Mechanisms of Airway Remodeling Airway Smooth Muscle JUDITH L. BLACK, MICHAEL ROTH, JINHEE LEE, STEPHEN CARLIN, and PETER R. A. JOHNSON Department of Pharmacology, and Institute of Respiratory Medicine, University of Sydney, Sydney, Australia
The airway smooth muscle cell can contract; relax; participate in allergic and inflammatory responses by expressing adhesion molecules, releasing cytokines, and producing matrix proteins and proteases; and, as has been reported, undergo migration. These properties enable the muscle cell to be a key component in the airway wall remodeling that accompanies persistent asthma. Evidence is emerging that identifies the pivotal steps in the signal transduction pathways that lead to the excessive proliferation of the muscle observed in vitro in airway smooth muscle cells from subjects with asthma. The contractile, biochemical, and growth characteristics of muscle from allergic subjects are different from those of nonallergic subjects. In addition, the allergic response impacts on the extracellular matrix in which the muscle is embedded, by altering the profile of matrix proteins released. Once the relationships between allergy and inflammation of the smooth muscle and its extracellular matrix are better defined, opportunities to prevent or reverse airway remodeling will become available.
sponse to histamine when in the presence of mast cell-derived tryptase than does tissue that does not contract in response to allergen (nonsensitized) (14). Sensitized airway smooth muscle contains more myosin light chain kinase than does nonsensitized muscle (15) and more mast cells are observed in the muscle layer of sensitized bronchi (16). Whether this translates to altered contractility or growth is not known. When human airway smooth muscle cells in culture are exposed to allergic serum and challenged with allergen, there is increased protein expression of the oncogenes c-fos and c-jun and cycling of the cells as measured by incorporation of tritiated thymidine (13). Exposure of muscle cells to allergic serum also specifically increases the release of some matrix proteins (4). Clearly, therefore, there is an important interaction between the allergic response and the properties of the muscle cell that are relevant to asthmatic airway wall remodeling.
The concept of the contribution of the airway smooth muscle cell to the pathophysiological changes that occur in asthma has markedly changed. From playing a passive role as a structural cell, implicated merely in the contraction producing immediate airway narrowing, the airway smooth muscle cell is now acknowledged as an active participant in the inflammatory and allergic events that accompany persistent asthma. This is based on the observations that this cell (1) undergoes proliferation in response to many growth factors and cytokines, resulting in an increase in muscle volume that is in part due to hyperplasia, (2) produces and releases a number of cytokines (1), (3) is capable of expressing on the cell surface adhesion molecules that engage inflammatory cells, which in turn results in the further elaboration of cytokines and increases in proliferation (2, 3), and (4) produces extracellular matrix proteins that can profoundly influence muscle cell function (4).
AIRWAY SMOOTH MUSCLE PROLIFERATION
AIRWAY SMOOTH MUSCLE AND ALLERGY The airway smooth muscle has the potential to contribute to and be influenced by the allergic response. This is true of both contractility and growth. Exposure of human bronchial segments to allergic asthmatic serum (passive sensitization) produces increases in contraction and decreases in relaxation responses to a number of agonists (5–13). Bronchial tissue which contracts in response to the application of allergen (i.e., which is “sensitized”—a response that is likely to be the airway in vitro equivalent of the skin prick test), contracts more in re-
(Received in original form June 15, 2001; accepted in final form July 13, 2001) Supported by the NH&MRC, Australia; the Medical Foundation of the University of Sydney; and AstraZeneca, Sweden. Correspondence and requests for reprints should be addressed to Judith L. Black, Department of Pharmacology, University of Sydney, Sydney NSW 2006, Australia. E-mail:
[email protected] Am J Respir Crit Care Med Vol 164. pp S63–S66, 2001 DOI: 10.1164/rccm2106059 Internet address: www.atsjournals.org
Since culture of human airway smooth muscle cells has become possible, there has been considerable focus on elucidating the signal transduction pathways leading to proliferation (1). The effects of mitogens are mediated through at least two distinct receptor systems: tyrosine kinase-linked receptors (such as platelet-derived growth factor, epidermal derived growth factor, and basic fibroblast growth factor) and G protein-coupled receptors (GPCRs) (stimulated by thrombin). Downstream of these two systems, activation of the Shc–Grb2–Sos complex occurs or phosphatidylinositol (PI) 3 kinase is activated. This leads to activation of p21 Ras, and, after activation, active Ras binds to GTP. Raf-1 is then activated and translocates to the plasma membrane, where it phosphorylates mitogen-activated protein kinase kinase (MEK). A group of key regulators of growth, the mitogen-activated protein (MAP) kinases, is then stimulated. Evidence is accumulating that one of the MAP kinases, extracellular signal-regulated protein kinase (ERK), plays a crucial role in human airway smooth muscle (HASM) cell growth (17). Mitogens activating both tyrosine kinase and GPCRs cause upregulation of active ERK (Figure 1) and this is associated with an increase in thymidine uptake, an indicator of cell proliferation. Inhibition of MEK with specific antagonists such as UO126 or use of an antisense sequence directed to ERK decreases ERK activation and also inhibits thymidine incorporation (17). Activated ERK activates transcription factors such as c-Jun, c-Fos, and c-Myc in the nucleus and this may occur via activation of p90 ribosomal S6 kinase (Figure 1). This would suggest that ERK activation is necessary and sufficient for proliferation of HASM cells. However, others have reported the existence of ERK-independent pathways to proliferation that are mediated via stimulation of PI 3-kinase (18). In addition, there is synergy between the two pathways (18). Inflammatory and contractile mediators that signal through G protein-coupled receptors and that do not themselves produce proliferation can significantly augment growth occurring through the receptor tyrosine kinase pathway. Although ERK and perhaps PI 3-kinase may afford opportunities for therapeutic targeting, other steps
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Figure 1. Mitogenic signaling pathways in human airway smooth muscle cells. Mitogens that signal through receptor tyrosine kinase or G protein-coupled receptors activate the Shc–Grb2–Sos complex or PI 3-kinase. This results in successive activation of p21 Ras, Raf-1, MEK1 and -2, ERK1 and -2, and p90rsk. PI 3-kinase activates p70rsk or PKC-. ERK1 and -2 as well as the S6 kinases, p90rsk and p90rsk, then activate transcription factors that are needed for DNA synthesis in the nucleus. Definitions of abbreviations: Sos son of sevenless; PI-3 kinase phosphatidylinositol 3-kinase; MEK1 and -2 mitogen-activated protein kinase (MAP kinase) kinase 1 and 2; ERK 1 and 2 extracellular signal regulated-kinase 1 and 2 p90rsk p90 ribosomal S6 kinase; p70rsk p70 ribosomal S6 kinase; Shc Src homology/collagen adaptor protein; Grb2 growth factor receptor-bound protein 2.
in the signaling pathway may need to be inhibited. Protein kinase C (PKC)–, one of the atypical isoforms of this enzyme, is specifically increased when HASM cells are stimulated with mitogens (19). Moreover, an antisense oligonuceotide directed to PKC- significantly inhibits proliferation of HASM cells induced by platelet-derived growth factor (PDGF) (20). PKC- can be activated by PI 3-kinase and by Ras and PKC- may directly activate Raf-1. The relationship between ERK, PI 3-kinase, and PKC- in the proliferative pathway in human airway smooth muscle cells and, in particular, in asthmatic cells remains to be determined, especially if targeted inhibition of proliferation to prevent or reverse remodeling is to be attempted.
AIRWAY SMOOTH MUSCLE AND THE EXTRACELLULAR MATRIX Although several studies have demonstrated an increase in the amount of smooth muscle in the airway wall, this is not the only component of remodeling, that is, architectural/structural changes, that can impact on airway function. The extracellular matrix (ECM), in particular that component which surrounds and embeds the airway smooth muscle, plays a pivotal role in modulating the proliferative and contractile properties of the smooth muscle. The ECM is an intricate network of macromolecules that have the potential to influence migration, proliferation and differentiation (21, 22) of human airway smooth
Figure 2. The effect of urokinase on human airway smooth muscle motility. The receptor for urokinase (uPAR) is present on human airway smooth muscle cells and the cells also produces urokinase (uPA). Binding of uPA to uPAR with integrins forms a complex that initiates cell migration.
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muscle. It can also influence the distribution and adhesion of inflammatory cells, fluid balance, and elasticity and act as an inflammatory mediator reservoir (23). In the airways of subjects with asthma, there is an increase in the amount of collagen I, III, and V, fibronectin, tenascin, hyaluronan, versican, and laminin 2/2 (reviewed in Johnson and coworkers [4]) and a decrease in collagen IV and elastin (24). Changes in ECM deposition within the airways of subjects with asthma may lead, or contribute, to the development of bronchial hyperresponsiveness. The causes of the change in matrix deposition are unknown; however, plasma leakage from the microvasculature as part of allergic and inflammatory processes may play a role. HASM cells in culture produce fibronectin, perlecan, elastin, laminin 1, 1, 2, and 1, thrombospondin, chondroitin sulfate, collagen types I, III, IV, and V, versican, and decorin. When these cells are exposed to allergic serum from subjects with asthma, production of fibronectin, laminin 1 chain, perlecan and chondroitin sulfate is increased compared with cells exposed to nonallergic serum from patients without asthma (4). Interestingly, these increases in specific proteins are not inhibited by prior exposure to either corticosteroids or leukotriene antagonists. The changes in the ECM in the asthmatic airway wall could also result from decreased activity of degrading enzymes—the matrix metalloproteinases (MMPs) or upregulation of the tissue-specific inhibitors of metalloproteinases (TIMPs). The influence of the allergic response is also apparent on these enzymes. HASM cells secrete the two gelatinases MMP-2 and MMP-9 as well as TIMP-1 but not TIMP-2 or -3. Exposure of asthmatic HASM cells to asthmatic serum increases the activity of MMP-9 and decreases the expression and activity of MMP-2. Thus the inflammatory/ allergic response with associated vascular leakage and subsequent exposure of muscle cells to serum components may contribute to changes in the extracellular matrix (25). These changes have the potential to produce profound alterations in the properties of the smooth muscle that lies within it. Allergic serum contains many potentially active components, however, and which of these is responsible for the observed increases in specific matrix proteins is not known. Attempts to attribute a role to the high concentration of IgE that characterizes the allergic serum have so far been unsuccessful (4).
AIRWAY SMOOTH MUSCLE CELL MIGRATION The similarities between the remodeling that occurs in the vasculature as an accompaniment to atherosclerosis and that in airways in asthma has been noted for some time (26). Both involve chronic inflammatory processes in hollow tubes and both are amenable to intervention. Until recently, one of the major differences between the two resided in the fact that one of the pivotal events in the remodeled vessel is the early migration of muscle cells from the media to form a neointima underneath the endothelium. Chemotaxis was first studied in human cells in 1976 by Postlethwaite and coworkers (27). Fibroblasts were shown to migrate through 8-m pores in a Boyden chamber toward a compartment containing a chemotactic agent, and the same procedure was used to demonstrate that PDGF is chemotactic for aortic smooth muscle cells. Skinner and coworkers (28) showed that migration of human vascular cells was dependent on the extracellular matrix in that the presence of the 21 integrin complex was necessary. Mukhina and coworkers (29) reported that human airway smooth muscle cells in culture are capable of migration, particularly in response to urokinase plasminogen activator. Plasminogen activators are of two types: urokinase plasminogen activator (uPA or urokinase), which is secreted by many cell
types, and tissue-type plasminogen activator (tPA). Of these, uPA is implicated in cell migration and proliferation (Figure 2). These actions are mediated via the high-affinity cell surface receptor – uPA receptor (uPAR), which is also designated CD87. The uPAR system is also implicated in the chemotactic responses initiated by growth factors that are linked to receptor tyrosine kinase. The uPAR is anchored to the extracellular membrane by a membrane lipid anchor and, because it does not cross the cell membrane, its intracellular actions may be mediated via integrins (30) and also the lipoprotein receptor (LPR). Upregulation of the uPA/uPAR system can occur in response to a variety of growth factors and cytokines (31) and the signal transduction events that mediate this are thought to be the classic mitogenic pathway involving phospholipase D, PKC, Ras, Raf, and ERK. Mukhina and coworkers (29) found that human airway smooth muscle cells migrate via an interaction involving the binding of the uPA kringle domain to the cell surface membrane and the association of uPA with the urokinase receptor. The significance of this finding that airway smooth muscle cells migrate lies in the fact that, just as occurs in remodeled vessels, in which muscle cells migrate to form a neointima underneath the endothelium, airway smooth muscle cells may migrate in response to a number of factors—perhaps arising from the injured epithelium or the presence of inflammatory mediators and growth factors, to take up a position underneath the epithelium. It is possible that myofibroblasts, which have been observed in this position in the asthmatic airway, could be muscle cells that have migrated lumenally. Whether this is true in the remodeled airway, and whether there are significant functional sequelae, requires further investigation.
ASTHMATIC AIRWAY SMOOTH MUSCLE CELLS Until more recently it has been difficult to obtain and culture airway smooth muscle cells from subjects with asthma. However, this opportunity has now arisen. We hypothesized that there is an intrinsic abnormality of the smooth muscle cell that contributes to the increased amount of smooth muscle in the asthmatic airway wall and that we would detect this abnormality in our culture system. Indeed, asthmatic airway smooth muscle cells grow at approximately twice the rate of cells from subjects without asthma (32). It is now crucial to determine at what point(s) in the signal transduction pathway this abnormality occurs. There is preliminary evidence that activation of ERK is altered in asthmatic cells. Although basal ERK activity is surprisingly lower in asthmatic cells, peak ERK activity in response to stimulation with a low concentration of mitogen is greatly increased over that in cells from subjects without asthma (33). Whether any abnormality exists in the PKC- or the PI 3-kinase pathways is not known. Differences between asthmatic and nonasthmatic airway smooth muscle cells in the effects of the long-acting -agonist formoterol and the corticosteroid budesonide on proliferation have also been observed in preliminary experiments (34). Whereas both drugs produce inhibition of mitogenesis in nonasthmatic cells, and formoterol inhibits growth in asthmatic cells, budesonide does not cause inhibition in asthmatic cells. Thus there may be an intrinsic abnormality in endogenous inhibitory pathways in the asthmatic airway smooth muscle that could contribute to increased proliferation. Exactly how remodeling in the airways, and in particular those changes that occur in relation to the smooth muscle, relate to changes in lung function is not known. What is known, however, is that increased amounts of smooth muscle are likely to lead to exaggerated airway narrowing. Moreover, pa-
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tients differ in the degree to which their airflow limitation is reversible. Whether this heterogeneity is related to the presence of airway wall remodeling remains to be investigated.
CONCLUSIONS AND FUTURE RESEARCH Now that it is possible to study airway smooth muscle cells from subjects with asthma, we have an opportunity to define the exact abnormality in the signal transduction pathways that lead to excessive proliferation. Further valuable information may be gained by examining an additional property of airway smooth muscle cells, namely, migration. As a consequence of this, it will be possible to devise strategies to intervene or reverse the processes that lead to airway wall remodeling. In addition, the mechanisms underlying the relationship between the allergic response and alteration in the properties of smooth muscle require elucidation. Acknowledgment : The authors acknowledge the collaborative efforts of Dr. Greg King, and of the Cardiopulmonary Transplant Team at St. Vincent’s Hospital.
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