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Pathobiology of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease

Division of Cardiopulmonary Pathology, Department of Pathology, and Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland

ABSTRACT

I. INTRODUCTION

II. RISK FACTORS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

A. Direct Cigarette Smoking and Environmental Tobacco Exposure: The Main Causes of COPD

B. The Adjuvant Role of Air Pollution

C. Tobacco Components and Nicotine Class Acetylcholine Receptors

III. AIRWAY DISEASE

A. Chronic Bronchitis

B. Airway Inflammation

C. Mucus

D. Small Airway Remodeling

IV. EMPHYSEMA

A. Inflammation

B. Inflammatory Cells

C. Cytokines and Chemokines

D. Extracellular Matrix Proteolysis: Protease/Antiprotease Imbalance

E. Disruption of Alveolar Cell and Molecular Maintenance Program: Role of Growth Factors and Bone Marrow

F. Oxidative Stress

G. Alveolar Cell Apoptosis and Proliferation

H. Aging and Senescence

V. MEDIATORS AND SIGNALING PATHWAYS LINKING INFLAMMATION, PROTEASE/ANTIPROTEASES, OXIDATIVE STRESS, AND APOPTOSIS

A. Ceramide

B. Nuclear Factor-kappaB

C. Histone Acetyltransferases and Histone Deacetylases

VI. CONCLUSIONS

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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Chronic obstructive pulmonary disease (COPD) represents the fourth leading cause of morbidity and mortality in North America, in excess of 110,000 yearly deaths. After decades of a relative lack of interest in the disease, there is increased realization that we are in the midst of an epidemic, with a major impact on the health care resources, involving both the developed world and developing countries. The realization of the urgent need of novel therapies, new clinical end points, and insights into its natural history led to a major mobilization of resources of the National Institutes of Health of the United States directed to COPD research (57). It is clear that these urgent needs will be addressed by a better understanding of the pathobiology of COPD. It is our intent to provide the readers with an updated review of the pathogenesis of COPD, with a special focus on molecular mechanisms and novel overarching concepts developed in the past few years. The readers can access the summaries of recent meetings in the field in References 263, 335, 345.

COPD classically involves two spectra of clinical or pathological presentations, chronic bronchitis, emphysema, and small airway disease. While chronic bronchitis is defined clinically based on mucus production leading to cough with expectoration, emphysema is a pathological process of alveolar destruction with no apparent fibrosis (297). Both chronic bronchitis and emphysema eventually share a reduction of amount of air leaving the lung in the first second of a forced expiration, also known as forced expiratory volume at 1 s (or FEV₁). The reduction of FEV₁ characterizes the airflow limitation in the disease, and probably occurs because of small airway disease. Its immense utility for screening, early diagnosis, and clinical follow-up overshadows the complexity of COPD, characterized by the existence of several clinically important phenotypes (potentially with underlying distinct pathogenesis), and most importantly, the lack of a clear understanding of the pathophysiological mechanisms underlying the decrement of FEV₁ in a given patient or even in a population with similar COPD phenotypes. It is clear that COPD is also a systemic disease with involvement of the cardiovascular system, skeletal muscle, bone marrow, and metabolism, among others (40). The lung compartment that is critically involved in the disease consists of airways and parenchyma. The large airways are the main site of chronic bronchitis, while the parenchymal changes underlie the process of emphysema. Notwithstanding this relatively simplistic regional stratification of the disease, it is clear that the small airways, particularly the terminal bronchioles (structures <2 mm in diameter and with extensive overall surface area given its branching pattern through 20–23 generations), are a critical site of disease involvement, with a potential role in the pathogenesis of both chronic bronchitis and emphysema. However, there is a lack of understanding of how the small airway disease interfaces with the involvement of large airways or the destruction of the lung parenchyma, and the extent to which large and small airways contribute to airflow limitation.

The pathogenesis of COPD involves several pathogenetic processes such as inflammation, alterations of cell growth, cellular apoptosis, abnormal cell repair, extracellular matrix destruction, and oxidative stress, caused by air pollutants, including cigarette smoke, and modified by genetic factor (polymorphism), senescence, and infection. These processes are mediated by a growing number of molecular players, many of whom affect more than just one of these pathogenetic processes. This review focuses on specific pathobiologically relevant processes and molecules that ultimately mediate these processes, based on human or animal data and cell culture experimentation.

	II. RISK FACTORS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

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A. Direct Cigarette Smoking and Environmental Tobacco Exposure: The Main Causes of COPD

Active exposure to cigarette smoke causes the vast majority of COPD cases and contributes to increased incidence of related pulmonary diseases such as asthma and allergic rhinitis. Passive or environmental smoking increases the risk of smoking-related lung diseases, particularly of cancer and possibly of COPD. Cigarette smoking likely accounts for 80–90% of COPD cases in the United States (283). Mainstream smoke, which represents 45% of the total biomass in the burning cigarette smoke, is inhaled by puffing, while 55% of the content of the burning cigarette constitutes the side stream smoke, which is released into the environment. Environmental tobacco exposure (ETS) or involuntary smoking is defined as exposure to a combination of exhaled mainstream smoke and side stream smoke released from the smoldering end of a cigarette (249, 283). The effect that household ETS exposure has on children has been a growing focus of concern given its broad epidemiological impacts. Eisner et al. (74) evaluated the association between lifetime ETS exposure and the risk of developing COPD using data from a population-based sample of 2,113 United States adults aged 55–75 yr (74). The prevalence of prior prenatal ETS exposure was higher among adults with COPD than in those without the disease, consistent with multiple hits to potentially injurious agents throughout the life of a patient. Moreover, the prevalence of any subsequent lifetime home or workplace ETS exposure was higher among those with COPD than among those without COPD. In school children residing in as geographically distant regions as Southern California communities or in Russian cities, maternal smoking was a risk factor for subsequent decreased lung function, airflow limitation, and chronic bronchitis (93, 138). Similar findings were reported by the European Community Respiratory Health Survey (306). The impact of cigarette smoke in the newborns and in children can be significant. In vivo experiments showed that rhesus monkeys when treated with nicotine or cigarette smoke during pregnancy and/or postnatally have compromised offspring, displaying emphysematous alterations and apoptosis, with caspase-3 activation in the lung (281, 372).

Smokers with congenital deficiency of serine protease inhibitor (or serpin) -1 antitrypsin are susceptible to emphysema largely because -1 antitrypsin is the main inhibitor of neutrophil elastase (303). School children with low levels of -1 antitrypsin were at risk of developing pronounced decrements in pulmonary function, particularly if exposed to ETS (337). Collectively, the results suggest that home and work ETS, including maternal smoking, synergistically contributes to the risk of COPD.

B. The Adjuvant Role of Air Pollution

The outdoor and indoor air pollution from an urban environment provides a common ground to respiratory diseases, such as asthma, allergy, and COPD. Driscoll et al. (72) reported an estimated 386,000 deaths (asthma, 38,000; COPD, 318,000; pneumoconiosis, 30,000) and nearly 6.6 million of disability-adjusted life years (DALYS) (asthma, 1,621,000; COPD, 3,733,000; pneumoconiosis, 1,288,000) due to exposure to occupational airborne particulates worldwide in the year 2000 (72). A case control-crossover study carried out in 36 United States cities evaluated the effect of ozone and particulate matter with an aerodynamic diameter of less than 10 µm (PM₁₀) on respiratory hospital admissions during unseasonably warm weather spells between 1986 and 1999. This study showed that the combination of a 2-day exposure to 5 parts per billion (ppb) of ozone or the exposure to an increase of PM₁₀ of 10 µg/m³ in particulate matter resulted in 0.27 and 1.47% increase in COPD rates, respectively (211). In consecutive cross-sectional studies conducted in the Rhine-Ruhr Basin of Germany between 1985 and 1994, an increase of 7 µg/m³ in 5-yr means of PM₁₀ carried a 5.1% decrease in FEV₁ for COPD women. Furthermore, women living <100 m from a busy road also had a significantly decreased lung function, and COPD was 1.79 times more likely than for control women (278). Decrements in lung function indices were associated with increasing concentrations of PM_2.5, NO₂, and some metals (especially zinc and iron) in COPD cases in a study reported by the Catholic University Hospital in Rome, Italy (170).

The impact of the indoor environment also remains a major public health issue in COPD. A cross-sectional assessment of indoor air quality in Nepal and its health effects revealed that solid biomass fuels (animal dung, crop residue, and wood) were the main sources of indoor air pollution affecting women's health (295). The average smoke level (PM₁₀) in kitchens using biomass fuels was about three times higher than in those using cleaner fuels (kerosene, liquified petroleum gas, and biogas). The prevalence of respiratory illnesses and symptoms was considerably higher in those living in mud and brick houses when compared with concrete houses, and higher in those living on hills and in rural areas when compared with flatland and urban areas. Regalado et al. (260) reported that women who used a stove burning biomass fuel showed moderate airflow obstruction with COPD at stage GOLD >II, in the village of Solis, close to Mexico City. Orozco-Levi et al. (240) found that most of their study population composed by women with COPD were exposed to wood and charcoal smoke during their childhood and youth, but remained free of exposure for more than 25 years prior to presenting with symptoms of the disease, in Barcelona, Spain between 2000 and 2003. The follow-up data at the COPD Clinic of the National Institute of Respiratory Diseases, Mexico, between 1996 and 2003 demonstrated that women exposed domestically to biomass developed COPD with clinical characteristics, quality of life, and increased mortality similar in degree to that of tobacco smokers (257). Particles originated from biomass may synergize with cigarette smoke in the causation of airway diseases, and in fact replace cigarette smoke as the main culprit of COPD with the present populational trends of decreased smoking.

Although there is ample epidemiological and clinical evidence that ongoing exposure to cigarette smoke or environmental pollutants is clearly the main cause of COPD, these toxic agents recruit cell signaling pathways, which eventually get amplified, becoming self-perpetuating and highly destructive. Once lung destruction has ensued and patients have become airflow limited, interruption of cigarette smoke exposure may not significantly prevent further lung damage. The elucidation of these pathophysiological processes is the main hope for the development of biomarkers and targets of novel therapies.

C. Tobacco Components and Nicotine Class Acetylcholine Receptors

Cigarette smoke contains thousands of chemical components, including 10¹⁵ reactive species in the gas phase alone (199), particularly of high levels of nitric oxide. The tar phase has an equally abundant number of reactive oxygen and nitrogen species (ROS, RNS), including phenols and quinone. Obot et al. (237) quantified several smoke constitutes in samples collected from experimental exposure chambers (237). The mean ratios of nicotine/total particular matter (TPM) (0.07), formaldehyde/TPM (0.002), acetaldehyde/TPM (0.1), acrolein/TPM (0.01), and propionaldehyde/TPM (0.005) were consistently present at all exposure concentrations. TPM levels of 250–600 µg/l increased neutrophils and/or cytokines in bronchoalveolar lavage (BAL) in cigarette smoke-exposed mouse lungs.

The tobacco components, nicotine, nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N'-nitrosonornicotine (NNN) stimulated signal transduction through the nicotine class acetylcholine receptors (nAChRs) (9, 279). Functional nAChR are composed of homopentamers of 7–10 subunits or heteropentamers derived from 5(2–6) and 3(2-b4) subunits. By RT-PCR, small airway epithelial cells expressed nAChR2 and 4, while human normal bronchial epithelial cells (HBEC) expressed 3 and 5; the subunits 7, 9, 10, 2, and 4 of nARCHRs were expressed in both cells (321, 349). In the developing lung of monkeys, the 7-subunit was detected by immunohistochemistry in alveolar type II cells, pulmonary neuroendocrine cells, submucosal cells, smooth muscle cells, fibroblasts, and alveolar macrophages (281). By in situ hybridization, the subunits 3–5, 7, 2, and 4 were demonstrated in human vascular endothelial cell (198) and accounted for a significant nicotine-triggered cell signaling (111). These tobacco components activated nuclear factor-B (NF-B) in several cell lines, mediated partly by extracellular signal-regulated kinase 1 and 2 (ERK1/2) through the nAChR (56, 118). Heeschen et al. (111) involved nAChR 7-dependent signaling in angiogenesis, based on testing the effects of the nAChR 7-antagonist -bungarotoxin in endothelial cells. 7-Mediated endothelial cell angiogenic networking depended partly on vascular endothelial cell growth factor (VEGF) and was completely reliant on signaling by phosphoinositide 3-kinase (PI3K), ERK, and p38 mitogen-activated protein kinase (MAPK), finally resulting in NF-B activation.

nAChR form ion channels permeable to either calcium or sodium. Carlisle et al. (35) showed that HBEC have functional receptors of muscle-type heteropentamer 1/1// and neuronal homopentamers 7 or 9, neuronal heteropentamers 3/5/2, 3/5/4, 6/2, or 6/4 receptors, while airway fibroblasts had muscle-type heteropentamers 1/1// and neuronal homopentamers 7 (35). Nicotine-induced calcium influx was mediated by protein kinase C (PKC) and p38 MAPK in HBEC and activated ERK1/2 in airway fibroblasts. Additionally, cigarette smoke increased the expression of subunit 5 protein in HBEC and 3 in a panel of airway fibroblasts obtained from active smokers, never smokers, and ex-smokers. It is noteworthy that 3-containing receptors, a sodium channel, undergoes inactivation upon long-term exposure to nicotine, leading to thickening of the airway wall in chronic bronchitis because of an imbalance of apoptosis and cell proliferation.

Recent studies indicated that the vagus nerve, which is the longest of the cranial nerves and innervates most of the peripheral organs, can modulate immune responses and control inflammation through a "nicotinic anti-inflammatory pathway" dependent on the 7nAChR (329). Nicotine inhibited more efficiently than acetylcholine proinflammatory cytokines, tumor necrosis factor- (TNF-), interleukin (IL)-1, IL-6, and IL-18, but not the anti-inflammatory cytokine IL-10 through a posttranslational mechanism in human peripheral blood mononuclear cells (23, 342). Nicotine also inhibited the production of prostaglandin E₂ (PGE₂), monocyte inflammatory factor (MIP)-1 and MIP-1 in these cells (309, 365). These in vitro data are likely consistent with in vivo data showing that cigarette smoke reduced bacterial clearance in female C57BL/6 mice after infection with Pseudomonas aeruginosa (71). Furthermore, 7nAChR-deficient mice produced greater amounts of proinflammatory cytokines than wild-type mice when treated with lipopolysaccharide (LPS), consistent with an anti-inflammatory role of nicotine/7nAChR signaling (342). Prolonged presence of bacteria and persistent inflammation is a critical event in the course of COPD, a central component leading to exacerbations.

Given the complexity of cell signaling triggered by components of cigarette smoke, it is often difficult to isolate the effects of a specific component with regards to pulmonary pathophysiology. Therefore, most of the studies rely on the pathobiological effects of cigarette smoke rather than on isolated components.

	III. AIRWAY DISEASE

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A. Chronic Bronchitis

The two main characteristics of chronic bronchitis are excessive mucus production and chronic inflammatory cell infiltration of the bronchial wall. The excessive mucus production with increased expectoration correlates but poorly with increased mucus gland mass in the wall of large airways, which is documented by the Reid index that rates the level of airway wall occupied by mucus glands in relation to total airway thickness (261). The increase in mucus gland cells is not specific to COPD as it also occurs due to smog exposure, asthma, or cystic fibrosis. There is both an increase in mucus cell numbers via proliferation (hyperplasia) and enhanced mucus synthesis and secretion (hypertrophy). Inflammation caused by cigarette smoke is a critical factor affecting both pathobiological processes. The dominant site of the airflow obstruction is now thought to reside in the smaller airways measuring <2 mm in diameter. In contrast, the dominant site of clinically relevant mucus secretion remains the larger airways. A distinction is often made between disease of the larger airways (chronic bronchitis) and disease of the noncartilagenous membranous bronchioles and terminal airways (small airway disease or remodeling; small airway disease is addressed subsequently in this review). In fact, recent evidence generated by three-dimensional computer tomography documented that FEV₁ values in COPD patients correlate with luminal area and inversely with percent wall area, particularly of smaller generation airways (3rd to 6th, i.e., closer to 2 mm in diameter airways) rather than larger airways (<3rd generation airways) (105). Animal models of experimentally induced chronic bronchitis were reviewed elsewhere (234).

B. Airway Inflammation

Chronic bronchitis is defined as a CD8+ cytotoxic T cell-dominant airway disease. CD8+ T cells were observed in sputum (47, 328), and infiltrating the bronchial tree, including epithelium (269), submucosa (172), bronchial glands (270), smooth muscle layer (14, 268), and around lymphoid follicles (121). Morphometric analysis of bronchial biopsies showed that the ratios of CD8+ to CD4+ T cells were 1.3, 11.8, and 4.3 (mean/mm³) in healthy smokers, patients with stable chronic bronchitis, and patients with exacerbated chronic bronchitis, respectively (373). The lower ratio of CD8+/CD4+ in patients with exacerbation might occur due to an enhanced recruitment of CD4+ cells by RANTES, and/or potential lysis of CD8+ cells due to enhanced viral infection. The role of CD8+ T cells is well documented in respiratory virus infections, a common complication of COPD patients. CD8+ T cells are important contributors to viral clearance, utilizing contact-dependent effector functions, mediated by perforin and Fas ligand (CD95L/FasL) as well as interferon (IFN)- and TNF-. These latter two cytokines are primary mediators of T-cell-mediated lung injury, particularly TNF- (28). CD8+ T cells isolated from the sputum of smokers with COPD were highly activated based on the expression of perforin (47), which is synthesized upon T-cell activation, and released through intercellular channels formed through the target cell membrane, causing activation of mediators of cell death (294). Cigarette smoke increased CD95L expression in bronchioles of male Wistar rats, in association with phosphorylation of c-Jun NH₂-terminal kinase (JNK) (355). In humans, the role of FasL is unclear because one study reported that plasma-soluble Fas was increased in severe COPD (362), while another group reported that serum-soluble FasL and plasma-soluble Fas were not changed in patients with COPD, when compared with controls (308). Increased levels of CD95L could be potentially linked to CD95-producing inflammatory cell influx, which could affect airway epithelial or alveolar cell survival by activating CD95 cell death receptor.

Cytokines and chemokines, their receptors, and functions described in this review are summarized in Table 1, and the pathways involved in cytokine regulation of mucin production are shown in Figure 1. The involvement of neutrophils containing high levels of myeloperoxidase (MPO) and leukotriene B₄ (LTB₄) is also a typical feature of chronic bronchitis, influencing disease progression and the risk of acute bacterial exacerbations (55, 95, 115). The airway epithelium is a rich source of cytokines/chemokines that recruit both neutrophils and macrophages. Chung (48, 49) summarized in detail the cytokines overexpressed in COPD (48, 49). IL-6, IL-1, TNF-, growth-related gene- (Gro-)/keratinocyte-derived chemokine (KC) (CXCL1), monocyte chemoattractant protein-1 (MCP-1), and IL-8 were increased in sputum, with further increases during exacerbations of COPD, and the bronchiolar epithelium overexpressed MCP-1, its receptor CCR2, MIP1, and IL-8. MCP-1 and CCR2 were involved in the recruitment of macrophages and mast cells into the airway epithelium in COPD (62). Airflow limitation (i.e., decreased FEV₁) correlated with increased expression of IL-8, MIP-1, MCP-1, and the CCR2 in airway epithelium (85). Although IL-8 is thought to be a chemoattractant for neutrophil and CD8+ T cells via binding to receptors CXCR1 and CXCR2, epithelial IL-8 expression did not necessarily correlate with the numbers of these cells in airways of smokers (62). Aqueous cigarette smoke extract (CSE, i.e., cigarette smoke-bubbled into PBS/medium) induced the release of IL-8 from human airway smooth muscle cells, and the effect was enhanced by TNF- (238). Mice, which lack the IL-8 gene, have two neutrophilic CXC chemokines, MIP-2 and Gro-/KC (356), which were significantly elevated in the BAL of smoke-exposed female C57BL/6 mice (314).

View larger version (63K): [in this window] [in a new window]   FIG. 1. Signal transduction involved in the regulation of mucin production. TNF-, tumor necrosis factor-; IL-1, interleukin-1; PFA, platelet activating factor; LPS, lipopolysaccharide; NF-B, nuclear factor-B; IB, inhibitor of NF-B; IKK, IB kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; AA, arachidonate; PLA2, phospholipase A2; COX2, cyclooxygenase-2; PGH2, prostaglandin H2; PGE2, prostaglandin E2; EPR2/4, E-prostanoid 2 and 4 receptors; PKA, protein kinase A; MSK1, mitogen- and stress-activated protein kinase 1; CREB, cAMP-response element-binding protein; TGF-, transforming growth factor-; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HB-EGF, heparin-binding epidermal growth factor; TACE, TNF--converting enzyme; Duox1, dual oxidase 1; NE, neutrophil elastase; PKC, protein kinase C; MEK1/2, ERK kinase 1/2; HA, hyaluronan; TK, tissue kallikrein; X/XO, xanthine/xanthine oxidase; ROS, reactive oxygen species; MMP-9, metalloproteinase-9; JNK1/2, c-Jun NH2-terminal kinase 1/2; AP-1, activator protein-1; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; STAT6, signal transducers and activators of transcription 6.

C. Mucus

The mucus layer forms a film coating the apical portion of the airway lining. The coordinated movement of cilia propels the mucus layer directionally from the periphery of the lung into the upper airways. Mucus glycoproteins (mucins), water, and peptides are the main constituents of these layers, all playing key roles in the clearance of foreign materials and infectious agents. In chronic bronchitis, there is an increase in luminal mucus due to enhanced production of mucins, increased secretion from goblet cells, goblet cell hyperplasia and/or metaplasia, accumulation of cell debris, and inflammatory cells.

At least 12 human mucin genes (MUC1, 2, 4, 5AC, 5B, 7, 8, 11, 13, 15, 19, and 20) are expressed at the mRNA level in the lower respiratory tract from healthy individuals. Goblet cells typically express MUCs 5AC and 2, while glandular mucosal cells express MUCs 5B, 8, and 19. MUC5AC and 5B, which are large-molecular-mass glycoproteins, and MUC19, a newly identified mucin, are secreted and have cysteine-rich motifs. MUC7, expressed in serous cells, corresponds to a low-molecular-mass mucin lacking a cysteine-rich domain (265). Airway mucus obstruction is shared among cystic fibrosis, asthma, and COPD. COPD is associated with increased expression of MUC5B in the bronchiolar lumen and of MUC5AC in the bronchiolar epithelium (33).

Human bronchial mucins consist of highly polydispersed O-glycosyl proteins, which protect the respiratory surface from inhaled particles and microorganisms. The diversity of the mucins is enhanced by posttranslational modifications, such as O-glycosylation and sulfation (on serine/threonine residues; 339), thus contributing to highly diverse O-linked carbohydrate chains (265). There is a distinct pattern of mucins in chronic bronchitis with and without bacterial infection. Di-sialylated oligosaccharide and sialylated or sulfated oligosaccharide bearing sialyl Lewis X epitope were not found in patients with chronic bronchitis with no evidence of heavy bacterial or viral infection (63, 191). In contrast, mucins from infected patients with chronic bronchitis were more sialylated and contained more sialyl-Lewis X epitopes (61). TNF- increased 2,3-sialyltransferase activity, which mediated the sialylation of the weakly sialylated mucins in a human respiratory glandular cell line MM-39 (64). Thus the degree of sialylation of bronchial mucin might indicate the protection status against inhaled particles and microorganisms in chronic bronchitis.

A variety of stimuli such as neutrophil elastase, LPS, TNF-, IL-1, cigarette smoke, or oxidative stress causes goblet cell metaplasia and mucus hypersecretion (339) (Fig. 1). Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability (340) mediated by ROS in HBEC (79). Male Balb/c mice showed MUC5AC mRNA and protein expression, and goblet cell metaplasia after 8 days of intratracheal instillation of pancreatic elastase. These changes were mediated by elastase proteolytic activity and subsequent initiation of an inflammatory process documented by increases in Gro-/KC and IL-5 (338). Pseudomonas LPS induced MUC5AC expression, associated with neutrophil infiltration (359), and metalloproteinase (MMP)-9 expression (152). LPS also increased MUC7 mRNA and glycoprotein products in HBEC (188). TNF--, IL-1-, and LPS-induced MUC5AC synthesis were mediated by 1) IKK- and NF-B signaling (192), 2) mitogen- and stress-activated protein kinase 1 (MSK1), 3) signaling cascades leading to activation of the cAMP-response element by the cAMP-response element binding protein (CREB) via ERK and p38 MAPK (299), 4) COX-2 activation of E-prastanoid receptor (EPR) 2 and/or 4, and 5) cAMP-PKA-mediated signaling (96) (Fig. 1).

Signaling initiated by epidermal growth factor receptor (EGFR) tyrosine phosphorylation (induced by cigarette smoke-derived ROS), epidermal growth factor (EGF), transforming growth factor (TGF)-, or heparin-binding (HB)-EGF has been found to play an important role in mucin production in human airway epithelial cells. Hyaluronan fragmentation due to ROS generated by the xanthine/xanthine oxidase system activated the serine protease tissue kallikrein, which then cleaved the transmembrane precursor of EGF. Thus the EGF-EGFR-dependent stimulation of Ras-MAPK/ERK kinase (MEK)/ERK seems to be central in goblet cell hyperplasia and increased MUC5AC gene expression (36, 37). Cigarette smoke caused EGFR activation and mucin production via ROS and activation of TNF--converting enzyme (TACE), which shed pro-TGF- in human airway epithelial cell NCI-H292 (285). The same group showed that the TACE-EGFR pathway was dependent on dual oxidase 1, a homolog of glycoprotein p91phox, activated by PKC (284). Acrolein, also a component of cigarette smoke, induced MUC5AC expression via an early ligand-dependent activation of EGFR, mediated by TACE and MMP-9. A prolonged effect of acrolein may be mediated by altering MMP-9 and tissue inhibitor of metalloprotease (TIMP)-3 balance (67). Human airway trypsin-like protease, which is a novel serine protease purified from the sputum of patients with chronic bronchitis and bronchial asthma, induced the production of amphiregulin by the action of the protease-activated receptor-2. Amphiregulin was then released by TACE, resulting in activation of EGFR signaling (46).

Furthermore, ROS derived from cigarette smoke activated JNK, via a Src-dependent signaling cascade. This downstream signaling triggered transcriptional upregulation of MUC5AC, mediated by the binding of the activator protein 1 (AP-1) response element by JunD and Fra-2 (90). Additionally, exposure to cigarette smoke significantly decreased the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), thus increasing AKT and EGFR-specific signaling, leading to MUC5AC mucin production (185). The finding that aqueous CSE synergized with LPS or TNF- in the induction of MUC5AC expression suggests that cigarette smoke potentially amplifies the expression of respiratory mucins by proinflammatory stimuli relevant to COPD pathogenesis (12).

The close interplay between inflammation, oxidative stress, and growth factors in the lung airways has been progressively unraveled using state of the art approaches in transgenic mice (Tables 2 and 3, and Figs. 1 and 2). CD4+ Th2 cells and their cytokines, including IL-4, -10, and -13, play a crucial role in the development of goblet cell hyperplasia/metaplasia in animal models (182, 195, 371). IL-13 overexpressing mice had increased expression of MUCs 5AC, 1, and 4, and Gob-5 (42, 184). In signal transducers and activators of transcription 6 (STAT6)-deficient mice, in which both STAT6 and IL-13 signaling were impaired, allergen-induced goblet cell metaplasia was largely inhibited (184). Administration of recombinant IL-13 to nonimmunized mice induced goblet cell hyperplasia with mucus hypersecretion (3, 296), via EGFR signaling (291). IL-13 altered ciliated cell differentiation and increased the proportion of secretary cells in human nasal epithelial cells (174). Using a mouse model of Sendai virus infection, Tyner et al. (327) proposed a dual signaling model driving ciliated cell differentiation into goblet cells in chronic mucus-producing airway epithelium (327). First, EGFR signaling activated AKT/PI3K, and thus inhibited apoptosis of ciliated cells. Subsequently, ciliated cells responded to IL-13/IL-13R signaling and activated MEK1/2, ERK1/2, and STAT6 to produce mucin, including MUC5AC. Furthermore, IL-13 made by CD8+ T cells (214) induced emphysematous changes in the lungs of mice (371).

View larger version (53K): [in this window] [in a new window]   FIG. 2. Inflammation, extracellular matrix proteolysis, and oxidative stress: the classical hypothesis. Air pollutants, including cigarette smoke, are a rich source of oxidants (i.e., O2–, NO), leading to the recruitment of macrophages and neutrophils. These inflammatory cells might be the source of cytokines (i.e., TNF-1, IL-1, IL-8, MCP-1, MIP-1), oxidants (i.e., O2–, NO, HOCl), and proteases (i.e., MMPs and neutrophil elastase). By stimulating dendritic cells, CD4+ and CD8+ T cells (Tc1/Th1-dominant) also release chemokines (i.e., IFN-, IL-4, IL-13) and perforins. These stimuli result in alveolar wall cell breakdown and matrix proteolysis. Further inflammation might be triggered by elastin fragments. Cell injury decreases antioxidant and antiprotease defense so as to favor protease and oxidant-mediated alveolar wall destruction. AEI and AEII, alveolar type I and II epithelial cells; EC, endothelial cells; IC, interstitial cells; AM, alveolar macrophages; PMN, neutrophils; DC, dendritic cells; CD4+/CD8+, CD4+/CD8+ T cells.

D. Small Airway Remodeling

The progressive increase in connective tissue in the smoker's bronchioles has been documented in numerous studies (1, 122, 208, 233). The group led by Hogg et al. (120) recently provided the most compelling evidence for remodeling and fibrous thickening, based on the analysis of lungs obtained by cancer resections or lung volume reduction surgery (120). With increased severity of COPD based on GOLD criteria (244), small airways have increased thickness, and heightened inflammatory infiltrate, including infiltration by neutrophils, macrophages, T lymphocytes (CD4+ and CD8+ T cells), and B lymphocytes. Lymphoid follicles accumulated within the walls of the affected bronchioles. The lumen of these airways was also obliterated more often by mucus.

The interaction between inflammation and small airway remodeling has been mechanistically addressed in rodent models. Overexpression of the Th2 cytokine IL-10 caused mucus cell metaplasia, B- and T-cell-rich inflammation, and subepithelial fibrosis of airways (182). Interestingly, these responses were mediated by multiple mechanisms. Mucus metaplasia was dependent on IL-13/IL-4 receptor-/STAT6 signaling, while inflammation and fibrosis were independent of these signaling pathways. Furthermore, overexpression of IL-1 induced peribronchial fibrosis (175). In rat trachea organ culture, cigarette smoke released active TGF-1 and induced nuclear localization of phospho-smad2 in epithelial and interstitial cells, subsequently leading to upregulation of the TGF--dependent procollagen gene (343).

Fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling seems to be associated with airway and vascular remodeling in chronic bronchitis. Immunohistochemical studies of lung tissues from COPD patients showed that FGF-1 and its receptor FGFR-1 are detected in vascular and airway smooth muscle as well as airway epithelial cells. Basic FGF/FGF-2 was localized in the cytoplasm of airway epithelium and in nuclei of airway, vascular smooth muscle, and endothelial cells (163). FGF-1 and/or FGF-2 increased steady-state mRNA levels of FGFR-1 and induced cellular proliferation of cultured human airway smooth muscle cells (164). Smokers with chronic bronchitis and airflow limitation had increased expression of FGF-2 in the central airways, which was mainly due to an enhanced expression in the bronchial gland compartment, suggesting that FGF-2 may have a role in promoting mucus hypersecretion in smokers (98).

Based on the assessment of bronchial biopsies of patients with airflow limitation, small airway remodeling and obstruction may be caused by submucosal hypercellularity of endothelial cells in response to overexpressed VEGF (30, 164). Enhanced vascularity of the inner region of medium-sized airways might contribute to airflow limitation in asthma as well (106). The molecular mechanisms accounting for the effects of VEGF-regulated vascular permeability may include Src-mediated signaling and an increase in both nitric oxide and prostacyclin. VEGF-overexpressing transgenic mice showed enhanced angiogenic responses in airways associated with inflammation, and mucus accumulation, dependent to some extent on enhanced production of nitric oxide (18).

	IV. EMPHYSEMA

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Emphysema has been defined pathologically as an irreversible destruction of alveolar structures with airspace enlargement, not associated with significant amount of fibrosis (297). The central elements of this definition are the concepts of destruction and irreversibility of the pathological process. The statement regarding the lack of fibrosis was warranted by the common finding of pericicatricial emphysema around areas of interstitial scarring. The following discussion highlights that several destructive mechanisms do not act in isolation, but rather converge and ultimately account for the destruction of alveolar structures. The term destruction implies that alveolar cells die, either by necrosis or apoptosis, the two prototypic terminal modes of cell death. But how do alveolar cells ultimately die in emphysema? Although alveolar lung tissue is exposed to similar environmental toxins facing the proximal airways, the responses of these two anatomic compartments are clearly distinct. Airways respond with cell proliferation, while alveolated lung tissue disappears. Three critically important pathophysiological processes interact in the periphery of the lung in COPD: oxidative stress, alveolar cell apoptosis, and extracellular matrix proteolysis. Inflammatory cells also actively participate in the pathogenesis of alveolar destruction. Furthermore, novel concepts pertaining to senescence and aging are currently of growing interest in this field. Current genetically altered mice prone to and resistant to pulmonary emphysema are summarized in Tables 1 and 2.

A. Inflammation

Lung inflammation continues to dominate the present thinking of the pathophysiology of COPD, including emphysema (Fig. 2). Cigarette smoke inhalation incites acute and chronic inflammatory responses, which potentially cause alveolar destruction. Lung retention of activated neutrophils has been documented after inhalation of cigarette smoke (200). Short exposures to cigarette smoke were associated with increased desmosine detection in BAL, indicative of elastin fiber breakdown in mouse lungs (50). Chronic cigarette smoke exposure was associated with an increase in inflammatory cells in the BAL and lung tissue of humans and experimental models (324). Most importantly, an increase in CD8+ T cells has been linked to worsened COPD (76), an effect that potentially overshadows the potential role for neutrophils. However, neutrophils and eosinophils predominate with progression of COPD, particularly during exacerbations, and may participate in the acceleration of tissue destruction or airway disease (243). The enhanced infiltration of inflammatory cells in COPD has been attributed to either cigarette smoke/pollutants or to oxidative stress as the result of the exposure to environmental hazards, causing enhanced recruitment of inflammatory cells. However, potential ground-breaking concepts were recently introduced that suggest that the inflammatory cells could be a result of an autoimmune attack to the lung tissue (2), or potentially linked to an associated lung aging process (325).

B. Inflammatory Cells

The smoker's lung has increased numbers of neutrophils, lymphocytes, and macrophages (352). The specific pathophysiological roles of each of these inflammatory cells in emphysema remain to be determined. Neutrophils could be a source of oxidants and elastases, while macrophages produce oxidants and potentially destructive extracellular matrix proteases. On the other hand, some lung injuries require neutrophils for proper repair (114). Whether inflammatory cells play a similar role (i.e., protective or destructive) early versus late in the disease also remains unknown. The finding that ex-smokers have persistent inflammation despite discontinuation of smoking argues for a proinflammatory environment set by cigarette smoke-induced chronic lung damage (176). Recent ground-breaking studies indicate that parenchymal inflammation in advanced emphysema contains oligoclones of CD4+ and CD8+ T cells, perhaps the first indication in support of a potential autoimmune attack (161, 305). A potential target to infiltrating lymphocytes is the adenovirus latent infection antigen, which can amplify inflammatory responses to cigarette smoke in the guinea pig model (212), and whose expression was enhanced in advanced emphysematous lungs compared with milder forms of emphysema (264).

C. Cytokines and Chemokines

The cytokines, their receptors, and functions involved in COPD are listed in Table 1. In exhaled breath condensate, healthy smokers had increased levels of IL-1, IL-6, IL-8, IL-10, IL-12p70, and TNF- when compared with nonsmokers, and all cytokines were increased on COPD (GOLD stages III and IV) exacerbations when compared with patients with stable COPD (92). Several rodent models exhibit increased cytokine profiles after acute and repeated (chronic) cigarette smoke. Guinea pigs had increased transcript levels of TNF-, IL-1, IL-8, and MCP-1 and decreased IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), TGF-, and eotaxin mRNA after a single exposure and/or 4 wk of cigarette smoke. These changes were associated with increases of mononuclear cells and neutrophils in the BAL (166). ICR and C57BL/6 mice had increased TNF-, IL-1, IL-5, IL-13, GRO/KC, IL-17, RANTES, MCP-1/JE, and thymus and activation-regulated chemokine (TARC) expression in BAL, along with an increase of neutrophils, but not macrophages when exposed to 7 consecutive days of cigarette smoke. These alterations were prominent after 2-h exposure and partly recovered after 12 h (237). After 6-mo exposure to cigarette smoke, AKR mice increased TNF-, IL-12 (p35 and p40 subunits), IL-10, MIP-1 and -1 mRNA expression, indicative of Th1-adaptive response, while C57BL/6 mice only had an increase in IL-10 (99). These AKR mice had a marked inflammatory infiltrate comprised of CD4+, CD8+, T cells, neutrophils, and macrophages in the lung. Despite these studies, the interpretation of cytokine changes in rodents is complicated by the discrepant response among different strains to the same stimulus (i.e., cigarette smoke) and a significant variability of responses by individual mice of the same strain (99). Recently, Bracke et al. (27) demonstrated that mice lacking CCR6, a receptor of MIP-3/CCL20, have attenuated cigarette smoke-induced emphysema, accompanied by a decrease of dendritic cells, CD8+ T cells, and granulocytes in the lung (27). These knockout mice showed lower levels of TNF- and no increase of MCP-1 in BAL, suggesting that MCP-1-CCR2 interaction was affected by CCR6 deficiency (27).

TNF- has been a leading cytokine linked to cigarette smoke-induced emphysema because of extensive documentation of increased levels of TNF- in smoker's serum and sputum samples (150). TNF- signals through the TNF- receptor type 2 (p55, CD120b) and the type 1 receptor (p75, CD120a). Transgenic overexpression of TNF- led to emphysema and exaggerated alveolar inflammation (82), while TNF- receptor knockout mice showed significant protection against cigarette smoke-induced emphysema (51). Enhanced TNF- can explain partly several of the specific airway and airspace pathologies seen in COPD. With relevance to emphysema pathogenesis, TNF- stimulated MMP synthesis by alveolar macrophages (49). In cultured macrophages, aqueous CSE induced TNF- via ERK1/2 in differentiated U937 cells (66). Furthermore, circulating TNF-, soluble receptor p55, and p75 were significantly increased in COPD patients compared with healthy controls (308). The link between TNF- and alveolar cell apoptosis remains unexplored. The TNF- receptor p55, rather than the p75, is critical to the development of cigarette smoke-induced emphysema (58). Lack of TNF- receptor p55 protected against cigarette smoke-induced emphysema, along with decreases of neutrophils, macrophages, and CD4+ and CD8+ T cells. Induction of alveolar wall apoptosis (predominantly involving type II epithelial cells) appeared to be dependent on the activation of both receptors. IL-1 also plays a role in the development of emphysema because lung-specific induction of human IL-1 caused emphysema, accompanied by expression of Gro/KC and MIP-1 (175). Inhibition with anti-IL-1 antibody attenuated alveolar macrophage influx in BAL after 7 days of cigarette smoke exposure (38). Furthermore, the double IL-1 receptor and TNF- receptor knockout mice were protected against elastase-induced emphysema (193). These double transgenic mice showed protection against inflammation and alveolar cell apoptosis caused by elastase instillation. IL-6 knockout mice showed reduced cell proliferation in terminal bronchiolar epithelium and proximal alveolar regions, and maintained Clara cell secretary protein expression after exposure to cigarette smoke and/or ozone (366). However, a role for IL-6 in human emphysema has not been studied in detail.

In airways of COPD patients, T lymphocytes preferentially expressed IFN- and the chemokine receptor CXCR3 (242). Lymphocytes obtained from advanced emphysematous lungs (from lung volume reduction surgeries) were strongly polarized. These cells secreted high levels of IFN-, CCR5, and CXCR3 and had increased expression of CXCR3 ligands, MIG and IP10 (97). These findings support a strong polarization of alveolar lymphocytes towards a Th1 phenotype in advanced emphysema. When treated with IP10 or MIG, cultured alveolar macrophages expressed MMP-12 protein, which could also be detected in alveolar macrophages in emphysematous lungs. These studies provided substantial insight into the nature of the parenchymal lymphocytic infiltrate in emphysema, and mechanistically linked the observations of increased chemokines in emphysema and activation of MMP-12.

The evidence of the role of cytokines/chemokines in the pathogenesis of emphysema was better delineated through the work of Elias and collaborators; these investigators showed that inducible and lung specific overexpression of the prototypic Th1 cytokine IFN- (344) or the Th2 cytokine IL-13 (371) produced emphysema associated with inflammation, variable degree of fibrosis with MMP, and cathepsin expression. In the IL-13-overexpressing mice, MMP-9 and -12 were responsible for the emphysema phenotype, since the combined MMP-9 and -12 knockout mice were partly protected against emphysema due to lung expression of IL-13 (173). Furthermore, inhibition of cathepsins also contributed to the protection against emphysema (370).

D. Extracellular Matrix Proteolysis: Protease/Antiprotease Imbalance

Destruction of the elastin framework of the lung has been a leading potential mechanism of alveolar destruction in emphysema (Fig. 2). Neutrophil elastase and MMPs are the most studied candidates that account for the protease/antiprotease imbalance in COPD.

The evidence in support of a significant role of neutrophil elastase in the pathogenesis of emphysema has relied on the finding of early emphysema in patients deficient in 1-antitrypsin, the major inhibitor of neutrophil elastase (303). Pallid mice, a strain with reduced -1 antitrypsin levels, developed emphysema earlier than C57BL/6 mice after cigarette smoke exposure (39, 311). Emphysema caused by lung instillation of porcine pancreatic elastase has been a disease model widely used in support of the protease/antiprotease imbalance over the past 30 years (80, 109, 124, 132–134, 146, 193, 289, 290). Furthermore, neutrophil elastase-null mice were significantly protected against chronic cigarette smoke-induced emphysema, which was associated with a decrease of MMP-12/TIMP-1 ratio (287). The treatment with a synthetic neutrophil elastase inhibitor ZD0892 reduced BAL neutrophils and BAL desmosine and hydroxyproline to levels similar to controls. This elastase inhibitor also reduced levels of MIP-2, MCP-1, and TNF- and decreased airspace enlargement (351).

There is substantial evidence of increased expression of several MMPs in emphysematous lungs (10, 324). MMPs comprise at least 20 proteolytic enzymes that play an essential role in tissue remodeling and repair associated with development and inflammation, by degrading collagen, laminin, and elastin. Depending on substrate specificity, amino acid similarity, and identifiable sequence modules, the MMP family can be classified into distinct subclasses as collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10, -11), membrane-type MMP (MMP-14 to MMP-25), matrilysin (MMP-7), and macrophage metalloelastase (MMP-12) (169). The major physiological inhibitors of the MMPs in vivo are -2 macroglobulin and the TIMP family, which are naturally occurring proteins specifically inhibiting these proteases and produced by different cell types. The TIMP family at present comprises four structurally related members, TIMP-1, -2, and -3, and the recently discovered TIMP-4.

Because it degrades elastin and is predominantly produced by alveolar macrophages, MMP-12 activation has been a leading candidate proteinase responsible for pulmonary emphysema. MMP-12 protein was observed in the sputum, BAL, bronchial biopsies (65, 218), and peripheral lung tissue of patients with advanced emphysema (33). Patients with COPD caused by cigarette smoke as well as wood smoke from domestic heating and cooking fuels showed higher expression levels of MMP-2, -9, and -12 transcripts in macrophages from BAL than control patients (219). CSE [prepared by dissolving the collected smoke particulates in dimethyl sulfoxide (DMSO)] or cytokine mix (TNF- and IFN-) induced MMP-12 mRNA in HBEC (177, 178). MMP-12 expression was associated with H₂O₂ production and was dependent on NADPH oxidase, p67phox, and p51 (NOXA1) (177). IL-1 induced the expression of MMP-12 mRNA via activation of JNK and PI3K (357). IL-1-mediated expression of MMP-9 and -12 was demonstrated in IL-1 overexpressing transgenic mice as well (175).

There are abundant data linking MMP-12 and experimental emphysema. MMP-12 immunoreactivity was evident in alveolar macrophages and septal macrophages after cigarette smoke exposure in male C57BL/6 mice (331). Chronic inhalation of cigarette smoke for 1, 3, and 6 mo caused significant increases of MMP-12 mRNA and protein in the lung and mRNA in BAL cells of male C57BL/6 mice. There were no differences in the expression levels of TIMP-1 and -2, indicating an increase of MMP-12/TIMP ratio (26). Shapiro and colleagues (107) demonstrated that MMP-12-null mice are protected from the development of cigarette smoke-induced emphysema. Infiltrating macrophages in wild-type mice exposed to cigarette smoke might have been attracted by elastin fragments released by MMP-12, as MMP-12 knockout mice did not show evidence of increased alveolar macrophage accumulation due to chronic cigarettes smoke inhalation (124). When exposed to cigarette smoke for 2–3 days, MMP-12-null mice had an attenuated number of lung neutrophils and decreased expression of MIP-2, MIP-1, and MMP-9 activity in BAL, suggesting that the MMP-9 response is dependent almost entirely on MMP-12 expression (180). MMP-12 is specifically involved in cigarette smoke-induced inflammation, because there was no difference in the number of neutrophils in BAL between knockout and wild-type mice treated with LPS. MMP-12 also acted as a TACE, promoting the release of active TNF- for subsequent endothelial activation, neutrophil influx, and elastase release in cigarette smoke-induced acute pulmonary injury (50). These acute lung changes were not dependent on NF-B activation, since both MMP-12-null mice and wild-type mice showed a rapid increase of NF-B DNA binding activity.

There has been increasing evidence that cells other than alveolar macrophages are the source of MMP-12. MMP-12 was expressed by dendritic cells sorted from nonlavaged lung, and bone marrow-derived dendritic cells upregulated MMP-12 expression after treatment by aqueous CSE or LPS (26). Although the role of MMP-12-expressing dendritic cells is unknown, the authors speculated that dendritic cells could harm the surrounding tissues during lung recruitment to orchestrate adaptive immunity responses.

Despite evidence of MMP-9 and -2 (which also degrade elastin) expression in COPD lungs, the evidence that either MMP has a central role in emphysema remains unclear. MMP-9 knockout mice were not protected against emphysema (10). On the other hand, experimental emphysema caused by lung overexpression of IL-13 or deletion of surfactant protein D involved both MMP-9 and MMP-12 (173, 348), as surfactant D-deficient mice expressed high levels of MMP-9 and -12 (108, 364). However, surfactant D-deficient mice lacking MMP-9 or -12 developed air space enlargement similar to surfactant D-deficient mice expressing both MMPs (368). Supporting evidence for the involvement of MMP-9 was provided by the finding of increased MMP-9 expression by tissue samples of COPD patients (280).

Collagenases (i.e., MMP-1) might have a significant role in emphysema. MMP-1 expression was increased in COPD lungs when compared with control lungs (128). Furthermore, there is growing support that human MMP-1 participates in the pathogenesis of emphysema (60, 81). Interestingly, despite the suggested lack of significant fibrosis in emphysema (297), advanced emphysematous lung exhibited a complex pattern of collagen deposition, which eventually replaced the elastin framework (77).

The remodeled extracellular matrix may modulate alveolar inflammation. Elastin fragmented by MMP-12 had chemoattractant activity for monocytes via interaction with the elastin-binding protein (124). In addition, the collagen-derived peptide N-acetyl Pro-Gly-Pro (PGP) originated from the breakdown of extracellular matrix, attracted neutrophil infiltration in lungs, and caused pulmonary inflammation dependent on activation of the chemokines receptor CXCR2 (347). Repeated intranasal dosing of PGP for 12 wk resulted in alveolar space enlargement of C57BL/6 mice, an effect putatively attributed to neutrophil-mediated lung destruction. Furthermore, PGP levels in the BAL were significantly increased in COPD patients when compared with control subjects. The authors pointed out that the basis for PGP activity lies in its molecular similarity to the GP motif present in all Glu-Leu-Arg (ELR motif)-CXC chemokines such as IL-8 (CXCL8), GRO-, -, and - (CXCL1–3) in humans and KC (CXCL1) and MIP-2 (CXCL2) in mice. Thus degraded products from extracellular matrix might represent critical factors in the pathogenesis of destructive air space enlargement.

TIMP-3-null mice developed spontaneous alveolar enlargement, which started at 2 wk of age and subsequently progressed with age (181, 203, 204). The mice exhibited reduced collagen and fibronectin, fragmented collagen in the peribronchiolar space, and disorganization of collagen fibrils in the alveolar walls with no changes of MMP-2,-7, -8, -9, and -12 expression, indicating a shift to MMP/TIMP imbalance to favor MMP-mediated destruction of lung extracellular matrix. Cigarette smoke exposure of male BALB/c mice increased MMP-12/TIMP-2 ratio after a 10-day exposure (59). An increase of MMP-9 and a decrease of TIMP-1 expression in emphysematous lung were reported in the senescent mice lacking the aging-protective protein Klotho (86). In humans, flow cytometry-sorted alveolar macrophages of patients with COPD produced lower levels of TIMP-1 than observed from healthy smokers and nonsmokers, therefore favoring a proteolytic environment in patients with alveolar destruction (251). Two polymorphisms of the TIMP-2 gene involving +853 G/A and –418 G/C nucleotide substitutions were identified in COPD patients (116). It is possible that the former is related to the downregulation of TIMP-2 activity, while the latter, located on the consensus sequence for Sp1 binding site, might downregulate the transcriptional activity of TIMP-2.

E. Disruption of Alveolar Cell and Molecular Maintenance Program: Role of Growth Factors and Bone Marrow

The finding that decreased VEGF or VEGF signaling caused experimental emphysema (149, 312) and the evidence that COPD lungs have decreased expression of VEGF and VEGF receptor-2 (VEGFR-2) led to the concept that alveolar maintenance (Fig. 3) was required for structural preservation of the lung. Cigarette smoke would then disrupt this maintenance program, causing emphysema (324). In fact, several experimental models underscored a potential disruption of alveolar maintenance via genetic manipulations of genes or alterations of the extracellular matrix (323).

View larger version (47K): [in this window] [in a new window]   FIG. 3. Breakdown of cellular and molecular maintenance program in emphysema. Air pollutants, including cigarette smoke, might inhibit the production and release of growth and survival factors (most notably VEGF and HGF) from alveolar epithelial cells (type I and II cells), endothelial cells, and interstititial cells (fibroblasts and myofibroblasts), resulting in apoptosis. Impairment of efferocytosis causes decreases of growth factor release from cells involved in apoptotic cell clearance (macrophages as well as epithelial and endothelial cells), and thus might also contribute to persistent inflammation. Decreased circulating progenitor cells might delay replenishment of damaged lung epithelial and endothelial cells. Reduced production and function of TGF- by cells involved in apoptotic cell clearance might delay repair processes and contribute to inflammatory cell activation. Apoptotic and inflammatory cells might be the sources of enzymes involved in extracellular matrix proteolysis.

Ito et al. (136) reported that, when compared with younger mice, older animals had decreased expression of VEGF-A, -B, and -C; VEGF-A isoforms 120, 164, and 188; and VEGFR-1, -2, and -3 mRNA. These molecules were then further downregulated by LPS injection (136). These results suggest that pulmonary expression of VEGF types and isoforms and VEGF receptors decline with age, and additional stimuli such as LPS, oxidative stress, and cigarette smoke further decrease their lung expression. The concept is supported by the finding of downregulation of VEGF protein in the emphysematous lungs of senescence-accelerated prone (SAMP)-1 mice, when compared with age-matched senescence-accelerated resistant (SAMR-1) mice. The expression of VEGF in SAMP-1 mice was further downregulated by cigarette smoke exposure (147).

Evidence of the critical role of apoptosis in emphysema caused by VEGFR blockade begs the question whether maintaining cell homeostasis by growth factors such as VEGF could rescue emphysema. VEGF treatment improved short-term survival in prematurely delivered mice with respiratory distress syndrome (RDS), attributed to an increase in surfactant production (54). Hyperoxia-induced bronchopulmonary dysplasia, which is characterized by air space enlargement and loss of lung capillaries, was associated with decreased lung VEGF and VEGFR-2 expression in newborn rats. Postnatal gene delivery of VEGF improved survival, promoted lung capillary formation, and preserved alveolar enlargement (315). However, no data are available on the potential benefits of VEGF gene therapy in emphysematous lung. VEGF is a tightly regulated gene in the lung, playing specific growth and differentiation roles in different structural and cellular compartments. While VEGF protein and VEGF189 mRNA contents in the lung were reduced in severe emphysema, VEGF gene expression was increased in pulmonary arteries of smokers and patients with moderate COPD and correlated with medial thickening of the pulmonary vascular walls (274). In guinea pigs exposed to cigarette smoke, pulmonary artery pressure and medial thickness were increased and associated with heightened gene and protein expression of VEGF, endothelin-1, and endothelial nitric oxide synthase (353). These findings suggest that, while loss of alveolar maintenance by VEGF might contribute to severe lung disease, the early induction of VEGF during cigarette smoke exposure may contribute to pulmonary vascular remodeling characteristically described in the disease (15).

Primary targeting of type II cells for apoptosis may trigger failure of alveolar maintenance as type II and lung endothelial cells rely on VEGF for growth, survival, and differentiation (336). Tsao et al. (317) proposed a unique mechanism of cell death in alveolar epithelial cells and endothelial cells, because transgenic mice with lung overexpression of placental-like growth factor (PLGF) (a homolog of VEGF, which reacts with VEGFR-1, but not VEGFR-2) showed pulmonary emphysema, starting at 6 mo of age and becoming prominent at 12 mo (317). In accordance with these changes, decreases of numbers of lung endothelial cells and VEGF mRNA expression were observed. In vitro treatment with PLGF promoted death of type II epithelial cells, leading the authors to suggest that overexpressed PLGF induces epithelial cell death via reduction of VEGF expression. A lower availability of VEGF might result in endothelial cell damage and impaired microcirculation, subsequently promoting further epithelial cell death.

Hepatocyte growth factor (HGF), which acts as a potent multifunctional pulmotrophic factor in repairing lung injury and promoting angiogenesis, may represent a promising approach to stimulate lung repair in emphysema (239, 272). Elastase instillation in rat lungs enhanced HGF mRNA and protein expression in plasma and BAL, followed by a significant decline to levels below the baseline. Reduced levels of HGF correlated with progressive emphysematous changes and deterioration in pulmonary physiology. Lung HGF replenishment using a hemagglutinating virus of Japan (HVJ)-mediated transduction in alveolar endothelial and epithelial cells improved pulmonary function by decreasing alveolar cell apoptosis and enhancing alveolar regeneration and angiogenesis (290). HGF likely induced proliferation of bone marrow-derived cells and resident endothelial cells in alveolar walls of mice treated with intratracheal instillation of pancreatic elastase (