Eugeny Patkin, Vadim Vasilyev, Larisa Pavlinova, Halmurat Upur, Denis Dubrovin,

(1Pavlov′s Institute of Experimental Medicine, Russian Academy of Science，Saint-Petersburg 197000, Russia； 2Xinjiang Medical University, Urumqi 830011, China)



New medical technologies in predictive assessment of the myocardial infarction risks: a systematic review

Eugeny Patkin1, Vadim Vasilyev1, Larisa Pavlinova1, Halmurat Upur2, Denis Dubrovin1,2

(1Pavlov′sInstituteofExperimentalMedicine,RussianAcademyofScience，Saint-Petersburg197000,Russia；2XinjiangMedicalUniversity,Urumqi830011,China)

Abstract:DNA methylation is involved in the regulation of cell’s chromatin organizing and remodeling, X-chromosome inactivation, genomic imprinting, chromosome stability, gene transcription etc. DNA methylation is an epigenetic factor, which is not affecting the genomic structure, but could play an important role in the development of cardio-vascular disease. In the recent researches there have been developed and proved by the authors their own test of whole-genome DNA methylation for the patients as the new biomarkers for cardiovascular diseases. It is important that these markers can serve as predictive personalized markers in screening, diagnostics and prognosis of various forms of cardiovascular diseases.

Keywords:DNA methylation；epigenetics；new biomarkers for cardiovascular diseases

It is well known that epigenetic modifications contribute to the structural organization of chromatin, chromosomes, and expression of genes. These modifications concern the activity of the highly expressed genes thereby involving every level of genome organization. The abnormalities of epigenetic modifications lead to development of a number of serious pathologies, including neurological, neurodegenerative, cardiovascular and cerebral diseases, especially those that occur in embryogenesis and aging[1-5]. Although these epigenetic changes do not affect the genetic code, the phenotype of separate cells, tissues, organs and of the entire organism may change, leading to the development of pathologies[6].

The main feature of epigenetic mechanisms that regulate gene expression is their high variability resulting from lifestyle, diet, geographic environment, psycho-emotional stress[7].Such variability suggests the importance of its, first of all, on an individual basis[8]. However, basic research of the role of epigenetics in human pathology is focused on various types of cancer, while the amount of works dedicated to the participation of epigenetic mechanisms in the development of cardio-vascular disease is surprisingly low. At the same time, many observations of clinicians and epidemiologists point out, albeit indirectly, the environmental risk factors which do not affect the genomic structure[9-10,5]. This suggests that environmental influences in early life can cause epigenetic changes in the genome that appear much later in ontogenesis at the level of metabolic processes, which can affect the state of cardiovascular system[11-15].

Methylation of DNA is a key covalent modification inherited during division of somatic cells and 5-methyl-cytosine (5MeC) constitutes 2-5% of cytosines in mammalian genomes, being found primarily in CpG dinucleotides[10,16]. DNA methylation is associated with covalent attachment of a methyl group to the C5 of cytosine residues in cytosine-guanine pairs (CpG) dinucleotide sequences. DNA methylation is involved in the regulation of many cellular processes, including chromatin organizing and remodeling, X-chromosome inactivation, genomic imprinting, chromosome stability and gene transcription[10,17-18]. Usually promoter hypermethylation is associated with a decreased gene expression[19]. It is necessary to take into consideration, that more than 90% of the genomic 5-methyl-cytosines are not directly associated with gene function, as they lie in CpG dinucleotides located in transposable repetitive elements, also known as transposons, as well as in different types of tandem repeats of DNA (macro-, mini- and microsatellites)[20]. DNA methylation can modulate the tertiary DNA structure, first of all, repetitive elements, and thus affect the availability of DNA promoter for transcription factors and many regulatory elements, which ultimately affect the transcription[21]. Modulation of gene expression by modification of the genome due to structural changes in the architecture of chromatin, without changing the associated genomic sequence of DNA, but with controlled access to the regulation of transcription factors and target gene, may be a part of the cellular response to environmental alteration.

It has become clear in recent years, that DNA methylation is important to not only to determine the identity of cells, but it can be transferred to future generations in the course of meiotic and mitotic divisions, so in certain cases this modification may function as the epigenetic code[22]. This phenomenon of intergenerational inheritance of epigenetic modifications explains the previously enigmatic increase of susceptibility to diseases in people who no longer face the damaging environmental factors, but are descendants of such sensitive individuals. It was found that the descendants of people, who survived the famine during the World War II, show an increased incidence of cardiovascular pathologies[23]. Epigenetic regulation in the ability of the myocardium to adapt to a changing environment is especially important because of rather limited ability of human cardiomyocytes to proliferate[24].

Despite the advances in prevention and treatment of cardiovascular disease, this group of multifactorial diseases remains the leading cause of death worldwide. The endpoint of various cardiovascular diseases is heart failure. The development of cardiovascular disease is provoked in some cases by a number of genetic and environmental factors, however, only few of those can be explained solely by the effects of such factors. It has recently been established that aging, diabetes, high blood cholesterol (atherosclerosis), smoking, high blood pressure (hypertension), obesity, physical inactivity, male gender, and family history are the risk factors that increase the susceptibility to the development of heart failure[25]. As it was already indicated, all these deviations are reflected by changes in the level of DNA methylation[26-27]. It is important, that in models of atherosclerosis in animals changes of level of DNA methylation in peripheral blood leukocytes are confirmed in blood of patients with this pathology.

Such epigenetic changes precede an appearance of any established symptoms of atherosclerosis, which were detected by biochemical, histological and cytological methods. Thus, this modification of the DNA may serve as a biomarker for the early stages of atherosclerosis and cardiovascular diseases[28-30]. Taking into account the obvious polygenic nature of atherosclerosis as well as of various types of cardiovascular and cerebrovascular disease, it may be assumed that the level of the global DNA methylation reflects the change of level of DNA methylation of genes in different cell types involved in immune or inflammatory responses during the development of these diseases[30-31]. At the same time there are evidences of the key role of epigenetic modifications in the regulation of endothelial gene expression and thus in the pathogenesis of vascular diseases, including atherosclerosis and vascular restenosis[32]. It is important to note that the largest number of potentially methylated / demethylated CpG sites is localized in retrotransposons and DNA tandem repeats. These both DNA types are characterized by potential instability and sequence breaks during changes of methylation level[33]. In turn, such instability induces deviations from normal gene expression[34].

Taken together all these data indicate the need for general assessment of genomic DNA methylation status as a potential biomarker for diseases. Indeed, recently it was shown that changes and disturbances of the endothelial responses are caused by changes in the whole genome methylation (methylome) together with differentially methylated DNA of specific regions of individual genes[35]. An example of such specific genes whose action is under the control of methylation / demethylation of DNA and whose expression, or rather disturbances in the expression lead eventually to the development of cardiovascular diseases, may be the genes encoding the expression of soluble mediators, and cell surface molecules linked adhesion and migration of leukocytes in the vascular tissue[36].

Moreover, currently it has been clearly shown, that the development of essential hypertension (EH), which relates to the important risk factors for heart failure, is closely connected with disturbance of methylation / demethylation of promoter DNA of several key genes[37]. In particular, it has been shown the increase of DNA methylation in the promoter of the gene encoding the 11-beta-hydroxysteroid dehydrogenase-2 (11beta-HSD2), the reduced activity of which is usually accompanied by the development of essential hypertension[38-39].

The reviews are devoted to connection of changes in the level of DNA methylation with diabetes that is also considered as risk factor for cardiovascular diseases[40-46]. The number of studies has noted an important role of DNA repeats LINE-1 methylation in increased risk of metabolic abnormalities, which are the negative factor for the possible development of cardiovascular disease[36,47]. This gave grounds to propose to use the methylation level of such repeats as a biomarker of risk of cardiovascular pathologies[48-49], though precise mechanisms of such modifications are still unclear.

Currently it was observed a certain inconsistency of total genomic DNA methylation and methylation of several genes data for dysfunction of blood vessels in cardiovascular diseases (see below). Apparently it is determined firstly by the complex polygenic nature of such diseases. Secondly, the various methodological approaches are used in the determination DNA methylation status. Thus, there were revealed high levels of homocysteine and adenosylhomocysteine and DNA hypomethylation of CpG sites in atherosclerosis[50]. It was also found that low level of methylation of LINE-1 sequence of DNA isolated from peripheral blood leukocytes, was correlated with mortality from coronary heart disease and stroke[51]. At the same time, the increased rather than decreased methylation level of Alu repeats in DNA in peripheral blood leukocytes was predominated in individuals with cardiovascular diseases and obesity in China[52]. There was also found elevated methylation of two specific genes encoding estrogen receptors in smooth muscle of the vascular system in atherosclerosis[53]. It was suggested that mosaic changes in the level of DNA methylation, for example, global hypomethylation and hypermethylation of the promoters of several genes play a role as a trigger in the development of age-related diseases, such as atherosclerosis[54], as a rule, lead to disturbances of the cardiovascular system. In human studies, methylation of LINE-1 DNA sequence or global methylation, measured in blood DNA, was reduced in people with obvious known risk factors for atherosclerosis as well in older people, with smoking, although the range of changes in healthy individuals was less pronounced[55-57]. The same reduction of methylation of LINE-1 sequence of DNA, and the increase of methylation of one of key genes encoding enzymes of fatty acids were associated with the presence of Type II diabetes[49]. Numerous studies have shown that already mentioned retrotransposons LINE-1 were activated through hypomethylation under conditions of cellular stress. These data indicate that changes in the level of LINE-1 methylation can serve as a marker for atherosclerosis and coronary heart disease[5,28,51]. It is noteworthy that the low level of LINE-1 methylation of genomic DNA is correlated with age[55]. At the same time peripheral blood DNA analysis of the level of DNA methylation at sites CCGG using methyl-sensitive restrictases HpaII-MspI reveals a more complex pattern of changes in global DNA methylation in various diseases of the cardiovascular system.

Genomic CCGG sequences are represented as more dense clusters of potentially methylated sites as compared with the individual CpGs[58]. So, whole-genome methylation analysis on CCGG sites may be more convenient than on CpG sites. This method demonstrated a connection of global DNA hypermethylation with vascular inflammatory response as a result of endothelial injury, which in turn correlated with mortality from cardiovascular disease[59]. Similar results of DNA hypermethylation in peripheral blood were obtained for patients with coronary artery disease[60]. The authors have also shown that in older age group (61-75 years) of patients with similar disease, significantly higher level of global DNA hypermethylation was observed in comparison with the control group. It was also shown, that strengthening symptoms of cardiomyopathy in cardiomyocytes was accompanied by decrease of CpG sequences methylation in the promoter and gene bodies regions of genes responsible for the increase in the expression, and did not change in those that were responsible for the decrease of the expression[61].

It should be kept in mind that cardiovascular disease often develops after a prolonged asymptomatic phase. So, it is necessary to have the maximum number of biomarkers of different nature. Obviously, the development of new markers for the diagnosis and prevention of cardiovascular disease is a major public health goal worldwide. New biomarkers for cardiovascular disease may enable the development of such approaches in the diagnosis, by means of which it will be possible to determine more accurately the risk of diseases development in patients. Thus, biomarkers may help in screening, diagnosis and prognosis of disease in patients with some form of cardiovascular disease, as well as those in which they have not yet been diagnosed. Peripheral blood leukocytes could be used with great success for the development of new biomarkers based on the changes of epigenetic status in cardiovascular disease especially with inflammatory or atherosclerotic etiology. With the help of epigenetic biomarkers, even before the onset of symptoms of a particular disease it will be possible to prevent patients with coronary artery disease from the myocardial infarction (MI) or to evaluate the success of the therapy.

To our opinion, an optimal strategy of using epigenetic and epigenomic markers should include at the first stage determination of whole-genome methylation, as it is clear from above mentioned data, that genomic DNA methylation is associated with various already known risk factors, which unlike of them, preceded the symptoms of pathology. At the next step it is necessary to study the methylation of individual genes and their individual regions (promoters, bodies of gene, introns) which are most likely associated with the risk of disease.

In this connection, the tandem repeats of genes directly related to vascular function may be of particular interest. In patients with coronary heart disease we have identified gender differences in DNA methylation of minisatellite B2-VNTR of receptor B2 bradykinin gene[62],which plays an important role in vascular homeostasis associated with the permeability and tone of blood vessels, inflammatory response, and pain sensitivity[63-65]. Depression, anxiety and other psychosocial factors are additional, independent risk factors for cardiovascular disease[66-68]. High frequency of comorbid depression is typical for patients with cardiovascular disease[69]. Impact depression in cardiovascular diseases is mediated through platelets receptors of catecholamines and serotonin[70].

Increased levels of catecholamines that have been identified in depression can cause platelet activation, including its aggregation and the development of acute coronary syndrome[71]. Depression in pathologies of the cardiovascular system after myocardial infarction was shown to be associated with the methylation status of minisatellite 5-HTTLPR, which is localized in the promoter of the serotonin transporter gene 5-HTT[72-73]. It was also shown that higher level of promoter methylation of 5-HTT in carriers of the protective genotype on allelic length polymorphism of 5-HTTLPR was associated with impaired expression of 5-HTT (reduction in mRNA level) and increased risk of mental disorders[74-77]. Our research also suggests the specific role of DNA methylation of minisatellites 5-HTTLPR and STin2 of 5HTT gene in the pathogenesis of coronary heart disease and myocardial infarction[78]. We found, that in patients with coronary heart disease and myocardial infarction, minisatellite 5-HTTLPR is only in the unmethylated state, whereas an increased frequency of methylated alleles for minisatellite STin2, localized in intron 2 of gene 5HTT, was found in patients with these diseases[78].The possible role of tandem repeats polymorphism of DRD4 and DAT genes may be associated with anxiety and risk of cardiovascular disease[79-82]. Therefore, the methylation status of minisatellite repeats of these genes may also be an indicator of the state of cardiovascular disease. Equally, the methylation status of minisatellite UPS29, localized in gene ACAP3, may reflect abnormalities in nerve cells in the pathology. This gene belongs to a family of genes that express metabolically important proteins involved in cell signaling, in cell survival and neurodegeneration, and probably connected with the development of neurological diseases[83-85]. In our preliminary studies we have analyzed whole-genome methylation and methylation of some tandem repeats of several genes in patients with coronary artery disease and myocardial infarct, which received appropriate treatment (unpublished data). These data together with cited data may serve as the basis for the proposed epigenetic approach. Developed survey takes into account analysis of family, lifestyle, ethnicity, and area of residence, diet, the presence of other pathologies, biochemical and cytological data upon hospitalization and after treatment in clinic. Such analysis will permit better understanding of mechanisms of cardiovascular diseases and to optimize treatment. Moreover, taking into consideration above mentioned interrelationship between cardiovascular and cerebrovascular diseases on the base of epigenomic and epigenetic mechanisms, epigenetic approach is expected to be able to significantly advance our current understanding of the etiology of cardiovascular and cerebrovascular diseases and can serve as powerful marker of the future risk of developing such diseases.

Conclusion Thus, determination of whole-genome DNA methylation for patients can be considered as new biomarker of cardiovascular diseases. Development of epigenetical approach in diagnostics, its expansion in study of methylation of several key genes, can help in screening, diagnostics and prognosis of various forms of CVD. To this end it is necessary to increase data set. Optimal strategy of using epigenetical and epigenomic markers has firstly to be consisting of determination of whole-genome DNA methylation, as this marker is connected with already known risk factors, but additionally precedes an appearance of pathology symptoms. To the next step it is necessary to study a level of methylation of separate genes and their regions (promoters, gene bodies, intrones) which are most likely connected with disease. In the past few years the development of epigenetical approach initiated the search for the new ways of cerebrovascular diseases’ treatment[9]. It seems that epigenetical approach will permit to adapt treatment personally for patients with cardiovascular diseases vs cerebrovascular diseases, as in both cases the vessels injury takes place. Epigenomic and/or epigenetic approaches are capable significantly improve current understanding of etiology of cardio- and cerebrovascular diseases, their interdependence. These markers can serve as predictive personalized markers of these diseases risk.

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[Date of reception:2016-03-14]

(Edited by: WANG Yan)

Foundation items: International Cooperation Project of China,France and Russia (87EZA0313397)

Corresponding Authors: Denis Dubrovin (1964-), male, professor. Research field: integrated Chinese medicine, Uighur medicine and Modern medicine development in complex diseases and syndromes. E-mail: silospradotm@yandex.ru

中图分类号：R29

文献标识码：A

文章编号：1009-5551(2016)08-1055-08

doi:10.3969/j.issn.1009-5551.2016.08.029

Biography: Eugeny Patkin (1944-), male, prof., PhD, Head of lab. of Molecular Cytogenetics. Research field: molecular genetics, molecular cytogenetics, epigenetics, DNA methylation, cardiovascular diseases, new biomarkers of susceptibility to CVD . E-mail :elp44@mail.ru.

·Silk Road Medicine Research·