Chronic obstructive pulmonary disease (COPD) is a preventable and treatable disease with some significant extrapulmonary effects that may contribute to its severity in individual patients. Its pulmonary component is char-acterized by airflow limitation that is not fully reversible. The airflow limitation is associated with an abnormal inflammatory response of the lung to noxious particles or gases [1].
Smoking is one of the main risk factors for COPD, but since not all smokers develop COPD, as well as the fact that the disease often develops in middle age, it is suggested that other factors may play a role in the pathogenesis such as genetic factors [2]. Inhalation of cigarette smoke, organic and/or inorganic dust, chemical agents and particle matters increase the risk of developing COPD. The presence of these irritants, may lead to chronic inflammation and structural changes in the lung due to repeated injury and repair [3]. Pathological changes characteristic for COPD are found in the proximal airways, peripheral airways, lung parenchyma and pulmonary vasculature [4].
One of the main roles of the epithelial cells is to provide a barrier against pathogens and to release antimicrobial products. By producing chemoattractants and adhesion molecules, epithelial cells contribute to the migration of the inflammatory cells to injury sites [5,6]. Epithelial damage is an important characteristic of several pulmonary diseases including COPD. Numerous enzymes, proteins and peptides are involved in the process of tissue repair and remodeling [7].
The matrix metalloproteinases (MMPs) family is composed of more than 25 zinc-dependent proteases that cleave the extracellular matrix and cell-surface proteins to regulate wound healing, physiological angiogenesis and immune response [8]. Matrix metalloproteinases can activate and increase the bioavailability of a variety of non matrix proteins, including cytokines, chemokines, recep-tors and antimicrobial peptides [5,9]. Matrilysin 1 (MMP-7), unlike many MMPs, is expressed by non injured, non inflamed mucosal epithelia in most adult human tissues (7). Besides extracellular matrix (ECM) components, MMP-7 processes cell surface molecules such as pro-defensin, Fas-ligand, pro-tumor necrosis factor (TNF), and E-cadherin [10,11].
The production of the enzyme is highly up-regulated by injury or exposure to bacteria, stimulating cell migration and coordinating the inflammatory response [12, 13, 14]. Thus, MMP-7 participates in the processes of defense, repair and inflammation.
In the promoter of the gene of MMP-7 an A>G transition has been identified as a functional polymorphism (
So far, there are only very limited studies concerning the role of MMP-7 and its genetic variants in lung diseases such as lung cancer [18], pneumoconiosis [19], bron-chiolitis obliterans syndrome (BOS) in patients after lung transplantation [20], and idiopathic pulmonary fibrosis (IPF) [21]. Concerning COPD, there was only one report showing increased serum level of MMP-7 in patients exposed to biomass and tobacco smoke compared with non smoking healthy controls, and the levels were negatively correlated with spirometric index of lung function (FEV1 %pr.) [22]. However, there has only been one study exploring the functional variants of MMP-7 in the development of COPD [23].
In this respect we aimed to evaluate the possible role of the
We have genotyped 191 patients with COPD and 215 healthy volunteers or individuals unaffected by lung or cancer diseases. The inclusion criteria for COPD were as follows: age higher than 40 years; forced expiratory volume in one second (FEV1) of <80.0%; forced expiratory volume in one second (FEV1)/ forced vital capacity (FVC) ratio of ≤70.0%; FEV1 reversibility after inhalation of 400 μg Salbutamol of <12.0%.
In both groups, the age of inclusion in the study and smoking status were noted; in the patients’ group: age of diagnosis, the spirometric indexes, duration and the stages of the disease (GOLD stages) were also reported. The available demographic and clinical data are presented in Table 1. Informed consent was obtained from patients and controls before the beginning of the study.
Demographic and clinical data of patients with chronic obstructive pulmonary disease and controls. Determined by the Determined by the Mann-Whitney U test. Determined by the Determined by the Mann-Whitney U test. Determined by the Mann-Whitney U test. COPD: chronic obstructive pulmonary disease; SD: standard deviation.Characteristics Patients with COPD (%) Controls (%) Number 191 215 males 146 (76.4) 109 (50.7) <0.001 females 45 (23.6) 106 (49.3) Age (years) at inclusion in the study median (range) 67 (36-88) 59 (18-80) <0.001 Age (years) at diagnosis of the disease median (range) 64 (30-86) – Smoking status non-smokers 54 (29.0) 97 (59.0) <0 001 ex-smokers 89 (48.0) 25 (15.0) current smokers 43 (23.0) 42 (26.0) FEV1 %pr (mean ± SD) (range) 50.60 ± 14.10 (15-79) 93.40 ± 11.90 (82-114) <0.001 FEV1/FVC % (mean ± SD (range 61.1 ± 8.6 (27.1-70.0) 80.4 ± 7.2 (76.3-93.0) <0.001 COPD staging (GOLD 2009) stage II (moderate) 102 (55.0%) – stage III (severe) 73 (39.0) – stage IV (very severe) 11 (67.0) –
Genomic DNA was isolated from 0.2 mL of whole blood using a commercial kit for isolation of genomic DNA from blood (GenElute™ Mammalian Genomic DNA Miniprep Kit, Sigma-Aldrich, St. Louis, MO, USA).
The genotyping for the
The temperature profile of the PCR reactions included primary denaturing of the template DNA for 3 min. at 94°C, followed by 30 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 53.6°C and poly-merization for 30 seconds at 72°C. The PCR reaction was terminated by a final extension for 5 min. at 72°C.
The restriction reaction was performed with 5U
Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Continuous variables were analyzed for normality of the distribution using the Kolmogorov-Smirnov test (One-Sample Kolmogorov-Smirnov D-Test in SPSS, version 16; SPSS Inc.). When the level of signif-icance in this test was lower than 0.05 (
The PCR product amplified with the primers for
The genotype distribution in both controls and patients did not deviate from HWE (
Genotype and allele frequencies of the OR: odds ratio; 95% CI: 95% confidence interval.Patients Controls OR (95% CI) Frequency Frequency AA 43 0.225 54 0.251 1.0 (referent) AG 99 0.518 119 0.554 1.465 (0.826-2.598) 0.243 GG 49 0.257 42 0.195 1.045 (0.647-1.687) 0.903 AG+GG 148 0.775 161 0.749 1.154 (0.731-1.823) 0.562 –181 A 185 0.484 227 0.528 1.0 (referent) –181 G 197 0.516 203 0.472 1.191 (0.904-1.569) 0.232
However, the carriers of the G allele (AG and GG genotypes) appeared to develop COPD significantly early compared to those with the AA genotype (61.01 ± 10.11
Genotype and allele and genotype frequencies of the The bold The bold The bold The bold The bold The bold OR: odds ratio; 95% CI: 95% confidence interval.Patients Controls OR (95% CI) OR (95% CI) Frequency Frequency AA 11 0.145 35 0.330 1.0 (referent) 1.0 (referent) AG 43 0.566 50 0.471 2.736 (1.252-5.962) 4.742 (1.426-15.768) GG 22 0.289 21 0.198 3.333 (1.363-8.148) 3.194 (0.862-11.830) 0.082 AG+GG 65 0.855 71 0.670 2.913 (1.380-6.133) 4.122 (1.318-12.890) –181 A 65 0.428 120 0.566 1.0 (referent) – – – –181 G 87 0.572 92 0.434 1.746 (1.147-2.657) – –
A common feature of COPD is the chronic in inflammation in the airways and the development of extensive tissue remodeling during the course of the disease process [25]. The matrix metalloproteinases are a family of zinc-containing enzymes with proteolytic activity against a wide range of extracellular proteins [10]. Under normal physiological conditions, the activities of MMPs are precisely regulated at the level of transcription, activation of the precursor zymogens and inhibition by endogenous inhibitors [26]. Due to their activity, MMPs participate in many physiological and pathological processes in the body such as development, involution, inflammation, tumor growth, and repair [27, 28].
Matrix metalloproteinases play an important role in the turnover of almost all extracellular matrix molecules and thus, participate in the pathogenesis of COPD [22, 29]. An A>G substitution in the promoter of the
So far, only one study has been found in the literature exploring the role of
It has been shown that the same variant is not a risk factor for IPF, but influences the plasma level of MMP-7 in patients, as carriers of the AA genotype had higher concentrations than carriers of other genotypes [21].
In COPD patients, the serum levels of MMP-7, as well as of some other MMPs, MMP-1 and MMP-9, has been found to be significantly higher in exposed to biomass and tobacco smoke when compared with unexposed healthy controls. Moreover, the levels of those enzymes have been found in negative correlation with the lung function indexes (FEV1 %pr) [22].
Matrilysin 1 has also been reported to associate with moderate panlobular emphysema as well as with severe and moderate centrilobular emphysema [30]. Matrilysin 1 efficiently cleaves the basement membrane protein entac-tin, which bridges laminin and collagen type IV, and sug-gested a potentially important role for MMP-7 in the dis-ruption of basement membranes by inflammatory cells [31]. Matrilysin 1 is produced by the epithelium of several uninjured, uninflamed tissues, such as lung, liver and breast. Except in intact tissues, matrilysin is expressed in migrating epithelium in injured airways [5].
It has been shown that MMP-7 mediates shedding of E-cadherin from alveolar epithelium during progression of bleomycin-induced pulmonary fibrosis [7]. In specimens of emphysema, strong immunoreactive signal for matrilysin protein has been detected in epithelial cells lining damaged alveoli, especially in cells bordering denuded epithelium [6]. The higher promoter activity of the G allele may contribute to ongoing epithelial activation by mediating persistent shedding of the E-cadherin ectodomain, altering cell-cell interactions. By cleaving the Fas ligand, matrilysin can regulate apoptosis and it may promote local coagulation by cleaving tissue factor pathway inhibitor [31]. Altogether, these data suggest that the carriers of the G allele may lead to disruption of ECM and cell-cell interactions, impairment of the process of tissue repair or fibrosis, and thus, to early development of COPD.
In conclusion, the results of our study suggest that