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1 University of Edinburgh, MRC Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh, United Kingdom; and 2 Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado at Denver, Aurora, Colorado
Correspondence and requests for reprints should be addressed to William MacNee, M.D., F.R.C.P., University of Edinburgh, MRC Centre for Inflammation Research, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. E-mail: w.macnee{at}ed.ac.uk
ABSTRACT
This paper reviews the potential participation of novel pathogenic mechanisms in chronic obstructive pulmonary disease (COPD) relating to aging, including oxidative stress and enhanced expression of markers of senescence in emphysematous lungs and the potential enhanced tissue destruction involving alveolar apoptosis. These insights provide new beginnings for future investigations in the pathobiology of COPD which may lead to future therapies for this condition.
Key Words: aging • oxidative stress • senescence • emphysema • apoptosis
A key mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD) is thought to be an abnormal inflammatory response in the lungs to the inhalation of toxic particles and gases, derived from tobacco smoke, air pollution, and/or occupational exposures (1). Although tobacco smoking elicits an inflammatory response in the lungs of all smokers, this response is not only enhanced but also it fails to resolve after quitting smoking in those who develop COPD (2). This suggests that in smokers who develop COPD there is abnormal regulation of the inflammatory response in the lungs. The susceptibility factors are still poorly understood and may involve genetic and epigenetic factors, altered immune regulation, impaired resolution of inflammation, and abnormal repair mechanisms (3). However, the relationship between the inflammatory responses in the lungs and the accelerated decline in FEV1, which characterizes this condition, is far from clear. Indeed, the hypothesis that inflammation accounts for all features of the pathobiological processes involved in alveolar destruction and airway remodeling in COPD oversimplifies the overall polymorphic and multifactorial nature of inflammation in this disease and consequently, it has not led to major therapeutic advances in treatments for COPD. It is also clear that COPD is a heterogeneous disease where the involvement of large and small airways (bronchitis/bronchiolitis) and lung parenchyma (emphysema) varies greatly between patients. The mechanisms resulting in these pathological changes are also likely to be different.
This review highlights the potential participation of several alternative pathogenetic processes, particularly involving the potential participation of biological and pathobiological processes related to aging, including oxidative stress and enhanced expression of markers of senescence/aging in emphysematous lungs, and the potential for enhanced tissue destruction involving alveolar cell apoptosis. These processes are intimately associated with the generation of interactive feedback loops, leading to excessive extracellular matrix proteolysis (i.e., protease/antiprotease imbalance) and oxidative stress (Figure 1). These insights provide a fresh view for future investigations in the pathobiology of COPD, in which inflammation can be framed as a more complex mechanism underlying tissue remodeling and destruction in the disease. Not only does cigarette smoke directly cause inflammation, but alveolar tissue damage further amplifies inflammatory processes, potentially leading to autoimmunity and self-perpetuating stimuli for enhanced influx of inflammatory cells in the damaged lung (4).
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An imbalance between oxidants and antioxidants occurs in COPD results in oxidative stress, which underlies many of the pathogenic mechanisms in COPD (5). There is considerable evidence for increased oxidative stress in the lungs of patients with COPD. Numerous studies have documented increased expression of markers of oxidative stress in the lungs of patients with COPD, compared with healthy subjects or smokers with a similar smoking history who have not developed COPD (5). This finding is exemplified by the expression of 4-hydroxy-2-nonenal (4HNE), a highly reactive lipid peroxidation end product, in COPD lungs. 4HNE reacts quickly with extracellular proteins to form adducts. These adducts have been shown to be present in greater quantities in airway epithelial and endothelial cells in the lungs of patients with COPD, compared with smokers with a similar smoking history who have not developed the disease (6). 4HNE can act as a chemoattractant for neutrophils (7) and is also involved in numerous cellular functions, such as cell proliferation/inhibition (8), T cell apoptosis (9), and activation of various signaling pathways (10). 4-hydroxy-2-nonenal has also been shown to activate the synthesis of the antioxidant glutathione by induction of the glutamate cysteine lygase gene as an antioxidant response to increased oxidative stress and also a variety of proinflammatory genes such as interleukin (IL)-8, monocyte chemoattractant protein-1, epidermal growth factor, and MUC5AC (9). The presence of increased oxidative stress in COPD lungs has been recently confirmed by the finding of increased expression of 8-hydroxy-2 deoxy-guanosine, which form due to the reaction of hydroperoxides with the DNA base guanosine (11).
There are many actions of oxidative stress that can potentially play a role in the pathogenic mechanisms in COPD. These include the inactivation of antiproteases (such as 
1-antitrypsin or secretory leukoprotease inhibitor) (12) or activation of metalloproteases (13) by oxidants, resulting in a protease/antiprotease imbalance in the lungs, which forms the basis of the protease/antiprotease theory of the pathogenesis of emphysema (14). Oxidants can directly damage components of the lung matrix (e.g., elastin and collagen) and can also interfere with elastin synthesis and repair (5).
Oxidative stress also influences the molecular mechanisms involved in proinflammatory gene expression. Gene activation by transcription factors is regulated by a number of factors, among which is the remodeling of DNA dependent on the nuclear histone acetylation/deacetylation balance (15), controlled by the activities of histone acetyl-transferases (HATs) and histone deacetylases (HDACs). DNA in the resting cell coiled around a nucleosome core of histone residues. This configuration suppresses the accessibility of transcription factors such as nuclear factor-
B to their cognate DNA sequences. Acetylation of the lysine residues in the core histones results in uncoiling of the DNA, increasing the accessibility of transcription factors and RNA polymerase 2 and hence increased gene transcription. Deacetylation, under the influence of HDACs, results in rewinding of the DNA around the histone proteins; decreasing gene transcription.
Macrophages from cigarette smokers show a decrease in histone deacetylases activity (16), as has also been shown in the lungs of smoke-exposed animals (17). In the lungs of rats exposed to cigarette smoke, HDAC2 activity and protein expression were significantly decreased. These changes were associated with nitrotyrosine, 4HNE, and aldehyde—modified HDAC proteins—resulting in their enhanced degradation in the proteosome system and thus decreased protein HDAC activity (17).
Studies of resected lung indicate that HDAC2 activity is reduced in lung tissue in COPD, associated with lower HDAC2 RNA levels and expression of HDAC2 protein as a result of modification of HDACs by oxidants (18, 19). This decrease in HDAC increased with disease severity and was associated with an increase in histone-4 acetylation at the IL-8 promoter and increased in IL-8 mRNA expression. Thus, molecular mechanisms such as transcription factor activation and chromatin remodeling, as a result of increased oxidative stress, may be responsible for perpetuating inflammation in COPD.
The most compelling data in support of the causal role of oxidative stress in the pathogenesis of emphysema has been provided by studies outlining the role of the master antioxidant transcription factor nuclear erythroid-related factor 2 (Nrf2) in the disease. Nrf2 controls the expression of more than 100 gene products, including several of the most important antioxidant enzymes (20). Human lungs have decreased expression of Nrf2 transcriptional activity, which has been linked to decreased expression of its coactivator DJ-1 (21), increased expression of its transcriptional repressor Bach-1, or decreased levels in alveolar macrophages (22). The protective role of Nrf2 in emphysema is underscored by the increased susceptibility to Nrf2-null mice to cigarette smoke– (20, 23) and elastase-induced emphysema (24). Furthermore, enhancement of Nrf2 expression using a small molecule activator protected wild-type mice against cigarette smoke–induced emphysema (25). In these mice, the protection afforded by the Nrf2 activator correlated with decreased alveolar cell death rather than inflammation.
As will be made apparent in the next section, oxidative stress is intimately linked to cell death, including alveolar cell apoptosis in the setting of human and experimental emphysema (26–29). Moreover, oxidative stress underlies several of the mechanisms thought to participate in aging, which may lower the threshold for lung injury to cigarette smoke (30).
ALVEOLAR CELL DEATH IN EMPHYSEMA
Studies over the past 5 years have documented that lung cell apoptosis occurs in emphysematous lungs, predominantly involving endothelial cells in the alveolar walls, compared with lungs from normal subjects or from smokers without COPD (27). Experimental emphysema in animals could be produced by decreased vascular endothelial growth factor (VEGF) or VEGF signaling (31), and studies in human lungs demonstrated decreased expression of VEGF and VEGF-receptor 2 expression in association with emphysema (27). The overall relevance of this signaling pathway in lung injury by cigarette smoke was highlighted in subsequent experimental studies in which cigarette smoke reduced expression levels of VEGF receptor 2 (32). The aggregate of these data led to the concept of an alveolar maintenance program that was required for structural preservation of the lungs. Cigarette smoke is thought to cause destruction of this maintenance program, thus causing emphysema.
Lung tissue destruction occurs due to the mutual interaction among alveolar cell apoptosis, oxidative stress, and protease/antiprotease imbalance (33). This concept was supported by the observations that antioxidant treatment prevented both apoptosis and emphysema induced by down-regulation of VEGF (34) and the findings that experimental cigarette smoke–induced emphysema requires cathepsin-S expression, as cathepsin-S knockout mice are protected from the disease and alveolar cell apoptosis (35).
It is clear that inflammation can be triggered and amplified by alveolar injury, including either enhanced alveolar cell apoptosis or defective apoptotic cell clearance (36). We have recently reported that targeted alveolar cell apoptosis using a chimeric peptide containing a lung homing sequence linked to a proapoptotic peptide led to emphysematous enlargement of murine lungs, associated with alveolar cell apoptosis, oxidative stress, macrophage influx, and increases in ceramide levels (37). Moreover, viral infections (such as influenza virus) or double-stranded RNA, via activation of the RING-helicase system of viral protection, can synergize with cigarette smoke to cause experimental emphysematous tissue destruction involving alveolar cell death and secondary lung inflammation (38).
ROLE OF AGING IN THE PATHOGENESIS OF EMPHYSEMA
There is ample evidence of shared features between pulmonary emphysema and lung aging, which led us to advance the hypothesis that both conditions share underlying mechanisms, including oxidative stress, inflammation, and apoptosis (39, 40). The concept of disposable soma predicates that aging results from the somatic damage to organismal macromolecules, resulting from the interaction between the host and environmental stresses, occurring well beyond the age of procreation (41).
The cellular equivalent of aging is senescence, which is characterized by a nonproliferative state in which cells are metabolically active and apoptosis-resistant. A number of molecular and cellular mechanisms are associated with cellular senescence including accumulation of DNA damage (42), impairment of DNA repair (43) epigenetic modifications in nuclear DNA (44), protein damage (45) from oxidative stress, and telomere attrition (46). Central in the "end replication senescence" is the erosion of telomeres, with the ensuing activation of DNA repair enzymes ATM/ATR (ataxia telengectasia gene products), and the cell cycle control kinase inhibitors p53, p21, and p16. These signaling processes converge on de-phosphoylated (active) retinoblastoma protein, which potently inhibits cell cycle progression. There are several lines of evidence indicating that cellular senescence also applies in vivo, particularly to conditions associated with aging. Indeed, several markers of cellular senescence are present in vivo, particularly the enzymatic detection of senescence-associated β-galactosidase (SA–β-gal), expression of cyclin-kinase inhibitors p16 and p21, and detection of DNA repair/damage responses, including the phosphorylated form of H2AX (47).
There is considerable evidence of senescence/aging caused by cigarette smoke and in emphysematous lungs. Cigarette smoke extract leads to increased SA–β-gal expression in cultured type II cells (48) or lung fibroblasts (49), which has also been shown in lung fibroblasts from emphysematous lungs (50). Oxidative stress enhances telomere shortening (51). Furthermore, alveolar epithelial and endothelial cells in emphysematous lungs also have increased expression of p21 in association with decreased telomere length (52). The decreased telomere length parallels similar findings in circulating peripheral blood leukocytes. An association has been shown between blood leukocyte telomere length and pack-years of smoking (53), and telomeres are shorter in blood leukocytes from patients with COPD compared with control subjects (54). It is therefore tempting to include COPD as one of the diseases associated with "premature" aging.
Several animal models of aging have associated emphysema. The klotho gene encodes a membrane protein that is a regulator of oxidative stress and cell senescence (55). Mice with a defect of the klotho gene develop a syndrome resembling aging including emphysema (56). Senescent marker protein 30 (SMP-30), which is expressed in early life and progressively decreases with age (57) SMP-30 knockout mice develop distal airspace enlargement indicative of emphysema (58), associated with increased oxidative stress in the lungs, and show enhanced development of emphysema after smoke exposure (59).
Metabolic nitocinamide adenines, nucleotide (NAD+)-dependent histone/protein deacetylases (sirtuins), are type III histone deacetylases (HDAC) and are structurally different from other HDACs and inhibited by different compounds (60); they play an important role in a variety of processes, including stress resistance, metabolism, apoptosis, senescence, differentiation and aging (61). HDACs act on histone residues in DNA and thereby mediate gene silencing. Sirtuin I (SIRT-1) is essential for maintaining silent chromatin via the deacetylation of histones, but also has a number of nonhistone targets such as the regulation nuclear factor-B–dependent transcription and cell survival in responses to tumor necrosis factor- (62). Activation or overexpression of SIRT-1 increases the lifespan of a number of species (63). Environmental stress, such as cigarette smoke exposure, decreases SIRT-1 levels in both macrophages in vitro and in rat lungs in vivo associated with increased expression of inflammatory cytokines (64). SIRT-1 has been shown to be decreased in lung cells from patients with COPD, compared with smokers who have not developed the disease, as a result of post-translational oxidative modification (65). This would accelerate the process of aging and also enhance inflammation.
Given the current expanded appreciation of the pathobiological processes potentially involved in alveolar destruction caused by cigarette smoke, the classical paradigms (i.e., inflammation and protease/antiprotease imbalance) can be analyzed more broadly, potentially leading to novel therapeutic tools against this untreatable disease. Senescence/aging can lead to decrease in stem/progenitor cells, thus limiting the potential for cell regenerative approaches and possibly justifying the limited results with retinoic acid treatment. Persistent increases in the proinflammatory cytokines IL-6 and IL-8, shown to be induced by cigarette smoke and present in increased levels in lungs of individuals with COPD, are also triggered by aging. More importantly, IL-8 engagement of its receptor CXCR2 reinforces cellular senescence (66). IL-6 overexpression can also lead to enhanced senescence (67). These cytokines interact with senescence creating positive feedback loops that amplify these processes.
CONCLUSIONS
The so-called traditional hypothesis states that COPD is initiated and maintained through the activation of inflammation by long-term cigarette smoking, leading to protease/antiprotease imbalance. Though this hypothesis carries compelling experimental and observational (clinical) support, it does not address several observations particularly related to COPD's progressive nature: why it takes decades to develop the disease, the persistence of inflammation despite smoking cessation, and the vexing observation that corticosteroids have little or no impact in COPD, except for their narrow use in exacerbation.
New and exciting alternatives to this hypothesis are being provided by the evidence of shared molecular features between cigarette smoke–induced emphysema and lung changes due to aging. The potential contribution of aging to cigarette smoke–induced lung injury encompasses the observations of increased expression of markers oxidative stress and apoptosis in emphysematous lungs. Indeed, aging may be characterized by altered lung alveolar cell maintenance due to decrease in growth factor signaling and an overall increased sensitivity to oxidative stress (30). It is therefore tempting to include COPD as one of the diseases associated with "premature" aging. Consequently, the role of lung inflammation in cigarette smoke–induced COPD has to be reconsidered in light of this overall pathobiological scenario. The significant injury imparted by cigarette smoke, as the trigger of the disease, and the supervening endogeneous destructive processes may underlie the involvement of autoimmune inflammation (68, 69).
These conceptual advances, anchored by extensive experimental and human data, have led to potential disease modifying therapeutic opportunities. The cell cycle suppressor p21CIP1/WAF1/SDI1, whose expression is increased in patients with COPD, appears to mediate alveolar inflammation due to cigarette smoke (70), as p21CIP1/WAF1/SDI1 knockout mice are protected against cigarette smoke–induced alveolar inflammation. Nrf2, a key antioxidant transcription factor whose activity is decreased in COPD lungs (21), appears to be a central player in the susceptibility to emphysema (20). More importantly, as its activity can be up-regulated experimentally, resulting in protection against cigarette smoke–induced alveolar cell apoptosis and alveolar enlargement, similar approaches can be extended to patients with COPD (25).
FOOTNOTES
Conflict of Interest Statement: W.M. served as a consultant for Pfizer Pharmaceuticals ($1,001 - $5,000) and served on the Board or Advisory Board for GlaxoSmithKline and Pfizer ($1,001 - $5,000). He received lecture fees from GlaxoSmithKline and AstraZeneca ($5,001 - $10,000), and has received grant support from GlaxoSmithKline and Pfizer a ($100,001 or more). R.M.T. has received grant support from the NHLBI ($100,001 or more) and from the Alpha 1 Foundation ($50,001 - $100,000).
(Received in original form May 11, 2009; accepted in final form August 6, 2009)
REFERENCES
-induced alveolar remodeling and pulmonary emphysema. J Immunol 2005;174:8106–8115.
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