Animal models of chronic obstructive pulmonary disease.

Review

Animal models of chronic obstructive pulmonary disease Michael Fricker, Andrew Deane & Philip M Hansbro† 1.

Why are animal models of chronic obstructive pulmonary disease needed?

2.

COPD: what (and how) should

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Queensland on 05/26/14 For personal use only.

we be modelling? 3.

CS-induced models of COPD

4.

What have we learned from animal models of COPD?

5.

Modelling COPD and bacterial/ viral exacerbations of COPD

6.

Modelling systemic co-morbidities of COPD

7.

Conclusion

8.

Expert opinion

†

University of Newcastle and Hunter Medical Research Institute, Priority Research Centre for Asthma and Respiratory Disease, New South Wales, Australia

Introduction: Chronic obstructive pulmonary disease (COPD) is a leading global cause of mortality and chronic morbidity. Inhalation of cigarette smoke is the principal risk factor for development of this disease. COPD is a progressive disease that is typically characterised by chronic pulmonary inflammation, mucus hypersecretion, airway remodelling and emphysema that collectively reduce lung function. There are currently no therapies that effectively halt or reverse disease progression. It is hoped that the development of animal models that develop the hallmark features of COPD, in a short time frame, will aid in the identifying and testing of new therapeutic approaches. Areas covered: The authors review the recent developments in mouse models of chronic cigarette smoke-induced COPD as well as the principal findings. Furthermore, the authors discuss the use of mouse models to understand the pathogenesis and the contribution of infectious exacerbations. They also discuss the investigations of the systemic co-morbidities of COPD (pulmonary hypertension, cachexia and osteoporosis). Expert opinion: Recent advances in the field mark a point where animal models recapitulate the pathologies of COPD patients in a short time frame. They also reveal novel insights into the pathogenesis and potential treatment of this debilitating disease. Keywords: animal model, chemokine, cigarette, chronic obstructive pulmonary disease, cytokine, emphysema, guinea pig, immune system, inflammation, lung, lymphocyte, macrophage, mast cell, model, mouse, neutrophil, oxidative stress, protease, pulmonary, rat, reactive oxygen species, rodent, smoke, therapeutic, tobacco Expert Opin. Drug Discov. (2014) 9(6):629-645

Why are animal models of chronic obstructive pulmonary disease needed?

1.

Chronic obstructive pulmonary disease (COPD) is an umbrella term describing a debilitating multi-systemic disease that develops in and emanates from the lungs. It is characterised by chronic bronchitis (pulmonary inflammation), cough, airway sensory nerve sensitivity, mucus hypersecretion, airway remodelling and/or emphysema, which result in reduced lung function [1]. It is the third leading cause of death globally and is predicted to become the fifth leading cause of chronic morbidity worldwide by 2030 [1-3]. Recent studies estimate that at least 80% of COPD cases are induced by the inhalation of tobacco smoke, especially from cigarettes, with other risk factors including exposure to second-hand tobacco smoke and the fumes from burning biomass fuels, as well as genetic factors [1,4]. Despite the weight of epidemiological evidence incriminating tobacco smoke as the predominant causative factor of COPD, its incidence is predicted to rise due to continued exposure over prolonged periods coinciding with increasing uptake in populous countries (e.g., India and China) and increasing average lifespans [1]. Once initiated, the patients’ clinical condition and features of disease often deteriorate further, even after the cessation of smoking. Currently, there are no treatments that effectively 10.1517/17460441.2014.909805 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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Article highlights. .

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Numerous independent research groups have developed cigarette smoke (CS)-exposure models in the guinea pig, rat and mouse in which animals develop the key pathological features of chronic obstructive pulmonary disease (COPD). The importance of the immune system is clear from the published findings using animal models of COPD, and several recent studies have identified novel mechanisms and drivers of the long-term disease. Progress has also been made in modelling and understanding the acute viral and bacterial exacerbations that contribute to COPD. Established CS-exposure models are now being put to good use in investigating the systemic co-morbidities associated with COPD.

This box summarises key points contained in the article.

halt the induction or progression of COPD. The use of inhaled glucocorticoids and bronchodilators to treat symptoms and exacerbations of COPD has limited clinical efficacy. Increasing our understanding of the molecular pathways and responses that contribute to the initiation and progression of COPD will facilitate the development of much-needed effective therapeutics [5]. The development of animal models of COPD that accurately recapitulate the complex multi-faceted features of this disease, particularly in a short time frame, will significantly enhance these ongoing efforts. In this review, we summarise recent advances in the development and use of animal models of experimental COPD as well as their potential limitations. This rapidly expanding field holds considerable promise, and we highlight a selection of areas of research that we consider of particular importance for future studies.

COPD: what (and how) should we be modelling? 2.

COPD is a complex condition principally defined by impaired lung function that is characterised by poorly reversible airflow limitation [1]. It is associated with a heterogeneous collection of pulmonary and extrapulmonary manifestations across patients diagnosed with various severities of disease [4]. Airflow limitation is caused by a number of pathological changes in the lungs, including chronic airway inflammation, mucus hypersecretion, airway remodelling and emphysema characterised by parenchymal destruction and loss of alveolar attachments. It is persistent chronic pulmonary and systemic inflammation that is thought to drive disease initiation and progression [5]. The decline in the condition of patients is exaggerated by exacerbations of the disease where acute increases in disease symptoms occur that are typically induced by respiratory infection with bacteria (e.g., Streptococcus 630

pneumoniae) and viruses (e.g., rhinovirus and influenza). In addition, COPD patients are more susceptible to these infections [6,7]. The chronic low-grade systemic inflammation is thought to underpin the systemic co-morbidities associated with COPD, which significantly contribute to increased morbidity rates and decreased quality of life of patients. They also have deleterious consequences for distal sites in the circulatory system, gut and skeletal muscle [8,9]. However, the mechanisms of pathogenesis in COPD are poorly understood and a further understanding of the processes involved may lead to the identification of therapeutic targets and the development of new therapies. Representative animal models are valuable in investigating these processes. They can be used to identify novel pathways of disease pathogenesis, increasing our understanding of the mechanisms and outcomes of acute bacterial and viral exacerbations and delineating the relationships between pulmonary and systemic manifestations in COPD. Many early efforts to understand the molecular basis of COPD focussed on developing animal models that reproduced individual elements of disease in isolation, such as emphysema or airway fibrosis and remodelling. These studies have identified candidate pathways that may play a role in development of these features of disease. However, it is necessary to accurately model all disease features to shed light on the true nature of their development and likely interplay [10]. An ideal animal model of COPD would reproduce a combination of the principal features of the human disease, namely chronic bronchitis, mucus hypersecretion, small airway remodelling, emphysema, impaired lung function and systemic co-morbidities. One feature that unifies the majority of COPD cases is the involvement of exposure to tobacco smoke, the major risk factor for COPD [1]. Thus, it is logical that tobacco smoke exposure would be the pertinent stimulus for modelling the induction and progression of a COPD-like state in animal models. Cigarette smoke (CS) contains a myriad of 4 -- 6000 components with known or potential modulatory effects and no other stimulus is likely to reproduce such a complexity of signals. In other diseases such as asthma, the causes are not known and must be induced by various stimuli to promote the development of features of different endotypes of the disease [11-13]. In contrast, the causes of COPD are known, and it is logical that the responses of healthy mammalian respiratory tracts of rodents and humans to CS would be similar. There is no single strong genetic predisposing factor identified and so models in wild-type animals would be ideal and development in a short time frame would be experimentally preferable. Thus, we focus the rest of our discussion on CS-induced animal models of COPD, in particular chronic CS-exposure models in which the major hallmark features of COPD, that is, airway remodelling, emphysema and impaired lung function, are induced. For discussion of alternative means of inducing COPD-like lung pathologies, readers are directed to the review by Wright et al. [10].

Expert Opin. Drug Discov. (2014) 9(6)

Animal models of COPD

Table 1. Features of COPD documented using chronic CS exposure. Species

Inflammation

Guinea pig

[18] [19] [20] [21] [28,29] [30,31] [32]

Rat

Mucus hypersecretion

Small airway remodelling

[24,25]

[22,23] [20] [21]

[26]

Emphysema

[18] [16] [20] [21] [28,29] [30,31] [32]

[32]

Impaired lung function

Smoking cessation

[22]

[17,25]


[139]

Mouse

[47,88] [54] [42] [140] [38] [45] [48] [127] [46]

[47,88] [54] [42] [140] [38] [45] [48] [127] [46]

[49] [84]

[46]

[46]

[47] [42] [38] [48] [127] [46]

[46]

Each row of references represents the features reported by individual research groups. COPD: Chronic obstructive pulmonary disease; CS: Cigarette smoke.

3.

CS-induced models of COPD

A number of factors must be considered with regard to the design of CS-induced models of COPD. The method of smoke production and exposure is a critical factor. ‘Mainstream’ smoke refers to the smoke that is produced and drawn back through a cigarette by the act of inhalation or ‘puffing’. This exposure occurs in active smokers. ‘Sidestream’ smoke refers to the smoke produced passively from the burning end of a cigarette that escapes into the surrounding atmosphere before being inhaled. Exposure to sidestream and exhaled mainstream smoke occurs during second-hand or passive smoke exposure. Although both types of smoke possess the same chemical composition, there is considerable difference in the relative concentrations of components [14,15]. These differences could ostensibly lead to altered cellular responses and subsequent development of COPD-like features but it remains unclear if this is indeed the case. Leberl et al.’s extensive review suggested that the two types of smoke may, in fact, not generate vastly different disease states in animal models [14]. The two systems in common use at present are ‘whole-body’ and ‘nose-only’ exposure systems. Both systems have been used extensively to provide valuable insights into COPD pathogenesis. Nose-only systems offer the benefit of reducing the potential confounding ingestion of nicotine, tar and other chemical agents by animals from their fur and are able to generate intermittent smoke exposures mimicking the puff profile of human smokers. Finally, although both systems have been reported to result in the induction of a number of different pathological features of COPD such as airway collagen deposition, emphysema and reduced lung function, Wright et al. highlighted

that nose-only exposure systems may offer an added benefit of the generation of more pronounced disease phenotypes which may make evaluation of the success of potential therapeutic interventions clearer [10]. Species, age and gender The species, age and gender of animals used are all critical considerations. Many studies have utilised guinea pigs, rats and mice as model mammals for the induction of CS-induced experimental COPD (Table 1). A limitation of the use of rodents in modelling COPD is that there are significantly fewer airway branches in their lungs compared to humans. However, as discussed below, CS-exposure studies in rodents have demonstrated important similarities in immune responses and the development of pathology to those observed in COPD patients. Thus, animal models of experimental COPD hold considerable promise for use in the development of novel therapeutic strategies. 3.1

Guinea pig Several groups have reported the development of COPD-like pathology following long-term exposure of guinea pigs to CS, with the majority of these studies originating from the laboratory of Wright and Churg. Guinea pigs develop pulmonary inflammation and increased pulmonary arterial blood pressure and muscularisation of pulmonary vessels reminiscent of that observed in COPD patients [16-21]. This is accompanied by the development of small airway remodelling and emphysema between 2 and 6 months of CS exposure and is associated with alterations in lung function such as increased resistance [21-23]. Altered mucus production in the airway epithelium has also been reported by two separate groups 3.1.1


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but this remains relatively unexplored [24-26]. Smoking cessation does not reverse pathology in the guinea pig, with increased presence of mucus secreting cells and emphysema preserved after 4 months rest following 4 months CS exposure [17,25]. Interestingly, the administration of simvastatin in this model prevented pulmonary hypertension and emphysema, but it did not affect small airway remodelling or altered lung function. This indicates that small airway remodelling and emphysema can be driven by discrete processes within the same model [27]. Rat Relatively few studies have utilised the rat as a model for the induction of CS-induced COPD-like pathology (Table 1). Several groups have demonstrated the induction of inflammation followed by emphysema [28-32]. In some cases, emphysema was observed after only 2 months of CS exposure, which represents a significantly shorter time period than commonly required in guinea pig and mouse studies [28,32]. Furthermore Wang et al. demonstrated that exposure of spontaneously hypertensive rats resulted in accelerated onset of CS-induced COPD pathology, with inflammation, increases in mucus producing cells, emphysema and impaired lung function observed within 4 weeks of CS exposure [33]. This could provide a valuable rapid onset model of disease for testing candidate therapeutic strategies, as the authors demonstrated with the phosphodiesterase-4 inhibitor rolipram. 3.1.2

Mouse Mice have been extensively studied [34] and are desirable for modelling due to their advantages of ease of breeding and maintenance, availability of genetically modified strains and reagents with which to assess molecular pathological processes as well as their relatively low cost. Studies where mice of various ages were exposed to CS demonstrated that aged mice showed reduced antioxidant responses and increased susceptibility to inflammation [35,36]. However, these studies used acute smoke exposure in which none of the chronic COPD pathologies were observed, with the exception of exaggerated inflammation. A recent study built on this early work by comparing the development of COPD pathologies in 3and 12-month-old mice exposed to CS over 6 months in a well-established model [37]. These studies confirmed a modest reduction in antioxidant responses in aged mice and demonstrated some differences in the inflammatory gene profile between young versus aged mice following smoke exposure. However, the development of COPD pathologies such as small airway remodelling and emphysema were unaffected by the age of the mice during smoke exposure [37]. Gender may also play a role in the development of the disease, as female mice appeared to develop COPD pathologies at a faster rate than male mice when exposed to identical doses of CS [38]. The differing susceptibilities of various mouse strains to CSinduced pathology have been well documented. Cavarra et al. 3.1.3

632

demonstrated that imprinting control region mice are resistant to CS-induced emphysema, and further studies supported a protective role for enhanced anti-oxidant responses in this strain [39,40]. They also showed that four susceptible strains, C57BL/6, DBA/2, B6C3F and A/J, of mice developed emphysema to different degrees following chronic CS exposure, with A/J mice being particularly susceptible [39,41-43]. These differing disease phenotypes could be viewed as an advantage as they may provide genetic clues as to why only a small portion (15%) of smokers develop COPD, as well as offer an opportunity to test the robustness of experimental findings in different strains [44]. Several research groups have now reported and characterised mouse models of CS-induced COPD (Table 1). The time required to develop COPD-like pathologies, including emphysema, varies, although the majority of studies require 6 months, and this is likely to be dependent on dose and delivery method [14,45]. Studies examining the acute inflammatory responses to short time frame (1 -- 4 week) CS exposures may provide clues as to the pathways involved in the nascent stages of COPD. However, as stand-alone studies, they do not elucidate the pathways involved in driving the long-term progression and maintenance of the disease state. Here, we focus on chronic CS-exposure models in which extensive COPD pathology is reported (e.g., emphysema, airway remodelling) and readers are directed to other recent reviews for discussion of acute CS-exposure studies [10,14,34]. We recently developed a novel mouse model of CS-induced experimental COPD. Using a nose-only system, acute and chronic onset of pulmonary inflammation was induced which lead to mucus hypersecretion and the development of airway remodelling, emphysema and impaired lung function along with systemic involvement (cachexia, loss of skeletal muscle mass, cardiac hypertrophy) [46]. We have since demonstrated airway fibrosis. This study represents a comprehensive characterisation of the induction of COPD-like pathology in mice. Importantly, emphysema and other pathological alterations were observed after only 8 weeks of CS exposure, which renders the investigation of potential mechanisms of disease and therapeutic interventions more feasible [46]. As in humans, disease persisted after the cessation of smoking (for 4 weeks). Other studies have demonstrated prolonged inflammation and emphysema following smoking cessation [38,47,48], including a study which implicated altered IL-12 and IL-10 signalling as mediators of disease maintenance and progression [48]. The smoke exposure in our model is representative of that in humans. Mice were exposed to puffs of smoke interspersed with rests from 12 cigarettes twice per day (equivalent to a pack-a-day smoker). Smoke is not forced into the mice, which breathed in the mouse lung volume equivalent doses of CS that would be comparable on a volume-to-weight basis to humans. In our model, also as in humans, features did not respond to steroid therapy and mice with experimental COPD were more susceptible to infection with S. pneumoniae and influenza.



Table 2. Immune cell involvement in experimental COPD. Cell type

Macrophages

Neutrophils


Mast cell NK cell APCs gd T-cell CD3+ T-cell (CD4+ and CD8+) CD4+ T-cell CD8+ T-cell TH17 T-cell Regulatory T cell

B cell

Role

Method used

Emphysema Inflammation and emphysema Impaired efferocytosis Elastin and collagen breakdown None Inflammation, emphysema and altered lung function NK hyper-responsive phenotype, inflammation, airway damage Emphysema (IL-17 dependent) Suppression of inflammation and emphysema Pathology driving None Inflammation and emphysema Emphysema Elevated in CS-exposed mouse lung. May reduce CS-induced B-cell infiltrates Suppression of B cells reduces lymphoid follicle development and partially reduces lung tissue destruction

Preventative or therapeutic inhibition

Anti-Mac antibody Clodronate

Preventative Preventative

Anti-neutrophil antibody

Preventative and therapeutic (acute) Preventative Preventative

Anti-neutrophil antibody mMCP-6-/NKG2D-/-

Ref.

[29] [46] [75-77] [53,54] [29,46] [46] [55,56]

APC transfer gd T-cell deficient mice

Preventative

T-cell transfer

[60] [60] [58,59]

CD4+-/CD8+-/-


[57] [57] [60] [64,65]

Cxcl13 blocking antibody

Preventative

[141]

Preventative inhibition refers to experiments in which the cell/factor has been inhibited throughout the experimental procedure to prevent the development of disease (i.e., factor-deficient mice or pharmacological agent), whereas therapeutic inhibition refers to those experiments in which the cell/factor was inhibited only for part of the experimental procedure (see references for details). APC: Antigen-presenting cell; COPD: Chronic obstructive pulmonary disease; CS: Cigarette smoke; mMCP: Mouse mast cell protease; NK: Natural killer.

3.2

Measurement of end points

The major features of COPD pathology should be evaluated in mouse models to investigate pathogenesis and the effects of interventions on the course of disease. Airway cellular inflammation can be assessed by enumerating leukocyte populations in bronchoalveolar lavage (BAL) and by FACS-based analysis of BAL cells and cell suspensions prepared directly from lung tissue [46]. This can be accompanied by determination of cytokine and chemokine mRNA and protein levels. Tissue remodelling can be evaluated using histological techniques (e.g., assessment of Masson’s Trichrome staining) to evaluate collagen deposition around airways and in the parenchyma and by measurement of breakdown products of extracellular matrix (ECM) factors [46,49]. Emphysema is commonly quantified in rodent models in terms of the mean linear intercept or destructive index, which provides discrete methods of estimating the extent of parenchymal tissue destruction [46]. A number of pathophysiologically relevant lung function changes in rodents can be measured using forced oscillation techniques (resistance, static and dynamic compliance) and forced manoeuvre techniques (FEV100: FVC that is representative of the FEV1:FVC ratio in humans, total lung capacity, work of breathing) [46]. Other less standard end points can be determined to answer specific

questions. For example, cough responses are typically assessed in guinea pigs, although a recent study demonstrated that such responses in mice can be measured using a combination of whole body plethysmography, observation and sensitive sound measurement [50].

What have we learned from animal models of COPD?

4.

Immune cell involvement COPD is characterised by chronic low-grade pulmonary and systemic inflammation, which is increased during bacterial and viral exacerbations. Inflammation is thought to underpin the progression of disease and so immune cell types and pathways have received much attention (Table 2 and Figure 1). Macrophages are well documented to be elevated in the BAL and pulmonary tissue of COPD patients, and this is also evident in animal models of CS-induced COPD [46,51]. Numerous studies have demonstrated that macrophage-associated factors such as MMP-12 [52] play critical roles in the development of pathology, implicating macrophages as important players in COPD pathogenesis (Table 3). The selective depletion of macrophages in mice provided confirmation of their importance in COPD pathogenesis, as clodronate-treated 4.1


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Model of pathogenesis

Experimental observations

Human

Rodent

Cigarette smoke Smoke components

RONS, toxins Increased cell death, DAMP release


RONS, proteases, cytokines Inflammationinnate & adaptive immune system

Cellular damage

B-cell, CD4+ T-cell, CD8+ T-cell, γδ T-cell, TH17 T-cell T-reg cell

DAMP release

Cytokines, proteases

Macrophages, neutrophils, APCs, NKs

Cytokines proteases, RONS

Increased cell death, DAMP release

Macrophages, neutrophils, APCs, NKs, mast cells

B-cell, CD4+ T-cell, CD8+ T-cell, γδ T-cell, TH17 T-cell T-reg cell

Cell death

Tissue remodeling

Emphysema

FEV1/FVC

Decreased lung function

10+ years

COPD

FEV100/FVC, resistance, TLC, compliance

2-6 months

Figure 1. Pathogenesis of human and experimental COPD induced in response to chronic CS exposure. APC: Antigen-presenting cell; COPD: Chronic obstructive pulmonary disease; CS: Cigarette smoke; NK: Natural killer; RONS: Reactive oxygen and nitrogen species.

animals were protected from small airway remodelling, emphysema and impaired lung function in experimental COPD [46]. Similar experiments in which antibody-based depletion of neutrophils was employed demonstrated that neutrophils may contribute to early events such as exaggerated breakdown of ECM components [53,54]. Other innate immune cell types have been implicated through the use of animal models. The pathogenic role of mast cells (MCs), which was hitherto unexplored, was revealed using mice that were genetically deficient for expression of the mouse MC protease (mMCP)-6 [46]. Finally, chronic exposure to CS results in a hyper-responsive natural killer (NK) cell phenotype, which is partially suppressed in mice deficient with the activating receptor NKG2D. These mice also show reduced inflammation and airway pathology [55,56]. Recent work has implicated T cells in the pathogenesis and progression of COPD. CD8+ (but not CD4+) T-cell-deficient mice were protected from cellular inflammation as well as 634

emphysema following chronic CS exposure [57]. Recent studies have provided further evidence for the potential role of T cells as drivers of COPD pathology. Transfer of CD3+ T cells harvested from smoke-exposed mice to non-CSexposed Rag2-deficient (-/-) recipient mice induced COPD-like pathology, including increased inflammation and emphysema [58]. A follow-up study demonstrated that the transfer of CD4+ and CD8+ T cells derived from CS-exposed mice was sufficient to induce COPD immunopathology in a manner that was dependent on antigen recognition pathways, highlighting the potential involvement of autoimmune mechanisms [59]. These studies suggest that the persistent generation of pathogenic T cells by CS exposure may contribute to the chronic nature of disease, including in those individuals where disease progression continues despite cessation of smoking. Similarly, Shan et al. demonstrated that the transfer of CD1a+ antigen-presenting cells (APCs) from CS-exposed mice to naı¨ve mice was sufficient to drive



Table 3. Factor/pathway involvement in experimental COPD. Factor/pathway

TNFR-I and -II TNFR-II


IL-1b

Role

Method used

Inflammation, emphysema, small airway remodelling Inflammation, emphysema and cell death

IL-1R, IL-1a

Inflammation, emphysema and small airway remodelling No role Inflammation, emphysema

IL-18/IL-18R

Inflammation, emphysema and cell death

IL-6 IFN-g EMAP-II

None Emphysema Emphysema and cell death

CCR5 CCR6

Inflammation, emphysema and cell death Lymphocyte, DC, neutrophil recruitment, emphysema, MMP-12 induction. B-cell recruitment, lymphoid aggregate development Inflammation, emphysema, lung function Inflammation, emphysema, MMP-12 induction Chemo-attraction, inflammation, emphysema Inflammation, emphysema

CXCL13 Adiponectin IL-17 ATP, P2R NE

MMP-9/-12

Inflammation, emphysema, small airway remodelling No role Inflammation, emphysema Inflammation, emphysema

Preventative

[54,68,49]

TNFR-II-/(TNFR-I-/- not protective) IL-1b-/-

Preventative

[69]

Preventative

[49]

IL-1b blocking antibody IL-1R-/-, IL-1a-/-, IL-1a blocking antibody IL-18R-/-, IL-18R blocking antibody IL-6-/IFN-g -/EMAP-II blocking antibody


[70] [70]

Preventative

[71]

Preventative Preventative Preventative and therapeutic Preventative Preventative

[73] [82] [81]

Preventative and therapeutic Preventative Preventative

[141]

Preventative

[74]

Preventative Preventative and therapeutic Preventative Preventative

[19] [87]

Preventative Preventative Preventative

[89] [88] [90]

Preventative and therapeutic Preventative Preventative

[97]

Preventative

[75]

CCR5-/CCR6-/CLCL13 blocking antibody Adiponectin-/IL-17 Tg, IL-17-/P2Y2R-/-, P2R blockade, ATP neutralisation Inhibitor ZD0892 Inhibitor AZD9668 NE-/Inhibitor AZ11557272

MPO

iNOS

Inflammation, emphysema, prolonged emphysema, PH

SOD1 Tg mice SOD3 Tg mice, SOD3-/-, SOD3 mimetic ICR mice, MnTBAP, extracellular SOD iNOS-/-, iNOS inhibitor L-NIL

Gpx-1 Nrf-2

Suppresses inflammation Suppresses oxidative stress, emphysema, inflammation, PH. Inflammation

Inflammation, small airway remodelling, emphysema, impaired lung function, PH Reactive oxygen and Inflammation, emphysema, impaired nitrogen species efferocytosis

ABC transporters COX-2 Statin targets mTOR pathway, Rtp801 Soluble guanylyl cyclase

Ref.

TNFR-I and -II-/-

MMP-9-/MMP-12-/Inhibitor CP-471,474 MPO Inhibitor

MMP-9 MMP-12 MMPs

Preventative or therapeutic dose

Preventative and therapeutic (post emphysema) Gpx-1-/-, Gpx-1 mimic ebselen Preventative Nrf-2 activator Preventative CDDO-imidazolide, Nrf-2-/MRP1, MDR1a/b triple Preventative knockout mice Celecoxib Preventative Simvastatin Preventative Therapeutic Rtp801 Tg mice, Rtp801-/Preventative

Inflammation, emphysema Inflammation, emphysema, PH but not small airway remodelling Suppression of mTOR, oxidative stress, inflammation, emphysema Suppresses bronchial hyper-responsiveness Activator BAY58-2667 is beneficial

[82,83] [84]

[125] [60]

[86] [91]

[93] [95]

[95]

[96] [100] [142] [31] [30] [27] [98] [134]

COPD: Chronic obstructive pulmonary disease; DC: Dendritic cell; EMAP: Endothelial monocyte-activating protein; ICR: Imprinting control region; iNOS: Inducible nitrogen oxide synthase; MPO: Myeloperoxidase; mTOR: Mammalian target of rapamycin; NE: Neutrophil elastase; P2R: Purinergic receptors; SOD: Superoxide dismutase; TNFR: TNF receptor. Expert Opin. Drug Discov. (2014) 9(6)

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COPD-like pathology, which occurred in an IL-17-dependent manner, identifying a potential therapeutic target for the treatment of chronic disease [60]. In contrast, it appeared that increases in the numbers of gd T cells and associated IL-17 production by CS exposure was important for the suppression of pathogenic TH17 responses, highlighting autoimmune aspects of COPD [60]. The link between gd T cells, IL-17 and inflammation in COPD was also described in a recent study [61]. Activation of T cells may also, in part, be mediated by the IL-1R/IL-1a-dependent accumulation of dendritic cells (DCs) in the lungs of CS-exposed mice [62]. Regulatory T cells control inflammation and are increased in humans with COPD and CS-exposed mice [63,64]. However, they clearly do not prevent pathogenesis. This suggests that these cells are dysfunctional and could be modified or further increased for therapeutic gain [65,66]. Myeloid-derived suppressor cells are also increased and altered in COPD, although the consequences of this and potential for modulation are not known [67]. Molecular drivers of COPD Given the demonstrated involvement of numerous immune cell types, it is unsurprising that numerous immune cellrelated factors have also been implicated in the pathogenesis of COPD-like disease in rodents (Table 3). 4.2

Cytokines and chemokines Churg et al. demonstrated in a series of studies that mice deficient in the expression of both TNF receptors (TNFR)-I and - II were protected from inflammation in both acute and chronic CS-exposure models and that small airway remodelling and emphysema were prevented following chronic CS exposure of TNFR double -/- mice [49,54,68]. These studies were complemented and refined independently by D’Hulst et al., who demonstrated that deletion of TNFR-II (but not TNFR-I) alone was sufficient to prevent inflammation, emphysema and associated cell death in a chronic CS-exposure model [69]. Other strongly implicated molecules are IL-17 (discussed above) and IL-1R. IL-1R is required for inflammation, small airway remodelling and emphysema following CS exposure [49,70]. Pathogenetic signalling via IL-1R involves IL-1a, as shown by using mice deficient in this factor and blocking antibodies [70]. IL-1b may also be involved in the induction and exacerbation of COPD features [46,49,70]. Expression of the IL-1 superfamily member IL-18 is elevated in the macrophages of smokers and COPD patients, a feature also observed in CS-exposed mice. Blockade of IL-18 function in IL-18R-/- mice partially suppressed inflammation, parenchymal cell death and emphysema [71]. IL-6 is increased in COPD patients and in mice exposed to CS and is required for the development of Th17 cells [72]. However, CS-induced inflammation was independent of IL-6 in mice deficient of this factor [73]. CS exposure in mice results in increased ATP release, potentially through induction of cell death, which acts as a neutrophil and macrophage chemoattractant acting 4.2.1

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via the purinergic receptors, including P2Y2R [74]. Genetic and pharmacological blockade of this pathway reduced inflammation and emphysema following CS exposure. CS exposure may also increase ATP release through reduction of the efferocytotic capacity of macrophages, allowing dying cells to undergo secondary necrosis and plasma membrane rupture following apoptosis [75]. Impaired phagocytic ability of macrophages has also been demonstrated in COPD patients and healthy smokers, as well as smoke-exposed mice, and may contribute to disease progression [76-78]. The GM-CSF is elevated and regulates innate immune responses and cellular influx in the lung in COPD models [79,80]. Antibody-mediated neutralisation of this cytokine inhibited macrophage and neutrophil influx into the lung and the production of TNF-a and MMP-2 and -12 [79]. Endothelial monocyte activating protein II (EMAP-II) is a cell deathpromoting ligand with increased expression in the BAL of COPD patients and smokers compared to controls. EMAPII was also increased in the lungs of CS-exposed mice, and forced expression of EMAP-II was sufficient to induce cellular injury and emphysema, whereas EMAP-II neutralising antibodies prevented emphysema and cell death following chronic CS exposure [81]. The chemokine receptors CCR5 and CCR6 are also required to drive inflammation in chronic CS-exposure models and their deletion results in protection from emphysema, but not from small airway remodelling [82-84]. Interestingly, CCR6 plays a role in promoting the pulmonary infiltration of DCs and T cells, both of which are thought to play a role in chronic disease. In contrast CCR7 appears largely dispensable for the development of COPD-like symptoms in CS-exposed mice [85]. Proteolytic factors Emphysema is thought to arise at least in part due to the development of a protease/anti-protease imbalance [8,10]. Genetic deletion and pharmacological inhibition of neutrophil elastase (NE) has generated strong evidence supporting a role for this molecule in driving COPD pathogenesis [19,86,87]. Notably, a recent study in which the orally available reversible NE inhibitor AZD9668 was administered using preventative or therapeutic dosing strategies demonstrated that this may be a viable therapeutic option [87]. A seminal study using deficient mice identified MMP-12 as a driver of COPD-like disease in rodents [88], whereas MMP-9 appears dispensable [89]. Broad spectrum (CP-471,474) and relatively MMP-9/-12-specific (AZ11557272) inhibitors reduced the development of the disease when administered in a preventative manner and further studies that examine therapeutic dosing options for such agents are warranted [90,91]. Finally, we recently demonstrated that expression of the mMCP-6 was required to drive inflammation, small airway remodelling and emphysema [46]. Numbers of MCs were increased in the lungs of CS-exposed mice, and given the numerous proteolytic enzymes harboured within their secretory granules, further investigation of the role of MC factors in COPD is an interesting prospect. 4.2.2




4.2.3

Oxidative stress

CS is a potent inducer of oxidative stress through the production of reactive oxygen and nitrogen species (RONS), as are the immune cells that are recruited to the lung in response to smoke exposure. RONS have important immune effector and signalling functions, but when aberrantly produced can mediate pathogenetic cellular damage, DNA alteration and cell death [92]. Several groups have investigated the use of RONS-targeting strategies for the suppression of COPDlike pathology in animal models, with promising results in some studies (Table 3). Mice overexpressing the cytosolic superoxide dismutase (SOD)1 were protected from inflammation and emphysema with reduced evidence of oxidative stress following CS exposure compared to normal air-exposed mice [93]. Yao et al. used several approaches to test the role of extracellular SOD3 to demonstrate its importance in limiting or preventing inflammation, ECM breakdown and emphysema in CS-exposed mice [94]. The implication of superoxide in oxidative stress-mediated pathology is complemented by a recent work that highlights the roles of inducible nitrogen oxide synthase (iNOS) and peroxynitrite (the production of which requires NO and superoxide) in COPD. Although several studies have shown that endothelial NOS does not play an important role in driving COPD-like pathology, iNOS knockout mice were protected from inflammation and emphysema, as well as pulmonary hypertension which developed prior to emphysema [95]. Importantly, administration of L-NIL, a selective iNOS inhibitor, prevented emphysema and pulmonary hypertension when delivered preventatively, and reversed these features when administered as a late intervention following the establishment of emphysema [95]. This finding supports the development of therapeutic agents that can suppress inflammation while stimulating regeneration of lung parenchyma. Similar to SOD, glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that also protects against ROS. Mice deficient in this factor had increased CS-induced inflammation, which could be reversed using the gpx mimetic ebselen [96]. Administration of MnTBAP, a relatively selective peroxynitrite-scavenging molecule, was able to prevent CSinduced reductions in macrophage efferocytosis function, indicating a further disease-causing mechanism associated with RONS [75]. In addition, a recent study demonstrated that administration of mannose-binding lectin was able to restore macrophage efferocytosis function in CS-exposed mice [78]. Myeloperoxidase (MPO) is a pro-oxidative enzyme involved in neutrophil and macrophage functions. MPO activity was increased following chronic CS exposure, and inhibition of MPO with both preventative and therapeutic dosing of an inhibitor (AZ1) prevented the development of several key COPD pathologies, including inflammation, small airway remodelling, emphysema and altered lung function in guinea pigs [97]. Rtp801, a suppressor of mammalian target of rapamycin (mTOR) signalling, was increased in emphysematous human lungs and in CS-exposed mice.

Overexpression of Rtp801 was sufficient to drive inflammation, oxidative stress and tissue damage in mouse lungs, and conversely Rtp801 deletion prevented CS-induced inflammation and emphysema, thus potentially uncovering an important new pathway involved in the induction of CS-induced injury [98]. In contrast, signalling by the transcription factor NF-kB, which can regulate expression of a number of immune proteins associated with COPD, was not required for inflammation in response to CS exposure [99]. Nuclear erythroid 2 p45 (Nrf2) is an oxidative stress-sensitive transcription factor that induces the expression of genes that code for antioxidants. Nrf2 pathways are reduced in COPD patients and genetic disruption of this factor in mice leads to emphysema. Treatment with an Nrf2-activating factor reduced oxidative stress, emphysema and pulmonary hypertension in CS-exposed mice, an effect which was ablated in the absence of Nrf2 expression [100]. As well as the direct effects on tissues, oxidative stress may also induce the development of auto-antibodies against host proteins such as those that are carbonyl-modified [101]. Thus, oxidative stress may also drive autoimmunity, a process that is increasingly recognised as an important component of COPD [102]. Further studies such as the recent comprehensive analysis of oxidative stress in a model of ozone-induced lung inflammation may lead to the identification of similar pathways in CS-induced disease [103]. In summary, recent studies using animal models of COPD have identified a number of exciting avenues for further exploration to develop future therapeutics.

Modelling COPD and bacterial/viral exacerbations of COPD

5.

COPD patients experience periodic exacerbations that contribute significantly to the progression of their disease. At least half of all exacerbations are caused by bacterial and viral respiratory tract infections [6,104]. Exacerbations are of particular concern, given that they correspond with increases in hospitalisations and subsequent burden on healthcare systems and are associated with increases in mortality rates [105,106]. The chronic inflammatory environment in the lungs of COPD patients is thought to alter the immune response to bacteria and viruses, resulting in acute periods of inflammation which in turn drive the progression of COPD by causing permanent tissue damage and decline in lung function. The mechanisms that cause increased susceptibility to bacterial and viral infections in COPD patients are poorly understood. Only a few studies have addressed the mechanisms associated with increased susceptibility to exacerbations in animal models of COPD (Table 4). Viral exacerbations of COPD Several groups have examined the effect of acute and chronic CS exposure on influenza virus infection. COPD patients 5.1


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Table 4. Effect of CS exposure on responses to infections. Pathogen

Influenza


Viral mimetic poly(I:C) Staphylococcus aureus enterotoxin B Mycobacterium tuberculosis Pseudomonas aeruginosa Haemophilus influenzae Streptococcus pneumoniae Pneumocystis murina

Smoke exposure 2 weeks 6 months 4 days 5 days 8 weeks 2 weeks 14 -- 25 days 6 8 8 2

-- 8 weeks weeks weeks -- 4 months

Effects of CS exposure

Inflammation, cell death, emphysema, dependent on RNase L Inflammation Inflammation, increased viral replication Increased mortality Inflammation dependent on IL-1R and IL-1a Increased viral replication Inflammation, small airway remodelling, emphysema. Driven by IL-18, IFN-g, MAVS and RNase L Increased lymphocyte and neutrophil accumulation, IL-17, IFN-g, mucus cell hyperplasia Increased bacterial burden Increased bacterial burden and inflammation Increased bacterial burden, inflammation, weight loss Increased bacterial burden Increased susceptibility, inflammation, emphysema, impaired lung function

Ref.

[112,113] [56] [109,110] [117] [70,84] [46] [112,113] [120] [117] [118] [119] [46] [143]

CS: Cigarette smoke.

have increased susceptibility to influenza infection and experience exaggerated inflammatory immune responses following infection [107,108]. The majority of published studies report increased pulmonary and systemic inflammation following influenza infection in acute and chronic CS-exposure models. In some cases, increased viral proliferation or reduced clearance was noted [46,109]. In other cases, the dynamics of viral proliferation were not affected by CS exposure, although exaggerated inflammatory responses to influenza infection were still present [56,110]. It is possible that these discrepancies may be related to the smoke exposure protocols and the dose of virus used to induce infection. Indeed, the differential effects of CS observed were dependent on viral dose used in a chronic exposure model [111]. Elias and colleagues reported a synergistic effect of 2 weeks of CS exposure followed by influenza on inflammation and also reported increased lung damage in the form of induction of small airway remodelling, parenchymal cell death and emphysema [112,113]. The amplified immune response elicited by CS exposure followed by influenza infection may be a result of increased Toll-like receptor (TLR)3 expression observed in the lungs of mice [114]. Indeed, Kang et al. demonstrated that mice deficient in TLR3 were protected against the exaggerated inflammation and lung damage induced by influenza infection with concomitant CS exposure [112]. In addition, increased production of IL-18 was observed in CS-exposed mice following influenza infection, and genetic deletion of IL-18R was sufficient to prevent features of exacerbation such as lung inflammation and damage [112]. The same group recently demonstrated that inducible expression of IL-18 in mice was sufficient to induce inflammation and emphysema in an IFN-g-dependent manner. This supports a previous observation using poly (I:C) administration as a surrogate for viral infection in which elevated IFN-g expression was required to mediate emphysema 638

observed in a CS-exposure model of COPD with viral exacerbation [115]. In addition, a recent study revealed that the innate antiviral immune 2¢-5¢ oligoadenylate synthetase/RNaseL system is activated synergistically by CS exposure in combination with influenza infection. Deletion of RNaseL was sufficient to prevent the increases in inflammation, fibrosis, cell death and emphysema in the lungs of CS-exposed mice infected with influenza [113]. The exaggerated production of MMP-2 and -9 following influenza infection in an acute CS-exposure model may provide further clues as to the potential mechanisms of enhanced lung damage that occur in viral exacerbations of COPD [109]. Bauer et al. reported that acute (5 days) CS exposure resulted in a corticosteroid-resistant pulmonary inflammatory response to infection, although IFN-related antiviral responses were similar between groups and viral replication appeared unaffected by smoke exposure [116]. Whereas glucocorticoids were ineffective in treating the symptoms of influenza infection in smoke-exposed mice, the PPAR-g agonist pioglitazone improved resistance to viral infection in CS-exposed mice. The same research team demonstrated that blockade of IL-1a or IL-1R by genetic deletion or blocking antibody was able to prevent acute and chronic CSinduced neutrophilia in lungs, and IL-1R deletion also inhibited the amplified inflammatory response to influenza infection following acute CS exposure [70]. Thus, it is possible that strategies targeting IL-1a/IL-1R signalling or those that prevent neutrophilia could be useful in the treatment of viral exacerbations of COPD patients. Bacterial exacerbations of COPD Work investigating bacterial exacerbations in COPD using animal models is less advanced from a mechanistic perspective. However, several studies have shown increased susceptibility or inflammatory responses to infections or infection-associated 5.2



molecules, including S. pneumoniae [46], Mycobacterium tuberculosis [117], Pseudomonas aeruginosa [118], Haemophilus influenzae [119] and Staphylococcus aureus enterotoxin B [120]. Further studies using these models are likely to uncover novel therapeutic approaches for bacterial exacerbations of COPD.


6.

Modelling systemic co-morbidities of COPD

It is increasingly recognised that COPD manifests beyond the pulmonary system as a multi-systemic disease. COPD patients are at increased risk of developing numerous systemic co-morbidities including pulmonary hypertension (for discussion, see above and Ref. [14]), cachexia, Crohn’s disease (for discussion, see Ref. [8]) and osteoporosis [4]. Although much of the initial research with animal models of COPD has understandably focussed on the pulmonary aspects of disease, several groups are now using these established models to begin to investigate the complex interrelationship between pulmonary manifestations and systemic co-morbidities of COPD. Cachexia Cachexia is a progressive, wasting loss of adipose and skeletal muscle associated with other severe disease manifestations [121]. Approximately 25% of COPD patients develop cachexia, which is associated with around a 50% reduction in mean survival time [122]. Compromise of muscle function can occur during the early stages of disease [123]. The majority of animal studies examining COPD and cachexia have focussed on muscle, although Chen et al. reported inflammation in and depletion of white and brown adipose tissue in a chronic CS-exposure model [124]. Further indications of the potential dysregulation of adipose tissue function came from a recent study by Miller et al., who demonstrated that adiponectindeficient mice had reduced inflammation, emphysema and improved lung function compared to control mice exposed to CS [125]. Some studies report insignificant reductions in muscle mass after chronic CS exposure of mice but note a shift from type II oxidative to glycolytic muscle fibre types, reminiscent of the fibre type shift that occurs in the limb muscles of COPD patients [126,127]. Tang et al. reported loss of muscle mass and function and linked this to increased systemic release of TNF-a causing the suppression of oxygen transport to muscle and increases in atrophy-related genes MuRF1 and Atrogin-1 [128]. Caron et al. reported similar findings, although the mechanistic contribution of altered atrophy-related gene expression and signalling to muscle atrophy was not tested [129]. TNFR-II, which is required for the development of pulmonary manifestations of COPD-like disease in mice, was shown to partially contribute to the atrophy of hind limb muscles in a model of chronic CS exposure [130]. It is unclear whether this protective effect was due to reduced pulmonary or muscleTNF signalling; nevertheless this remains one of the few studies to directly test the contribution of a signalling pathway to COPD-associated cachexia.

Osteoporosis COPD patients have an increased incidence of osteoporosis, which is thought to be caused by exposure to known risk factors, including CS and systemic inflammation [131]. Akhter et al. reported reduced bone strength in mice exposed to CS over 3 months, whereas another recent study reported minor decreases in bone strength accompanied by alterations in osteoclast and osteoblast morphology and function in CS-exposed rats [132,133]. These preliminary reports suggest that animal models of CS-induced COPD could be useful in the investigation of COPD-associated osteoporosis and this merits further investigation. Many groups have developed and established models of CS-induced lung pathology. The relatively few studies examining systemic co-morbidities associated with smoking and COPD indicate that these models will likely be useful for revealing the complex mechanisms by which CS exposure and pulmonary inflammation can affect and compromise organ function throughout the body. 6.2

6.1

7.

Conclusion

Numerous studies have established chronic CS exposure as the best current method of modelling COPD pathology in rodents, although acute studies have produced valuable information and are sufficient to answer specific questions. Improvements in techniques and technologies have enabled the development of models that mimic the hallmark features of COPD pathology, including pulmonary and systemic inflammation, small airway remodelling, emphysema and impaired lung function, some within the relatively short time frames of 8 weeks. These and other models have enabled the role of several immune cells and related factors to be explored in more detail, highlighting potential new therapeutic targets. In addition, important mechanistic insights into how acute infectious exacerbations contribute to disease have also been reported. With these models firmly established, it will now be possible to investigate the molecular complexities underlying the development of systemic co-morbidities, as well as develop novel therapeutic agents for the treatment of COPD. 8.

Expert opinion

Great strides have been made in the development of animal models of CS-induced COPD. These models variously reproduce many of the pathophysiological features of COPD, including aberrant inflammation, small airway remodelling, emphysema and impaired lung function, as well as dysfunctional immune responses in response to viral or bacterial infection. However, some studies are limited in terms of the scope of aspects of disease reported. There is unlikely to be complete standardisation of protocols, equipment and animals between laboratories [14]. Nevertheless, recent models


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have been developed that accurately reproduce the hallmark features of human disease in a reasonable time frame [46]. This is perhaps unsurprising as, unlike in other diseases, the causes of COPD are known and it is logical that exposure of the healthy murine respiratory tract to CS will elicit similar responses and lead to the development of similar disease features compared to those in humans. The field appears to be at a point now where researchers are no longer simply striving to replicate disease pathology, and are in a position to utilise these models to provide genuine insights into the pathogenesis of COPD and identify novel therapeutic strategies. The use of adoptive transfer of immune cell types from CS-exposed to non-CS-exposed mice has offered some tantalising hints about the roles of the adaptive immune system and autoimmunity in driving chronic COPD [58,59]. Furthermore, the identification of novel cell types and the factors they produce that may contribute to pathogenesis such as (MCs) and TH17 cells opens up many possibilities for further investigation [46,60]. The use of a pharmacological agents to not only halt but also reverse emphysema in mouse CS-exposure models highlights their exciting potential for drug discovery and target identification [95]. This also raises the prospect of focussing on pathways that might be activated for therapeutic gain, as has recently been shown with a small molecule activator of soluble guanylyl cyclase and in the administration of adiposederived stem cells to suppress CS-induced inflammation and Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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injury [134,135]. Another area in which animal models will be valuable is in the investigation of corticosteroid insensitivity that characterises inflammation associated with COPD. Several studies have shown that CS-induced inflammation (both acute and chronic) in rodents is steroid-insensitive [46,136]. Investigations of acute CS-exposure mouse models have identified phosphatidylinositol 3-kinase d (PI3Kd) to play an important role. Inhibition of PI3Kd prevents CS exposure-induced decreases in histone deacetylase 2 resulting in the restoration of steroid responsiveness of airway inflammation [136]. Recent data also implicates the elevation of serum amyloid A as a contributing factor to the generation of corticosteroid refractory inflammation in COPD [137]. Further studies are required to investigate other avenues for restoring steroid sensitivity, and the long-term efficacy of such approaches could be assessed in chronic CS-exposure models [138].

Declaration of interest The authors were supported by the National Health and Medical Research Council, Australia. The authors have no other relevant affiliations or financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Affiliation Michael Fricker1 PhD, Andrew Deane1 MSc & Philip M Hansbro†2 PhD † Author for correspondence 1 University of Newcastle and Hunter Medical Research Institute, Priority Research Centre for Asthma and Respiratory Disease, New Lambton Heights, New South Wales, Australia 2 Professor, University of Newcastle and Hunter Medical Research Institute, Priority Research Centre for Asthma and Respiratory Disease, New Lambton Heights, New South Wales, Australia E-mail: [email protected]

645

Animal models of chronic obstructive pulmonary disease.

Animal models in chronic obstructive pulmonary disease-an overview.

Chronic obstructive pulmonary disease.

Underrecognized comorbidities of chronic obstructive pulmonary disease.

Predictors of chronic obstructive pulmonary disease exacerbations.

Systemic manifestations of chronic obstructive pulmonary disease.

Skeletal Implications of Chronic Obstructive Pulmonary Disease.

Integrative genomics of chronic obstructive pulmonary disease.

Assessment of pulmonary rehabilitation efficacy in chronic obstructive pulmonary disease patients using the chronic obstructive pulmonary disease assessment test.

Management of stable chronic obstructive pulmonary disease.

Takotsubo cardiomyopathy and chronic obstructive pulmonary disease.

Bronchoscopic interventions for chronic obstructive pulmonary disease.

Extracellular Vesicles in Chronic Obstructive Pulmonary Disease.

Exacerbation phenotyping in chronic obstructive pulmonary disease.

Phosphodiesterase inhibitors in chronic obstructive pulmonary disease.

Excretion urgency in chronic obstructive pulmonary disease.

Tuberculosis in Chronic Obstructive Pulmonary Disease.

Smoking cessation and chronic obstructive pulmonary disease.

Selective polypharmacy for chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease in rheumatoid arthritis.

Osteoporosis Associated with Chronic Obstructive Pulmonary Disease.

Allergy and Chronic Obstructive Pulmonary Disease.

Arterial stiffness in chronic obstructive pulmonary disease.

Osteoporosis in chronic obstructive pulmonary disease.