Palahniuk: Ratio of Endothelin-1 and C-Type Natriuretic Peptide Concentrations in Men with Hypertensive Disease of Different Severity. The Regulatory Role of Polymorphism of the Endothelin-1 Gene



Problem statement and analysis of the recent research

Hypertensive disease (HD) is one of the most common diseases both in Ukraine and all over the world. According to Center for Health Statistics of the Ministry of Health of Ukraine, more than 12 million people with high blood pressure were registered in 2014 in Ukraine constituting within the range of 30% of the adult population [5].

Endothelial dysfunction (imbalance between production of vasodilator, angioprotecive, antiproliferative factors on the one hand and vasoconstrictor, prothrombotic, proliferative factors on the other hand) is regarded as one of the mechanisms of HD development and progression. Recently, the data concerning more meaningful value of essential hypertension (EH) as a risk factor for the development of chronic heart failure (CHF) has increased [2].

According to different authors’ views, genetic factors determine HD development in 30%-80% of cases. One of these factors is the polymorphism of the endothelin-1 (ET-1) gene (Lys198Asn), which has a direct effect on ET-1 plasma concentration [1, 3, 6]. Concentration of C-type of natriuretic peptide (CNP) as direct antagonist of the peptide is no less important than possible genetic control of ET-1 concentration in patients with HD and CHF. CNP can vary in different ways depending on the plasma levels of ET-1 in carriers of the different genotypes of the ET-1 genes in healthy persons and in case of cardiovascular diseases. However, studies of ET-1gene structural changes in patients with HD and impact of its polymorphism on the plasma levels of ET-1 and CNP are understudied and have not been studied in Ukraine at all yet.

The objective of the research was to improve diagnosis of HD severity in male citizens of Podillia region being at the age of 40-69 by determining the plasma levels of vasoactive peptides ET-1 and CNP and their ratio in case of ET-1 gene (Lys198Asn) polymorphism.

Materials and methods

191 men 40-60 years old living in Podillia region in Ukraine were examined. 79 men were included into the control group. Group I consisted of 62 men with II stage HD. Group II included 50 patients with HD complicated by CHF II-III functional classes by NYHA Classification. Men from all study groups were representative by age. All patients during the examination were treated at Vinnytsia Regional Specialized Dispensary of Radiation Protection of the Ministry of Health of Ukraine, Military Medical Center of the Central Region of Air Force of Ukraine and were observed outpatiently from December 2013 to June 2014. The control group included individuals without pathological changes of the cardiovascular system. The criteria for inclusion of patients with HD into the study were verified HD diagnosis, with the obligatory inclusion of symptomatic arterial hypertension, the absence of the data about past HD complications such as myocardial infarction, acute cerebrovascular accident, and the presence of symptoms and anamnestic indications for coronary heart disease, the development of which preceded HD. HD diagnosis was made on the basis of the patients’ complaints, past medical history, physical examination, laboratory and instrumental methods of investigation according to the order №384 of the Ministry of Health of Ukraine from 24.05.2012 and guideline of the European Society of Hypertension (ESH) and the European Society of Cardiology (ESC) in 2013.

CHF diagnosis was made on the basis of patients’ complaints, anamnesis, physical, laboratory and instrumental methods, in accordance with the protocols of care for patients with CHF, approved by the Ministry of Health of Ukraine №436 from 03.03.2006 and recommendations of the Association of Cardiologists of Ukrainian Association of heart failure (2012) and recommendations for the diagnosis and treatment of heart failure of the European Society of Cardiology (2012).

Exclusion criteria for the study were chronic obstructive pulmonary disease, neoplasms, renal and liver dysfunction, endocrine disorders, hemic system diseases.

Genotyping of ET-1 gene was conducted using polymerase chain reaction. This study was conducted in consort with the Research Institute of the genetic and immunological bases of pathology and pharmacokinetics “Ukrainian Medical Stomatological Academy” (Poltava, the head is professor I. P. Kaidashev). ET-1 concentration in plasma was determined using ELISA method on enzyme-linked immunosorbent analyzer “Humareader single” (Germany) and differential filter 630 nm. Standard set of “DRG” firm (USA) was used to determine ET-1 plasma concentration. The mathematical processing was performed on a personal computer using a standard statistical package STATISTICA 6.0.

Results of the research

The distribution of ET-1 gene genotype frequencies among male patients of the control group and patients with HD corresponded to Hardy-Weinberg equilibrium.

Among patients of the control group Lys/Lys genotype of ET-1 gene was detected in 65.82% of patients (n=52), Lys/Asn genotype was found in 27.85% individuals (n=22) and Asn/Asn genotype was observed in 6.33% cases (n=5) (рLys/Asn-Lys/Lys<0.0001; рAsn/Asn-Lys/Lys<0.0001; рAsn/Asn-Lys/Asn<0.001). The frequency of Lys allele in male patients of the control group constituted 79.75%, Asn allele was observed in 20.25% of cases (рLys-Asn<0.0001).

Lys/Lys genotype of ET-1 gene was found to dominate in patients with stage II HD constituting 56.45% (n=35), Lys/Asn genotype was observed in 33.87% of patients (n=21) and Asn/Asn genotype was detected in 9.68% of cases (n=6) (рLys/Asn-Lys/Lys<0.01; рAsn/Asn-Lys/Lys<0.0001; рAsn/Asn-Lys/Asn<0.001). The frequency of Lys allele in male patients of the main group constituted 73.39%, Asn allele was detected in 26.61% of cases (рLys-Asn<0.0001).

Among patients with HD complicated by CHF Lys/Lys genotype of ET-1 gene was found 66.00% of cases (n=33), Lys/Asn genotype was observed in 28.00% of patients (n=21) and Asn/Asn genotype was noted in 6.00% of cases (n=3) (рLys/Asn-Lys/Lys<0.0001; рAsn/Asn-Lys/Lys<0.0001; рAsn/Asn-Lys/Asn<0.01).

Men with Lys/Asn and Asn/Asn genotypes in each group were united as carriers of Asn allele due to the low frequency of genotype Asn/Asn carriership (Table 1).

Table 1.

Distribution of ET-1 gene genotypes frequencies in men of the control group and the patients with stage II HD and with HD complicated by CHF, (%)

Group Genotype Lys/Lys carriers Carriers of the Asn allele р
Control group (n=79) 65.82% (n=52) (1) 34.18% (n=27) (4) р4-1<0.0001
Stage II HD (n=62) 56.45% (n=35) (2) 43.55% (n=27) (5) р5-2>0.05
HD complicated by CHF (n=50) 66.00% (n=33) (3) 34.00% (n=17) (6) р6-3<0.001
р р2-1>0.05;
р3-1>0.05;
р3-2>0.05
р5-4>0.05;
р6-4>0.05;
р6-5>0.05

Gene polymorphism of ET-1 is known to lead to changes in ET-1 concentration in plasma. Therefore, it was interesting to determine the level of this peptide, its antagonist – CNP and their ratio in men carriers of different ET-1 gene genotypes (Table 2).

Table 2.

Plasma ET-1 levels in the control group and in the patients with stage II HD and HD complicated by CHF in case of different genotypes of the ET-1 gene carriership (М±m, fmol/ml)

Group Genotype Lys/Lys Carriers Carriers of Asn allele р
Control group (n=79) 1.41±0.05 (n=52) (1) 2.53±0.12 (n=27) (4) р4-1<0.0001
Stage II HD (n=62) 11.58±0.23 (n=35) (2) 13.90±0.22 (n=27) (5) р5-2<0.0001
HD complicated by CHF (n=50) 12.89±0.08 (n=33) (3) 14.07±0.18 (n=17) (6) р6-3<0.0001
р р2-1<0.0001;
р3-1<0.0001;
р3-2<0.0001
р5-4<0.0001;
р6-4<0.0001;
р6-5>0.05

Plasma concentration of ET-1 was found to be significantly higher in men with HD of different severity in case of any genotype of ET-1 gene carriership in comparison with the control group. The highest concentration of peptide among all study groups was observed in patients with HD complicated by CHF as the carriers of Lys/Lys genotype of ET-1 gene. However, ET-1 level was not different in Asn allele carriers between patients with HD. Comparing the plasma concentration of ET-1 in each group, Asn allele was found to be associated with higher level of peptide compared to genotype Lys/Lys carriers.

The next step was to determine CNP plasma concentrations in carriers of different genotypes of the ET-1 gene (Table 3).

Table 3.

CNP plasma concentration in male patients of the control group, patients with stage II HD and in patients with HD complicated by CHF in case of different genotypes of ET-1 gene carriership (М±m, pmol/ml)

Group Genotype Lys/Lys carriers Carriers of Asn allele р
Control group (n=79) 2.02±0.29 (n=52) (1) 2.98±0.08 (n=27) (4) р4-1<0.0001
Stage II HD (n=62) 4.68±0.12 (n=35) (2) 5.90±0.11 (n=27) (5) р5-2<0.0001
HD complicated by CHF (n=50) 4.88±0.09 (n=33) (3) 5.93±0.18 (n=17) (6) р6-3<0.0001
р р2-1<0.0001;
р3-1<0.0001;
р3-2>0.05
р5-4<0.0001;
р6-4<0.0001;
р6-5>0.05

CNP level was significantly higher in men with stage II HD and in patients with HD complicated by CHF than in the control group in case of all genotypes of ET-1 gene carriership. However, the significant difference in plasma concentrations of peptide between patients with HD of different severity as carriers of Lys/Lys genotype and Asn allele was not detected. Asn allele carriers had a significantly higher level of CNP than genotype Lys/Lys carriers in patients with stage II HD and HD complicated by CHF and in individuals without cardiovascular diseases.

CNP/ET-1 coefficient in men-carriers of polymorphic genotypes of ET-1 gene was calculated to assess the balance of vasodilator/vasoconstrictor (Table 4).

Table 4.

CNP/ET-1 coefficient in male patients of the control group, patients stage II HD and in patients with HD complicated by CHF in case of different genotypes of ET-1 gene carriership (M±m, c.u.)

Group Genotype Lys/Lys carriers Carriers of the Asn allele р
Control group (n=79) 1.49±0.04 (n=52) (1) 1.22±0.05 (n=27) (4) р4-1<0.0001
Stage II HD (n=62) 0.40±0.003 (n=35) (2) 0.42±0.004 (n=27) (5) р5-2>0.05
HD complicated by CHF (n=50) 0.38±0.006 (n=33) (3) 0.42±0.007 (n=17) (6) р6-3>0.05
р р2-1<0.0001;
р3-1<0.0001
р3-2>0.05
р5-4<0.0001;
р6-5>0.05
р6-4<0.0001

Analysis of the data showed that CNP/ET-1 coefficient was significantly lower in patients with HD among genotype Lys/Lys and Asn allele carriers in comparison with the control group. However, no significant difference in the ratio of peptides between men with stage II HD and complicated CHF was found. CNP/ET-1 coefficient was significantly lower in the control group in case of Asn allele carriership in comparison with genotype Lys/Lys carriers. However, Asn allele carriership in the other study group was not associated with a change in CNP/ET-1 coefficient in plasma.

Discussion of the results

Lys/Lys genotype and Lys allele of ET-1 gene was found to dominate in men without cardiovascular diseases and in patients with HD. Similar results were obtained in the research of F. A. Treiber et. al. (UK) of healthy individuals of Negro and white races [14]. A. U. Dzholdasbekova, A. E. Gaipov (Kazakhstan) showed that genotype Lys/Lys dominated in men from the control group and patients with HD [9]. Similar results were elucidated in male and female living in Sumy (Ukraine) [4]. A. Barath et. al. (Hungary) detected the following results – healthy young people of different sexes were mostly carriers of Lys/Lys genotype and people with arterial hypertension were more commonly carriers of Lys/Lys genotypes, Lys/Asn and Lys allele than Asn/Asn genotype and Asn allele [8]. Investigations of Jin J. J. et. al. (Japan) demonstrated Lys/Lys genotype of ET-1 gene also prevailed in men and women with HD [10]. However, the study which was conducted in French cities and on the territory of Northern Ireland and Turkey showed that significant difference in the frequency of genotypes and alleles of ET-1 gene between patients of the control group and persons with myocardial infarction was not established [11, 13].

Asn allele carriership was investigated to be associated with higher levels of ET-1 in plasma compared with Lys/Lys genotype of ET-1 gene in patients of all study groups. Plasma level of ET-1 was significantly higher in individuals with HD in case of both Lys/Lys genotype and Asn allele of ET-1 gene carriership in comparison with those in the control group. Similar results were obtained by E. M. Berezykova (Russia), where men and women as carriers of Asn/Asn genotype had significantly higher levels of ET-1 than Lys homozygotes [1]. According to E. V. Shcheglova (Vladikavkaz, Russia) the carriership of Asn allele was associated with higher levels of ET-1 in plasma than in patients with Lys/Lys genotype [6]. A. E. Barden et. al. (Australia) observed that Asn/Asn genotype in pregnant women led to the highest plasma concentration of ET-1 compared with Lys/Asn and Lys/Lys genotypes [7]. However, there are opposing studies. S. Tanaka et. al. (Japan) indicated that individuals with HD with Asn/Asn genotype tended to increase the level of ET-1 in plasma compared with patients with Lys/Lys genotype. However, significant differences were not detected [12].

CNP plasma concentrations were identified for the first time in male carriers of different genotypes of ET-1 gene, citizens of Podillia region in Ukraine. CNP plasma level in men with stage II HD and HD complicated by CHF was significantly higher than in the control group carriers of any genotype of ET-1 gene. However, difference between patients with HD was not found. CNP plasma concentration was significantly higher in Asn allele carriers among three study groups. That could be explained by CNP compensatory hyper-production on synthesis of powerful vasoconstrictor ET-1 in men with HD.

The last step was to estimate the coefficient of two vascular antagonists, namely CNP and ET-1, in patients without cardiovascular diseases and in patients with HD as the carriers of polymorphic genotypes of ET-1 gene. CNP/ET-1 coefficient was found to be significantly lower in men with stage II HD and HD complicated by CHF in case of Lys/Lys and Asn allele genotype carriership than in the control group. However, the difference between patients with HD was not observed. Such changes of vasoactive peptides indicated the imbalance between vasoconstrictor and vasodilatator towards decrease in the first one. It is one of the first elements of HD pathogenesis. Asn allele carriership in men of the control group was associated with lower CNP/ET-1 coefficient compared to Lys/Lys genotype of ET-1 gene. Similar studies have not been performed in Ukraine and in the world yet.

Conclusions

  1. Lys/Lys genotype and Lys allele of ET-1 gene dominated among control group and patients with HD, residents of Podillia region in Ukraine, at the age of 40-60.

  2. ET-1 and CNP plasma levels were significantly higher and CNP/ET-1 coefficient was lower in men with HD than in patients without cardiovascular diseases in case of any genotype of ET-1 gene carriership. Registration of Asn allele of ET-1 gene was associated with the highest plasma concentration of ET-1 (2.53±0.12 fmol/ml, 13.90±0.22 fmol/ml and 14.07±0.18 fmol/ml, respectively) and CNP (2.98±0.08 pmol/ml, 5.90±0.11 pmol/ml and 5.93±0.18 pmol/ml, respectively) in males without cardiovascular diseases and in patients with stage II HD and HD complicated by CHF compared with carriers of Lys/Lys genotype (the plasma level of ET-1 constituted 1.41±0.05 fmol/ml, 11.58±0.23 fmol/ml and 12.89±0.08 fmol/ml, respectively, the plasma concentration of CNP constituted 2.02±0.29 pmol/ml, 4.68±0.12 pmol/ml and 4.88±0.09 pmol/ml). This fact demonstrated the necessity to take into account individual genetic characteristics when determining levels of ET-1 and CNP during clinical and scientific investigations.

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