Chronic obstructive pulmonary disease (COPD) is a common respiratory health problem characterized by persistent respiratory symptoms, such as dyspnoea, cough and airflow limitations (1). COPD represents one of the leading causes of death worldwide. According to the World Health Organization (WHO), COPD retains the III, IV and V places in upper-middle-, lower-middle- and high-income countries respectively, and by 2020 will attain the third place all over the world (2).
The frequency of comorbidities in COPD strongly varies between studies, given the heterogeneity of the definitions used, the study populations, patient COPD severity and age (3). Despite these differences, most studies conclude to a high prevalence of COPD patients with associated comorbidities, from 70% to virtually all patients (4). Cardiovascular diseases (CVDs) are arguably the most important comorbidities in COPD. CVDs are common in people with COPD, and their presence is associated with an increased risk for hospitalization, longer length of stay and all-cause and CVD-related mortality (5). The mechanisms that underlie the association between COPD and CVDs are not well understood, but several processes are thought to be important and may interact with each other. These include lung hyperinflation, hypoxaemia, pulmonary hypertension, systemic inflammation and oxidative stress, exacerbations, shared risk factors and shared genetics, as well as COPD phenotype (6).
Endothelial dysfunction fundamentally contributes to the development of atherosclerosis that finally leads to coronary heart disease (CHD). The process is further accelerated by systemic inflammation and oxidative stress (7). As a result, approximately one out of six COPD patients suffers from concomitant CHD (8). Endothelial function is known to be impaired in subjects with chronic heart failure (CHF), which is a syndrome rather than a disease (9). Left heart failure was diagnosed first time in one out of five COPD patients after extensive cardiovascular work-up (10). Vice versa, one out of three heart failure patients suffers from obstructive ventilation disorders (11). Arterial hypertension (AH) is comparably frequent in COPD patients and the general population. The prevalence of AH is about 50% and increases with age (12). Nevertheless, the overall elevated cardiovascular risk in COPD may be a product of this common condition and potentiating effects of other risk factors such as diabetes mellitus and cigarette smoking. Moreover, higher central blood pressure values and arterial stiffness are also found indicating premature atherosclerosis (8).
The coexistence of COPD, CVDs and major risk factors for CVD highlights the crucial need for the development of strategies to screen for and reduce cardiovascular risks associated with COPD. Both AH and COPD are genetically determined conditions with multiple genes, combinations of genes, inter-gene interactions and epigenetic processes responsible for their occurrence. When adverse genetic and external factors combine, a disease is formed (13).
The relationship between the polymorphism of the gene encoding angiotensin-converting enzyme (ACE) and COPD has been addressed in numerous prior studies; however, these studies have yielded ambivalent results (14,15,16). Prior studies have demonstrated the role of genetic factors in susceptibility to COPD; in part, susceptibility to COPD was found to be associated with polymorphism of proteinase-activated receptor-1 (17), plasminogen activator inhibitor-1 (18) and β2-adrenergic receptors (19). A study of relationship between COPD and ACE activity has shown the activity of ACE to depend on oxygen concentrations in the blood; therefore, an increase in ACE levels due to COPD-associated hypoxia may cause severe tissue damage (20).
Thus, the aim of this study was to establish the role of ACE gene polymorphism in the occurrence of AH in patients with COPD.
The study group consisted of 96 patients admitted to the Ternopil University Hospital. We stratified patients into three groups: Group 1 (25 patients with COPD), Group 2 (23 patients with AH) and Group 3 (28 patients with COPD + AH). The control group consisted of 20 healthy subjects.
The inclusion criteria were male patients 40 to 60 years of age at screening with a diagnosis of COPD and/or AH and informed consent form signed by patients prior to their participation in any study-related procedures. COPD was diagnosed according to Order 555 of MoH of Ukraine dated 27 June 2013 and according to the guidelines published by the American Thoracic Society and European Respiratory Society (GOLD, 2013). Airway obstruction was assessed using GOLD classification, 2008. The diagnosis of COPD with moderate (Stage 2) airway obstruction was confirmed with compatible clinical features concurrent with airflow limitation defined as forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) less than 0.70 (FEV1/FVC ratio of 50–79% predicted).
The diagnosis of AH (Stage I) was made according to 2018 ESC/ESH Guidelines for the management of AH (21). Systolic (140–159 mmHg) blood pressure and/or diastolic (90–99 mmHg) blood pressure were considered as the presence of Stage I AH. Left ventricular hypertrophy was confirmed by an electrocardiogram.
Patients with bronchial asthma; α1-antitrypsin deficiency; active tuberculosis; lung cancer; significant bronchiectasis; sarcoidosis; pulmonary fibrosis; interstitial lung disease; signs and symptoms of clinically significant neurological, psychiatric, renal, hepatic, immunological, gastrointestinal and urogenital disorders; musculoskeletal conditions; disorders of the skin and sensory organs; endocrine disorders (uncontrolled diabetes or thyroid disease) or uncontrolled haematological disease; unstable liver disease; unstable or life-threatening heart disease; cancer not completely disease free for a minimum of 5 years and any drug, substance or alcohol abuse were excluded.
Sampling of venous blood for genotyping was performed under sterile conditions into 2.7 mL Monovettes with potassium salt of ethylenediaminetetraacetic acid (EDTA) as an anticoagulant; the samples were frozen and stored at −20°С. Molecular genetic studies were performed with extraction of DNA and with use of polymerase chain reaction (PCR) and further analysis for the length of restriction fragments. DNA was extracted from peripheral blood leukocytes using a standard salt precipitation method. Genotyping for the ACE I/D was performed using PCR-based restriction fragment length polymorphism (RFLP). The primers used were 5′-GATGCGCACAAGGTCCT-GTC-3′ (forward) and 5′-CAGGGTGCTGTCCAC-ACTGG ACCCC-3′ (reverse). The PCR products were digested with 3 U of Tth111I (Fermentas), and the fragments were separated on a 3% agarose gel containing ethidium bromide and visualized with UV light. To assess genotyping reliability, we performed double sampling RFLP–PCR in more than 20% of the samples and found no differences (22).
Statistical data analysis was carried out using Statistica 7.0 software. Assessment of genotypes of the selected sample for conformity to general population sample was guided by the Hardy–Weinberg principle. The observed frequencies and the expected frequencies calculated from the expression p2 + 2
The frequency distribution of polymorphic genotypes of the gene encoding ACE and assessment of compliance with the Hardy–Weinberg population equilibrium were carried out in groups of patients with COPD, AH and COPD + AH combination. The frequencies of the genotype responsible for I/D polymorphism of the ACE gene in the control and experimental groups were not found to deviate significantly from the Hardy–Weinberg equilibrium (
Hardy–Weinberg equilibrium of the ACE gene I/D polymorphism in COPD, AH and their combination
Common homozygotes | I/I | 7.8 | 7 | 7.3 | 7 | 8 | 9 | 6.1 | 5 |
Heterozygotes | I/D | 14.6 | 14 | 11.3 | 12 | 13.9 | 12 | 9.9 | 12 |
Rare homozygotes | D/D | 2.6 | 4 | 4.4 | 4 | 6.1 | 7 | 4 | 3 |
Chi-square, χ2 | χ2 = 0.86, df = 2, | χ2 = 0.09, df = 2, | χ2 = 0.52, df = 2, | χ2 = 0.89, df = 2, |
ACE, angiotensin-converting enzyme; COPD, chronic obstructive pulmonary disease; AH, arterial hypertension.
Genotype frequencies of the ACE gene I/D polymorphism in COPD, AH and their combination
I/I | 7 | 28.0 | 7 | 30.4 | 9 | 32.1 | 5 | 25.0 |
I/D | 14 | 56.0 | 12 | 52.2 | 12 | 42.9 | 12 | 60.0 |
D/D | 4 | 16.0 | 4 | 17.4 | 7 | 25.0 | 3 | 15.0 |
Fisher’s exact | – |
ACE, angiotensin-converting enzyme; COPD, chronic obstructive pulmonary disease; AH, arterial hypertension.
The frequencies of alleles for the ACE gene in patients with COPD, AH and COPD + AH and in control group patients are given in Table 3. In the COPD group, the established distribution was 56.0% for ACE I allele and 44.0% for ACE D allele; in the AH group, 56.5% and 43.5% and in the COPD + AH group, 53.6% and 46.4%. However, these data significantly did not differ from the control group. The results of the study given in Table 4 have demonstrated absence of a statistically significant relationship between the factor (presence of I or D alleles) and occurrence of the disease (
Allele frequencies of the ACE gene I/D polymorphism
ACE I allele | 28 | 56.0 | 26 | 56.5 | 30 | 53.6 | 22 | 55.0 |
ACE D allele | 22 | 44.0 | 20 | 43.5 | 26 | 46.4 | 18 | 45.0 |
Pearson’s chi-square, χ2 (disease/control group) | χ2 = 0.01, df = 1, | χ2 = 0.02, df = 1, | χ2 = 0.02, df = 1, | – |
ACE, angiotensin-converting enzyme; COPD, chronic obstructive pulmonary disease; AH, arterial hypertension.
OR for alleles in different study groups
COPD | 1.04 | 0.45–2.40 | >0.05 | 0.96 | 0.42–2.22 | >0.05 |
AH | 1.06 | 0.45–2.50 | >0.05 | 0.94 | 0.40–2.21 | >0.05 |
COPD + AH | 0.94 | 0.42–2.13 | >0.05 | 1.26 | 0.47–2.39 | >0.05 |
OR, odds ratio; ACE, angiotensin-converting enzyme; CI, confidence interval; COPD, chronic obstructive pulmonary disease; AH, arterial hypertension.
OR for genotypes in different study groups
COPD | 1.17 | 0.31–4.44 | 0.85 | 0.26–2.80 | 1.08 | 0.21–5.50 |
AH | 1.31 | 0.34–5.05 | 0.73 | 0.22–2.44 | 1.19 | 0.23–6.11 |
COPD + AH | 1.42 | 0.39–5.14 | 0.50 | 0.16–1.61 | 1.89 | 0.42–8.43 |
OR, odds ratio; CI, confidence interval; COPD, chronic obstructive pulmonary disease; AH, arterial hypertension.
Analysis of dominant and recessive types of inheritance for the ACE gene (alleles I and D) did not establish any significant differences in the groups of COPD, AH, and their combination (
ACE is an endopeptidase consisting of two catalytic domains; this enzyme is usually expressed by endothelial, epithelial and neuronal cells (23). It exists both in a membrane-bound form (ACE) and in a soluble form (sACE), the latter produced through exposure to zinc metalloprotease (referred to as “ACE secretase”), which cleaves the mature membrane-bound ACE in the juxtamembrane extracellular domain to release the large extracellular part of the enzyme (23, 24). The known function of ACE is associated with the renin–angiotensin system, wherein ACE catalyzes the synthesis of angiotensin II vasoconstrictor from its non-vasoactive precursor, angiotensin I, and is also responsible for inactivation of vasodilator bradykinin (25). Association between
Kanazawa et al. show insertion/deletion (
The probable association between the D allele and D/D genotype of the ACE gene with the incidences of COPD + AH combination presented in results is attributable to the fact that, along with the altered endothelial responses, the
The present study has some limitations that should be considered in the interpretation of our results: sample size is too small, that is why it is difficult to find significant relationships from the data; the inclusion in the study group only of patients with Stage 2 COPD and Stage I AH and the patients were not randomly selected, generating a potential selection bias. Therefore, we cannot rule out the hypothesis that the patients evaluated do not represent the whole population of COPD patients in the Ternopil region of Ukraine. However, our results reflect a more heterogeneous real-world population, representative of clinical practice.
We did not observe a significant correlation between I/D polymorphism in the ACE gene and the risk of AH in COPD patients; therefore, comprehensive studies on Ukrainian patients are required. In addition, the trend towards increased D/D genotype frequency in patients with COPD and AH (although not significantly increased) suggests that this genotype may be associated with a higher risk of AH occurrence in COPD patients.
The study protocol was approved by the Medical Ethics Committees of I. Horbachevsky Ternopil National Medical University (No 47-25/02/2017), and the study was conducted in accordance with the Declaration of Helsinki of 1975, as revised in 1983. Informed consent was obtained from all patients.