Responsiveness of the isolated airway during simulated deep inspirations: effect of airway smooth muscle stiffness and strain.

In vivo, breathing movements, including tidal and deep inspirations (DIs), exert a number of beneficial effects on respiratory system responsiveness in healthy humans that are diminished or lost in asthma, possibly as a result of reduced distension (strain) of airway smooth muscle (ASM). We used bronchial segments from pigs to assess airway responsiveness under static conditions and during simulated tidal volume oscillations with and without DI and to determine the roles of airway stiffness and ASM strain on responsiveness. To simulate airway dilations during breathing, we cycled the luminal volume of liquid-filled segments. Volume oscillations (15 cycles/min) were set so that, in relaxed airways, they produced a transmural pressure increase of approximately 5-10 cmH(2)O for tidal maneuvers and approximately 5-30 cmH(2)O for DIs. ACh dose-response curves (10(-7)-3 x 10(-3) M) were constructed under static and dynamic conditions, and maximal response and sensitivity were determined. Airway stiffness was measured from tidal trough-to-peak pressure and volume cycles. ASM strain produced by DI was estimated from luminal volume, airway length, and inner wall area. DIs produced substantial ( approximately 40-50%) dilation, reflected by a decrease in maximal response (P < 0.001) and sensitivity (P < 0.05). However, the magnitude of bronchodilation decreased significantly in proportion to airway stiffening caused by contractile activation and an associated reduction in ASM strain. Tidal oscillations, in comparison, had little effect on responsiveness. We conclude that DI regulates airway responsiveness at the airway level, but this is limited by airway stiffness due to reduced ASM strain.     

Deep Inspiration and the Emergence of Ventilation Defects during Bronchoconstriction: A Computational Study

Deep inspirations (DIs) have a dilatory effect on airway smooth muscle (ASM) that helps to prevent or reduce more severe bronchoconstriction in healthy individuals. However, this bronchodilation appears to fail in some asthmatic patients or under certain conditions, and the reason is unclear. Additionally, quantitative effects of the frequency and magnitude of DIs on bronchodilation are not well understood. In the present study, we used a computational model of bronchoconstriction to study the effects of DI volumes, time intervals between intermittent DIs, relative speed of ASM constriction, and ASM activation on bronchoconstriction and the emergence of ventilation defects (VDefs). Our results showed a synergistic effect between the volume of DIs and the time intervals between them on bronchoconstriction and VDefs. There was a domain of conditions with sufficiently large volumes of DIs and short time intervals between them to prevent VDefs. Among conditions without VDefs, larger volumes of DIs resulted in greater airway dilation. Similarly, the time interval between DIs, during which the activated ASM re-constricts, affected the amplitude of periodic changes in airway radii. Both the relative speed of ASM constriction and ASM activation affected what volume of DIs and what time interval between them could prevent the emergence of VDefs. In conclusion, quantitative characteristics of DIs, such as their volume and time interval between them, affect bronchoconstriction and may contribute to difficulties in asthma. Better understanding of the quantitative aspects of DIs may result in novel or improved therapeutic approaches.     

Effect of Loading History on Airway Smooth Muscle Cell-Matrix Adhesions

Integrin-mediated adhesions between airway smooth muscle (ASM) cells and the extracellular matrix (ECM) regulate how contractile forces generated within the cell are transmitted to its external environment. Environmental cues are known to influence the formation, size, and survival of cell-matrix adhesions, but it is not yet known how they are affected by dynamic fluctuations associated with tidal breathing in the intact airway. Here, we develop two closely related theoretical models to study adhesion dynamics in response to oscillatory loading of the ECM, representing the dynamic environment of ASM cells in vivo. Using a discrete stochastic-elastic model, we simulate individual integrin binding and rupture events and observe two stable regimes in which either bond formation or bond rupture dominate, depending on the amplitude of the oscillatory loading. These regimes have either a high or low fraction of persistent adhesions, which could affect the level of strain transmission between contracted ASM cells and the airway tissue. For intermediate loading, we observe a region of bistability and hysteresis due to shared loading between existing bonds; the level of adhesion depends on the loading history. These findings are replicated in a related continuum model, which we use to investigate the effect of perturbations mimicking deep inspirations (DIs). Because of the bistability, a DI applied to the high adhesion state could either induce a permanent switch to a lower adhesion state or allow a return of the system to the high adhesion state. Transitions between states are further influenced by the frequency of oscillations, cytoskeletal or ECM stiffnesses, and binding affinities, which modify the magnitudes of the stable adhesion states as well as the region of bistability. These findings could explain (in part) the transient bronchodilatory effect of a DI observed in asthmatics compared to a more sustained effect in normal subjects.     

Cyclic stretch enhances reorientation and differentiation of 3-D culture model of human airway smooth muscle

Activation of airway smooth muscle (ASM) cells plays a central role in the pathophysiology of asthma. Because ASM is an important therapeutic target in asthma, it is beneficial to develop bioengineered ASM models available for assessing physiological and biophysical properties of ASM cells. In the physiological condition in vivo, ASM cells are surrounded by extracellular matrix (ECM) and exposed to mechanical stresses such as cyclic stretch. We utilized a 3-D culture model of human ASM cells embedded in type-I collagen gel. We further examined the effects of cyclic mechanical stretch, which mimics tidal breathing, on cell orientation and expression of contractile proteins of ASM cells within the 3-D gel. ASM cells in type-I collagen exhibited a tissue-like structure with actin stress fiber formation and intracellular Ca²⁺ mobilization in response to methacholine. Uniaxial cyclic stretching enhanced alignment of nuclei and actin stress fibers of ASM cells. Moreover, expression of mRNAs for contractile proteins such as α-smooth muscle actin, calponin, myosin heavy chain 11, and transgelin of stretched ASM cells was significantly higher than that under the static condition. Our findings suggest that mechanical force and interaction with ECM affects development of the ASM tissue-like construct and differentiation to the contractile phenotype in a 3-D culture model.     

Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Tidal Stretches Differently Regulate the Contractile and Cytoskeletal Elements in Intact Airways

Recent reports suggest that tidal stretches do not cause significant and sustainable dilation of constricted intact airways ex vivo. To better understand the underlying mechanisms, we aimed to map the physiological stretch-induced molecular changes related to cytoskeletal (CSK) structure and contractile force generation through integrin receptors. Using ultrasound, we measured airway constriction in isolated intact airways during 90 minutes of static transmural pressure (Ptm) of 7.5 cmH₂O or dynamic variations between Ptm of 5 and 10 cmH₂0 mimicking breathing. Integrin and focal adhesion kinase activity increased during Ptm oscillations which was further amplified during constriction. While Ptm oscillations reduced β-actin and F-actin formation implying lower CSK stiffness, it did not affect tubulin. However, constriction was amplified when the microtubule structure was disassembled. Without constriction, α-smooth muscle actin (ASMA) level was higher and smooth muscle myosin heavy chain 2 was lower during Ptm oscillations. Alternatively, during constriction, overall molecular motor activity was enhanced by Ptm oscillations, but ASMA level became lower. Thus, ASMA and motor protein levels change in opposite directions due to stretch and contraction maintaining similar airway constriction levels during static and dynamic Ptm. We conclude that physiological Ptm variations affect cellular processes in intact airways with constriction determined by the balance among contractile and CSK molecules and structure.     

Maintenance of airway caliber in isolated airways by deep inspiration and tidal strains.

Deep inspirations (DIs) are large periodic breathing maneuvers that regulate airway caliber and prevent airway obstruction in vivo. This study characterized the intrinsic response of the intact airway to DI, isolated from parenchymal attachments and other in vivo interactions. Porcine isolated bronchial segments were constricted with carbachol and subjected to transmural pressures of 5-10 cmH2O at 0.25 Hz (tidal breathing) interspersed with single DIs of amplitude 5-20 cmH2O, 5-30 cmH2O, or 5-40 cmH2O (6-s duration) or DI of amplitude 5-30 cmH2O (30-s duration). Tidal breathing was ceased after DI in a subset of airways and in control airways in which no DI was performed. Luminal cross-sectional area was measured using a fiber-optic endoscope. Bronchodilation by DI was amplitude dependent; 5-20 cmH2O DIs produced less dilation than 5-30 cmH2O and 5-40 cmH2O DIs (P=0.003 and 0.012, respectively). Effects of DI duration were not significant (P=0.182). Renarrowing after DI followed a monoexponential decay function to pre-DI airway caliber with time constants between 27.4+/-4.3 and 36.3+/-6.9 s. However, when tidal breathing was ceased after DI, further bronchoconstriction occurred within 30s. This response was identical in both the presence and absence of DI (P=0.919). We conclude that the normal bronchodilatory response to DI occurs as a result of the direct mechanical effects of DI on activated ASM in the airway wall. Further bronchoconstriction occurs by altering the airway wall stress following DI, demonstrating the importance of continual transient strains in maintaining airway caliber.     

The Importance of Synergy between Deep Inspirations and Fluidization in Reversing Airway Closure

Deep inspirations (DIs) and airway smooth muscle fluidization are two widely studied phenomena in asthma research, particularly for their ability (or inability) to counteract severe airway constriction. For example, DIs have been shown effectively to reverse airway constriction in normal subjects, but this is impaired in asthmatics. Fluidization is a connected phenomenon, wherein the ability of airway smooth muscle (ASM, which surrounds and constricts the airways) to exert force is decreased by applied strain. A maneuver which sufficiently strains the ASM, then, such as a DI, is thought to reduce the force generating capacity of the muscle via fluidization and hence reverse or prevent airway constriction. Understanding these two phenomena is considered key to understanding the pathophysiology of asthma and airway hyper-responsiveness, and while both have been extensively studied, the mechanism by which DIs fail in asthmatics remains elusive. Here we show for the first time the synergistic interaction between DIs and fluidization which allows the combination to provide near complete reversal of airway closure where neither is effective alone. This relies not just on the traditional model of airway bistability between open and closed states, but also the critical addition of previously-unknown oscillatory and chaotic dynamics. It also allows us to explore the types of subtle change which can cause this interaction to fail, and thus could provide the missing link to explain DI failure in asthmatics.     

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (E(max)) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in E(max) in BTSM strips and their proliferative responses in BSTM cells and for E(max) and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.     

Sevoflurane Inhibits Nuclear Factor-κB Activation in Lipopolysaccharide-Induced Acute Inflammatory Lung Injury via Toll-Like Receptor 4 Signaling

Background

Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR₄) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms.

Methods

In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR₄ and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR₄ and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards.

Results

The mRNA and protein levels of NF-κB and TLR₄ in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR₄ and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol.

Conclusions

This study demonstrates the crucial role of TLR₄ activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo and in vitro via inhibition of TLR₄/NF-κB pathway.     

The laminin β1-competing peptide YIGSR induces a hypercontractile,hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma

Background

Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established.

Methods

Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro.

Results

Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline- and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR.

Conclusion

These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide.     

Differential Regulation of Extracellular Matrix and Soluble Fibulin-1 Levels by TGF-β1 in Airway Smooth Muscle Cells

Fibulin-1 (FBLN-1) is a secreted glycoprotein that is associated with extracellular matrix (ECM) formation and rebuilding. Abnormal and exaggerated deposition of ECM proteins is a hallmark of many fibrotic diseases, such as chronic obstructive pulmonary disease (COPD) where small airway fibrosis occurs. The aim of this study was to investigate the regulation of FBLN-1 by transforming growth factor beta 1 (TGF-β₁) (a pro-fibrotic stimulus) in primary human airway smooth muscle (ASM) cells from volunteers with and without COPD. Human ASM cells were seeded at a density of 1×10⁴ cells/cm², and stimulated with or without TGF-β₁ (10 ng/ml) for 72 hours before FBLN-1 deposition and soluble FBLN-1 were measured. Fold change in FBLN-1 mRNA was measured at 4, 8, 24, 48, 72 hours. In some experiments, cycloheximide (0.5 µg/ml) was used to assess the regulation of FBLN-1 production. TGF-β₁ decreased the amount of soluble FBLN-1 both from COPD and non-COPD ASM cells. In contrast, the deposition of FBLN-1 into the ECM was increased in ASM cells obtained from both groups. TGF-β₁ did not increase FBLN-1 gene expression at any of the time points. There were no differences in the TGF-β₁ induced FBLN-1 levels between cells from people with or without COPD. Cycloheximide treatment, which inhibits protein synthesis, decreased both the constitutive release of soluble FBLN-1, and TGF-β₁ induced ECM FBLN-1 deposition. Furthermore, in cycloheximide treated cells addition of soluble FBLN-1 resulted in incorporation of FBLN-1 into the ECM. Therefore the increased deposition of FBLN-1 by ASM cells into the ECM following treatment with TGF-β₁ is likely due to incorporation of soluble FBLN-1 rather than de-novo synthesis.     

Bronchodilation response to deep inspirations in asthma is dependent on airway distensibility and air trapping

In healthy individuals, deep inspirations (DIs) have a potent bronchodilatory ability against methacholine (MCh)-induced bronchoconstriction. This is variably attenuated in asthma. We hypothesized that inability to bronchodilate with DIs is related to reduced airway distensibility. We examined the relationship between DI-induced bronchodilation and airway distensibility in 15 asthmatic individuals with a wide range of baseline lung function [forced expired volume in 1 s (FEV(1)) = 60-99% predicted]. After abstaining from DIs for 20 min, subjects received a single-dose MCh challenge and then asked to perform DIs. The effectiveness of DIs was assessed by the ability of the subjects to improve FEV(1). The same subjects were studied by two sets of high-resolution CT scans, one at functional residual capacity (FRC) and one at total lung capacity (TLC). In each subject, the areas of 21-41 airways (0.8-6.8 mm diameter at FRC) were matched and measured, and airway distensibility (increase in airway diameter from FRC to TLC) was calculated. The bronchodilatory ability of DIs was significantly lower in individuals with FEV(1) <75% predicted than in those with FEV(1) ≥75% predicted (15 ± 11% vs. 46 ± 9%, P = 0.04) and strongly correlated with airway distensibility (r = 0.57, P = 0.03), but also with residual volume (RV)/TLC (r = -0.63, P = 0.01). In multiple regression, only RV/TLC was a significant determinant of DI-induced bronchodilation. These relationships were lost when the airways were examined after maximal bronchodilation with albuterol. Our data indicate that the loss of the bronchodilatory effect of DI in asthma is related to the ability to distend the airways with lung inflation, which is, in turn, related to the extent of air trapping and airway smooth muscle tone. These relationships only exist in the presence of airway tone, indicating that structural changes in the conducting airways visualized by high-resolution CT do not play a pivotal role.     

The Cellular and Molecular Basis of Bitter Tastant-Induced Bronchodilation

Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca²⁺ signals, as revealed in cultured ASM cells, to activate large-conductance Ca²⁺-activated K⁺ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca²⁺ events nor alter spontaneous local Ca²⁺ transients. Interestingly, they increase global intracellular [Ca²⁺]_i, although to a much lower level than bronchoconstrictors. We show that these Ca²⁺ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca²⁺]_i induced by bronchoconstrictors, and this lowering of the [Ca²⁺]_i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca²⁺ channels (VDCCs), resulting in reversal in [Ca²⁺]_i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca²⁺ signaling pathways via Gβγ to increase [Ca²⁺]_i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca²⁺]_i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.     

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

Background

In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM) contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction.

Methods

Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX) to these reponses was established, using the inhibitors Y-27632 (1 μM), U-0126 (3 μM) and indomethacin (3 μM), respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂) was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM) and the selective EP₁-antagonist AH-6809 (10 μM) on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF_2α-and PGE₂-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay.

Results

Epidermal growth factor (EGF)-and platelet-derived growth factor (PDGF)-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF_2α-and PGE₂-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF_2α-and PGE₂-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM) significantly reduced (approximately 50 %) and the EP₁-antagonist AH-6809 (10 μM) abrogated growth factor-induced contractions, similarly in intact and epithelium-denuded preparations.

Conclusion

The results indicate that growth factors induce ASM contraction through contractile prostaglandins – not derived from the epithelium – which in turn rely on Rho-kinase for their contractile effects.     

Endobronchial Ultrasound Reliably Quantifies Airway Smooth Muscle Remodeling in an Equine Asthma Model

Endobronchial ultrasonography (EBUS) revealed differences in the thickness of the layer representing subepithelial tissues (L2) between human asthmatics and controls, but whether this measurement correlates with airway smooth muscle (ASM) remodeling in asthma is unknown. In this study, we sought to determine the ability of EBUS to predict histological ASM remodeling in normal and equine asthmatic airways. We studied 109 isolated bronchi from the lungs of 13 horses. They underwent EBUS examination using a 30 MHz radial probe before being processed for histology. ASM remodeling parameters were evaluated in EBUS images (L2 thickness, L2 area, L2 area/internal perimeter [Pi] and L2 area/Pi²) and histological cuts (ASM area/Pi²), and compared. EBUS was then performed ex vivo on the lungs of 4 horses with heaves, an asthma-like condition of horses, and 7 controls to determine whether central bronchial remodeling could be detected with this technique. An optimized approach was developed based on data variability within airways, subjects, and groups, and then validated in 7 horses (3 controls, 4 with heaves) that underwent EBUS in vivo. L2 area was significantly associated to ASM area in isolated lungs (p<0.0001), in the absence of significant bias related to the airway size. Bronchial size significantly affected EBUS ASM-related parameters, except for L2 area/Pi². L2 area/Pi² was increased in the airways of asthmatic horses compared to controls, both ex vivo and in vivo (p<0.05). Bronchial histology confirmed our findings (A_ASM/Pi² was increased in asthmatic horses compared to controls, p<0.05). In both horses with heaves and controls, L2 was composed of ASM for the outer 75% of its thickness and by ECM for the remaining inner 25%. In conclusion, EBUS reliably allows assessment of asthma-associated ASM remodeling of central airways in a non-invasive way.     

TNF-[alpha] modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl.

Although the mechanisms that underlie airway hyperresponsiveness in asthma are complex and involve a variety of factors, evidence now suggests that intrinsic abnormalities in airway smooth muscle (ASM) may play an important role. We previously reported that TNF-alpha, a cytokine involved in asthma, augments G-protein-coupled receptor (GPCR) agonist-evoked calcium responses in cultured ASM cells. Here we have extended our previous studies by investigating whether TNF-alpha also modulates the contractile and relaxant responses to GPCR activation using cultured murine tracheal rings. We found that in tracheal rings treated with 50 ng/ml TNF-alpha, carbachol-induced isometric force was significantly increased by 30% compared with those treated with diluent alone (P < 0.05). TNF-alpha also augmented KCl-induced force generation by 70% compared with rings treated with diluent alone (P < 0.01). The enhancing effect of TNF-alpha on carbachol-induced isometric force generation was completely abrogated in the tracheal rings obtained from TNF-alpha receptor (TNFR)1-deficient mice and in control rings treated with a TNF-alpha mutant that solely activates TNFR2. TNF-alpha also attenuated relaxation responsiveness to isoproterenol but not to PGE2 or forskolin. TNF-alpha modulatory effects on GPCR-induced ASM responsiveness were completely abrogated by pertussis toxin, an inhibitor of Gialpha proteins. Taken together, these data suggest that TNF-alpha may participate in the development of airway hyperresponsiveness in asthma via the modulation of ASM responsiveness to both contractile and beta-adrenoceptor GPCR agonists.