The current pathogenetic theories for the development of COPD include an imbalance between the protease and antiprotease system, dysregulation of oxidant‐antioxidant activity and chronic airway inflammation, processes that lead to the progressive destruction and abnormal repair of the lung connective tissue matrix.⁵ Recent studies have suggested that increased apoptosis of the alveolar wall accounts in part for the loss of lung tissue that characterises emphysema.^6,7 Transgenic and null mutant mouse studies have identified a number of genes and pathways that, when altered, result in the morphological changes of emphysema.^8,9,10

Although COPD primarily affects the lungs, it is associated with important systemic consequences which include malnutrition with a low body mass index (BMI)¹¹ and impaired peripheral muscle function.¹² These clinically relevant expressions of the disease have been associated with detectable systemic changes including evidence of increased oxidative stress, activation of circulating inflammatory cells and increased levels of proinflammatory cytokines.^13,14 The multidimensional expression of COPD can be expressed by a clinical score including BMI, degree of obstruction (O), perception of dyspnoea (D) and exercise capacity (E) by the 6‐minute walk distance known as the BODE index.¹⁵ This index predicts mortality better than the forced expiratory volume in 1 s (FEV₁).

We reasoned that the pathobiological processes that occur in the lungs and possibly in systemic tissues such as the peripheral muscles of patients with COPD could be associated with systemic biomarker levels detectable in the systemic circulation. Despite the many studies aimed at identifying the pathogenesis of COPD, to our knowledge only one study¹⁶ has explored the potential value of high‐density microarray technology to systematically define the serum protein expression profile in patients with COPD. Using a novel protein microarray platform (PMP) technology, we compared the serum proteomic profile of 143 serum biomarkers in patients with COPD with that of age and sex‐matched controls. We also explored the relationship between a selected subset of 24 biomarkers with clinically important outcome variables in COPD including lung function, the BODE index and its components and the frequency of exacerbations.

Several groups have shown an increase in a number of circulating inflammatory biomarkers in COPD,^24,25,26 suggesting that it might be possible to characterise patients with COPD using systemic biomarkers. To address this question we used a novel technology that simultaneously evaluated analytes covering diverse potential processes including inflammation, chemo‐attraction, cell activation, tissue destruction and repair. Based on a collaborative effort of statistical results and scientific plausibility, a subset of 24 biomarkers was identified and selected for subsequent testing against a variety of clinically important parameters. Many studies have been published on the association between a specific marker and COPD disease status, with both positive and negative results being reported.^{24,25,26,27,28,29,30,31} The disagreement in results can be attributed to different factors, including the heterogeneity of COPD phenotypes, low sample size, or the use of different methodologies and assays. The development of a panel of biomarkers addressing preconceived multiple pathophysiological pathways may provide a more specific tool to serve as an intermediate end point reflecting the natural history of the disease.

One obvious limitation of this preliminary dataset is that the biomarkers identified are limited by the pool of analytes that were available for the primary assessment. Clearly, use of an “open” proteomic platform would give information about a much broader range of proteins and might provide additional insights into biomarker selection and disease processes. However, a recent report¹⁶ using a panel developed from the one reported here shows that the panel as developed is valid and capable of reflecting changes induced by exacerbations. Recognising the fact that the discussion is valid only for the analytes explored, our findings may help to shed light on the underlying pathogenetic processes involved in this disease.

It has been proposed that various proteases break down lung connective tissue components to cause emphysema,^5,6 leading to aberrant remodelling and/or degradation of the extracellular matrix. In our study, several proteins (table 3​3)) related to the protease‐antiprotease mechanism were clearly different between patients with COPD and controls. Thus, metalloproteinases 7, 8, 9 and 10 (MMP‐7, MMP‐8, MMP‐9 and MMP‐10) were among the proteins with large differences between groups. Of these, MMP‐9 showed the strongest association with FEV₁ and Tlco, which is interesting because MMP‐9 has been implicated in the experimental genesis of emphysema.^32,33 The tissue inhibitor of metalloproteinase 1 (TIMP‐1), a collagenase inhibitor, was also different between patients and controls, providing evidence that the final expression of the disease may rest upon the appropriate balance of the system.³³

Differences were also found in enzymes other than the metalloproteinases that are related to tissue destruction, as well as proteins related to repair, that deserve some comments. While the fold increase of neutrophil elastase in COPD was not as great as that found for the metalloproteinases, the difference was still statistically significant. Previous studies of experimental emphysema produced by pancreatic or neutrophil elastase showed that increased levels of elastase enzymes lead to the degradation of connective tissue components and, thus, enlargement of distal airspaces.³⁴ While both elastin and collagen are rapidly re‐synthesised in these animal models and mRNA levels for both are increased, the connective tissue remodelling process is ineffective and lung mechanical properties remain abnormal.³⁵

The differences in tissue growth factor alpha (TGFα), amphiregulin (AR), brain‐derived neurotropic factor (BDNF) and nerve growth factor β (βNGF) and their association with low FEV₁ and Tlco (​(figsfigs 1 and 2​2)) suggest that connective tissue remodelling continues even in severe advanced COPD in humans, but the process fails effectively to restore the mechanical properties of the diseased lung. The role of TGFα is something of a mystery. Mice genetically manipulated to overexpress TGFα develop emphysema postnatally,³⁶ yet an in vitro model of alveolar re‐epithelialisation showed that TGFα induced faster wound repair.³⁷ The presence of significant associations between BDNF and lung function and the BODE index (fig 3​3)) is particularly interesting. Recent evidence indicates that BDNF decreases conversion from oxygen to hydrogen peroxide in experimental cell cultures,³⁸ suggesting a role in the modulation of oxidative stress, and makes this an interesting marker to study. Furthermore, similar to results seen with AR, exogenous BDNF can protect cells from serum deprivation‐induced cell death.³⁹

It has been suggested that angiogenesis and apoptosis of the alveolar wall may have a role in emphysema. While little is known about the role of the EGF family member AR in the aetiology of COPD, one study has found that AR can inhibit apoptosis of non‐small cell lung cancer cell line.⁴⁰ Blockade of vascular endothelial growth factor R2 (VEGF‐R2) receptor in rats induces apoptosis of the alveolar cell wall and results in an emphysema‐like pathology.^41,42 Several studies have found decreased expression of VEGF in induced sputum or bronchoalveolar lavage (BAL) fluid from patients with obstructive lung disease in comparison with normal subjects.^43,44 These studies have also shown a direct association between the reduction in VEGF and FEV₁. While our study showed an increase in VEGF serum content that was inversely associated with FEV₁, this difference could be due to differential expression of VEGF in lung tissue and serum. Studies of VEGF expression in human lung tissue by immunohistochemistry have shown increased VEGF in pulmonary and airway smooth muscle in subjects with COPD that correlated with decreased FEV₁.⁴⁵ Furthermore, patients with cystic fibrosis show an inverse relationship in the level of VEGF in serum and BAL fluid compartments. These patients had a higher level of VEGF in serum and a lower level of VEGF in BAL fluid compared with controls.⁴⁶ The role of apoptosis and its relationship to inflammation and repair seem supported by our findings.

Current thinking places inflammation at the centre of the pathogenetic mechanisms of COPD. The inflammation is characterised by increased numbers of alveolar macrophages, neutrophils and T lymphocytes, together with the release of multiple inflammatory mediators that result in a high level of oxidative stress. Multiple proteins related to inflammation were detected in the serum of patients with COPD (table 4​4).). These included interleukin (IL)‐12, IL‐15, IL‐17, IL‐1 receptor antagonist (IL‐1ra), tumour necrosis factor α (TNFα), tumour necrosis factor receptor 1 (TNF R1), interferon γ (IFNγ), IL‐12p40 and IL‐2Rγ. There is experimental evidence for the participation of all of these proteins in the inflammation that characterises COPD, and raises the possibility that the systemic manifestations of COPD may be intimately related to this process. Indeed, the association between inflammatory markers and exacerbation rate (fig 4​4)) suggests that this manifestation of the disease could be modulated by amplification of the inflammatory cascade. In this regard, eotaxin‐2—which had one of the strongest associations with the exacerbation rate in our patients—is a strong chemotactic cytokine for eosinophils,⁴⁷ cells that have been found to be increased in airway biopsy tissue from patients with COPD exacerbations.⁴⁸ Indeed, although the inflammatory pathways of COPD appear to be more related to lymphocytes expressing a T helper 1 (Th1) bias,⁴⁹ a high level of Th2 chemokines have been reported in experimental models of emphysema induced by cigarette smoking.¹⁰

There were several novel proteins that differed between patients with COPD and controls. We selected two of them—plasminogen activator inhibitor type 2 (PAI‐II) and prolactin—because of their presence in one of the eight clusters with the strongest association with COPD. PAI‐II belongs to the serpine class of protease inhibitors and is involved in the thrombogenic cascade. Known to be produced by activated monocytes in the peripheral blood,⁵⁰ this protein (together with PAI‐I) may have a role in tissue remodelling in airways disease.⁵¹ These data warrant further investigation to explore the possible role of serpines in COPD.⁵² Prolactin upregulation presents an enigma. Prolactin receptor has recently been reported to be upregulated in the lungs of mice exposed to lipopolysaccharide,⁵³ and prolactin can activate the inflammatory natural killer (NF)‐κβ cascade in pulmonary fibroblasts.⁵⁴ It is therefore plausible that prolactin may play a role in the inflammatory environment in COPD.

There are a number of important limitations to our study. Not all of the possible proteins that participate in the complex mechanism of COPD were tested. Absent were some with a known relationship to COPD such as C‐reactive protein and fibrinogen, and some of potential importance such as MMP‐12. The reason for their omission was not any preconceived mechanistic bias. Our study was designed as a proof of principle rather than a totally comprehensive evaluation of all of the markers that could potentially be explored. Many complex diseases have components related to inflammation, tissue remodelling, apoptosis and chemoattraction of specific cell types. This observation suggests that a panel of analytes might provide insight into the pathobiology of the disease under study in the absence of, or in conjunction with, novel “disease‐specific” biomarkers. We also acknowledge that not all phenotypic expressions of COPD were analysed; for example, it would have been interesting to have related the biomarkers to changes in the CT scan of patients with emphysema, but unfortunately the technique needed to quantitatively express CT changes was not available. However, the Tlco does relate to the phenotypic expression of emphysema. We believe that this study represents a proof of concept and opens a window for hypothesis testing and perhaps the discovery of yet to be described pathway interactions and targets.

For the correlation analyses we attempted to address the issue of many proteins representing the same pathophysiological mechanism by empirically grouping them according to their statistical strength and their presumed pathobiological role. We acknowledge the latter to be empirical, but it is based on the data currently available and aimed at simplifying the prospective testing. Furthermore, the inclusion of too many proteins may be intellectually desirable but may cause important cross‐correlative noise that may actually cloud the interpretation of the results. We also acknowledge that the patients included in the study do not represent the large population of patients with COPD since all of them had severe disease. However, the patients included represent those likely to be seen by clinicians and to benefit from new therapeutic strategies. On the other hand, this study is unique in that patients and controls were phenotypically well characterised and matched by age, sex and—very importantly—by smoking habits to minimise the hypothetical influence of these confounding factors. Indeed, the inclusion or exclusion of smokers in each of the groups did not affect the results. In addition, the evaluation of important associations of the panel markers with clinical markers of COPD such as the BODE index and its individual components offers a more comprehensive picture of the value of the technique. The association with exacerbation frequency is particularly interesting because exacerbations constitute an extremely important outcome and one where elucidation of the factors that may help prevent their occurrence would prove extremely useful. Finally, we also acknowledge that the stability of biomarker levels in serum samples is not well characterised and that we did not repeat the tests at different times. However, the recent report by Hurat and colleagues¹⁶ using the panel derived from this study independently validated our findings.