Epigenetic control of natriuretic peptides: implications for health and disease

Abstract

The natriuretic peptides (NPs) family, including a class of hormones and their receptors, is largely known for its beneficial effects within the cardiovascular system to preserve regular functions and health. The concentration level of each component of the family is of crucial importance to guarantee a proper control of both systemic and local cardiovascular functions. A fine equilibrium between gene expression, protein secretion and clearance is needed to achieve the final optimal level of NPs. To this aim, the regulation of gene expression and translation plays a key role. In this regard, we know the existence of fine regulatory mechanisms, the so-called epigenetic mechanisms, which target many genes at either the promoter or the 3′UTR region to inhibit or activate their expression. The gene encoding ANP (NPPA) is regulated by histone modifications, DNA methylation, distinct microRNAs and a natural antisense transcript (NPPA-AS1) with consequent implications for both health and disease conditions. Notably, ANP modulates microRNAs on its own. Histone modifications of BNP gene (NPPB) are associated with several cardiomyopathies. The proBNP processing is regulated by miR30-GALNT1/2 axis. Among other components of the NPs family, CORIN, NPRA, NPRC and NEP may undergo epigenetic regulation. A better understanding of the epigenetic control of the NPs family will allow to gain more insights on the pathological basis of common cardiovascular diseases and to identify novel therapeutic targets. The present review article aims to discuss the major achievements obtained so far with studies on the epigenetic modulation of the NPs family.

Introduction

The natriuretic peptides (NPs) family includes two circulating hormones (atrial and brain natriuretic peptides, ANP and BNP) produced by cardiac cells, and one local hormone (C-type natriuretic peptide, CNP) produced by the endothelium [1].

ANP derives from a gene (NPPA) located in the distal arm of chromosome 1(1p36.2). Upon synthesis, following cleavage of the pre-signal, the pro-hormone (proANP_1–126) is released into the cytosol and conserved within granules. When needed, proANP is further processed by corin (a cardiac transmembrane serine protease) and released into the circulation in the forms of ANP_1–98 and ANP_99–126 [2, 3]. The latter is the biological active peptide that undergoes clearance after few minutes from its secretion, mostly due to neprilysin (NEP) enzymatic activity and through internalization by the type C natriuretic peptide receptor (NPRC). ANP is secreted by atrial myocytes in response to volume or pressure overload. By acting through the type A natriuretic peptide receptor (NPRA), it plays diuretic, natriuretic and vasorelaxant effects and is a major player in the control of cardiovascular homeostasis [4]. It also contributes to vascular remodeling by performing anti-proliferative effects on cardiac, endothelial and smooth muscle cells through the same receptor [4]. Due to its key relevance within the cardiovascular system, functional derangements dependent on circulating peptide level or peptide structure are associated with several pathological conditions [5].

Brain natriuretic peptide (BNP) is secreted by ventricular myocytes in response to hemodynamic stress. BNP derives from a gene (NPPB) located nearby NPPA. The pro-hormone (proBNP_1–126) is further processed by furin into the BNP_1–76 and BNP_77–108 circulating peptides [2]. The clearance of BNP takes place through NEP and NPRC, similarly to ANP. BNP exerts, through NPRA, systemic functions (diuresis, natriuresis, vasorelaxation) and autocrine/paracrine functions, such as anti-hypertrophic and anti-fibrotic properties, contributing to both cardiovascular homeostasis and the remodeling process within the cardiovascular system [5]. Remarkably, lack of BNP leads to a relevant cardiac fibrosis [6]. Most importantly, BNP has been assessed as a key marker for establishing diagnosis and prognosis of heart failure patients and to monitor the course of the disease [7].

The circulating ANP and BNP levels are the result of a balance between gene regulation, peptide release and clearance. As expected, the regulation of NPPA and NPPB expression is a crucial step for the maintenance of the cardiovascular functions. Increased volume overload and myocyte stress are the main known determinants of the ANP and BNP synthesis and release [8]. Additional factors able to control NPs secretion are angiotensin II, endothelin-1 and phenylephrine, as part of a neuroendocrine control [8], and several growth factors and cytokines [3].

Novel mechanisms of gene regulation have been discovered, the so-called epigenetic mechanisms. The term epigenetic defines changes in gene expression that are not explained by changes in DNA sequence [9]. In these circumstances, alterations related to packaging and/or translation of genetic information explain the gene expression regulation [10]. These mechanisms include DNA methylation, histone modifications and RNA-based mechanisms regulating gene silencing or activation. DNA methylation, performed by DNA methyltransferase (DNMT) enzymes, is generally associated to gene silencing by hampering the accessibility of cis-DNA binding elements present in the promoter regions of genes to the transcription factors [11]. Acetylation of histones, mediated by histone acetyltransferases (HATs), increases gene expression; in turn, histone deacetylation, mediated by histone deacetylases (HDACs), inhibits gene expression. A deregulation of the equilibrium between the activities of these two types of enzymes leads to several pathological conditions [12]. Methylation of histones is mediated by histone methyltransferases (HMTs) and histone demethylases (HDMs) [13]. Unlike acetylation, histone methylation can induce either activation or repression of gene expression depending on the target sites and the degree of methylation [14]. The RNA-based mechanisms include the non-coding RNAs: microRNAs (miRNAs), long noncoding RNAs (LncRNAs), small interfering RNAs and Piwi-interacting RNAs.

The discovery of these new mechanisms of gene regulation has brought novel insights into our understanding of physiological and pathological processes underlying both health and disease conditions [15]. Interestingly, the epigenetic mechanisms have also been reported to modulate hormones of the cardiovascular system, including components of the NPs family. In the specific, it was reported that an interplay between multiple regulatory elements and dynamic epigenetic states distributed across a 60-kb regulatory domain centered around NPPB regulates NPs gene expression in development and under stress conditions, such as during pressure overload [16]. Thus, NPPA and NPPB are frequent targets of epigenetic mechanisms. In addition, NPRA, CORIN, NPRC and NEP may undergo epigenetic regulation.

The present article provides an overview of current knowledge on the epigenetic regulation of the main components of the NPs family, and its involvement in physiological and pathological cardiovascular conditions.

Epigenetic regulation of NPPA and NPPB by histone modifications

NPPA and NPPB descend from a common ancestral gene by duplication and share common chromatin-regulatory mechanisms [17]. Both NPPA and NPPB are abundantly expressed in the atrial and ventricular myocardium during embryonic and fetal stages. After birth, both genes remain expressed in the heart. However, postnatal expression of NPPA is strongly downregulated in the ventricles [18].

A reactivation of the fetal gene program takes place in response to mechanical stretch. Hence, the ventricular expression of both NPPA and NPPB is strongly increased in the cardiomyocytes, the pro-peptides are released by the heart and their level is increased into the circulation to protect from stress stimuli. In pathological states, including cardiac hypertrophy and heart failure, epigenetic changes, such as histone acetylation and methylation, occur in adult cardiomyocytes (Fig. 1a, b). A genome wide analysis of the epigenetic signature of hypertrophied hearts of mice showed that multiple genes implicated in hypertrophic cardiomyopathy and associated enhancers were modified through histone-3 lysine-27 acetylation (H3K27ac), a modification associated with gene activation [19]. An increased acetylation of H3 and H4 histones at both NPPA and NPPB is associated with increased pressure overload in the left ventricle [20]. Moreover, the histone demethylase JMJD2A has been shown to modulate NPPB in angiotensin II and endothelin 1 induced hypertrophy in human induced pluripotent stem cells [21].

Fig. 1.

NPPA/NPPB histone modifications and DNA methylation. Schematic representation of changes in histone acetylation (a) and in histone/DNA methylation (b) occurring at NPPA/NPPB during cardiac pathological conditions. As shown, the epigenetic modulation of both genes leads to an increase of their expression and of the circulating atrial and brain natriuretic peptides (ANP and BNP) levels. The latter are required to protect the cardiac function in cardiomyopathies. HDAC inhibition attenuates development of hypertrophy and fibrosis while reducing the NPPA and NPPB expression level and promoter activity (a). LVAD is able to modulate the expression of NPPA/NPPB, in association with a modulation of the epigenetic state, and to improve cardiac function (b). H3-4, histone 3 and 4; H3K9, lysine 4 on H3; H3K9, lysine 9 on H3; H3K27, lysine 27 on H3; H3K4me3, Tri-methylation of lysine 4 on H3; H3K9me2-3, di- and tri- methylation of lysine 9 on H3; HDAC (histone deacetylase); LVAD, left ventricular assist device; NPPA, natriuretic peptide A gene; NPPB, natriuretic peptide B gene; p300, histone acetyltransferase. See text for details

In patients with heart failure, the reactivation of NPPA and NPPB (as part of the reactivation of fetal gene program) is correlated with demethylation of H3K9 at their promoter regions, although a modest increase in H3K27ac could also be observed [17]. The cofactor p300, a transcriptional coactivator that is important in acetylation of histones, is involved in the hypertrophic response in cardiac myocytes [22] and promotes cardiac remodeling in infarcted mouse hearts through an interaction with the transcription factor GATA4 [23]. Remarkably, p300 was found to be recruited to the NPPA and NPPB promoter that is associated with increased histone acetylation such as H3K27ac [24].

Epigenetic mechanisms are involved in chronic heart failure caused by dilated cardiomyopathy. Here, transcriptional regulation of NPPA and NPPB is associated with DNA hypomethylation of 5′-flanking cytosine-phosphate-guanine (CpGs) islands. As a consequence, an upregulation of the ANP and BNP transcripts is observed. In this condition, the same direction of dysmethylation was detected in cardiac tissue and in peripheral blood [25].

In a study performed in patients affected by advanced heart failure related to non-ischemic dilated cardiomyopathy, three histone methylation–related molecules (H3K4me3, H3K9me2, and H3K9me3) were found to be less expressed in the left ventricle as compared with normal left ventricle. In these patients, cardiac function was substantially recovered by left ventricular assist device (LVAD) support in association with a reverse expression of NPPA and NPPB related to the cardiac disease. Interestingly, LVAD support modulated gene expression of cardiac myocytes and improved global function in association with a modulation of the epigenetic state. In fact, the expression of NPPA and NPPB was negatively correlated with that of H3K4me3, H3K9me2 and H3K9me3 in the LVAD-supported left ventricle [26]. This evidence provides an excellent demonstration of the role that modulation of epigenetic changes may have for a valid therapeutic approach.

In line with the previous observation, it was reported that HDAC inhibition prevented cardiac hypertrophy and attenuated the expression level and promoter activity of NPPA [27]. The HDAC inhibitor trichostatin A reduced cardiac hypertrophy through the acetylation and deacetylation of target genes and blunted the increase of NPPA and NPPB expression induced by pressure overload [28]. Moreover, HDAC inhibition improved cardiac function through a direct anti-fibrotic activity in an experimental model of heart failure [29] and in a model of hypertension [30]. A reduced expression of NPPA was confirmed in the latter experimental setting.

Histone modifications were reported in association with the development of other cardiomyopathies. A recent study analyzed the role of histone acetylation of NPs in diabetic cardiomyopathy and nephropathy [31]. In this study, a significant increase of histone acetylation at promoter regions of NPPA, NPPB and NEP was found in the heart and kidney of diabetic rats, suggesting that inhibitors of histone acetylation can be developed as therapeutic targets against the diabetic cardiomyopathy and nephropathy.

Of note, it is known that cardiac chambers show specific distinct gene expression profiles [32]. This evidence goes along with the demonstration that the two ventricular cavities are epigenetically different due to their distinct pattern of acetylation [25]. In fact, NPPA and NPPB expression in the left ventricle is associated with increased H3 and H4 histones hyperacetylation. The close correlation between the increased histone acetylation and changes in genes expression is likely due to the recruitment of the p300 histone acetyltransferase in the NPPA and NPPB promoters in the left ventricular chamber [24].

Histones modifications of NPPA and NPPB in other conditions

It is interesting to note that epigenetic mechanisms regulating NPPA expression have been identified in other situations, such as smoking and alcohol dependence. An increased DNA methylation of NPPA promoter was reported during withdrawal from smoking in tobacco-dependent subjects as compared to controls, despite the lack of any difference at baseline condition between the two groups [33]. A significant reduction of promoter-related DNA methylation of NPPA was reported during alcohol detoxification [34]. Similarly, a GATA4 methylation of NPPB was found during alcohol withdrawal and it significantly increased during detoxification. In parallel, the BNP level was significantly decreased [35]. The impaired regulation of NPs during withdrawal from smoking habit or alcohol dependence may have implications related to the modulation of the hypothalamic–pituitary–adrenal axis by this hormonal system [36].

Finally, significantly lower level of NPPA mRNA along with a hypermethylation status of NPPA promoter was found in patients with eating disorders (bulimia), likely contributing to the altered homeostasis of patients with anorexia or bulimia nervosa [37].

MicroRNAs-dependent NPPA regulation

The miRNAs are a group of small single-stranded non-coding RNAs composed by 18 to 22 nucleotides and they act by the recognition of specific mRNA targets within the 3′-untraslated region (3′-UTR) to induce either the degradation or the repression of the gene translation [38]. Due to their ability to suppress gene translation, miRNAs are involved in many biological processes including cell proliferation and differentiation, cell apoptosis and death. They can be dysregulated in several pathological conditions including cardiovascular diseases [39].

It is known that mRNAs tipically contain multiple predictive miRNA-binding sites. Binding of different miRNAs to the same mRNA may either independently or synergistically regulate the expression of any given gene. With regard to NPPA, three miRNAs are known to modulate its expression: miR-425, miR-155, miR-105 [40–42]. The miR-425 was the first one to be characterized for its ability to target NPPA [40] (Fig. 2a). This miR interacts with the single nucleotide polymorphism (SNP) rs5068 major allele (A), but not with the mutant allele (G) at this site [40]. The impact of miR-425 on ANP regulation was characterized in individuals exposed to either low or high salt diet. The subjects carrying the mutant G allele, who are resistant to the downregulation of ANP gene by miR-425, maintained higher ANP levels, as compared to carrier of the wild type allele, under either low or high salt diet [40]. Moreover, higher levels of miR-425 are present in blacks contributing to the known state of ANP deficiency of this race [43]. In fact, in response to a high-carbohydrate challenge, which is known to stimulate miR-425 and to reduce ANP level, the MRproANP plasma level decreased by 34% in whites and only by 23% in blacks [43]. Consistently with these findings, subsequent observations obtained in different cohorts of the general population underlined a protective effect of the rs5068 minor allele toward hypertension and metabolic syndrome occurrence [44]. In particular, the minor G allele at rs5068 was related to higher plasma level of ANP, lower blood pressure level and consequent lower risk of hypertension in general community-cohorts of whites from the United States and the Northern Europe [45]. In addition, carriers of rs5068 G minor allele showed lower body mass index and waist circumference, lower prevalence of obesity and metabolic syndrome, higher plasma high-density lipoprotein cholesterol and lower C-reactive protein levels in a North-American population [46]. The lower occurrence of a cardio-metabolic phenotype was replicated in a general community from the Mediterranean area [47]. Interestingly, the lower prevalence of diabetes and metabolic syndrome and of abnormalities of lipid metabolism was confirmed in carriers of the G minor allele from other countries and ethnicity (Northern Europeans [48], and African-Americans [49]). It is recognized that the protective factor of these individuals is represented by the higher circulating ANP level due to the rs5068 minor allele. In fact, the relevant metabolic phenotypes observed in the above mentioned studies are consistent with the known lipolytic and anti-inflammatory effects of ANP [50].

Fig. 2.

MicroRNAs involved in NPPA regulation and BNP processing. a microRNA-425 (miR-425), microRNA-155 (miR-155) and microRNA-105 (miR-105) reduce NPPA expression. These miRNAs are unable to regulate levels of ANP in the presence of specific genetic variants at their binding sites (rs5068, rs61764044). b Cardiomyocytes of failing hearts show decreased expression level of microRNA-30 (miR-30) resulting in the increase of GalNac-transferase (GALNT) 1/2 expression, consequent increase of proBNP glycosylation and decrease of proBNP processing. See text for further details

The miR-155 interacts with the SNP rs61764044 major allele of NPPA in human cardiomyocytes [42]. The combined action of miR-425 and miR-155 enhances the downregulation of NPPA expression [51]. The miR-155 is highly expressed in linfocytes, monocytes, macrophages, and is involved in the processes of immunity and inflammation. It plays a role in several pathological conditions [52, 53]. In fact, its inhibition protects from cerebral ischemia and attenuates the myocardial hypoxia-reoxygenation injury [54, 55].

The miR-105 interacts with NPPA 3′ UTR at the SNP rs61764044 in an allele-specific manner; in fact, it binds the wild type but not the mutant allele at this site (Fig. 2a) [42].

Contrarily to the miR-425, a demonstration of a tight relationship between miR-155 and miR-105 with NPPA regulation in specific pathological conditions is still lacking.

In summary, subjects carrying the minor allele at the binding sites of all three miRNAs mentioned above are resistant to the NPPA epigenetic regulation. Based on the experience gained with miR-425, we can expect that targeting these miRNAs with anti-miRs (mimicking the effect of a single base-pair variant at the miRNA binding site) may represent an attractive strategy to increase ANP level in the circulation and to achieve a better control of cardiac function, of blood pressure level and of metabolic parameters in the context of heart failure and of hypertension.

MicroRNAs as mediators of ANP functions

Almost a decade ago, a paper by Kotlo et al. [56] reported about the ability of ANP to exert some of its local functions in vascular smooth muscle cells (VSMCs) through the modulation of few miRNAs (miRs-21, -26b, -98, and -1826). In the specific, it was found that miR-21 was downregulated by ANP through the guanylyl cyclase pathway. Since this miR can promote contraction and proliferation of VSMCs [57, 58], it is reasonable to speculate that ANP exerts, at least in part, vasorelaxant and anti-proliferative effects in VSMCs through a partial downregulation of miR-21. Later on, we had the opportunity to investigate the deleterious effects of a common ANP variant (T2238C, with 14% frequency in the general population) in VSMCs in vitro and we found that the CC2238/ANP mutant peptide suppressed miR-21 expression along with consistent changes of its molecular targets (PDCD4, PTEN, Bcl2) [59]. As a result, CC2238/ANP reduced cell viability and increased apoptosis and necrosis, thus further supporting the proliferative role of miR-21 in VSMCs. Of note, opposite to the wild type ANP, the CC2238/ANP exerted these effects through the cAMP/PKA/CREB pathway driven by NPR-C activation [59]. Both NPRC gene silencing and miR-21 overexpression allowed the recovery of the normal condition in the cell line under study [59]. In a subsequent work, we described the downregulation of Apolipoprotein-E by the CC2238/ANP through Egr1-miR-199a activation in VSMCs, with consequent negative effects on cell viability and function [60]. Also in this experimental context, both NPRC gene silencing and miR-199a inhibition recovered the deleterious effects of the ANP variant.

Our studies document that the effects of ANP and of its molecular variant depend, at least in part, by the modulation of specific miRNAs. As a consequence, physiological functions of ANP may be enhanced by targeting the “good” miRNAs, whereas pathological functions of the ANP variant can be rescued by correcting the “bad” miRNAs modulation.

Antisense regulation of NPPA expression

The NPPA locus contains several natural antisense transcripts (NATs). They are lncRNAs that overlap protein-coding genes but are transcribed from the antisense strand [61]. They exert an inhibitory effect on the transcription of the corresponding sense mRNA. A recent work by Celik et al. [62] reported that the NPPA-AS1, as a natural negative feedback mechanism, has relevance during mammalian fetal heart development, in conditions of biomechanical stress and in heart failure patients. Most importantly, the in vivo NPPA-AS1 inhibition produced increased plasma ANP levels, increased renal cGMP levels and reduced blood pressure levels in mice [62]. Therefore, a pharmacological modulation of NPPA-AS1 can be developed as a valid approach for the treatment of cardiovascular diseases in humans. As a matter of fact, this strategy may be able to avoid the adverse effects of less specific inhibitors of the NPs system, such as neprilysin.

MicroRNA-dependent proBNP processing

It is known that a state of resistance to NPs characterizes heart failure and explains at least in part the disease progression despite the higher circulating NPs level. Several mechanisms have been proposed to explain the resistance to NPs in heart failure patients, one of which is the increased ratio of inactive proBNP versus active BNP [63, 64]. This phenomenon may be attributed to an increased rate of proBNP glycosylation that hampers the pro-peptide processing [65]. Based on this evidence, a recent study by Nakagawa et al. [66] analyzed a cohort of heart failure patients and found that those carrying a higher proportion of proBNP had a higher glycosylation rate at threonines 48 and 71 of proBNP with consequent attenuation of its processing. The GalNac-transferase (GALNT) 1 and 2 mediates the glycosylation-regulated increase of proBNP secretion in these subjects. Furthermore, the suppression of miR30 detected in the myocardium of failing hearts favors an enhanced expression of cardiac GALNT1, thus causing a higher proBNP glycosylation. In fact, the miR30-GALNT1/2 axis was identified as a mechanism responsible of the increased secretion of inactive proBNP in heart failure, likely contributing to the state of NPs resistance and to the progression of the disease (Fig. 2b).

Epigenetic control of other NPs family components

An epigenetic regulation of CORIN, encoding the enzyme processing the ANP precursor into the biological active form, has been described by Celik et al. [67]. In fact, miRNA-1-3p modulates the ANP circulating level through a potent inhibition of corin activity by a specific interaction with a target site in the 3′ UTR of CORIN. Of interest, this miR also modulates the expression of several transcriptional regulators of NPPA expression (FN1, TBX20), thus reinforcing its negative impact on the ANP circulating level. In a human study, miR495 has been proposed, by in silico analyses, as a potential regulator of CORIN through the interaction with the rs3749585 SNP located within the 3′ UTR of the gene. Consistently with the latter evidence, the minor allele of this SNP, ultimately responsible of lower ANP level, was significantly associated with higher susceptibility to hypertension in a Chinese population [68].

The NPRA, encoding the main receptor responsible of the ANP and BNP effects, may undergo histone deacetylation with consequent reduced gene expression. In fact, inhibition of histone deacetylases (HDAC1/2) led to increased NPRA expression. The phenomenon was associated with increased histones acetylation [69]. Apart from HDAC inhibition, an epigenetic upregulation of NPRA was achieved with the use of all-trans retinoic acid and led to a reduction of renal fibrosis and systolic blood pressure in mice. This strategy further underscores the potential implication of epigenetic regulatory mechanisms as targets for the treatment of cardiovascular diseases [70].

Type C natriuretic peptide receptor (NPRC) is devoted to the natriuretic peptides clearance and it also exerts biological functions on its own [71]. An increased expression of NPRC leads to lower circulating NPs level and to the consequent reduced physiological effects of the peptides. Abnormalities of its expression were reported in few pathological contexts, mainly hypertension and obesity [72, 73]. Studies exploring the potential epigenetic regulation of NPRC found two miRNAs, miR100 [74] and miR143 [75], able to modulate its expression. An upregulation of miR100 was found in hypoxic cells and in rat hearts after myocardial infarction, as well as in the blood of heart failure patients [74]. Increased level of miR143 and decreased expression of NPRC were detected in hypoxia-treated human cardiac cells and in left ventricular tissue from rats undergoing experimental myocardial infarction [75]. Once again, the studies of epigenetic modulation of NPRC suggest potential therapeutic targets to achieve the optimal tuning of NPs levels in cardiovascular diseases.

Apart from the study of Malek et al. [31] describing a significant increase of histone acetylation at the promoter region of NEP in a model of diabetic rat, the mRNA and protein expression of NEP were found to be reduced in a neurological disease. In fact, Chu et al. [76] described a miR26b dependent regulation of NEP in a cellular model of Alzheimer disease. This phenomenon was associated with inhibition of neurite growth and with increased cell apoptosis. This evidence reinforces previous data showing a remarkable decrease of NEP mRNA expression in Alzheimer disease patients compared with normal controls [77] and that upregulation of NEP expression reduces the concentration of amiloid β in the mouse brain [78]. Despite the relevance of the result, this study did not provide clear evidence of an implication of NPs, through NEP dysregulation, in the delay of disease progression in Alzheimer disease. Further studies may better investigate the role of NEP regulation through miR26b in cardiovascular diseases.

Summary and outlook

The deeper understanding of the epigenetic regulatory mechanisms governing the expression and translation of many genes allows a more detailed comprehension of several physiological and pathological conditions. Most importantly, the growing knowledge gained on these mechanisms may provide interesting molecular tools for the development of novel therapeutic targets.

With regard to the NPs system, a substantial amount of data already exists to support the previous statements. The histone acetylation and DNA methylation of NPPA and NPPB are critically associated with the development of several cardiomyopathies, and, in particular, of hypertrophy, dilated cardiomyopathy and heart failure. Furthermore, the miRNAs modulation of NPPA is able to explain few traits linked to race, and some pathological traits related to hypertension, obesity and metabolic disturbances, cerebral ischemia and myocardial infarction. By expanding our knowledge on this field of research, we may greatly improve the available strategies to combat cardiovascular diseases.

Funding

This work was supported by a Grant from the Italian Ministry of Health.

Compliance with ethical standards

Conflicts of interest

The authors declare no competing interests.