Chairman's Summary
http://www.100md.com
《美国胸部学报》
Division of Pulmonary and Critical Care, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
The extracellular matrix (ECM) of the lung is composed of a cable network of highly inert elastic fibers that are not distributed uniformly in the lung parenchyma. They loop around alveolar ducts, form rings at the mouths of alveoli, and penetrate as wisps into alveolar septa, concentrating at bends and junctions. Elastin is a highly proteinase-resistant protein that normally lasts the human lifespan. Collagen forms a continuous sheath around alveolar units and larger bundles. Interstitial collagens (types I and III) provide the tensile strength that keeps the lung intact. One can think of elastin as a strong rubber band, and collagen as ropes, together providing elasticity and structural stability to the lung. The basement membrane separating the alveolar endothelium and epithelium is principally composed of a type IV collagen reticular network that binds to laminin through entactin bridges. Proteoglycans are also highly expressed in the basement membrane and interstitium, perhaps shielding other matrix components from destruction.
In addition to providing structural stability and serving as a barrier partitioning the lung into discrete compartments, the ECM also participates in a variety of additional cellular functions. Indeed, the cell senses its external environment through its interaction with the ECM. Hence, many critical cellular functions, including cell death and proliferation, are regulated by the ECM and integrins on the cell surface to which they bind. The ECM is also a reservoir for growth factors, including transforming growth factor;(TGF-?). Hence, activation and release of growth factors from the ECM is an important mode of regulation of cell behavior. Finally, proteolytic fragments of the ECM themselves have discrete biological functions, the most-studied example being the chemotactic activity imparted by elastin, collagen, fibronectin, and laminin fragments.
Given the need to maintain structural support and barrier function, it is not surprising that alterations in the ECM play a profound role in lung disease. However, the additional roles that the ECM plays in cell homeostasis suggest that alterations in ECM may be involved in disease pathogenesis in ways not previously recognized. Moreover, the ability of the ECM to appropriately repair itself, and provide scaffolding for cellular repair, will likely turn out to be a major factor in determining whether an insult results in a chronic disease.
ECM changes have dominated concepts regarding most major pulmonary disorders. In the lung parenchyma, loss of elastin and other ECM components is central to the pathogenesis of emphysema. Accumulation of interstitial collagen is the central lesion in pulmonary fibrosis. Within the airway, subepithelial fibrosis is a key component of airway remodeling in asthma, and submucosal fibrosis in small airways is also observed in chronic obstructive pulmonary disease (COPD). Obliterative bronchiolitis (OB), the main cause of death after lung transplantation, is a result of total obliteration of small airways by collagen.
These pathologic findings lead to a variety of fascinating yet unanswered questions. Are mechanisms leading to airway fibrosis in asthma and COPD related? Why do we observe subepithelial fibrosis in asthma and COPD yet total obliteration of the airways in OB? Why is idiopathic pulmonary fibrosis (IPF) peripheral sparing the central airways, whereas these other diseases have fibrosis focused in the airways? Is this due to different disease mechanisms or a result of the location of the instigating abnormality.
To further define the spectrum of biological functions of the ECM and the capacity for ECM repair, and understand the similarities and differences of ECM remodeling in different pulmonary disease processes, the organizers of the Transatlantic Airway Conference brought together a small but talented group of scientific experts in different pulmonary diseases, combined with a group of discussants known for their profound knowledge of lung pathobiology and their ability to ask probing questions. We now share with you the insights gained from this meeting.
APPLICATION OF LESSONS LEARNED FROM THE CONFERENCE TO ECM DESTRUCTION, FIBROSIS, AND REPAIR IN COPD
This meeting has highlighted the need for precise control of ECM turnover in the lung. Either depletion or excess of ECM will disrupt the structural and functional integrity of the lung, with too little ECM leading to emphysema, while too much results in fibrosis. In addition to providing structural support, many additional functions of the ECM were discussed, including the involvement of the ECM and their proteolytic fragments in cell signaling and homeostasis. COPD highlights the complexity of ECM turnover. The manifestations of cigarette smoke, inflammation, and perhaps airway infection lead to airway fibrosis with ECM accumulation, whereas smoke-related inflammatory proteinase activity in the lung parenchyma leads to ECM degradation. Manifestations of ECM turnover in COPD will be used as an example to summarize concepts regarding ECM pathobiology, genetics, and even stem cell biology, which were discussed during lectures and discussions of this meeting.
COPD: Complex Effects of Cigarette Smoke Exposure on the ECM
COPD is defined as a disease state characterized by airflow limitation that is not fully reversible in a patient who has been exposed to noxious agents, which in our society is almost exclusively cigarette smoke. The effect of cigarette smoke on a given individual depends on one's genetic make-up as well as additional environmental insults. Cigarette smoke causes prominent and distinct changes in the large airways, small airways, and alveolar space.
Studies spanning five decades have shown that, in lungs of humans and experimental animals with COPD, total elastin content is unchanged or decreased, whereas total collagen content is increased. With the loss of an alveolar unit in emphysema, all ECM components are destroyed. This includes elastin, which is most prominent in the "mouth" of the alveolus (and therefore accounts for centriacinar changes), as well as collagen, which is more uniformly distributed throughout the alveolus. Although increases in elastin in the vessels and pleura may compensate for the loss in the alveolus, newly deposited elastic fibers in the emphysematous lung are disorganized and probably nonfunctional. This could be due to inability to form the complex microfibrillar network required for the alignment and efficient cross-linking of tropoelastin to form functional elastic fibers.
Collagen turnover is complex, but data tend to support the "inflammation repair hypothesis," which states that inflammation/degradation leads to overproduction of collagen and scarring in small airways results in submucosal fibrosis. Collagen is also augmented in the lung parenchyma, surrounding enlarged, coalesced alveolar units after destruction of individual alveoli. Despite the development of "fibrosis," there is a net loss of recoil and increase in airflow limitation, as opposed to a classic fibrotic restrictive defect.
Airway ECM Accumulation
In COPD, one observes a myriad of changes in small airways that result in airflow obstruction. Within a given lung there is marked heterogeneity, with some airways appearing normal, others with inflammation, some inspissated with mucus, some with deposition of collagen around the airway, and yet others show a combination of changes. In humans, clinical severity of COPD correlates with pathologic evidence of small airway inflammation and remodeling (1).
The process of airway remodeling has been more extensively studied in asthma. Airway remodeling refers to small airway changes associated with asthma, including mucus hyperplasia, smooth muscle hypertrophy, and subepithelial fibrosis (2). In asthma, chronic inflammation interacting with damaged epithelium and abnormal mechanical forces leads to release of growth factors and cytokines, including TGF-?, which results in myofibroblast activation and, among other things, deposition of ECM beneath the epithelium (3).
Whether similar mechanisms are involved in COPD is not clear; however, TGF-? does appear to be a critical regulator of inflammation and fibrosis in COPD as well. Too little TGF-?, as in the v?6 gene–targeted mouse, and the inflammatory brakes are released, resulting in macrophage accumulation and matrix metalloproteinase (MMP)-12–mediated emphysema (4). Too much TGF-?, and airway fibrosis occurs (5). In the interleukin-13 transgenic mouse, this fibrosis is due to MMP-9 activation of TGF-? (6).
Although the contribution of subepithelial fibrosis to asthma and COPD is not fully understood, airway fibrosis as a manifestation of chronic lung transplant rejection termed "obliterative bronchiolitis" (OB) is devastating. In OB, the basement membrane and epithelial layer are dissolved. No longer able to serve as a barrier, myofibroblasts and their collagenous matrix invade and totally obliterate the airway. Why this inflammatory process obliterates the airway while it is localized beneath the intact epithelial layer in asthma and COPD is an intriguing question.
ECM Depletion in Emphysema
Emphysema is characterized by destruction of gas-exchanging airspaces—that is, the respiratory bronchioles, alveolar ducts, and alveoli. Their walls become perforated and later obliterated with coalescence of small distinct airspaces into abnormal and much larger airspaces. A pathogenetic scheme that is emerging for the pathogenesis of emphysema is that cigarette smoke (and occasionally other toxic particulates) leads to inflammation with activation and release of elastases and other matrix-degrading proteinases. Cell death also occurs either as a result of altered ECM attachment or as a direct effect of cigarette smoke. In the absence of normal cellular and matrix repair, the disrupted airspaces disappear and coalesce, resulting in airspace enlargement that defines emphysema.
The importance of elastin was noted over 40 years ago with the advent of the elastase:antielastase theory for the pathogenesis of emphysema, which, remarkably, remains the prevailing hypothesis today (7). What is it about elastin that is so critical to emphysema? Although all ECM components are degraded by inflammatory cell proteinases in COPD, elastin is distinguished by the difficulty in restoring a functional elastic fiber in an adult. With injury, although most matrix components undergo physiologic turnover, the longer lived elastic fibers may be less capable of normal repair. Despite bursts of elastin synthesis in animal models, elastic fibers are disorganized. This is not surprising considering the complexity of elastic fiber assembly. Assembly requires tropoelastin to be chaperoned to the cell surface, secreted, and aligned on a "scaffold" of microfibrils including fibrillins, fibulins, microfibril-associated glycoproteins, and latent TGF-? binding proteins. Lysyl oxidases then cross-link monomers to form elastin, a highly insoluble rubberlike polymer. Perhaps this complex temporal and spatial coordination of elastic fiber constituents is not efficient or even possible in many adults, explaining why elastic fiber disruption is the critical factor for the development of emphysema.
In addition, proteolytic fragments of elastin serve as monocyte chemokines, fueling continued macrophage accumulation in COPD. In the early 1980s, independent studies by Senior and coworkers (8, 9) and Hunninghake and colleagues (10) demonstrated that elastase-generated fragments were chemotactic for monocytes and fibroblasts. The importance of elastin fragments as important chemokines in vivo was not appreciated until years later. The first hint was our report in a murine model of cigarette smoke–induced emphysema that, whereas smoke exposure in wild-type (MMP-12+/+) mice led to inflammatory cell recruitment followed by alveolar space enlargement, mice deficient in macrophage elastase (MMP-12–/–) were protected from development of emphysema despite long-term exposure (11). Moreover, MMP-12–/– mice failed to recruit monocytes into their lungs in response to cigarette smoke. Because MMP-12 and most other MMPs are only expressed on differentiation of monocytes to macrophages, we believed it was unlikely that monocytes require MMP-12 for transvascular migration. The working hypothesis was that cigarette smoke induces constitutive macrophages present in lungs of MMP-12 mice to produce MMP-12, which in turn cleaves elastin into fragments chemotactic for monocytes. This positive feedback loop perpetuates macrophage accumulation and lung destruction. This hypothesis has been supported by the recent finding that, in the porcine pancreatic elastase (PPE) model of emphysema, PPE initiates emphysema and elastin breakdown, whereas subsequent emphysema over time in this model requires elastin-fragment–mediated macrophage inflammation (12). Whether this system is operative in human COPD remains to be determined. However, it would help explain the fact that after cigarette smoke cessation, particularly late in the disease, inflammation persists.
Cell Death versus ECM Destruction as Initiator of Emphysema
As investigators have made great strides defining inflammatory cell interactions and networks leading to emphysema, new models that use apoptosis to drive airspace enlargement do not involve inflammation at all. Obviously, to lose an alveolar unit requires loss of both the cellular and extracellular components. Thus, the fact that apoptosis occurs was expected. The fact that it can drive the process was not.
Traditionally, we believed that inflammatory cell proteinases induced by cigarette smoke (initially at least) destroy the ECM, and on loss of cell–matrix interactions, the cells die; this process has been termed "anoikis." The apoptosis theory proposes that the initiating event is cigarette smoke, likely its oxidants that induce structural cell death. The apoptosis theory has gained remarkable acceptance despite the fact that there are only a handful of original manuscripts demonstrating that direct killing of either endothelial cells (13, 14) or epithelial cells (15–17) results in airspace enlargement.
There are several problems with the apoptosis model for emphysema. First, from a theoretical standpoint, to date all experimental models that initiate alveolar cell death, such as bleomycin and Fas ligand, result in acute lung injury and fibrosis rather than emphysema (18, 19). Also, the initial model was based on use of a vascular endothelial growth factor (VEGF) receptor 2 inhibitor, a drug that has been used for almost 2 years in patients as treatment for cancer, without adverse effects. A more serious conceptual flaw in the model is that the injury is reversible. Emphysema in humans and in animal models (e.g., pancreatic elastase and cigarette exposure) is irreversible. This is where the importance of the ECM comes into play.
As discussed above, we believe that it is the limited capacity to repair elastic fibers that is the basis for the fixed, irreversible nature of COPD. Taking this concept a bit further, as we determine the genetic factors that determine why a minority of smokers develop COPD, we predict that the genes involved will be related to elastic fiber assembly and repair. In other words, most smokers will get inflammation and proteolytic injury, but the susceptible minority will be those with genetic defects related to ECM repair.
Going back to the apoptosis models, preliminary results in our laboratory demonstrate complete repair within a week, and we have been unable to detect any ECM damage (M. Mouded and S.D. Shapiro, unpublished data). We suspect that loss of alveolar cells acutely (before surfactant is depleted) diminishes tissue forces, leading to increased lung volumes with fixed-pressure inflation relative to controls, but since ECM is intact, cellular repair is efficient.
Whether or not apoptosis is the initiating event has therapeutic implications. If it is, then apoptosis inhibitors should be effective in preventing emphysema. However, if it is initiated by ECM proteolysis, apoptosis inhibitors should either have no effect or worsen the lesion because cells destined to anoikis will be converted to necrosis. Indeed, in the pancreatic elastase model, composed of both inflammation and apoptosis, apoptosis inhibition worsens injury in preliminary studies (Mouded and Shapiro, unpublished data).
Repair in COPD: ECM and Stem Cell Considerations
To re-form a functional gas exchange unit after alveolar disruption, fibroblasts may form a provisional matrix; epithelial progenitor cells must proliferate, migrate to the alveolus, and differentiate. They must follow or lay down an ECM, and endothelial cells must either follow (or precede) epithelial repair in close juxtaposition. In emphysema, the lost and disordered ECM, with coalescence of damaged alveolar units into larger ones, makes repair a daunting task. In other words, the geometric complexity of the lung and need for a stable scaffold might limit the ability of cells to migrate to their appropriate place.
Nevertheless, there is evidence of limited repair in emphysema in animal models and humans. Years ago, Kuhn and colleagues reported that inhibition of lysyl oxidases in the PPE model led to greater emphysema than PPE alone (20), suggesting that some cross-linked and functional elastin and collagen must be formed. A classic manuscript by Massaro and Massaro (21) demonstrated restoration of alveoli after elastase-induced emphysema by administration of all-trans retinoic acid, an agent known to stimulate elastin synthesis in vitro (22). Human clinical trials have not led to obvious lung repair. Nevertheless, this remains an important and encouraging finding. In humans, the rise in FEV1 that ensues after discontinuation of smoking is associated with improved DLCO, suggesting true repair rather than just loss of nonspecific hyperreactivity.
Long-lived, quiescent lung epithelial cells may not be easily replaced. It is also possible that chronic cigarette smoke exposure leads to stem cell failure. The first questions that need to be answered, however, are whether there are multipotent lung progenitor/stem cells, and what/where is their origin. The bone marrow is a likely source for fibroblast and endothelial cell precursors. It has been more difficult to detect bone marrow–derived epithelial cells. Although bone marrow–derived lung epithelial cells had been reported, subsequent studies have failed to detect them. In fact, using deconvolution, confocal microscopy, all dual-positive cells for both bone marrow and epithelial origin actually turned out to be two cells closely juxtaposed (23, 24).
Recently, Kim and colleagues described a population of stem cells in the terminal bronchiole that they termed "bronchoalveolar stem cells" or BASCs (25), which seem to serve as distal airway and alveolar epithelial stem cells. These cells, characterized by Clara cell protein 10 (CC10)+, surfactant protein C (SP-C)+, and Sca-1+ markers, displayed self-renewal and were multipotent in clonal assays. They also underwent proliferation in vivo after airway injury with napthalene, and after alveolar injury with bleomycin. BASCs are also precursors for adenocarcinoma. Some investigators have hypothesized that the Clara cell is a progenitor cell for bronchiolar epithelium. BASCs certainly do have some Clara cell markers unifying these concepts (26). Whether BASCs represent epithelial cell precursors in emphysema is unknown, but is an attractive hypothesis. Alternatively, BASCs may replenish both type II and type I cells either in parallel or in series (II to I).
Combining the stem cell and ECM hypotheses, we propose that, after alveolar injury, distal small airway stem cells proliferate, migrate to the alveolus, and differentiate in an attempt to repair the damage. The success of repair depends on whether injury is strictly cellular in nature or includes ECM damage, with damage to elastin being most repair limiting. Moreover, the reason that repair in apoptosis models is so rapid and complete is because injury is cellular, sparing the matrix. Epithelial cells may be able to replace basement membrane as they migrate and differentiate along the alveolus; however, elastin limits this potential. Figure 1 demonstrates predicted results from ongoing lineage tagging experiments that test these hypotheses. If these concepts are correct, then we must either determine how to spatially and temporally bring elastic fiber components together to re-form the scaffold or add an artificial scaffold that will allow progenitor cells to migrate and re-form a new functional gas exchange unit.
FOOTNOTES
Conflict of Interest Statement: S.D.S. has in the past 3 years participated on advisory boards for Boehringer Ingelheim, GlaxoSmithKline, Millenium, Pfizer, Wyeth, and ICOS. His laboratory has performed research in collaboration with Pfizer, Arriva, ONO, and Taisho. Advisory board activity did not exceed $10,000/year per company. No personal income was obtained from research grants. S.D.S. receives a fixed stipend as Editor of the AJRCMB.
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Aoshiba K, Yokohori N, Nagai A. Alveolar wall apoptosis causes lung destruction and emphysematous changes. Am J Respir Cell Mol Biol 2003;28:552–562.
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The extracellular matrix (ECM) of the lung is composed of a cable network of highly inert elastic fibers that are not distributed uniformly in the lung parenchyma. They loop around alveolar ducts, form rings at the mouths of alveoli, and penetrate as wisps into alveolar septa, concentrating at bends and junctions. Elastin is a highly proteinase-resistant protein that normally lasts the human lifespan. Collagen forms a continuous sheath around alveolar units and larger bundles. Interstitial collagens (types I and III) provide the tensile strength that keeps the lung intact. One can think of elastin as a strong rubber band, and collagen as ropes, together providing elasticity and structural stability to the lung. The basement membrane separating the alveolar endothelium and epithelium is principally composed of a type IV collagen reticular network that binds to laminin through entactin bridges. Proteoglycans are also highly expressed in the basement membrane and interstitium, perhaps shielding other matrix components from destruction.
In addition to providing structural stability and serving as a barrier partitioning the lung into discrete compartments, the ECM also participates in a variety of additional cellular functions. Indeed, the cell senses its external environment through its interaction with the ECM. Hence, many critical cellular functions, including cell death and proliferation, are regulated by the ECM and integrins on the cell surface to which they bind. The ECM is also a reservoir for growth factors, including transforming growth factor;(TGF-?). Hence, activation and release of growth factors from the ECM is an important mode of regulation of cell behavior. Finally, proteolytic fragments of the ECM themselves have discrete biological functions, the most-studied example being the chemotactic activity imparted by elastin, collagen, fibronectin, and laminin fragments.
Given the need to maintain structural support and barrier function, it is not surprising that alterations in the ECM play a profound role in lung disease. However, the additional roles that the ECM plays in cell homeostasis suggest that alterations in ECM may be involved in disease pathogenesis in ways not previously recognized. Moreover, the ability of the ECM to appropriately repair itself, and provide scaffolding for cellular repair, will likely turn out to be a major factor in determining whether an insult results in a chronic disease.
ECM changes have dominated concepts regarding most major pulmonary disorders. In the lung parenchyma, loss of elastin and other ECM components is central to the pathogenesis of emphysema. Accumulation of interstitial collagen is the central lesion in pulmonary fibrosis. Within the airway, subepithelial fibrosis is a key component of airway remodeling in asthma, and submucosal fibrosis in small airways is also observed in chronic obstructive pulmonary disease (COPD). Obliterative bronchiolitis (OB), the main cause of death after lung transplantation, is a result of total obliteration of small airways by collagen.
These pathologic findings lead to a variety of fascinating yet unanswered questions. Are mechanisms leading to airway fibrosis in asthma and COPD related? Why do we observe subepithelial fibrosis in asthma and COPD yet total obliteration of the airways in OB? Why is idiopathic pulmonary fibrosis (IPF) peripheral sparing the central airways, whereas these other diseases have fibrosis focused in the airways? Is this due to different disease mechanisms or a result of the location of the instigating abnormality.
To further define the spectrum of biological functions of the ECM and the capacity for ECM repair, and understand the similarities and differences of ECM remodeling in different pulmonary disease processes, the organizers of the Transatlantic Airway Conference brought together a small but talented group of scientific experts in different pulmonary diseases, combined with a group of discussants known for their profound knowledge of lung pathobiology and their ability to ask probing questions. We now share with you the insights gained from this meeting.
APPLICATION OF LESSONS LEARNED FROM THE CONFERENCE TO ECM DESTRUCTION, FIBROSIS, AND REPAIR IN COPD
This meeting has highlighted the need for precise control of ECM turnover in the lung. Either depletion or excess of ECM will disrupt the structural and functional integrity of the lung, with too little ECM leading to emphysema, while too much results in fibrosis. In addition to providing structural support, many additional functions of the ECM were discussed, including the involvement of the ECM and their proteolytic fragments in cell signaling and homeostasis. COPD highlights the complexity of ECM turnover. The manifestations of cigarette smoke, inflammation, and perhaps airway infection lead to airway fibrosis with ECM accumulation, whereas smoke-related inflammatory proteinase activity in the lung parenchyma leads to ECM degradation. Manifestations of ECM turnover in COPD will be used as an example to summarize concepts regarding ECM pathobiology, genetics, and even stem cell biology, which were discussed during lectures and discussions of this meeting.
COPD: Complex Effects of Cigarette Smoke Exposure on the ECM
COPD is defined as a disease state characterized by airflow limitation that is not fully reversible in a patient who has been exposed to noxious agents, which in our society is almost exclusively cigarette smoke. The effect of cigarette smoke on a given individual depends on one's genetic make-up as well as additional environmental insults. Cigarette smoke causes prominent and distinct changes in the large airways, small airways, and alveolar space.
Studies spanning five decades have shown that, in lungs of humans and experimental animals with COPD, total elastin content is unchanged or decreased, whereas total collagen content is increased. With the loss of an alveolar unit in emphysema, all ECM components are destroyed. This includes elastin, which is most prominent in the "mouth" of the alveolus (and therefore accounts for centriacinar changes), as well as collagen, which is more uniformly distributed throughout the alveolus. Although increases in elastin in the vessels and pleura may compensate for the loss in the alveolus, newly deposited elastic fibers in the emphysematous lung are disorganized and probably nonfunctional. This could be due to inability to form the complex microfibrillar network required for the alignment and efficient cross-linking of tropoelastin to form functional elastic fibers.
Collagen turnover is complex, but data tend to support the "inflammation repair hypothesis," which states that inflammation/degradation leads to overproduction of collagen and scarring in small airways results in submucosal fibrosis. Collagen is also augmented in the lung parenchyma, surrounding enlarged, coalesced alveolar units after destruction of individual alveoli. Despite the development of "fibrosis," there is a net loss of recoil and increase in airflow limitation, as opposed to a classic fibrotic restrictive defect.
Airway ECM Accumulation
In COPD, one observes a myriad of changes in small airways that result in airflow obstruction. Within a given lung there is marked heterogeneity, with some airways appearing normal, others with inflammation, some inspissated with mucus, some with deposition of collagen around the airway, and yet others show a combination of changes. In humans, clinical severity of COPD correlates with pathologic evidence of small airway inflammation and remodeling (1).
The process of airway remodeling has been more extensively studied in asthma. Airway remodeling refers to small airway changes associated with asthma, including mucus hyperplasia, smooth muscle hypertrophy, and subepithelial fibrosis (2). In asthma, chronic inflammation interacting with damaged epithelium and abnormal mechanical forces leads to release of growth factors and cytokines, including TGF-?, which results in myofibroblast activation and, among other things, deposition of ECM beneath the epithelium (3).
Whether similar mechanisms are involved in COPD is not clear; however, TGF-? does appear to be a critical regulator of inflammation and fibrosis in COPD as well. Too little TGF-?, as in the v?6 gene–targeted mouse, and the inflammatory brakes are released, resulting in macrophage accumulation and matrix metalloproteinase (MMP)-12–mediated emphysema (4). Too much TGF-?, and airway fibrosis occurs (5). In the interleukin-13 transgenic mouse, this fibrosis is due to MMP-9 activation of TGF-? (6).
Although the contribution of subepithelial fibrosis to asthma and COPD is not fully understood, airway fibrosis as a manifestation of chronic lung transplant rejection termed "obliterative bronchiolitis" (OB) is devastating. In OB, the basement membrane and epithelial layer are dissolved. No longer able to serve as a barrier, myofibroblasts and their collagenous matrix invade and totally obliterate the airway. Why this inflammatory process obliterates the airway while it is localized beneath the intact epithelial layer in asthma and COPD is an intriguing question.
ECM Depletion in Emphysema
Emphysema is characterized by destruction of gas-exchanging airspaces—that is, the respiratory bronchioles, alveolar ducts, and alveoli. Their walls become perforated and later obliterated with coalescence of small distinct airspaces into abnormal and much larger airspaces. A pathogenetic scheme that is emerging for the pathogenesis of emphysema is that cigarette smoke (and occasionally other toxic particulates) leads to inflammation with activation and release of elastases and other matrix-degrading proteinases. Cell death also occurs either as a result of altered ECM attachment or as a direct effect of cigarette smoke. In the absence of normal cellular and matrix repair, the disrupted airspaces disappear and coalesce, resulting in airspace enlargement that defines emphysema.
The importance of elastin was noted over 40 years ago with the advent of the elastase:antielastase theory for the pathogenesis of emphysema, which, remarkably, remains the prevailing hypothesis today (7). What is it about elastin that is so critical to emphysema? Although all ECM components are degraded by inflammatory cell proteinases in COPD, elastin is distinguished by the difficulty in restoring a functional elastic fiber in an adult. With injury, although most matrix components undergo physiologic turnover, the longer lived elastic fibers may be less capable of normal repair. Despite bursts of elastin synthesis in animal models, elastic fibers are disorganized. This is not surprising considering the complexity of elastic fiber assembly. Assembly requires tropoelastin to be chaperoned to the cell surface, secreted, and aligned on a "scaffold" of microfibrils including fibrillins, fibulins, microfibril-associated glycoproteins, and latent TGF-? binding proteins. Lysyl oxidases then cross-link monomers to form elastin, a highly insoluble rubberlike polymer. Perhaps this complex temporal and spatial coordination of elastic fiber constituents is not efficient or even possible in many adults, explaining why elastic fiber disruption is the critical factor for the development of emphysema.
In addition, proteolytic fragments of elastin serve as monocyte chemokines, fueling continued macrophage accumulation in COPD. In the early 1980s, independent studies by Senior and coworkers (8, 9) and Hunninghake and colleagues (10) demonstrated that elastase-generated fragments were chemotactic for monocytes and fibroblasts. The importance of elastin fragments as important chemokines in vivo was not appreciated until years later. The first hint was our report in a murine model of cigarette smoke–induced emphysema that, whereas smoke exposure in wild-type (MMP-12+/+) mice led to inflammatory cell recruitment followed by alveolar space enlargement, mice deficient in macrophage elastase (MMP-12–/–) were protected from development of emphysema despite long-term exposure (11). Moreover, MMP-12–/– mice failed to recruit monocytes into their lungs in response to cigarette smoke. Because MMP-12 and most other MMPs are only expressed on differentiation of monocytes to macrophages, we believed it was unlikely that monocytes require MMP-12 for transvascular migration. The working hypothesis was that cigarette smoke induces constitutive macrophages present in lungs of MMP-12 mice to produce MMP-12, which in turn cleaves elastin into fragments chemotactic for monocytes. This positive feedback loop perpetuates macrophage accumulation and lung destruction. This hypothesis has been supported by the recent finding that, in the porcine pancreatic elastase (PPE) model of emphysema, PPE initiates emphysema and elastin breakdown, whereas subsequent emphysema over time in this model requires elastin-fragment–mediated macrophage inflammation (12). Whether this system is operative in human COPD remains to be determined. However, it would help explain the fact that after cigarette smoke cessation, particularly late in the disease, inflammation persists.
Cell Death versus ECM Destruction as Initiator of Emphysema
As investigators have made great strides defining inflammatory cell interactions and networks leading to emphysema, new models that use apoptosis to drive airspace enlargement do not involve inflammation at all. Obviously, to lose an alveolar unit requires loss of both the cellular and extracellular components. Thus, the fact that apoptosis occurs was expected. The fact that it can drive the process was not.
Traditionally, we believed that inflammatory cell proteinases induced by cigarette smoke (initially at least) destroy the ECM, and on loss of cell–matrix interactions, the cells die; this process has been termed "anoikis." The apoptosis theory proposes that the initiating event is cigarette smoke, likely its oxidants that induce structural cell death. The apoptosis theory has gained remarkable acceptance despite the fact that there are only a handful of original manuscripts demonstrating that direct killing of either endothelial cells (13, 14) or epithelial cells (15–17) results in airspace enlargement.
There are several problems with the apoptosis model for emphysema. First, from a theoretical standpoint, to date all experimental models that initiate alveolar cell death, such as bleomycin and Fas ligand, result in acute lung injury and fibrosis rather than emphysema (18, 19). Also, the initial model was based on use of a vascular endothelial growth factor (VEGF) receptor 2 inhibitor, a drug that has been used for almost 2 years in patients as treatment for cancer, without adverse effects. A more serious conceptual flaw in the model is that the injury is reversible. Emphysema in humans and in animal models (e.g., pancreatic elastase and cigarette exposure) is irreversible. This is where the importance of the ECM comes into play.
As discussed above, we believe that it is the limited capacity to repair elastic fibers that is the basis for the fixed, irreversible nature of COPD. Taking this concept a bit further, as we determine the genetic factors that determine why a minority of smokers develop COPD, we predict that the genes involved will be related to elastic fiber assembly and repair. In other words, most smokers will get inflammation and proteolytic injury, but the susceptible minority will be those with genetic defects related to ECM repair.
Going back to the apoptosis models, preliminary results in our laboratory demonstrate complete repair within a week, and we have been unable to detect any ECM damage (M. Mouded and S.D. Shapiro, unpublished data). We suspect that loss of alveolar cells acutely (before surfactant is depleted) diminishes tissue forces, leading to increased lung volumes with fixed-pressure inflation relative to controls, but since ECM is intact, cellular repair is efficient.
Whether or not apoptosis is the initiating event has therapeutic implications. If it is, then apoptosis inhibitors should be effective in preventing emphysema. However, if it is initiated by ECM proteolysis, apoptosis inhibitors should either have no effect or worsen the lesion because cells destined to anoikis will be converted to necrosis. Indeed, in the pancreatic elastase model, composed of both inflammation and apoptosis, apoptosis inhibition worsens injury in preliminary studies (Mouded and Shapiro, unpublished data).
Repair in COPD: ECM and Stem Cell Considerations
To re-form a functional gas exchange unit after alveolar disruption, fibroblasts may form a provisional matrix; epithelial progenitor cells must proliferate, migrate to the alveolus, and differentiate. They must follow or lay down an ECM, and endothelial cells must either follow (or precede) epithelial repair in close juxtaposition. In emphysema, the lost and disordered ECM, with coalescence of damaged alveolar units into larger ones, makes repair a daunting task. In other words, the geometric complexity of the lung and need for a stable scaffold might limit the ability of cells to migrate to their appropriate place.
Nevertheless, there is evidence of limited repair in emphysema in animal models and humans. Years ago, Kuhn and colleagues reported that inhibition of lysyl oxidases in the PPE model led to greater emphysema than PPE alone (20), suggesting that some cross-linked and functional elastin and collagen must be formed. A classic manuscript by Massaro and Massaro (21) demonstrated restoration of alveoli after elastase-induced emphysema by administration of all-trans retinoic acid, an agent known to stimulate elastin synthesis in vitro (22). Human clinical trials have not led to obvious lung repair. Nevertheless, this remains an important and encouraging finding. In humans, the rise in FEV1 that ensues after discontinuation of smoking is associated with improved DLCO, suggesting true repair rather than just loss of nonspecific hyperreactivity.
Long-lived, quiescent lung epithelial cells may not be easily replaced. It is also possible that chronic cigarette smoke exposure leads to stem cell failure. The first questions that need to be answered, however, are whether there are multipotent lung progenitor/stem cells, and what/where is their origin. The bone marrow is a likely source for fibroblast and endothelial cell precursors. It has been more difficult to detect bone marrow–derived epithelial cells. Although bone marrow–derived lung epithelial cells had been reported, subsequent studies have failed to detect them. In fact, using deconvolution, confocal microscopy, all dual-positive cells for both bone marrow and epithelial origin actually turned out to be two cells closely juxtaposed (23, 24).
Recently, Kim and colleagues described a population of stem cells in the terminal bronchiole that they termed "bronchoalveolar stem cells" or BASCs (25), which seem to serve as distal airway and alveolar epithelial stem cells. These cells, characterized by Clara cell protein 10 (CC10)+, surfactant protein C (SP-C)+, and Sca-1+ markers, displayed self-renewal and were multipotent in clonal assays. They also underwent proliferation in vivo after airway injury with napthalene, and after alveolar injury with bleomycin. BASCs are also precursors for adenocarcinoma. Some investigators have hypothesized that the Clara cell is a progenitor cell for bronchiolar epithelium. BASCs certainly do have some Clara cell markers unifying these concepts (26). Whether BASCs represent epithelial cell precursors in emphysema is unknown, but is an attractive hypothesis. Alternatively, BASCs may replenish both type II and type I cells either in parallel or in series (II to I).
Combining the stem cell and ECM hypotheses, we propose that, after alveolar injury, distal small airway stem cells proliferate, migrate to the alveolus, and differentiate in an attempt to repair the damage. The success of repair depends on whether injury is strictly cellular in nature or includes ECM damage, with damage to elastin being most repair limiting. Moreover, the reason that repair in apoptosis models is so rapid and complete is because injury is cellular, sparing the matrix. Epithelial cells may be able to replace basement membrane as they migrate and differentiate along the alveolus; however, elastin limits this potential. Figure 1 demonstrates predicted results from ongoing lineage tagging experiments that test these hypotheses. If these concepts are correct, then we must either determine how to spatially and temporally bring elastic fiber components together to re-form the scaffold or add an artificial scaffold that will allow progenitor cells to migrate and re-form a new functional gas exchange unit.
FOOTNOTES
Conflict of Interest Statement: S.D.S. has in the past 3 years participated on advisory boards for Boehringer Ingelheim, GlaxoSmithKline, Millenium, Pfizer, Wyeth, and ICOS. His laboratory has performed research in collaboration with Pfizer, Arriva, ONO, and Taisho. Advisory board activity did not exceed $10,000/year per company. No personal income was obtained from research grants. S.D.S. receives a fixed stipend as Editor of the AJRCMB.
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