Atherosclerosis is an epigenetic disease

Abstract

Atherosclerosis, is a chronic inflammatory and lipid-depository disease that eventually leads to acute cardiovascular events. Emerging evidence supports that epigenetic processes such as DNA methylation, histone modification, and non-coding RNAs play an important role in plaque progression and vulnerability, highlighting the therapeutic potential of epigenetic drugs in cardiovascular therapeutics.

Introduction

Atherosclerotic plaque development and rupture is responsible for major clinical cardiovascular events (such as myocardial infarction and stroke) worldwide [1]. Understanding the molecular mechanisms that drive atherosclerosis and plaque destabilization are indispensable for developing new therapeutic strategies [1]. Since 1990s, atherosclerosis has been considered as a chronic inflammatory disease. It initially starts from endothelial response to injury, followed by lipid buildup in the vessel wall, impaired resolution of persistent inflammation, as well as plaque rupture and thrombosis [1–4]. Based on the “lipid” and “inflammation” hypothesis of atherosclerosis development, lipid-lowering statins and anti-inflammatory agents are the cornerstone of atherosclerosis management. However, residual cardiovascular risk in certain groups of patients remain very high and some patients have poor responsiveness to statins [5]. Thus, additional therapies are needed. More recently, the role of epigenetics in atherosclerosis has been increasingly recognized [6]. Epigenetic mechanisms include DNA methylation/demethylation, histone methylation/demethylation, histone acetylation/deacetylation and non-coding RNAs [6]. Increasing evidence has shown that these epigenetic processes are involved in the initiation and progression of atherosclerosis. More importantly, epigenetic processes, DNA and histone modification in particular, have specific “writers” (introducing epigenetic marks) and “erasers” (removing epigenetic marks) that regulate gene expression. Epigenetic processes are highly dynamic, reversible and hence drug targetable, giving an excellent opportunity to treat atherosclerosis by targeting these epigenetic processes [7] (Figure 1).

Figure 1: Targeting epigenetics in atherosclerosis.

Epigenetic mechanisms, such as DNA methylation/demethylation, histone methylation/demethylation, and histone acetylation/deacetylation are involved in the development of atherosclerosis. For simplicity, other forms of histone modifications, such as phosphorylation, sumoylation, ubiquitination, and ADP-ribosylation as well as non-coding RNAs are not described here. Multiple pathological stimuli/risk factors, such as disturbed hemodynamic forces, classical cardiovascular risk factors (hyperlipidemia, hyperglycemia, hyperhomocysteinemia, and hypertension), cigarette smoking, and dietary and environmental factors, are well-known epigenetic regulators that contribute to atherosclerosis development. DNA and histone modifications are exerted through the combined effects of specific “writers” and “erasers” of epigenetic marks. The concerted actions of these epigenetic “writers” and “erasers” determine the the state of ‘openness’ (euchromatin) or ‘closed-ness’ (heterochromatin) of chromatin structure and final outcome of gene expression.

Abbreviations:

CV, cardiovascular; DNAme, DNA methylation; DNMT, DNA methyltransferase; EC, endothelial cells; EZH2, enhancer of zeste homolog 2; HATs, histone acetyltransferses; HDAC, histone deacetylases; JMJD3, Jumonji domain containing 3; Me3, trimethylation; Mφ, macrophages; SIRT, sirtuin; TET2, tet methylcytosine dioxygenases 2; VSMC, vascular smooth muscle cells; UTX, ubiquitously transcribed X chromosome tetratricopeptide repeat protein

Epigenetic mechanisms of atherosclerosis

The complex of DNA and histone proteins (H2A, H2B, H3 and H4) forms the chromatin, which is the basic unit critical for gene transcription/silencing, signal transduction, DNA repair, and DNA replication etc [8]. Chromatin can undergo a remodeling process by switching from a tightly packed condensed state (heterochromatin) to an open conformation state (euchromatin), allowing nuclear transcription factors or DNA binding proteins to access DNA and thus alter gene expression [8]. Chromatin modifications, such as DNA methylation and histone modification, are common in mammalian cells. DNA methylation is an epigenetic event that covalently transfer a methyl group to the cytosines, mainly on CpG dinucleotide site, causing transcriptional repression. Conversely, DNA hypomethylation are commonly observed in gene bodies and enhancer regions of genes with active transcription [6]. Histone modification is another important manner of chromatin alternation that regulates gene expression depending on the balanced effects of epigenetic “writers” and “erasers” [6]. Recent evidence has suggested that DNA methylation and histone modification (methylation and acetylation in particular) play important roles in the development of various forms of cardiovascular diseases.

It has been well established that multiple pathological stimuli/risk factors contribute to the complexity of atherosclerosis. These factors include disturbed hemodynamic forces, classical cardiovascular risk factors (hyperlipidemia, hyperglycemia, hyperhomocysteinemia, and psychological stress), cigarette smoking, and dietary and environmental factors (Figure 1). For example, smoking has a broad impact on DNA methylation at genome-wide scale, even after smoking cessation [9]. DNA methylation is catalyzed by DNA methyltransferase 1 (DNMT1), 3a (DNMT3a) and 3b (DNMT3b), and is reversed by Tet methylcytosine dioxygenases (TET1, 2, and 3). The role of DNMT1 and DNMT3a mediated DNA methylation in atherosclerosis has been increasingly recognized [7]. More recently, two landmark studies from the Walsh laboratory [10] and the Ebert laboratory [11], have independently demonstrated the important role of hematopoietic DNA demethylating enzyme TET2 in preventing atherosclerosis, which does so by repressing the upregulation of pro-inflammatory cytokines and chemokines as well as inflammasome activation. In line with both reports, hematopoietic or myeloid cellspecific TET2 deletion also aggravates cardiac dysfunction in heart failure, with activation of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3)/IL-1β (interleukin-1β) pathway [12]. A recent report has also shown that TET2 overexpression reduces, while TET2 depletion increases atherosclerosis development in apolipoprotein E deficient (ApoE^−/−) mice via modulating Beclin1-dependent autophagic processes [13]. These findings raise the concept that TET2 is a promising therapeutic target for treating atherosclerosis.

As a second major epigenetic mechanism, the effect of histone modification (methylation and acetylation) on target gene expression is cell type and epigenetic mark-specific. For example, histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 9 trimethylation (H3K9me3) are silencing epigenetic marks, while, histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 27 acetylation (H3K27ac) are epigenetic marks that leads to “open” chromatin and activates gene transcription [6]. The emerging role of histone acetyltransferases (HATs) and histone deacetylases (HDACs, including sirtuins) in atherosclerosis has been reviewed recently [6]. Recent evidence has suggested that enhancer of zeste homolog 2 (EZH2), the catalytic subunit in polycomb group repressive complex 2 (PRC2), also plays an important role in atherosclerosis. Specifically, adenoviral EZH2 overexpression promotes atherosclerosis development in ApoE^−/− mice. The mechanism is related to promoting DNMT1-mediated ABCA1 methylation, thereby reducing macrophage cholesterol efflux and promoting foam cell formation [14]. Direct evidence showing the involvement of H3K27me3 “erasers”-UTX (Ubiquitously Transcribed X Chromosome Tetratricopeptide Repeat Protein) and JMJD3 (Jumonji Domain containing 3) in atherosclerosis remains to be elucidated. Given the availability of several specific EZH2 inhibitors in clinical trials for potential cancer therapeutics [15], it warrants to be examined whether these EZH2 inhibitors would confer atheroprotection in various experimental animal models of atherosclerosis.

Last but not least, RNA-based mechanisms including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are emerging as important epigenetic regulators of cardiovascular diseases as well. miRNAs are involved in both DNA methylation and different histone modifications [16]. LncRNAs have been reported to affect a plethora of biological processes regulating mRNA stability, RNA splicing, miRNA-mediated gene regulation, and chromatin structure [17]. Furthermore, studies have demonstrated that lncRNAs coordinate many epigenetic regulatory processes including chromatin dynamics, DNA methylation, stability of mRNA and other non-coding RNAs, as well as epigenetic substrate availability [17]. The up-to-date functions of atherosclerosis related miRNA [16] and lncRNA[17] has been reviewed elsewhere. Therefore, DNA methylation, histone modifications, and RNA-based mechanisms are not independent, but rather reciprocally impacts each other and cooperatively regulate gene expression in vascular cells.

Therapeutic potential of epigenetic cardiovascular drugs

The epigenetic mechanisms of atherosclerosis suggest the necessities to develop small-molecule epigenetic drugs targeting chromatin architecture to combat atherosclerosis (Table 1). These epigenetic drugs are already widely used in cancer therapy or cancer-related clinical trials, such as HDAC inhibitors, sirtuin activating compounds (such as resveratrol), DNA methylation inhibitors (such as 5-Azacytidine and its nucleoside analogs) and histone methylation inhibitors (such as EZH2 inhibitors). Among these epigenetic drugs, HDAC inhibitors (such as SAHA), 5-aza-2′-deoxycytidine and others have already demonstrated pre-clinical efficacy in attenuating experimental atherosclerosis [18, 19]. Some anti-atherosclerotic nutritional supplements/phytochemicals (such as resveratrol, curcumin, and EGCG) have also shown promises in modulating epigenetic enzymes in vascular cells and atherosclerosis [20]. Lipid-lowering statins has been shown to reduce EZH2 expression in cancer cells [21], as well as human endothelial cells (Xu et al, unpublished observation). In addition, vitamin C, an anti-atherosclerotic antioxidant, has been reported to promote TET-mediated DNA demethylation [22] (Table 1). It remains unknown whether there are epigenetic drugs that modulate atherosclerosis-related lncRNA (“Atherolinc”) to date.

Table 1:

Examples of epigenetic drugs that delay atherosclerosis.

DRUG NAME	EPIGENETIC DRUG CATEGORY
Vitamin C	TET2 activator
5-Aza-2′-deoxycytidine	DNMT inhibitor
Statins	EZH2 inhibitor
SAHA	HDAC inhibitor
Quercetin	DNMT inhibitor
Curcumin	Broad-spectrum epigenetic modulator
EGCG	DNMT inhibitor
Resveratrol	SIRT1 activator

Abbreviations: EGCG, Epigallocatechin gallate; EZH2, enhancer of zeste homolog 2; SAHA, suberoylanilide hydroxamic acid; TET2, Tet methylcytosine dioxygenases 2; HDAC, histone deacetylases; DNMT, DNA methyltransferase; SIRT1, sirtuin 1

However, we have to mention that different vascular cell types have different expression abundance of epigenetic enzyme isoforms. Moreover, the function of individual isoform may vary in different cell types, making it necessary to target individual cell type specific isoform of enzyme in treating atherosclerosis. In addition, DNA methylation appears frequently in synergism with histone modification, further complicating our understanding of disease pathogenesis. Fortunately, our knowledge of the epigenetic basis of atherosclerosis was deepened by technical advances in systems biology approaches such as total RNA-sequencing (RNA-seq), single cell RNA-seq, chromatin-immunoprecipitation-sequencing (ChIP-seq), and DNA methylation profiling. These technologies will certainly provide further information and valuable tools for further understanding of epigenetic alternations in atherosclerosis [6].

Taken together, recent basic and translational evidence has revealed the therapeutic potential of epigenetic drugs to reduce and stabilize atherosclerotic plaques. This potentially new category of anti-atherosclerotic drugs will complement current lipid-lowering and anti-inflammatory therapies of atherosclerosis. In light of the involvement of epigenetic mechanisms in all phases of atherosclerosis development, we can envisage the era of epigenetic therapy of atherosclerosis is around the corner. More and more epigenetic drugs will be developed/repurposed and validated both pre-clinically and clinically to treat atherosclerosis in the coming decades.