Airway Smooth Muscle Tissues Research Articles

On the pathogenesis of COVID-19: the role of transforming growth factor beta

Proteins of the transforming growth factor beta (TGF-β) family regulate numerous cellular processes that are essential in the pathogenesis of acute respiratory distress syndrome (ARDS), contributing to increased alveolar epithelial permeability, activation of fibroblasts, and extracellular matrix remodeling. TGF-β is involved in the pathogenesis of inflammatory respiratory diseases during the development of COVID-19. SARS-CoV-2 leads to complex immune responses that include the release of inflammatory cytokines, increased activity of mast cells, and the release of mast cell secretome, in particular profibrotic enzymes and cytokines, including TGF-β.Tryptaseand chymase-positive mast cells play a major role in pulmonary fibrosis and embolism in COVID-19. Mast cell chymase is angiotensin-converting enzyme 2-independent due to extracellular formation of angiotensin II in the interstitium; it also activates TGF-β and other molecules, thereby playing a role in tissue remodeling. Mast cell β-tryptase increases the secretion of TGF-β1 by airway smooth muscle tissue and the expression of α-smooth muscle actin (α-SMA). TGF-β also induces the generation of mitochondrial reactive oxygen species (ROS), which enhances the production of ROS in lung fibroblasts. TGF-β is crucial for induing the synthesis of extracellular matrix components by fibroblasts.The review is devoted to the structure of TGF-β, the sources of its secretion and functions, the mechanism of its involvement in the pathogenesis of COVID-19, and the possibility of its use as a prognostic marker of COVID-19 severity.

Mechanism analysis of essential oil from Radix Bupleuri for the treatment of asthma through regulation of ectopic olfactory receptor

Ethnopharmacological relevanceRadix Bupleuri is the root of Bupleurum chinense DC. (BC) and a classic aromatic traditional Chinese medicine. The traditional pharmacological effects of Radix Bupleuri are alleviating bronchial spasms, dilating airways, and promoting the resolution of respiratory inflammation, thereby reducing asthma symptoms. Aim of the studyStudies have demonstrated the efficacy of water extracts from BC in asthma treatment. However, the potential role of volatile oil, another active constituent in BC, remains unexplored with asthma. Notably, volatile oil is renowned for its ease of absorption and direct targeting of affected areas, offering distinct advantages in alleviating airway inflammation. This study aims to explain the anti-asthmatic mechanism of BC-oil through in vivo and in vitro pharmacological experiments. Materials and methodsFirstly, the OVA-induced SD rat asthma model was utilized to evaluate the pharmacological effect of BC-oil by lung function monitoring, HE staining, flow cytometry, ELISA, and RT-qPCR. The anti-asthmatic mechanism was further analyzed by combining transcriptomic analysis of lung tissue from rat model and airway smooth muscle tissue from public database. Initially, GC-MS was used to analyze the components of BC-oil. The anti-asthmatic activity was evaluated in 16-HBE, RBL-2H3, and ASMC cells using CAMKII inhibitors to explore of the critical signal transduction regulated by BC-oil. Furthermore, molecular docking and calcium flow assay were utilized to screen and identify the active components from BC-oil. ResultsOral administration of BC-oil significantly enhanced pulmonary function in asthmatic SD rats by reducing airway resistance and elastic resistance. Additionally, BC-oil inhibited inflammatory cytokines, including serum IL-2, pulmonary Il1b, Tnf, and Cxcl13, demonstrating potent anti-inflammatory and immunomodulatory effects. In this study, we analyzed the significant role of OR2W3 in asthma using public transcriptomic data. Furthermore, we indicated that BC-oil regulated the expression of Olr1433 and GNAL in rat lung tissue. BC-oil reduced degranulation and inhibited gene expression of Il3 and Tnf in RBL-2H3 cells and suppressed gene expression of IL8 and TNF in 16-HBE cells. BC-oil also attenuated airway smooth muscle cell proliferation and expression of Acta2 and Ccnd1. Furthermore, BC-oil regulates asthma-related cellular processes by activating CAMKII. GC-MS analysis identified 11 components of BC-oil, and n-hexadecanoic acid, linoleic acid and oleic acid from BC-oil were identified to interact with OR2W3 by molecular docking. The calcium flow assay revealed linoleic acid as a significant activator of OR2W3 and indicated that BC-oil alleviated asthma through the ectopic olfactory signaling pathway. ConclusionsThe mechanism of BC-oil in treating asthma through signal transduction of OR2W3 is revealed at the molecular and cellular levels.

Inhibition of airway smooth muscle contraction and proliferation by LIM kinase inhibitor, LIMKi3

PurposeCurrent medical treatment for asthma aims to inhibit airway smooth muscle (ASM) contraction and proliferation, however, the efficacy of available treatment options is unsatisfactory. Therefore, we explored the effect of LIM domain kinase (LIMK) inhibitor - LIMKi3, on ASM to improve the understanding of ASM contraction and proliferation mechanisms, and to investigate new therapeutic targets. Materials and methodsAsthma model was induced in rats by intraperitoneal injection of ovalbumin. Using phospho-specific antibodies, we examined LIMK, phosphorylated LIMK, cofilin and phosphorylated cofilin. ASM contraction was studied in organ bath experiments. ASM cells proliferation was studied with cell counting kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays. ResultsImmunofluorescence indicated that LIMKs are expressed in ASM tissues. Western blot revealed that LIMK1 and phospho-cofilin were significantly elevated in asthma ASM tissues. The LIMK inhibitor, LIMKi3 (1 ​μM) could reduce cofilin phosphorylation and therefore inhibit contraction of ASM tissues, and induce actin filament breakdown as well as cell proliferation reduction in cultured human ASM cells. ConclusionsASM contraction and proliferation in asthma may underlie the effects of LIMKs. Small molecule LIMK inhibitor, LIMKi3, might be a potential therapeutic strategy for asthma.

Discovery of 2-Aminopyrimidines as Potent Agonists for the Bitter Taste Receptor TAS2R14.

The bitter taste receptor TAS2R14 is a G protein-coupled receptor that is found on the tongue as well as in the human airway smooth muscle and other extraoral tissues. Because its activation causes bronchodilatation, TAS2R14 is a potential target for the treatment of asthma or chronic obstructive pulmonary disease. Structural variations of flufenamic acid, a nonsteroidal anti-inflammatory drug, led us to 2-aminopyridines showing considerable efficacy and potency in an IP1accumulation assay. In combination with an exchange of the carboxylic moiety by a tetrazole unit, a set of promising new TAS2R14 agonists was developed. The most potent ligand 28.1 (EC50 = 72 nM) revealed a six-fold higher potency than flufenamic acid and a maximum efficacy of 129%. Besides its unprecedented TAS2R14 activation, 28.1 revealed marked selectivity over a panel of 24 non-bitter taste human G protein-coupled receptors.

Membrane adhesion junctions regulate airway smooth muscle phenotype and function.

The local environment surrounding airway smooth muscle (ASM) cells has profound effects on the physiological and phenotypic properties of ASM tissues. ASM is continually subjected to the mechanical forces generated during breathing and to the constituents of its surrounding extracellular milieu. The smooth muscle cells within the airways continually modulate their properties to adapt to these changing environmental influences. Smooth muscle cells connect to the extracellular cell matrix (ECM) at membrane adhesion junctions that provide mechanical coupling between smooth muscle cells within the tissue. Membrane adhesion junctions also sense local environmental signals and transduce them to cytoplasmic and nuclear signaling pathways in the ASM cell. Adhesion junctions are composed of clusters of transmembrane integrin proteins that bind to ECM proteins outside the cell and to large multiprotein complexes in the submembranous cytoplasm. Physiological conditions and stimuli from the surrounding ECM are sensed by integrin proteins and transduced by submembranous adhesion complexes to signaling pathways to the cytoskeleton and nucleus. The transmission of information between the local environment of the cells and intracellular processes enables ASM cells to rapidly adapt their physiological properties to modulating influences in their extracellular environment: mechanical and physical forces that impinge on the cell, ECM constituents, local mediators, and metabolites. The structure and molecular organization of adhesion junction complexes and the actin cytoskeleton are dynamic and constantly changing in response to environmental influences. The ability of ASM to rapidly accommodate to the ever-changing conditions and fluctuating physical forces within its local environment is essential for its normal physiological function.

Airway smooth muscle function in asthma.

Known to have affected around 340 million people across the world in 2018, asthma is a prevalent chronic inflammatory disease of the airways. The symptoms such as wheezing, dyspnea, chest tightness, and cough reflect episodes of reversible airway obstruction. Asthma is a heterogeneous disease that varies in clinical presentation, severity, and pathobiology, but consistently features airway hyperresponsiveness (AHR)—excessive airway narrowing due to an exaggerated response of the airways to various stimuli. Airway smooth muscle (ASM) is the major effector of exaggerated airway narrowing and AHR and many factors may contribute to its altered function in asthma. These include genetic predispositions, early life exposure to viruses, pollutants and allergens that lead to chronic exposure to inflammatory cells and mediators, altered innervation, airway structural cell remodeling, and airway mechanical stress. Early studies aiming to address the dysfunctional nature of ASM in the etiology and pathogenesis of asthma have been inconclusive due to the methodological limitations in assessing the intrapulmonary airways, the site of asthma. The study of the trachealis, although convenient, has been misleading as it has shown no alterations in asthma and it is not as exposed to inflammatory cells as intrapulmonary ASM. Furthermore, the cartilage rings offer protection against stress and strain of repeated contractions. More recent strategies that allow for the isolation of viable intrapulmonary ASM tissue reveal significant mechanical differences between asthmatic and non-asthmatic tissues. This review will thus summarize the latest techniques used to study ASM mechanics within its environment and in isolation, identify the potential causes of the discrepancy between the ASM of the extra- and intrapulmonary airways, and address future directions that may lead to an improved understanding of ASM hypercontractility in asthma.

Polyethylene microplastics adversely affect airway patency

Human exposure to microplastics through inhalation has been widely reported in recent years. There is a paucity of work focusing on the direct effect of accumulation of microplastics in airways and how it may impact the respiratory function. This study aimed to investigate whether the exposure of microplastic would change the contractility of isolated airway smooth muscle tissue. Microplastics were obtained through milling of high-density polyethylene (HDPE) pellets by using a centrifugal mill. To confirm that the milled microplastic particle size range fell within the definition of microplastic, field-emission scanning electron microscope (FESEM) was employed. The milled microplastics particle size ranged from 44.2 µm to 552.4 µm. The organ bath technique was employed to study the direct change of tissue contractility of rat isolated tracheal rings. Tracheal rings were incubated with polyethylene microplastics of different concentrations (0.3 mg/ml to 10 mg/ml) for a minimum of 18 hours in physiological Krebs buffer, followed by the construction of concentration-response curves to a contractile agent, carbachol (muscarinic agonist). Exposure to all concentrations of polyethylene microplastics enhanced the contractile responses of the tissues to carbachol. However, the effect was only statistically significant in tissues incubated at 3 mg/ml and above (p < 0.05). Findings from this study provide preliminary evidence that exposure to polyethylene microplastics adversely affects airway function. Heightened contractile responses of airways mimic the pathophysiological responses in respiratory diseases such as asthma, chronic cough and chronic obstructive pulmonary disease. Further experiments focusing on the possible mechanism of actions of these microplastics affecting the airway tissue function are now needed.

Sambucus nigra: A traditional medicine effective in reducing inflammation in mice

Ethnopharmacological relevanceSambucus nigra L. is a plant of European origin and popularly known as elder, elderberry, black elder, European elder, European elderberry, and European black elderberry, being described in pharmacopoeia of several countries. Its flowers and berries have been used in folk medicine to treat feverish conditions, coughing, nasal congestion, and influenza besides its popular use as anti-inflammatory, analgesic, and diuretic agent. Aim of the studyThe aim of this investigation was to elucidate the anti-inflammatory and the relaxant effect of the lyophilized aqueous extract obtained from S. nigra's flowers on in vivo and in vitro inflammation assays and on the isolated rat vascular and airway smooth muscle tissue. Material and methodsThe anti-inflammatory activity of the extract was investigated using carrageenan-induced inflammation model in the subcutaneous tissue of male Swiss mice orally treated with S. nigra extract (30, 100, 300 or 600 mg/kg). Leukocyte influx and the secretion of chemical mediators were quantified in the inflamed exudate. Additionally, histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1β, IL-10 and NO production, and adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry) were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with LPS and treated with the extract (1, 10 or 100 μg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. Finally, the effect of the extract was evaluated in isolated rat aorta and trachea rings. ResultsThe oral treatment with S. nigra promoted reduction in the neutrophil migration as well as the decrease of TNF, IL-1β and IL-6 levels in the inflamed exudate. In vitro treatment with S. nigra decreased NO2−, TNF, IL-1β and IL-6 and promoted increase of IL-10 in LPS-stimulated neutrophils. Similarly, the extract reduced the NO2−, TNF and IL-6 in LPS-stimulated macrophages. Rutin, the major constituent of S. nigra extract reduced NO2−, TNF, IL-1β, and IL-6 and promoted the increase of IL-10 in LPS-stimulated neutrophils supernatant. The extract also shed CD62L and CD18 expressions. The extract was able to increase the efferocytosis of apoptotic neutrophils by increasing the IL-10 and decreasing the TNF levels. Additionally, the extract reduced the hypersensitivity induced by carrageenan and promoted a relaxant effect in isolated vascular and non-vascular rat tissue. ConclusionsS. nigra flowers extract presents anti-inflammatory effect by modulating macrophage and neutrophil functions including the production of inflammatory mediators and cell migration, by promoting efferocytosis and consequently the resolution of acute inflammation, besides exerting antinociceptive effects, scientifically proving its popular use as medicinal plant. Allied to the relaxant effect in both vascular and non-vascular smooth muscle tissue, S. nigra extract represents an important tool for the management of acute inflammation.

Acetylation of Abelson interactor 1 at K416 regulates actin cytoskeleton and smooth muscle contraction.

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine‐416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with neuronal Wiskott‐Aldrich syndrome protein (N‐WASP), an important actin‐regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser‐19 and vimentin phosphorylation at Ser‐56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non‐histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist‐induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N‐WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation‐dependent regulation of the actin cytoskeleton in smooth muscle.

The proprotein convertase furin inhibits IL-13-induced inflammation in airway smooth muscle by regulating integrin-associated signaling complexes

Furin is a proprotein convertase that regulates the activation and inactivation of multiple proteins including matrix metalloproteinases, integrins and cytokines. It is a serine endoprotease that localizes to the plasma membrane and can be secreted into the extracellular space. The role of furin in regulating inflammation in isolated canine airway smooth muscle tissues was investigated. The treatment of airway tissues with recombinant furin (rFurin) inhibited the activation of Akt and eotaxin secretion induced by IL-13, and it prevented the IL-13 induced suppression of smooth muscle myosin heavy chain expression. rFurin promoted a differentiated phenotype by activating β1 integrin proteins and stimulating the activation of the adhesome proteins vinculin and paxillin by talin. Activated paxillin induced the binding of Akt to β-parvin IPP (ILK, PINCH, parvin) complexes, which inhibits Akt activation. Treatment of tissues with a furin inhibitor or the depletion of endogenous furin using shRNA resulted in Akt activation and inflammatory responses similar to those induced by IL-13. Furin inactivation or IL-13 caused talin cleavage and integrin inactivation, resulting in the inactivation of vinculin and paxillin. Paxillin inactivation resulted in the coupling of Akt to α-parvin IPP complexes, which catalyze Akt activation and an inflammatory response. The results demonstrate that furin inhibits inflammation in airway smooth muscle induced by IL-13, and that the anti-inflammatory effects of furin are mediated by activating integrin proteins and integrin-associated signaling complexes that regulate Akt-mediated pathways to the nucleus. Furin may have therapeutic potential for the treatment of inflammatory conditions of the lungs and airways.

Detecting the Multiomics Signatures of Factor-Specific Inflammatory Effects on Airway Smooth Muscles.

Smooth muscles are a specific muscle subtype that is widely identified in the tissues of internal passageways. This muscle subtype has the capacity for controlled or regulated contraction and relaxation. Airway smooth muscles are a unique type of smooth muscles that constitute the effective, adjustable, and reactive wall that covers most areas of the entire airway from the trachea to lung tissues. Infection with SARS-CoV-2, which caused the world-wide COVID-19 pandemic, involves airway smooth muscles and their surrounding inflammatory environment. Therefore, airway smooth muscles and related inflammatory factors may play an irreplaceable role in the initiation and progression of several severe diseases. Many previous studies have attempted to reveal the potential relationships between interleukins and airway smooth muscle cells only on the omics level, and the continued existence of numerous false-positive optimal genes/transcripts cannot reflect the actual effective biological mechanisms underlying interleukin-based activation effects on airway smooth muscles. Here, on the basis of newly presented machine learning-based computational approaches, we identified specific regulatory factors and a series of rules that contribute to the activation and stimulation of airway smooth muscles by IL-13, IL-17, or the combination of both interleukins on the epigenetic and/or transcriptional levels. The detected discriminative factors (genes) and rules can contribute to the identification of potential regulatory mechanisms linking airway smooth muscle tissues and inflammatory factors and help reveal specific pathological factors for diseases associated with airway smooth muscle inflammation on multiomics levels.

S100A4 is activated by RhoA and catalyses the polymerization of non-muscle myosin, adhesion complex assembly and contraction in airway smooth muscle.

S100A4 is expressed in many tissues, including smooth muscle (SM), but its physiologic function is unknown. S100A4 regulates the motility of metastatic cancer cells by binding to non-muscle (NM) myosin II. Contractile stimulation causes the polymerization of NM myosin in airway SM, which is necessary for tension development. NM myosin regulates the assembly of adhesion junction signalling complexes (adhesomes) that catalyse actin polymerization. In airway SM, ACh (acetylcholine) stimulated the binding of S100A4 to the NM myosin heavy chain, which was catalysed by RhoA GTPase via the RhoA-binding protein, rhotekin. The binding of S100A4 to NM myosin was required for NM myosin polymerization, adhesome assembly and actin polymerization. S100A4 plays a critical function in the regulation of airway SM contraction by catalysing NM myosin filament assembly. The interaction of S100A4 with NM myosin may also play an important role in the physiologic function of other tissues. S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), although its role in the regulation of their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalysing the assembly of integrin-associated adhesome complexes that activate pathways that catalyse actin polymerization. ACh stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalysed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using short hairpin RNA or small interfering RNA prevented NM myosin II polymerization as well as the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SM myosin regulatory light chain phosphorylation. The results show that S100A4 plays a critical role in tension development in airway SM tissue by catalysing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types.

S100A4 is secreted by airway smooth muscle tissues and activates inflammatory signaling pathways via receptors for advanced glycation end products.

S100A4 is a low-molecular-mass (12 kDa) EF-hand Ca2+-binding S100 protein that is expressed in a broad range of normal tissue and cell types. S100A4 can be secreted from some cells to act in an autocrine or paracrine fashion on target cells and tissues. S100A4 has been reported in the extracellular fluids of subjects with several inflammatory diseases, including asthma. Airway smooth muscle plays a critical role in airway inflammation by synthesizing and secreting inflammatory cytokines. We hypothesized that S100A4 may play an immunomodulatory role in airway smooth muscle. Trachealis smooth muscle tissues were stimulated with recombinant His-S100A4, and the effects on inflammatory responses were evaluated. S100A4 induced the activation of Akt and NF-κB and stimulated eotaxin secretion. It also increased the expression of RAGE and endogenous S100A4 in airway tissues. Stimulation of airway smooth muscle tissues with IL-13 or TNF-α induced the secretion of S100A4 from the tissues and promoted the expression of endogenous receptors for advanced glycation end products (RAGE) and S100A4. The role of RAGE in mediating the responses to S100A4A was evaluated by expressing a mutant nonfunctional RAGE (RAGEΔcyto) in tracheal muscle tissues and by treating tissues with a RAGE inhibitor. S100A4 did not activate NF-κB or Akt in tissues that were expressing RAGEΔcyto or treated with a RAGE inhibitor, indicating that S100A4 mediates its effects by acting on RAGE. Our results demonstrate that inflammatory mediators stimulate the synthesis and secretion of S100A4 in airway smooth muscle tissues and that extracellular S100A4 acts via RAGE to mediate airway smooth muscle inflammation.

Disruption of AKAP-PKA Interaction Induces Hypercontractility With Concomitant Increase in Proliferation Markers in Human Airway Smooth Muscle.

With the ability to switch between proliferative and contractile phenotype, airway smooth muscle (ASM) cells can contribute to the progression of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), in which airway obstruction is associated with ASM hypertrophy and hypercontractility. A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules in various tissues, including ASM cells. AKAPs can anchor the regulatory subunits of protein kinase A (PKA), and guide cellular localization via various targeting domains. Here we investigated whether disruption of the AKAP-PKA interaction, by the cell permeable peptide stearated (st)-Ht31, alters human ASM proliferation and contractility. Treatment of human ASM with st-Ht31 enhanced the expression of protein markers associated with cell proliferation in both cultured cells and intact tissue, although this was not accompanied by an increase in cell viability or cell-cycle progression, suggesting that disruption of AKAP-PKA interaction on its own is not sufficient to drive ASM cell proliferation. Strikingly, st-Ht31 enhanced contractile force generation in human ASM tissue with concomitant upregulation of the contractile protein α-sm-actin. This upregulation of α-sm-actin was independent of mRNA stability, transcription or translation, but was dependent on proteasome function, as the proteasome inhibitor MG-132 prevented the st-Ht31 effect. Collectively, the AKAP-PKA interaction appears to regulate markers of the multi-functional capabilities of ASM, and this alter the physiological function, such as contractility, suggesting potential to contribute to the pathophysiology of airway diseases.

P116Rip promotes myosin phosphatase activity in airway smooth muscle cells.

Myosin phosphatase-Rho interacting protein (p116Rip ) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116Rip in cellular contractile processes, we suppressed the expression of p116Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116Rip expression in asthma pathogenesis.

In vivo multifunctional optical coherence tomography at the periphery of the lungs.

Remodeling of tissue, such as airway smooth muscle (ASM) and extracellular matrix, is considered a key feature of airways disease. No clinically accepted diagnostic method is currently available to assess airway remodeling or the effect of treatment modalities such as bronchial thermoplasty in asthma, other than invasive airway biopsies. Optical coherence tomography (OCT) generates cross-sectional, near-histological images of airway segments and enables identification and quantification of airway wall layers based on light scattering properties only. In this study, we used a custom motorized OCT probe that combines standard and polarization sensitive OCT (PS-OCT) to visualize birefringent tissue in vivo in the airway wall of a patient with severe asthma in a minimally invasive manner. We used optic axis uniformity (OAxU) to highlight the presence of uniformly arranged fiber-like tissue, helping visualizing the abundance of ASM and connective tissue structures. Attenuation coefficient images of the airways are presented for the first time, showing superior architectural contrast compared to standard OCT images. A novel segmentation algorithm was developed to detect the surface of the endoscope sheath and the surface of the tissue. PS-OCT is an innovative imaging technique that holds promise to assess airway remodeling including ASM and connective tissue in a minimally invasive, real-time manner.

Molecular Mechanisms for the Mechanical Modulation of Airway Responsiveness.

The smooth muscle of the airways is exposed to continuously changing mechanical forces during normal breathing. The mechanical oscillations that occur during breathing have profound effects on airway tone and airway responsiveness both in experimental animals and humans in vivo and in isolated airway tissues in vitro. Experimental evidence suggests that alterations in the contractile and mechanical properties of airway smooth muscle tissues caused by mechanical perturbations result from adaptive changes in the organization of the cytoskeletal architecture of the smooth muscle cell. The cytoskeleton is a dynamic structure that undergoes rapid reorganization in response to external mechanical and pharmacologic stimuli. Contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins into large macromolecular signaling complexes (adhesomes) that undergo activation to mediate the polymerization and reorganization of a submembranous network of actin filaments at the cortex of the cell. Cortical actin polymerization is catalyzed by Neuronal-Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, which are activated by pathways regulated by paxillin and the small GTPase, cdc42. These processes create a strong and rigid cytoskeletal framework that may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. This model for the regulation of airway smooth muscle function can provide novel perspectives to explain the normal physiologic behavior of the airways and pathophysiologic properties of the airways in asthma.

Maintenance of contractile function of isolated airway smooth muscle after cryopreservation.

Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

High Pressure Freezing Airway Smooth Muscle Tissue at Physiological Length for Analysis of Contractile Filaments

High Pressure Freezing Airway Smooth Muscle Tissue at Physiological Length for Analysis of Contractile Filaments

Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle.

The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASp). N-WASP transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase-inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We found that ROCK regulates airway smooth muscle contraction by mediating activation of p21-activated kinase (Pak), a serine-threonine kinase, to promote actin polymerization. Pak catalyses paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signalling module to paxillin, which activates the guanine nucleotide exchange factor (GEF) activity of βPIX towards Cdc42. Cdc42 is required for the activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp), which transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and Cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle.