Keywords

1 Background

Cardiovascular system responsible for transportation of nutrients as well as cellular waste products comprises of the heart, blood and blood vessels. The term cardiovascular diseases (CVDs) refer to any disease of cardiovascular system. CVDs inflict major burden on mortality worldwide making it global epidemic even after the advances made in their prevention and management. CVDs are not only public health issue but also accounts for massive societal costs as healthcare expenditure. They as a group of multifactorial disorders are associated with various genetic and acquired risk factors. But the known genetic and environmental influences cannot fully explain the variability in CVD risk among different populations contributing to the major road blocks in its treatment and prevention. Though thorough studies pertaining to pathophysiology, epidemiology, gene polymorphism, genetic linkage maps and various environmental stresses have paved a way for better diagnosis and treatment of cardiovascular medicine, but still there is lacuna in our understanding of interaction between environment and genome, in development of CVD. As inherited genome contribute to only a part of individual’s risk profile, ‘epi’genomics has emerged as a promising area of interest that can bridge gaps in our understanding pathophysiology to therapeutics of diseases. Epigenetic changes or modifications are the ‘heritable changes in the genome that does not include changes in DNA sequences’ which includes various mechanisms like DNA methylation, demethylation, histone modifications, RNA mediated alterations (microRNAs, circulatory RNAs, long non-coding RNAs) etc. Epigenetic mechanisms collectively empower the cell to quickly respond to environmental changes. Various established risk factors for CVD such as stress, pollution, smoking, nutrition, circadian rhythm, hypoxia etc. are associated with modifications in epigenetic markers. Therefore, the examination of these markers as regulators in CVD may emerge as early preventive and novel therapeutic approach. This book chapter therefore focuses on acquainting the vascular biology community with the advent filed of epigenetics and its role in cardiovascular medicine. The chapter consists of a brief introduction of cardiovascular disease, epigenetic mechanisms and evidences linking various epigenetic mechanisms with CVD. The main emphasize of this chapter would be on epigenetic regulator of CVDs to understand their translational potential and clinical reality in cardiovascular medicine.

2 Cardiovascular Diseases (CVDs)

CVDs are wide spectrum of disorders that comprises of abnormalities related to heart and blood vasculature. They include: Coronary artery diseases (CAD) like angina and myocardial infarction (heart attack), stroke, heart failure, coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease cardiomyopathy, heart arrhythmia, congenital heart disease, congenital heart disease, thromboembolic disorders and thrombosis. The underlying molecular mechanism pertaining to different CVDs vary considerably as majority of CVDs are regulated by multiple factors. For example, CAD and stroke involve atherosclerosis and rheumatic heart disease involves damage to heart muscle and valves caused by streptococcal bacteria. Even with different molecular mechanisms, nevertheless, most CVDs share common risk factors that are, high blood pressure, high blood cholesterol, diabetes mellitus, smoking, obesity, lack of exercise, alcohol consumption, hypoxia, to name a few.

CVDs are the leading cause of death globally accounting for most of annual mortality as compared to any other disease [1]. Over the period of last 100 years the prevalence of CVDs has increased tremendously and for the coming two decades it is likely to remain a major health issue even with sophisticated techniques and improved medical care [2,3,4]. The deaths caused by CVDs have increased over the course of time from 12.3 million (25.8%) in 1990 to 17.9 million (32.1%) in 2015 [5, 6]. In 2016, 31% of the global deaths that reached up to 17.9 million people were due to Different CVDs. Heart attack and stroke contributed to 85% of these deaths [7]. Studies estimate that 90% of the CVDs may be preventable [8, 9] by improving the known risk factors like hypertension, diabetes, smoking, alcohol consumption by healthy eating, avoidance of smoke and alcohol, exercise and appropriate medical counselling [10]. Thus, people who are at high cardiovascular risk need early detection and management in order to prevent the occurrence of disease.

The CVDs appeared as a huge public health hazard during 1940s, that triggered the research associated with the factors that influence occurrence of the disease. The research programmes like Framingham Heart Study were initiated to find out common genetic, biochemical, environmental and life style related factors that lead to occurrence or predisposition to various CVDs [11]. This resulted in identification of several conditions like obesity, hypertension and diabetes to detect individuals at risk of CVD and their early treatment. It in turn lead to significant decline in CVD mortality in USA and Western Europe over the past 3 decades. Therefore, it is important to understand the risk factors associated with CVDs.

2.1 Types of Cardiovascular Diseases

CVDs are group of disorders involving heart, blood vessels or both. Thus, they are characterised either as vascular diseases that majorly affects blood vasculature or heart diseases that majorly affects heart.

2.1.1 Vascular Diseases

The common vascular diseases are coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, stroke, thrombosis.

2.1.2 Heart Diseases

The common CVDs affecting heart are cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, inflammatory heart disease, valvular heart disease, congenital heart disease, rheumatic heart disease.

2.2 The Risk Factors Associated with CVDs

The most contributing risk factors for stroke and heart disease are genetic predisposition, unhealthy diet, age, sex, excessive use of alcohol, obesity, hypertension, diabetes, hyperlipidemia, stress, air pollution etc. Though it is very difficult to access the individual contribution of each risk factor among different ethnic group or populations, but the overall impact of these risk factors is quite consistent [12].

2.2.1 Genetic Predisposition

Most CVDs are multifactorial in nature i.e. there are several genes or genetic factors that contribute to development and progression of the disease. There are many identified single nucleotide polymorphisms (SNPs) make individual susceptible to the disease [13, 14]. Even with the sophisticated techniques the contribution of all genetic factors and their influence of vascular diseases is poorly understood.

2.2.2 Age

Age is one of the most important contributing risk factors towards the development of heart diseases, with each passing decade it triples the risk of disease [15]. The case studies show that 82% coronary heart patients that die are either of 65 years or older [15]. Similarly, the risk of death by stroke doubles after every 10 passing years after the age of 55 [17]. There are multiple theories that explain the influence of age on CVDs, one relates to increased serum cholesterol level with age [18] other with the changes in vascular wall [15]. Age decreases the arterial elasticity and reduced arterial compliance that subsequently causes coronary artery disease.

2.2.3 Sex

Gender influences the prevalence of heart diseases. Men are more susceptible than pre-menopausal women [16]. Middle aged men are at 2–5 times higher risk of coronary heart diseases at compare to women [19]. A study by World Health Organisation (WHO) reveals that sex contributes to roughly 40% difference in the mortality rate caused by coronary heart diseases [20]. The influence of gender in CVDs may be due to hormonal differences between men and women [20].

2.2.4 Excessive Use of Alcohol

The effect of alcohol consumption on CVDs is complex and depends largely on the quantity of alcohol consumed. The high level of drinking is directly related to susceptibility to the disease [21].

2.2.5 Obesity

Another risk factor for developing CVDs is being overweight or obesity. An unhealthy diet and physical inactivity lead to high body mass index (BMI), that falls outside the normal range making a person obese. This may lead to diseases like coronary heart disease and congestive heart failure [22].

2.2.6 Hypertension

High blood pressure or hypertension cause stress on blood vasculature making them weak or cause them to clog. It is a potent risk factor that can lead to atherosclerosis as it causes narrowing of blood vessels which make them more prone to be blocked by blood clots or fatty deposits [23].

2.2.7 Diabetes

High blood glucose levels as experienced in diabetes make individual more vulnerable for developing CVDs. The high levels of glucose have damaging effect on arterial walls and more likely cause the build-up of fat deposits called atheroma. These fat depositions if occur in coronary arteries may lead to coronary heart disease and heart attacks [24].

2.2.8 Hyperlipidemia

Hyperlipidemia is presence of abnormal level of lipids (fats) in blood stream and is one of the major contributory risk factors for CVDs. The high levels of “bad cholesterol” i.e. low-density lipoprotein (LDL) cholesterol is associated with range of heart diseases as it results in fatty substance deposition on the vasculature leading to various complications [23].

2.2.9 Diet

Diet plays a crucial role in prevention and development of CVDs. A diet rich in saturated fatty acids increases the risk of stroke and heart diseases. It is estimated that 11% of stroke and 31% of coronary heart diseases worldwide are caused by high dietary saturated fatty acids [23].

2.2.10 Air Pollution

The effect of particulate matter present in the air for its short- and long-term exposure on CVDs has been accessed. It was found in recent studies that the long-term exposure of PM2.5 results in increased 8–18% CVD mortality risk [24].

The majority of above-mentioned CVD risk factors are manageable and can be reduced by opting alternative lifestyle. For example, reduction of alcohol consumption, fatty food, salt, regular physical exercise, consumption of vegetables and fruits reduce the risk of CVDs drastically. In addition, the treatment of hypertension, diabetes and hyperlipidemia is essential for reducing the risk of heart diseases. Thus, there are choices by which people can adopt and sustain healthy way of living, but we need health policies and conducive environments that can make these choices affordable and available to masses. The other major forces driving occurrence of CVDs are stress, poverty, urbanization, socio-economic and cultural changes. With all the knowledge on contributing factors and advancement of genomic era, we still fail to answer the variability in CVD risk, role of individual risk factors, their cumulative effect. Also, the clinical utility of identified genetic markers has been limited in both prevention and prediction of diseases [25].With the translational disappointment of genomics field, the vascular community is now exploiting epigenomics as new avenue for assessing risk stratification, preventive measures and treatment. Epigenetics is the heritable changes in the genome that are not coded by the DNA sequence [26]. There are three broad mechanisms that define epigenetic changes: DNA methylation, post- translational histone modifications and RNA based mechanisms (microRNAs, circular RNAs and non-coding RNAs) [27]. DNA methylation is the most common epigenetic modification in the mammalian genome [28, 29]. The recent studies though suggest that all the three epigenetic modifications contribute to the pathogenesis of CVDs [30]. The international projects like Human Epigenome Project (HEP) and International Human Epigenome Consortium (IHEC) have been initiated to study and catalogue human epigenome along with correlation of its pathophysiology with several diseases including CVDs [31].The focus of this chapter is epigenetics in CVDs, and will be discussed under the following sections.

3 Leads from Genetics

Almost three decades ago the research on the genetic factors and genes that predispose an individual to CVDs has started. It was thought that the SNPs and mutations in the genome could be analogous to already known CVD risk factors. It was anticipated that they could be included in risk assessment model like Framingham score [30] in order to calculate and determine the risk of an individual to develop CVD and adopt apt preventive therapeutic approaches. The traditional approach was to study the role of candidate gene as risk factor, but now the genome-wide association studies grouped with availability of large patient cohorts are used to uncover the novel genes and genetic factors that act like risk factors for various CVDs [31]. The genetic research has enabled the cardiovascular community to better understand the disease origin and its pathophysiology. It has progressively also helped in the risk stratification, direct and indirect diagnosis and therapeutic interventions. The novel GWAS has widened the understanding of pathophysiology of various CVDs. The genetic studies have also led to discovery of new drug targets that may considerably improve the current therapeutic approaches for CVDs. The genotyping studies have been used for diagnostic and prognostic assessment by clinicians. But still genetic studies are unable to find the “holy grail” for diagnosis of CVDs. So, there is a need of more profound knowledge that can answer dynamics of disease. ‘Epi’genetics promises to deepen the present insights as it can well explain the influence of environment on the genome. It can unveil the interaction between the genetic variants and environment changes; thus, can elucidate the clinical drug responsiveness and patient outcome.

4 Epigenetic Revolutions – Genomics to Post-Genomic Era

Epigenetics has emerged as a post genomic era that can reveal the lacuna in our understanding of inheritance of common traits. It provides the link between environment and its effect on genome, thus deciphering the mechanism by which cells are able to respond quickly to environmental stimuli. The phenotypic variations seen in humans that cannot be solely defined by genotype, can be better explained in the light of epigenetic modifications [32]. Epigenetic mechanisms are stable cellular memory, which let the propagation of gene activities generation after generation without the changes in the DNA sequence. There are three broad mechanisms that define epigenetic changes: DNA methylation, post- translational histone modifications and RNA based mechanisms (microRNAs, circular RNAs and long non-coding RNAs). The simple and static evaluation of the genetic code has limited the understanding of genetic influence on CVDs. In this respect, epigenetics as a dynamic mechanism can explain and modify the genome’s functionality under the influence of exogenous stimuli or environment condition. This would in turn help in identification of novel targets and mechanism of gene regulation with considerable acquisitions in CVD knowledge related to genetic risk and pathophysiology [33,34,35]. The environmental changes like pollution, diet, stress etc. leads to epigenetic modifications and impact the susceptibility to CVD (Fig. 6.1). There are reports that validate the role of epigenetic in various processes underlying CVDs like hypertension, inflammation and atherosclerosis [35].

Fig. 6.1
figure 1

Epigenetic modifications: The figure depicts the three most common epigenetic modifications that occur in mammalian genome namely, histone modification and chromatin remodelling, DNA methylation and modifications mediated by non-coding RNA (ncRNA) family like miRNA, circRNA and lncRNA. All these modifications are interlinked and enable cells to quickly respond to changing environment. The established risk factors of CVD like stress, pollution, age, diet, pollution etc. can act as environmental stimuli and triggers epigenetic modifications resulting in release of various regulatory molecules that can be used as biomarkers. The changes also influence the CVD outcomes

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The following sections of the chapters will deal with different epigenetic modifications and their role in progression, prevention, treatment and diagnosis of CVDs.

4.1 DNA Methylation

DNA methylation is the addition of the methyl group at the cytosine bases of the dinucleotide CpGs of eukaryotic DNA. As a result they are converted to 5-methylycytosine by de novo DNA methyltransferase (DNMT) enzymes such as DNMT1, DNMT3A and DNMT3B. In mammals, methylation is found sparsely but globally with the exception of CpG islands. Vertebrate CpG islands are short interspersed DNA sequences (>500 bp) generally found in the 5′ end of the gene that deviate significantly from the average genomic pattern by being GC-rich (G + C percentage greater than 55%), CpG rich (observed CpG/expected CpG of 0.65) and predominantly non-methylated [36].

Out of the several ways, the methylation of DNA is one of the most common ways of regulating gene expressions in eukaryotes [37]. It has emerged as an important process in numerous cellular processes like genomic imprinting, embryonic development, X-chromosome inactivation and many more. The first clue of the role of methylation in gene expression was provided by 5-azacytidine experiments in mouse studies [38]. The integration of 5-azacytidine in the growing strand of DNA severely inhibited the actions of DNMT enzymes to normally methylate DNA. Therefore, the comparisons of the cells before and after the treatment of 5-azacytidine allowed to see the impact of loss of methylation on gene expression [39]. The exact role of methylation in gene expression is unknown, perhaps it play a crucial role in repressing gene expression by blocking the promoters at which activating transcription factors bind. Approximately, 70% of annotated gene promoters are associated with CpG islands, making it the most common promoter type in the genome [36]. Not all CpG islands found are associated with the promoter. Recent works have found a large class of islands that are remote from the transcription start sites (TSSs) but still show evidence for the promoter function. The lack of CpG dinucleotides in the vertebrate genome except the CpG island is thought to be due to the loss of genomic CpGs due to deamination of methylated sequences [39].

Given the critical role of DNA methylation in gene expression and cell differentiation, it seems obvious that the errors in methylation could give rise to a number of devastating consequences, including various diseases. As a result, a growing number of human diseases have been found to be associated with aberrant DNA methylation [40]. The methylation of the promoter region bearing transcriptional start sites of many genes encoding tumor suppressors such as tumor protein p53, retinoblastoma-associated protein 1, tumor protein p16, breast cancer 1 and many more resulting in the reduced expression of these genes have been found in a large number of cancers like retinoblastoma, colon, lung and ovarian [40, 41]. 5-methylcytosine (5-mC) is spontaneously converted to thymine by deamination and is thought to be responsible for about one-third of all disease-causing mutations in the germline [39]. The mechanisms for establishing, maintaining and removing the methyl group are dependent on nucleosomal DNA and the histone modifications within the nucleosome [39]. Global methylation studies are the first ever epigenetics studies in the area of CVD research. The outcome of these studies though were conflicting as some studies highlighted decreased global DNA methylations with CVDs [40] while some associated increased global methylations with these disorders [41, 42]. Coronary heart disease have been associated with elevated homocystein and increased methylations [43]. DNA methylation studies include investigation of repetitive sequences across the genome such as Alu elements and long interspersed nucleotide element-1 (LINE)-1. Hypomethylation of LINE-1 was associated with cardiovascular risk factors such as higher serum vascular cell adhesion molecule [44] and higher low-density lipoprotein (LDL) and lower high-density lipoprotein (HDL) cholesterol [47]. LINE-1 hypomethylation has also been associated with higher prevalence of metabolic syndrome, and therefore elevated risk for CVD [48]. There are several genes responsible for DNA methylation and investigation of these candidate genes demonstrated some interesting results. Fat mass and obesity-associated protein (FTO), a prominent obesity-associated gene has been linked to levels of DNA methylation [49] and CVD risk independently of its effect on body mass index [50].Similarly, other candidate genes such as F2R Like Thrombin Or Trypsin Receptor 3, Insulin, GNAS Antisense RNA 1, Phospholipase A2 Group VII, Insulin Like Growth Factor 2, to name a few has been associated with various CVDs [51,52,53,54]. With the advent of whole epigenome array several markers and novel distinct patterns of DNA methylations have been identified in the context of CVDs [55,56,57,58]. The outcome of all these studies could result in the novel therapeutic interventions in cardiovascular diseases.

4.2 Post-translation Histone Modifications and Chromatin Remodelling

Histone modification is a covalent post-translational modification, which includes methylation, phosphorylation, acetylation, sumoylation and ubiquitylation, to histone proteins. These modifications are known to play an important role in replication, transcription, heterochromatin formation, chromatin compaction, and DNA damage repair. Investigation of histone modifications in CVDs reveals the crucial role of histone deacetylases (HDACs). It is the most extensively studied family of histone-modifying enzymes in the cardiovascular system [59]. Studies have demonstrated the effect of its inhibition in attenuating hypertension [60] and in prevention of proliferation of vascular smooth muscle cells [61, 62]. Class III HDACs also known as sirtuins have been shown to participate in cardiac hypertrophy and myocardial ischemia [63]. The other class of enzyme, histone acetyl transferase has been associated in the settings of atherosclerosis through regulation of genes that inhibit endothelial cell inflammation [64]. Similarly, the loss-of-function studies of histone methyltransferase in the adult heart showed hypertrophy, dilation, and derepression of some cardiac disease genes [65]. The polycomb repressive complex, one of the best-studied gene silencing complexes, has been implicated in a wide variety of phenotypes in the cardiovascular system. They have been shown to be differentially involved in cardiac development and regeneration [66].

4.3 RNA Based Modifications (miRNAs, circRNAs, lncRNAs)

The RNA based modifications include microRNAs (miRNAs), circular RNAs (circRNAs) and recently discovered long non-coding RNAs (lncRNAs). They all are non-coding endogenous RNAs that regulate the genome without being translated into proteins. The recent studies show the implication of these non-coding RNAs in CVD pathophysiology. There is large number of growing evidences that show role of miRNAs and lncRNAs in both animal and cellular models of CVD. In the following sections, we will be studying the role of these non-coding RNAs in CVDs and how they can be used as either biomarker or for treatment in CVDs.

4.3.1 miRNAs in CVD Scenario

miRNAs are class of endogenous interfering RNAs that are coded by human genome. Mature miRNAs are 20–25 nucleotide long RNA sequence that are synthesised in canonical pathway from a large RNA precursor, pri-miRNA. In the nucleus pri-miRNA is transcribed and matured by RNA polymerase II. It is then subsequently cleaved by RNase III Drosha and associated protein, giving a 60–70 nucleotide long pre-miRNA, which is released into the cytoplasm. In cytoplasm it is cleaved by RNaseIII Dicer resulting in 21–25 nucleotide long double stranded miRNA/miRNA∗ duplex. This duplex is unwounded by RNA induced-silencing complex (RISC), which carries mature single stranded miRNA to target messenger RNA causing subsequent gene silencing. Thus, miRNAs decrease the expression of gene by binding to them and causing translational repression [67]. Non-coding RNAs like miRNAs have emerged as prime candidates for discovering novel biomarkers for CVDs. As there is correlation between the pathologies and blood miRNA levels, miRNAscan be great tool for being non-invasive biomarkers. The general hypothesis is that miRNAs would be differentially expressed in people having CVD and are at risk of CVD and in normal population. So, plasma miRNA levels can be promising avenue for evaluating early CVD risk and patient outcome, along with assessing individual patient response towards various surgical procedures or treatment. The pioneering work in this field is done by Dimmeler et al., they highlighted the change in serum miRNA levels of CAD patients Vs. control [42]. There are other studies that have shown miRNAs as potential biomarkers to predict CVD outcome. For instance, the work done by Seronde et al. has shown that low serum levels of miR-423-5p are associated with a poor long-term outcome in acute heart failure patients, emphasising the role of miR-423-5p as a prognostic biomarker for predicting acute heart failure [43]. The cardiovascular deaths caused by acute coronary syndrome were precisely predicted by the sericlevels of miR-132, miR-140-3p, and miR-210 in the study consisting of 1114 patients [44]. Several miRNAs are very sensitive to even small exogenous stimuli like changes in blood pressure that can be predictive of circulatory stress or hypertension (established risk factor for CVD) [45]. miR-22 is a potential biomarker candidate for predicting CVD in elderly patients asmiR-22 regulates cardiac autophagy specially in myocardium of elderly patients thus circulating levels this miRNA gives prognostic clue on eventual progression of disease ultimately leading to heart failure in elderly [46]. Regular exercise can reduce the outcomes of CVDs and several miRNAs have been proposed as biomarkers to monitor physiological effect of regular exercise in different populations. For example, decreased level of miR-146a andmiR-221, and increased level of miR-149 are seen after acute exercise [47]. Whereas the altered level of expression of miR-1, miR-133, and miR-206 is seen after the endurance exercise [48]. A recent study has highlighted the role of miR-145 in impeding the thrombus formation in vivo by targeting tissue factor in the case of venous thrombosis [49].

4.3.2 circRNAs in CVD Scenario

circular RNAs are single stranded RNAs that form a covalently closed continuous loop. They are expressed in mammalian tissues as transcriptional and translational regulators [50]. Just like miRNAs, circRNAs are stable and detectable in blood stream, making it possible to use them as non-invasive biomarker. It is seen that in aged human hearts the level of circAmotl1decreases dramatically [51]. The risk of atherosclerotic vascular diseases is correlated with the expression of the increased expression circANRILin CAD patients [52]. The diagnostic biomarkers for various CVDs identified in peripheral blood mononuclearcells (PBMCs) are Hsa_circ_0005836, hsa_circRNA_025016, andhsa_circ_0124644 [53, 54].

4.3.3 Long Non-coding (lnc)-RNAs in CVD Scenario

lncRNAs are the RNA sequences that exceed more than 200 nucleotides and don’t code for any functional transcript. They differ from other non-coding RNAs like miRNAs as they are poorly conserved across the species also, they regulate gene expression at both transcription and post-transcriptional level. They allow microRNAs’ target mRNA escape degradation as they act as decoy for microRNA [55]. The advancement in sequencing techniques like deep sequencing has made it possible to study the lncRNA profiles in different CVDs as well as normal healthy population. And it is found that lncRNAs regulate multiple biological pathways in aging and cardiac development [56]. The 9 lncRNAs namely CDKN2BAS1/ANRIL, RMRP, RNY5, SOX2-OT, SRA1 EGOT, H19, HOTAIR, and LOC285194/TUSC7 were significantly modulated in non-ischemic myocardial biopsies of patients suffering from heart failure and dilated ischemic cardiomyopathy. Also, in mouse model the expression level of RMRP, H19, and HOTAIRlncRNAs were upregulated when measured in hypertrophic heart sample [57]. MIAT (myocardial infarction-associated transcript) and SENCR (smooth muscle and endothelial cell enriched migration/differentiation-associated lncRNA), are other lncRNA markers that are associated with dysfunction and myocardial infarction [58, 59]. Nodal lncRNAs act as key regulators of cardiomyocte cell cycle as revealed by the data of single cardiomyocyte nuclear transcriptome analysis of normal and failing heart [60]. Interestingly, the severity of fibrosis in human diseased heart is directly correlated with a cardiac fibroblast enriched lncRNA- Wisper (Wisp2 super enhancer-associated RNA) [61]. Growing evidences show that majority of lncRNAs are localised in nucleus and are capable of triggering chromatin remodelling by recruitment of various epigenetic factors, thereby causing activation or repression of genes. The genes involved in cardiac hypertrophy are regulated by a cardiac-enriched lncRNA via its direct interaction with catalytic subunit of PRC2 causing inhibition of methylation of histone H3 lysine 27 [62].

At last we can conclude that non-coding RNAs play a role in controlling gene expression and shaping genome organisation. With increasingly more functions and roles of non-coding RNAs being discovered, we need to use better biochemical approaches along with deep sequencing analysis coupled with novel bioinformatics strategies so that a comprehensive understanding of their role in CVDs can be provided.

5 Perspective

The role of epigenetic alterations in the capacity of DNA methylation, chromatin remodelling and non coding-RNAs has been emerging in the development of CVDs. However, the results of epigenetic studies are inconsistent and contradictory. Epigenetic phenomenon, as we know, is not a stable or heritable event. It is dynamic in the sense that it gets altered by environmental factors and can vary with time. These all features have to be taken into considerations while designing the studies for any type of epigenetics research. Not just the nature of samples and the conditions they are exposed to are important but the numbers of times the samples are taken are equally critical. There are various upcoming combinatorial technologies through which researchers can explore epigenetic landscapes. Bisulfite-sequencing combined to ChIP, de novo methylation simultaneously examining nucleosome occupancy and CpG methylation can be utilized for studying multiple epigenetic marks simultaneously. Research in epigenetics is a relatively new approach but it has a remarkable potential to identify new biomarkers in the CVD field. These biomarkers would not be just helpful in CVD diagnosis, outcome, prognosis and treatment but could lead us to new avenues for novel targeted CVD therapies. However, more epigenomic studies are warranted that will help to decipher the complex link between genetics, epigenetics, and CVDs.