription. These data suggest that RA metabolism is decreased in asthmatic lung and that enhancing RAR signaling using ATRA or RARγ agonists may mitigate airway remodeling associated with asthma.

The Impact of Monoclonal Antibodies on Airway Smooth Muscle Contractility in Asthma: A Systematic Review

Airway hyperresponsiveness (AHR) represents a central pathophysiological hallmark of asthma, with airway smooth muscle (ASM) being the effector tissue implicated in the onset of AHR. ASM also exerts pro-inflammatory and immunomodulatory actions, by secreting a wide range of cytokines and chemokines. In asthma pathogenesis, the overexpression of several type 2 inflammatory mediators including IgE, IL-4, IL-5, IL-13, and TSLP has been associated with ASM hyperreactivity, all of which can be targeted by humanized monoclonal antibodies (mAbs). Therefore, the aim of this review was to systematically assess evidence across the literature on mAbs for the treatment of asthma with respect to their impact on the ASM contractile tone. Omalizumab, mepolizumab, benralizumab, dupilumab, and tezepelumab were found to be effective in modulating the contractility of the ASM and preventing the AHR, but no available studies concerning the impact of reslizumab on the ASM were identified from the literature search. Omalizumab, dupilumab, and tezepelumab can directly modulate the ASM in asthma, by specifically blocking the interaction between IgE, IL-4, and TSLP, and their receptors are located on the surface of ASM cells. Conversely, mepolizumab and benralizumab have prevalently indirect impacts against AHR by targeting eosinophils and other immunomodulatory effector cells promoting inflammatory processes. AHR has been suggested as the main treatable trait towards precision medicine in patients suffering from eosinophilic asthma, therefore, well-designed head-to-head trials are needed to compare the efficacy of those mAbs that directly target ASM contractility specifically against the AHR in severe asthma, namely omalizumab, dupilumab, and tezepelumab.

MiR-182/Sestrin2 affects the function of asthmatic airway smooth muscle cells by the AMPK/mTOR pathway.

Asthma is a chronic inflammatory airway disease and brings heavy economic and spiritual burdens to patients' families and the society. Airway smooth muscle cells (ASMCs) afect the development of asthma by secreting cytokines, growth factors, and prostates. The stress-inducing protein, Sestrin2, plays a vital role in antioxidant defense. The aim of this study is to investigate the role of Sestrin2 in asthma and its corresponding molecular mechanism. Airway remodeling was induced by construction of asthma rat model. Primary ASMCs were isolated through combining tissue block adherence and enzymatic digestion and identified by immunofluorescence staining. Gene expression was measured by quantitative real-time PCR (qPCR) and western blot (WB) experiments. Cell viability, proliferation, migration, and calcium flow of ASMCs were measured by Cell Counting Kit-8 (CCK-8), 5-ethynyl-deoxyuridine (EdU), Transwell, and Fluo-3AM, respectively. The binding of miR-182 and Sestrin2 3'-untranslated region (3'-UTR) was measured by luciferase reporter system and RNA-binding protein immunoprecipitation (RIP) analysis. Sestrin2 expression was upregulated in asthma rat model and cell model. Overexpression of Sestrin2 enhanced the growth, migration, and calcium flow, and inversely, repression of Sestrin2 was reduced in ASMCs from the asthma group. MiR-182, one of the microRNAs (miRNAs) that possesses the potential to regulate Sestrin2, was downregulated in ASMCs from the asthma group. Further experiments revealed that Sestrin2 was inhibited by miR-182 and that overexpression of Sestrin2 reversed the miR-182-induced inhibition of the cellular progression of ASMCs from the asthma group. This study further investigated the downstream signaling pathway of Sestrin2 and found that increased expression of Sestrin2 activated 5'-adenosine monophosphate-activated protein kinase (AMPK), leading to the inactivation of mammalian target of rapamycin (mTOR) and thus promoting the growth, migration, and calcium flow of ASMCs from the asthma group. This study investigated the role of Sestrin2 for the first time and further dissected the regulatory factor of Sestrin2, ultimately elucidating the downstream signaling pathway of Sestrin2 in asthma, providing a novel pathway, and improving the understanding of the development and progression of asthma.

MiR-182/Sestrin2 affects the function of asthmatic airway smooth muscle cells by the AMPK/mTOR pathway

Abstract Background and Objectives Asthma is a chronic inflammatory airway disease and brings heavy economic and spiritual burdens to patients’ families and the society. Airway smooth muscle cells (ASMCs) afect the development of asthma by secreting cytokines, growth factors, and prostates. The stress-inducing protein, Sestrin2, plays a vital role in antioxidant defense. The aim of this study is to investigate the role of Sestrin2 in asthma and its corresponding molecular mechanism. Materials and Methods Airway remodeling was induced by construction of asthma rat model. Primary ASMCs were isolated through combining tissue block adherence and enzymatic digestion and identified by immunofluorescence staining. Gene expression was measured by quantitative real-time PCR (qPCR) and western blot (WB) experiments. Cell viability, proliferation, migration, and calcium flow of ASMCs were measured by Cell Counting Kit-8 (CCK-8), 5-ethynyl-deoxyuridine (EdU), Transwell, and Fluo-3AM, respectively. The binding of miR-182 and Sestrin2 3′-untranslated region (3′-UTR) was measured by luciferase reporter system and RNA-binding protein immunoprecipitation (RIP) analysis. Results Sestrin2 expression was upregulated in asthma rat model and cell model. Overexpression of Sestrin2 enhanced the growth, migration, and calcium flow, and inversely, repression of Sestrin2 was reduced in ASMCs from the asthma group. MiR-182, one of the microRNAs (miRNAs) that possesses the potential to regulate Sestrin2, was downregulated in ASMCs from the asthma group. Further experiments revealed that Sestrin2 was inhibited by miR-182 and that overexpression of Sestrin2 reversed the miR-182–induced inhibition of the cellular progression of ASMCs from the asthma group. This study further investigated the downstream signaling pathway of Sestrin2 and found that increased expression of Sestrin2 activated 5′-adenosine monophosphate-activated protein kinase (AMPK), leading to the inactivation of mammalian target of rapamycin (mTOR) and thus promoting the growth, migration, and calcium flow of ASMCs from the asthma group. Conclusion This study investigated the role of Sestrin2 for the first time and further dissected the regulatory factor of Sestrin2, ultimately elucidating the downstream signaling pathway of Sestrin2 in asthma, providing a novel pathway, and improving the understanding of the development and progression of asthma.

Knockdown of NOVA1 inhibits inflammation and migration of asthmatic airway smooth muscle cells to regulate PTEN/Akt pathway by targeting PTBP1

NOVA1 (neuro-oncological ventral antigen 1) is a neuron specific RNA binding protein, belonging to the Nova family, which plays an important role in various diseases. However, the role of NOVA1 in childhood asthma remains unclear. This study was aimed to investigate the role of NOVA1 in TGF-β1-induced ASMCs proliferation and migration as well as the potential mechanisms. In our study, the NOVA1 expression was significantly increased in asthmatic tissues and TGF-β1-induced ASMCs. Inhibition of NOVA1 significantly inhibited TGF-β1-induced ASMCs cell proliferation and migration, and alleviates TGF-β1-induced inflammation. NOVA1 positively regulated the PTBP1 expression and si-NOVA1 inhibited the activation of PTEN/AKT signal pathway. Importantly, the overexpression of PTBP1 partially reversed the effect of NOVA1 on cell viability, migration, inflammation and the activation of PTEN/AKT signal pathway. Generally, our study demonstrated that si-NOVA1 inhibited TGF-β1-induced inflammation and migration in ASMCs through PTBP1/PTEN/AKT pathway. Therefore, inhibition of NOVA1 may be useful for the prevention or treatment of asthma airway remodeling.

Network and co-expression analysis of airway smooth muscle cell transcriptome delineates potential gene signatures in asthma

Airway smooth muscle (ASM) is known for its role in asthma exacerbations characterized by acute bronchoconstriction and remodeling. The molecular mechanisms underlying multiple gene interactions regulating gene expression in asthma remain elusive. Herein, we explored the regulatory relationship between ASM genes to uncover the putative mechanism underlying asthma in humans. To this end, the gene expression from human ASM was measured with RNA-Seq in non-asthmatic and asthmatic groups. The gene network for the asthmatic and non-asthmatic group was constructed by prioritizing differentially expressed genes (DEGs) (121) and transcription factors (TFs) (116). Furthermore, we identified differentially connected or co-expressed genes in each group. The asthmatic group showed a loss of gene connectivity due to the rewiring of major regulators. Notably, TFs such as ZNF792, SMAD1, and SMAD7 were differentially correlated in the asthmatic ASM. Additionally, the DEGs, TFs, and differentially connected genes over-represented in the pathways involved with herpes simplex virus infection, Hippo and TGF-β signaling, adherens junctions, gap junctions, and ferroptosis. The rewiring of major regulators unveiled in this study likely modulates the expression of gene-targets as an adaptive response to asthma. These multiple gene interactions pointed out novel targets and pathways for asthma exacerbations.

Glial-derived neurotrophic factor in human airway smooth muscle.

Airway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain-derived neurotrophic factor (BDNF). The glial cell-derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+ ]i . Serum-deprived ASM cells from non-asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura-2 loaded cells, acute GDNF or NRTN alone induced [Ca2+ ]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1-Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+ ]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1-Fc chelator markedly decreased [Ca2+ ]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine-induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+ ]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Angiogenic regulatory influence of extracellular matrix deposited by resting state asthmatic and non-asthmatic airway smooth muscle cells is similar.

The extracellular matrix (ECM) is the tissue microenvironment that regulates the characteristics of stromal and systemic cells to control processes such as inflammation and angiogenesis. Despite ongoing anti‐inflammatory treatment, low levels of inflammation exist in the airways in asthma, which alters ECM deposition by airway smooth muscle (ASM) cells. The altered ECM causes aberrant behaviour of cells, such as endothelial cells, in the airway tissue. We therefore sought to characterize the composition and angiogenic potential of the ECM deposited by asthmatic and non‐asthmatic ASM. After 72 hours under non‐stimulated conditions, the ECM deposited by primary human asthmatic ASM cells was equal in total protein, collagen I, III and fibronectin content to that from non‐asthmatic ASM cells. Further, the matrices of non‐asthmatic and asthmatic ASM cells were equivalent in regulating the growth, activity, attachment and migration of primary human umbilical vein endothelial cells (HUVECs). Under basal conditions, asthmatic and non‐asthmatic ASM cells intrinsically deposit an ECM of equivalent composition and angiogenic potential. Previous findings indicate that dysregulation of the airway ECM is driven even by low levels of inflammatory provocation. This study suggests the need for more effective anti‐inflammatory therapies in asthma to maintain the airway ECM and regulate ECM‐mediated aberrant angiogenesis.