Similar gene expression profiles in smokers and patients with moderate COPD

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by multiple cellular and structural changes affecting the airways, lung parenchyma and vasculature, some of which are also identified in smokers without COPD. The molecular mechanisms underlying these changes remain poorly understood. With the aim of identifying mediators potentially implicated in the pathogenic processes that occur in COPD and their potential relationship with cigarette smoking, we evaluated the mRNA expression of genes involved in inflammation, tissue remodeling and vessel maintenance.

Lung tissue samples were obtained from 60 patients who underwent lung resection (nonsmokers, n = 12; smokers, n = 12; and moderate COPD, n = 21) or lung transplant (severe-to-very severe COPD, n = 15). PCR arrays containing 42 genes coding for growth factors/receptors, cytokines, metalloproteinases, adhesion molecules, and vessel maintenance mediators were used.

Smoking-induced changes include the up-regulation of inflammatory genes (IL-1β, IL-6, IL-8, CCL2, and CCL8) and the decreased expression of growth factor/receptor genes (BMPR2, CTGF, FGF1, KDR and TEK) and genes coding for vessel maintenance factors (EDNRB). All these genes exhibited a similar profile in moderate COPD patients. The up-regulation of MMP1 and MMP9 was the main change associated with COPD. Inflammatory genes as well as the endothelial selectin gene (SELE) were down-regulated in patients with more severe COPD. Clustering analysis revealed a closer relationship between moderate COPD and smokers than between both subsets of COPD patients for this selected set of genes.

The study reveals striking similarities between smokers and COPD patients with moderate disease emphasizing the crucial role of cigarette smoking in the genesis of these changes, and provides additional evidence of the involvement of the matrix metalloproteinase’s in the remodeling process of the lung in COPD.

Introduction

Chronic obstructive pulmonary disease (COPD) is characterized by a progressively worsening airflow capacity, associated with an abnormal inflammatory response of the lungs to noxious particles or gases [1]. Primarily caused by cigarette smoking, it is a highly prevalent respiratory disease and a major cause of mortality and morbidity worldwide [2]. Although it has long been recognized that smoking is the main risk factor and that exposure to cigarette smoke (CS) per se can elicit an inflammatory response [2], [3], not all smokers develop the disease. The factors determining the development of COPD in susceptible smokers are still poorly understood but may involve genetic and epigenetic factors, altered immune regulation and abnormal repair mechanisms among others [4], [5].

Pathological changes producing airflow limitation in COPD involve inflammation and remodeling of peripheral airways [6], as well as the emphysematous lung parenchyma destruction [6], [7]. But apparent structural and functional alterations also occur in the pulmonary arteries (vascular remodeling) during the initial stages of the disease [8], [9], which can produce pulmonary hypertension in advanced COPD [10]. The pathobiology of this complex disease, therefore, encompasses multiple cellular and structural changes involving all compartments of the lung [11]. Importantly, some of these changes can already be present in the lungs of “normal” smokers, i.e. smokers with normal lung function, indicating that smoking itself is able to damage the lung even before airflow limitation occurs. While those structural alterations have been well characterized, the molecular mechanisms underlying these changes remain poorly understood [12].

These marked structural changes are presumably due to chronic inflammation and the release of cytokines, growth factors, proteases, and other factors by different inflammatory and structural cells, all of which ultimately contribute to the remodeling and destruction observed in the lung tissue [13]. The release of multiple inflammatory mediators also results in a high level of oxidative stress [14]. Although some of these mediators have been identified and characterized in different samples from COPD patients (i.e., induced sputum, bronchoalveolar lavage (BAL), lung tissue), this is still insufficient for a full comprehension of the molecular mechanisms underlying the pathogenesis of COPD. One approach for identifying new molecules and pathways involved has been to perform unbiased high-throughput gene expression analysis in association with smoking and COPD. A number of studies have analyzed gene expression changes induced by cigarette smoking in alveolar macrophages [15], in the airway epithelium [16], [17] or in both [18] comparing phenotypically normal smokers and nonsmokers. Changes in the airway transcriptome have also been studied in patients with COPD [19]. The few genome-wide expression studies in the lung tissue of COPD patients published to date have identified common biological processes underlying COPD pathogenesis, however little overlap has been revealed among differentially expressed genes [20], [21]. Clearly, more information is needed to gain further insight into the molecular basis of this disease and to develop better and more tailored treatments.

We have observed similar expression of genes related to vascular function and maintenance, in particular eNOS [22] and VEGF [23], in smokers with normal lung function and patients with mild-to-moderate COPD. In the present study, we sought to investigate to what extent a selected set of genes involved in inflammation, growth factor activity, tissue repair/remodeling and vessel function and maintenance express differently in smokers and COPD patients, and whether or not gene expression varies according to COPD severity. We performed this analysis on peripheral lung tissue, which is thought to be the main site of the disease process, employing PCR arrays, a technology based on TaqMan real-time quantitative polymerase chain reaction that allows for the simultaneous gene expression assessment of multiple candidate genes in a highly reproducible and sensitive manner. The preliminary results of our study have been previously reported in abstract form [24].