Asthmatic Muscle Research Articles

Research Highlights

Research Highlights

Cyclic GMP-dependent protein kinase regulation of airway smooth muscle in asthma

The mechanisms regulating altered airway smooth muscle (ASM) cell tone and airway reactivity in asthma remain incompletely understood. For vascular smooth muscle (VSM), the most important endogenous relaxation pathway is the nitric oxide (NO)-guanylate cyclase-cyclic GMP-dependent protein kinase type I (PKGI) pathway in which endogenous NO from NO synthases or from exogenous nitrovasodilators activate guanylate cyclase, increasing cGMP levels, and activating PKGI. PKGI in turn targets specific VSM molecules that mediate smooth muscle relaxation. Recent evidence supports that in ASM, like in VSM, activators of guanylyl cyclases have significant cGMP-mediated relaxant and antiproliferative effects. PKGI is therefore a prime but unexplored candidate molecule to mediate ASM relaxation. Recently, we have used mice harbouring a mutation in the leucine zipper N-terminal interaction domain (LZM mice [1]) to test the hypothesis that PKGIα is a critical mediator of ASM relaxation that normally protects against airway hyperresponsivity via interactions with specific molecular targets, including ASM myosin phosphate and Rho/Rho-kinase, and that this regulatory system is perturbed in the setting of asthma. Using this knock-in mouse harboring a discrete mutation in the protein-protein interaction domain of PKGI, which disrupts NO-PKG-mediated smooth muscle relaxation by disrupting the PKGI-myosin phosphatase complex we have begun to explore the mechanism of PKGI-mediated ASM relaxation and the role of PKGI in models of asthma. Several asthma studies in LZM mice and WT littermates have now been performed and analyzed using both the acute and the chronic House Dust Mite (HDM) asthma model in WT and LZM mice. Endpoints examined include airway responsiveness to methacholine; quantification of inflammatory cell populations from Bronchoalveolar lavage (BAL); and studies of airway smooth muscle in the mice. In the LZM mice, there is a significant increase in bronchospasm noted in the airways of mutant mice in both acute and chronic asthma models. No differences between WT and LZM mice in the extent of increase in either total cells or in eosinophilia in BALF samples following HDM was noted in the either the acute or chronic models. The extent and volume of Airway SMC a-actin was also measured in the WT and LZM mice following HDM-induced asthma in the acute model. SMC labeling in the airways was quantified using immunohistochemistry and computerized morphometrics and for each measure, a significant increase in SMC number and total volume was noted in the LZM mice. Studies of PKG expression level and of SMC proliferation in the asthma models are underway. These experiments will provide better understanding of ASM relaxation at the molecular level and have the potential to identify novel gene targets for the development of new therapeutic approaches to bronchoconstriction in asthma.

Modulation of epidermal growth factor receptor binding to human airway smooth muscle cells by glucocorticoids and β2-adrenergic receptor agonists

EGF receptors (EGFRs) are increased in airway smooth muscle in asthma, which may contribute to both their hyperproliferation and hypercontractility. Lysophosphatidic acid (LPA) is a candidate pathological agent in asthma and other airway diseases, and LPA upregulates EGFRs in human airway smooth muscle (HASM) cells. We tested whether therapeutic glucocorticoids and/or beta(2)-adrenergic receptor (beta(2)AR) agonists also alter EGFR binding in HASM cells. Exposure to glucocorticoids for 24 h induced a twofold increase in EGFR binding similar to that with LPA; fluticasone was markedly more potent than dexamethasone. The increase in EGFR binding by glucocorticoids required 24-h exposure, consistent with transcription-mediated effects. Although the increase in EGFR binding was blocked by the protein synthesis inhibitor cycloheximide for LPA, fluticasone, and dexamethasone, only LPA induced a significant increase in EGFR protein expression detected by immunoblotting. In contrast to the increased binding induced by the glucocorticoids, the beta(2)AR agonists isoproterenol, albuterol, and salmeterol all induced a decrease in EGFR binding. beta(2)AR agonist effects were multiphasic, with an initial decline at 2-4 h that reversed by 6 h and a second, somewhat greater decrease by 18-24 h. In cells pretreated with glucocorticoids, the decreases in EGFR binding by subsequent beta(2)AR treatment were not statistically significant; glucocorticoid upregulation of EGFRs also prevented further increases by LPA. Similar increases by glucocorticoids and decreases by beta(2)AR agonists were found in HFL-1 human lung fibroblasts. These complex and opposing effects of clinically relevant glucocorticoids and beta(2)AR agonists on airway mesenchymal cell EGFRs likely contribute to their overall therapeutic profile in the diseased airway.

IS5-5 Altered properties of airway smooth muscle in asthma(Session 5: Therapeutic strategies targeting inflammatory cells,Eosinophils, other Inflammatory Cells and Molecules in Allergy)

IS5-5 Altered properties of airway smooth muscle in asthma(Session 5: Therapeutic strategies targeting inflammatory cells,Eosinophils, other Inflammatory Cells and Molecules in Allergy)

Airway smooth muscle in asthma: Phenotype plasticity and function

Clinical asthma is characterized by reversible airway obstruction which is commonly due to an exaggerated airway narrowing referred to as airway hyperresponsiveness (AHR). Although debate exists on the complex etiology of AHR, it is clear that airway smooth muscle (ASM) mediated airway narrowing is a major contributor to airway dysfunction. More importantly, it is now appreciated that smooth muscle is far from being a simple cell with only contractile ability properties. Rather, it is more versatile with the capacity to exhibit numerous cellular functions as it adapts to the microenvironment to which it is exposed. The emerging ability of individual smooth muscle cells to undergo changes in their phenotype (phenotype plasticity) and function (functional plasticity) in response to physiological and pathological cues is an important and active area of research. This article provides a brief review of the current knowledge and emerging concepts in the field of ASM phenotype and function both under healthy and asthmatic conditions.

Correlation between airway inflammation and loss of deep-inhalation bronchoprotection in asthma

One of the characteristic features of the hyperresponsive airway smooth muscle in asthma is the loss of deep-inhalation bronchoprotection and bronchodilation. The airway of individuals with asthma is also characterized by inflammation. To evaluate whether the loss of deep-inhalation bronchoprotection is correlated with the degree of inflammation in the asthmatic airway. Eighteen study participants performed 2 methacholine challenges (identical doses), 1 with deep inhalations and 1 without, separated by at least 24 hours. Airway inflammation was evaluated by measurement of fraction of exhaled nitric oxide (FE(NO)) and induced sputum eosinophils. A significant negative correlation was found between the degree of deep-inhalation bronchoprotection and airway inflammation when measured by FE(NO) (P = .02, r = .54, n = 18) and by percentage of eosinophils (P = .002, r = .76, n = 12). A significant positive correlation was also found between the FE(NO) and percentage of eosinophils (P = .009, r = .68, n = 12). Deep-inhalation bronchoprotection was significantly impaired in individuals with greater airway inflammation. This finding suggests that therapy directed at decreasing airway inflammation may promote the recovery of normal deep-inhalation bronchoprotection.

Extracellular matrix components and regulators in the airway smooth muscle in asthma

There is an intimate relationship between the extracellular matrix (ECM) and smooth muscle cells within the airways. Few studies have comprehensively assessed the composition of different ECM components and its regulators within the airway smooth muscle (ASM) in asthma. With the aid of image analysis, the fractional areas of total collagen and elastic fibres were quantified within the ASM of 35 subjects with fatal asthma (FA) and compared with 10 nonfatal asthma (NFA) patients and 22 nonasthmatic control cases. Expression of collagen I and III, fibronectin, versican, matrix metalloproteinase (MMP)-1, -2, -9 and -12 and tissue inhibitor of metalloproteinase-1 and -2 was quantified within the ASM in 22 FA and 10 control cases. In the large airways of FA cases, the fractional area of elastic fibres within the ASM was increased compared with NFA and controls. Similarly, fibronectin, MMP-9 and MMP-12 were increased within the ASM in large airways of FA cases compared with controls. Elastic fibres were increased in small airways in FA only in comparison with NFA cases. There is altered extracellular matrix composition and a degradative environment within the airway smooth muscle in fatal asthma patients, which may have important consequences for the mechanical and synthetic functions of airway smooth muscle.

Gene Expression in Asthmatic Airway Smooth Muscle

Airway smooth muscle abnormalities are central to the pathophysiology of asthma. These airway smooth muscle cell abnormalities may include changes in cell number, size, phenotype, or function. Gene expression studies performed using asthmatic airway smooth muscle cells represent one approach to identifying the abnormalities of airway smooth muscle that occur in asthma in vivo. However, due to the technical challenges involved, only two studies have been performed to date using freshly obtained tissue from subjects with asthma. The first of these studies suggested increased expression of myosin light-chain kinase in airway smooth muscle from patients with asthma, whereas the second study found no difference in myosin light-chain kinase expression, nor any difference in other markers of smooth muscle phenotype in asthma. Studies performed in cell culture through the application of gene expression microarrays to profile airway smooth muscle cells exposed to potential mediators of asthma yield more consistent results, including induction by IL-13 of tenascin, the H1 histamine receptor, and IL-13 receptor subunits. However, the significance of these microarray findings for smooth muscle function is uncertain. Furthermore, gene expression studies have a fundamental limitation in that many functional properties of airway smooth muscle are regulated at other levels (e.g., protein phosphorylation). Thus, gene expression studies ultimately must be integrated with other methodological approaches to adequately study airway smooth muscle in asthma in vivo.

Response to C3a, mast cells, and asthma

This is in response to a “Letter to the Editor (1)” by Dr. Bradding regarding our manuscript entitled “Airway smooth muscle cells enhance C3a-induced mast cell degranulation following cell-cell contact” that was published in your journal recently (2). Dr. Bradding contends that our manuscript, which utilizes a C3a-responsive human mast cell line, is misleading because “it ignores wealth of consistent literature demonstrating that human lung mast cells and human lung fragments do not exhibit any response to C3a or C5a.” We disagree with this view for the reasons discussed below. Although studies performed in the 1980s and 1990s, showed that dispersed human lung mast cells are unresponsive to C3a and C5a (3–5), these studies did not characterize the expression of C3a receptor in mast cells and failed to consider the possibility that lung tissue may contain a subpopulation of mast cells that could be responsive to anaphylatoxins. Human mast cells (MC) consist of a mixture of at least two cell types that can be distinguished based on the protease content of their granules. It is well documented that MCTC cells (tryptaseand chymase-positive) that are found predominantly in the skin are responsive to C3a and C5a whereas MCT cells (tryptase positive) present in other tissues are unresponsive to these anaphylatoxins (6). The previous demonstration that dispersed lung mast cells did not respond to C3a or C5a probably reflects the fact that 90% of these cells are MCT cells (7). Indeed, a recent report by Oskeritzian et al. (8) showed that MCTC cells purified from a mixture of human lung mast cells are highly responsive to C5a for histamine release and leukotriene C4 generation, mediators that promote airway hyperreactivity. Most importantly, mast cells that infiltrate into the airway smooth muscle in asthma are positive for both tryptase and chymase (9). Given that MCTC cells are also responsive to C3a for mediator release (10, 11), we argue that a C3a responsive human mast cell line is an appropriate in vitro cellular model to delineate the signaling pathways involved in mast cell-airway smooth cell interaction relevant to asthma. REFERENCES

Remodelling of airway smooth muscle in asthma: what sort do you have?

Shortening of smooth muscle around airways is the basis of symptoms and abnormal lung function in asthma. The airway wall is increased in thickness in small and large airways asthma, in relation to the clinical severity of asthma and the airway smooth muscle layer is the main contributor to this thickening. The relative contributions of more airway smooth muscle cells, bigger cells or more extracellular matrix to the increased thickness of the smooth muscle layer in asthma is not clear and has been examined in only a small number of cases, Studies of the natural history of asthma suggest that the clinical severity of asthma is relatively constant over time, deficits in lung function compared with nonasthmatic subjects occur early in the course of the disease and the decline in lung function with age is increased in asthma. The observations from studies of the quantitative pathology and the natural history of asthma might be combined in the hypothesis that the severity of asthma is determined early (in its natural history) and is related mainly to increased volume density of airways smooth muscle cells (hyperplasia) and that later deposition of extracellular matrix from larger, hypertrophic smooth muscle cells results in fixed and increasing deficits in lung function. The relative contribution of more smooth muscle cells, bigger cells and extracellular matrix will be determined by unbiased stereological measurements in many cases of asthma of varying severity. The outcomes of such studies will be methods of monitoring and of treatment that will be tailored to the sort of smooth muscle modelling that is present in individual cases.

The CXCL10/CXCR3 Axis Mediates Human Lung Mast Cell Migration to Asthmatic Airway Smooth Muscle

Mast cell microlocalization within the airway smooth muscle bundle is an important determinant of the asthmatic phenotype. We hypothesized that mast cells migrate toward airway smooth muscle in response to smooth muscle-derived chemokines. In this study, we investigated (1) chemokine receptor expression by mast cells in the airway smooth muscle bundle in bronchial biopsies from subjects with asthma using immunohistology, (2) the concentration of chemokines in supernatants from stimulated ex vivo airway smooth muscle cells from subjects with and without asthma measured by enzyme-linked immunosorbent assay, and (3) mast cell migration toward these supernatants using chemotaxis assays. We found that CXCR3 was the most abundantly expressed chemokine receptor on human lung mast cells in the airway smooth muscle in asthma and was expressed by 100% of these mast cells compared with 47% of mast cells in the submucosa. Human lung mast cell migration was induced by airway smooth muscle cultures predominantly through activation of CXCR3. Most importantly, CXCL10 was expressed preferentially by asthmatic airway smooth muscle in bronchial biopsies and ex vivo cells compared with those from healthy control subjects. These results suggest that inhibition of the CXCL10/CXCR3 axis offers a novel target for the treatment of asthma.

Brightling CE, Bradding P, Symon FA, et al. Mastcell infiltration of airway smooth muscle in asthma. N Engl J Med. 2002;346:1699-1705.

Brightling CE, Bradding P, Symon FA, et al. Mastcell infiltration of airway smooth muscle in asthma. N Engl J Med. 2002;346:1699-1705.

Airway smooth muscle in asthma: flirting with disaster.

Airway smooth muscle in asthma: flirting with disaster.

Synthetic functions of airway smooth muscle in asthma.

Studies now show that the airway smooth muscle cell, in addition to its contractile function, can participate in and coordinate the inflammatory response. In response to inflammatory cytokines, the airway smooth muscle cell produces cytokines, chemokines, growth factors and cell adhesion molecules leading to inflammatory cell chemotaxis, hyperplasia and hypertrophy. Anti-inflammatory mediators are also produced suggesting a dual role in inflammation. Moreover, in response to growth stimuli the cells may adopt a phenotype more suited to 'synthetic' function. These issues are discussed in this article by Simon Johnson and Alan Knox, who also point to ways in which some of these pathways offer future therapeutic potential in asthma.

Airway smooth muscle in asthma.

One of the factors that may contribute to the exaggerated airway narrowing in asthma is an abnormality of the airway smooth muscle. This abnormality could take the form of an increase in the amount of muscle or an alteration in its pharmacological reactivity. The former could be due to either hypertrophy (an increase in individual muscle cell size) or hyperplasia (an increase in cell number). Changes in pharmacological reactivity that could be relevant to altered airway calibre could result from an increase in contraction or alternatively, a decrease in relaxation. Based on available evidence, the increase in smooth muscle bulk is probably the consequence of both hyperplasia and hypertrophy and several growth factors, inflammatory mediators and cytokines have been implicated. Asthmatic airway tissue is rarely available for in vitro pharmacological studies and evidence for enhanced contraction is limited. Recent evidence suggests that an abnormality in beta adrenoceptor function may contribute to impairment of relaxation, but further work needs to be done. Passive sensitization of non-asthmatic airways in vitro provides a good model for the study of the mechanisms underlying airway hyperresponsiveness, and will be the subject of more intensive study in the future.

Simultaneous measurement of Ca 2+ transients and changes in the cell volume and microviscosity of the plasma membrane in smooth muscle cells : Evaluation of the effect of formoterol

The effects of the β 2-adrenoceptor agonist formoterol (50 nM) on the angiotensin II (20 nM)-induced Ca 2+ response and changes in the cell volume and microviscosity of the plasma membrane of vascular smooth muscle cells were studied. Applied as a model substance for the stimulation of the phosphoinositide-phospholipase C pathway, angiotensin II has been used to simulate the bronchospasm of smooth muscle in asthma. Our results demonstrated that angiotensin II-induced smooth muscle contraction primarily involves an InsP 3-mediated release of Ca 2+ from intracellular stores and, to a minor extent, an enhanced influx of Ca 2+ through the plasma membrane. Both the Ca 2+ response and the contractile reaction were strongly antagonized by pretreatment of the cells with 50 nM formoterol. The protective effect of formoterol on smooth muscle contractions is proposed to be mainly related to a direct stimulation of β 2-adrenoceptor-coupled cAMP generation. Moreover, it is predicted that the interaction between the β 2-adrenoceptor glycoprotein and adenylate cyclase will be enhanced following a formoterol-associated decrease in the microviscosity of the plasma membrane.

Is Asthma a Vascular Disorder?

Is Asthma a Vascular Disorder?

A Mechanism for Impaired Relaxation of Airway Smooth Muscle in Asthma

A Mechanism for Impaired Relaxation of Airway Smooth Muscle in Asthma

Functional significance of increased airway smooth muscle in asthma and COPD.

Using a computational model, we investigated the effect of the morphologically determined increased airway smooth muscle mass, adventitial mass, and submucosal mass observed in patients with asthma and chronic obstructive pulmonary disease (COPD) on the increase in airway resistance in response to a bronchoconstricting stimulus. The computational model of Wiggs et al. (J. Appl. Physiol. 69: 849-860, 1990) was modified in such a way that smooth muscle shortening was limited by the maximal stress that the muscle could develop at the constricted length. Increased adventitial thickness was found to increase constriction by reducing parenchymal interdependence. Increased submucosal thickness led to greater luminal occlusion for any degree of smooth muscle shortening. Increased muscle thickness allowed greater smooth muscle shortening against the elastic loads provided by parenchymal interdependence and airway wall stiffness. We found that for constant airway mechanics, as reflected by the passive area-pressure curves of the airways, the increased muscle mass is likely to be the most important abnormality responsible for the increased resistance observed in response to bronchoconstricting stimuli in asthma and COPD. For a given maximal muscle stress, greater muscle thickness allows the development of greater tension and thus more constriction of the lumen.

Role of airway smooth muscle in asthma: Possible relation to the neuroendocrine system

Though not yet firmly established, it appears likely that the neuroendocrine system (NES) regulates airway smooth muscle function. As it is the latter which is altered in asthma, the importance of the role of the NES in this disease is clear. The fact that transmitters from the NE cells are released from their basal aspect, and are in close proximity to the subjacent airway smooth muscle, further indicates an interaction. The question then arises as to what are the experimental desiderata for conducting studies of the ASM. These should constitute what Sergei Sorokin has called the "Koch's postulates of airway smooth muscle research." As human tissues from asthmatics are difficult to obtain, animal models have been developed. The requirements are that, in these animals, the allergy be IgE based, that a congenital or familial factor be operative, that a noncholinergic nonadrenergic inhibitory system be a component of the neural regulatory system, and that the antigen for immunization be of a type commonly found in human asthmatics. Ideally, evidence of clinical asthma and exercise-induced asthma and nocturnal attacks should also be present. Unfortunately, no ideal animal models exist and one cannot talk about asthmatic animals, but only of animals with allergic bronchospasm. If in vitro research is to be conducted, there are additional requirements. The tissue should be from a relevant location. The tracheal smooth muscle which has been the favorite, purely because of its convenience, is not a good model. For the early asthmatic attack, central bronchi (3-5 mm diameter) should be used. Muscle strips obtained from them should be parallel-fibred and the cartilage plaques should be carefully dissected away, otherwise they contribute unwanted frictional forces when velocity is measured. Care should be taken to ensure that the epithelial cell layer is intact, as evidence indicates that it may regulate airway muscle function, though this has not been established for all the animal species used in asthma research. The isolated muscle strip should be in a steady state, particularly with respect to the functional variable under study, before definitive data are collected. Most importantly, it is shortening capacity that must be studied, as this is the in vitro analogue to in vivo narrowing of airways. Isometric force development provides information about wall stiffness and is of very little relevance to the elucidation of the mechanism of bronchospasm.(ABSTRACT TRUNCATED AT 400 WORDS)