Athero-thrombosis of the arteries supplying the brain and lower limb are the main causes of stroke and limb loss. New therapies are needed to improve the outcomes of athero-thrombosis. Recent evidence suggests a role for epigenetic changes in the development and progression of ischaemic injury due to atherosclerotic occlusion of peripheral arteries. DNA hypermethylation have been associated with cardiovascular diseases. Histone post-translational modifications have also been implicated in atherosclerosis. Oxidized low-density lipoprotein regulated pro-inflammatory gene expression within endothelial cells is controlled by phosphorylation/acetylation of histone H3 and acetylation of histone H4 for example. There are a number of challenges in translating the growing evidence implicating epigenetics in atherosclerosis to improved therapies for patients. These include the small therapeutic window in conditions such as acute stroke and critical limb ischaemia, since interventions introduced in such patients need to act rapidly and be safe in elderly patients with many co-morbidities. Pre-clinical animal experiments have also reported conflicting effects of some novel epigenetic drugs, which suggest that further in-depth studies are required to better understand their efficacy in resolving ischaemic injury. Effective ways of dealing with these challenges are needed before epigenetic approaches to therapy can be introduced into practice.

INTRODUCTION

Atherosclerosis commonly affects the arteries supplying the heart, brain and peripheries resulting in conditions which are commonly referred to as coronary artery disease (CAD), cerebro-vascular disease (CVD) and peripheral arterial disease (PAD), respectively. Most patients have atherosclerosis involving more than one site. Common causes of death listed for patients with PAD are CAD (40–60%) and CVD (10–20%) for example [1].

Clinical presentations of CVD include transient ischaemic attack (TIA) and stroke. Stroke is a clinical syndrome characterized by an acute loss of neurological function, with symptoms lasting greater than 24 h [2]. TIA is a more transient (lasting <24 h) neurological dysfunction [2]. Stroke is the second leading cause of death and long-term disability world-wide according to the World Health Organization statistics [3]. Ischaemic stroke and TIA are usually secondary to thrombosis or embolization of the arteries supplying the brain. The impact of such athero-thrombo-embolism depends in part on the site of the arterial occlusion and the collateral blood supply to the part of the brain affected. The patency of the circle of Willis plays an important role in determining the effect of occlusions of the basilar and carotid arteries for example. The main current therapy for stroke is clot busting medicine or thrombolysis however this has to be administered within a few hours of the stroke to have any value and in some patients has limited benefit.

Common clinical presentations of PAD include pain in the leg on walking, known as intermittent claudication (IC), and rest pain or gangrene, often referred to collectively as critical limb ischaemia (CLI), when the viability of the limb is at risk [4]. The main therapies used to treat IC and CLI are endovascular or surgical revascularization, however, these interventions are frequently ineffective at achieving long term functional improvement in the limb. Amputation is the only option for those patients with severe progression of limb ischaemia where revascularization is not possible [5,6]. The incidence of lower limb amputation varies from ~120 to 500/million per year [7].

There is an urgent need for alternative therapies for patients with CVD and PAD. The complications of CVD and PAD largely result from tissue ischaemia. Therapies that are successful in resolving this ischaemic insult could potentially benefit both these diseases. Improved outcomes of arterial occlusions could result from growth of pre-existing collateral networks and also increased number and quality of newly formed smaller collateral vessels [8]. Factors contributing to the development of arteriolar collateral networks and other responses to ischaemia are poorly understood. Improved understanding of the responses to ischaemic injury is needed in order to design novel therapies for CVD and PAD.

Heritability estimates suggest that inherited genetic factors account for ~30% of the variation in the incidence of most cardiovascular diseases [9]. The remaining variation is believed to be explained by life style factors such as dietary and exercise habits, environmental exposure to toxins, and drug usage. The role of epigenetics in determining a range of processes that are believed to be critical in the development and outcome of CVD and PAD is being increasingly appreciated [10]. Epigenetics has been implicated in influencing atherosclerosis, angiogenesis, ischaemia/reperfusion damage, and the cardiovascular response to hypoxia and fluid shear stress. In this review, we first briefly describe epigenetic control of gene expression, then outline evidence linking epigenetics with CVD and PAD and describe how epigenetics may influence processes important in complications and outcomes of CVD and PAD.

EPIGENETIC REGULATION OF GENE EXPRESSION

Epigenetic variation can be acquired or inherited and constitutes a means by which interactions between genes and environment can occur. Three important epigenetic regulators of gene expression are controlled by: (i) methylation of CpG islands, mediated by DNA methyltransferases (DNMTs), (ii) histone post translational modifications (PTMs) and (iii) microRNAs (miRNAs) (Figure 1). These epigenetic regulators can control expression of various genes ultimately leading to gene activation or gene silencing (Table 1).

Epigenetic control of gene expression

Figure 1
Epigenetic control of gene expression

There are three key epigenetic mechanisms that regulate gene expression (i) methylation of CpG islands, mediated by DNA methyltransferases, (ii) histone protein modifications and (iii) microRNAs. The various epigenetic modifications control gene activation and silencing affecting gene expression. Abbreviations: CpG, cytosine phosphate guanine; meCpG, methylated CpG island; DNMTs, DNA methyltransferases.

Figure 1
Epigenetic control of gene expression

There are three key epigenetic mechanisms that regulate gene expression (i) methylation of CpG islands, mediated by DNA methyltransferases, (ii) histone protein modifications and (iii) microRNAs. The various epigenetic modifications control gene activation and silencing affecting gene expression. Abbreviations: CpG, cytosine phosphate guanine; meCpG, methylated CpG island; DNMTs, DNA methyltransferases.

Table 1
Summary of important epigenetic mechanisms that control gene expression
Epigenetic regulation mechanismPurpose of markEnzyme adding the markEnzyme removing the markMark locationReferences
DNA methylation  Inactivation DNA methyl transferase (DNMT) Methyl CpG binding domain protein-2 or excision/repair CpG islands on DNA [11
Post-translational modifications of histones Histones modified    Amino acids  
Acetylation H3, H4, H2A and H2B Activation Histone acetyl transferase (HAT) Class I HDAC 1–3 and 8 Lysine (K) [11
    Class II HDAC 4–7, 9 and 10   
    Class III (sirtuins 1–7)   
    Class IV HDAC 11   
Methylation H3, H4 and H1 Inactivation/activation Histone methyl transferase (HMT) Histone demethylase (HDM) Lysine (K), mono, di and tri, arginine (R) mono and di (symmetric/asymmetric) [11
Phosphorylation H2A, H3 and H1 Inactivation/activation Phosphorylase Kinase Serine (S) and threonine (T) and tyrosine (Y) [12
RNA mechanism       
microRNAs Not applicable Inactivation/activation Not applicable Not applicable Not applicable [13
Epigenetic regulation mechanismPurpose of markEnzyme adding the markEnzyme removing the markMark locationReferences
DNA methylation  Inactivation DNA methyl transferase (DNMT) Methyl CpG binding domain protein-2 or excision/repair CpG islands on DNA [11
Post-translational modifications of histones Histones modified    Amino acids  
Acetylation H3, H4, H2A and H2B Activation Histone acetyl transferase (HAT) Class I HDAC 1–3 and 8 Lysine (K) [11
    Class II HDAC 4–7, 9 and 10   
    Class III (sirtuins 1–7)   
    Class IV HDAC 11   
Methylation H3, H4 and H1 Inactivation/activation Histone methyl transferase (HMT) Histone demethylase (HDM) Lysine (K), mono, di and tri, arginine (R) mono and di (symmetric/asymmetric) [11
Phosphorylation H2A, H3 and H1 Inactivation/activation Phosphorylase Kinase Serine (S) and threonine (T) and tyrosine (Y) [12
RNA mechanism       
microRNAs Not applicable Inactivation/activation Not applicable Not applicable Not applicable [13

A key element of such epigenetic control is that these regulators are mostly dynamic and therefore can potentially be manipulated to prevent or reverse the effects of aberrant expression of genes. The expression of a range of specific genes has been shown to be under epigenetic control and can potentially influence a variety of processes important in CVD and PAD complications and outcomes (Table 2).

Table 2
Examples of how epigenetic changes influence vascular cells
Epigenetic changeTarget genes for epigenetic changeCell typeEffectReferences
DNA methylation Matrix metalloproteinase-1 and -8; tissue inhibitor of metalloproteinases-1; collagen type Iα 1 Cultured smooth muscle cells and neutrophils-like cell line (HL-60) Vascular dysfunction [14
 Suppressor of cytokine signalling-3 Smooth muscle cells Intimal hyperplasia and restenosis [15
 Mitofusin-2   [16
 Extracellular superoxide dismutase  Cell-specific expression; protection from oxidative stress and modulation of vascular tone [17
 Sixth CTCF-binding site upstream of H19; insulin like growth factor-2  Homocysteine-induced atherosclerosis [18
 Homeobox A5 and Kruppel-like factor-3 Endothelial cells Flow-dependent atherosclerosis [19
 Vascular endothelial growth factor receptors 2 and 3  Angiogenesis and lymphangiogenesis [20
 Platelet-derived growth factor  Homocysteine-induced atherosclerosis, endothelial dysfunction, smooth muscle cell activation [21
 Dimethylarginine dimethylaminohydrolase-2  Homocysteine-induced apoptosis and atherosclerosis [22
 CD31 von Willebrand factor; vascular endothelial-cadherin; intercellular adhesion molecule-2  Cell-specific gene expression [23
 Vascular endothelial growth factor receptor 2  Angiogenesis and survival [24
 Thymocyte differentiation antigen-1 Fibroblasts Fibrosis [25
 α-Smooth muscle actin Fibroblasts; myofibroblasts Fibrosis; differentiation [26,27
 Endothelial nitric oxide synthase Endothelial cells Angiogenesis and survival; cell-specific expression and maintaining cardiovascular homoeostasis [24
 Homeobox protein-B7, -C6, -C8l; transgelin Stem cells Differentiation, neointima formation and tumour vascularization [28
 Thrombospondin-1 Cerebral endothelial cells Angiogenesis [29
 Smooth muscle 22α Smooth muscle cells Vascular calcification [30
Histone modifications Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases-4 Endothelial cells Reactive oxygen species generation in the vascular wall [31
 Endothelial Kruppel-like Factor-2  Vascular homoeostasis; low-density lipoprotein cholesterol-mediated endothelial dysfunction [32
 Mothers against decapentaplegic homologue 2 Smooth muscle cells Human thoracic aortic aneurysms [33
 Stromal cell-derived factor-1 Endothelial cells Cell-specific expression [34
microRNA Vascular cell adhesion molecule-1  Inflammation [35
 Endothelial nitric oxidase synthase and E-26  Angiogenesis, inflammation [36
 Thrombospondin-1  Angiogenesis [37
 EphrinA3   [38
 Matrix metalloproteinase-2 and -9 Smooth muscle cells Cell migration, atherosclerosis [39
 Angiotensin II type 1 receptor Endothelial cells and smooth muscle cells Inflammation [36
Epigenetic changeTarget genes for epigenetic changeCell typeEffectReferences
DNA methylation Matrix metalloproteinase-1 and -8; tissue inhibitor of metalloproteinases-1; collagen type Iα 1 Cultured smooth muscle cells and neutrophils-like cell line (HL-60) Vascular dysfunction [14
 Suppressor of cytokine signalling-3 Smooth muscle cells Intimal hyperplasia and restenosis [15
 Mitofusin-2   [16
 Extracellular superoxide dismutase  Cell-specific expression; protection from oxidative stress and modulation of vascular tone [17
 Sixth CTCF-binding site upstream of H19; insulin like growth factor-2  Homocysteine-induced atherosclerosis [18
 Homeobox A5 and Kruppel-like factor-3 Endothelial cells Flow-dependent atherosclerosis [19
 Vascular endothelial growth factor receptors 2 and 3  Angiogenesis and lymphangiogenesis [20
 Platelet-derived growth factor  Homocysteine-induced atherosclerosis, endothelial dysfunction, smooth muscle cell activation [21
 Dimethylarginine dimethylaminohydrolase-2  Homocysteine-induced apoptosis and atherosclerosis [22
 CD31 von Willebrand factor; vascular endothelial-cadherin; intercellular adhesion molecule-2  Cell-specific gene expression [23
 Vascular endothelial growth factor receptor 2  Angiogenesis and survival [24
 Thymocyte differentiation antigen-1 Fibroblasts Fibrosis [25
 α-Smooth muscle actin Fibroblasts; myofibroblasts Fibrosis; differentiation [26,27
 Endothelial nitric oxide synthase Endothelial cells Angiogenesis and survival; cell-specific expression and maintaining cardiovascular homoeostasis [24
 Homeobox protein-B7, -C6, -C8l; transgelin Stem cells Differentiation, neointima formation and tumour vascularization [28
 Thrombospondin-1 Cerebral endothelial cells Angiogenesis [29
 Smooth muscle 22α Smooth muscle cells Vascular calcification [30
Histone modifications Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases-4 Endothelial cells Reactive oxygen species generation in the vascular wall [31
 Endothelial Kruppel-like Factor-2  Vascular homoeostasis; low-density lipoprotein cholesterol-mediated endothelial dysfunction [32
 Mothers against decapentaplegic homologue 2 Smooth muscle cells Human thoracic aortic aneurysms [33
 Stromal cell-derived factor-1 Endothelial cells Cell-specific expression [34
microRNA Vascular cell adhesion molecule-1  Inflammation [35
 Endothelial nitric oxidase synthase and E-26  Angiogenesis, inflammation [36
 Thrombospondin-1  Angiogenesis [37
 EphrinA3   [38
 Matrix metalloproteinase-2 and -9 Smooth muscle cells Cell migration, atherosclerosis [39
 Angiotensin II type 1 receptor Endothelial cells and smooth muscle cells Inflammation [36

DNA methylation

The most studied aspect of epigenetics is methylation of DNA at CpG islands. Methylation within the promoter is generally considered to represent an inactive or silenced gene. As a result, the DNA structure becomes more compact which is thought to prevent the necessary transcription factors accessing their binding sites on the DNA required for gene activation. DNA methylation involves the covalent addition of a methyl group to the fifth position in the six-atom ring of the nucleotide cytosine (5-methylcytosine; 5meC). A group of enzymes known as DNMTs add the methyl group to the cytosine whereas the process of removing the methyl group (often referred to as a mark) is more complex. Bird demonstrated for the first time that the methyl group on DNA can be removed enzymatically by methyl-CpG-binding domain protein 2 (MBD2) [40]. Alternatively the methyl group can be removed by DNA excision and repair where the methyl cytosine is excised and replaced by an unmodified cytosine [41]. This demonstrates that DNA methylation is not static and can be manipulated during gene regulation [42]. DNMTs can be subdivided into two groups. During maintenance methylation DNMT1 copies the methyl group on to newly synthesized DNA (hemi-methylated) after replication, whereas during de novo methylation DNMT 3a and 3b add novel methylation marks to the DNA. DNA methylation can result in gene repression by physically compacting the chromatin and excluding transcription factors or by methyl binding proteins (MBPs) that bind methylated DNA and subsequently recruit histone deacetylases (HDACs) and co-repressors.

Histone post-translational modifications

Histone regulation of gene transcription is more complex due to the different types of PTMs. The main ones are acetylation, phosphorylation and methylation [43] (Table 1). The effect that these modifications have on gene regulation can depend on which residue has been modified and to what extent. Combinations of the various histone marks can provide binding sites for the effector proteins that generate folding of the chromatin which ultimately leads to gene regulation generally referred to as the ‘histone code’ [44].

Histone acetylation

Histone acetylation is by far the most studied PTM and is currently the best understood. Histone acetylation occurs at lysine residues and it changes the charge of the histone from positive to neutral and therefore weakens the interaction between the histones along the chromatin causing the chromatin (?) to ‘relax’ and become more open. The acetylation is regulated by histone acetyl transferases (HATs) and deacetylation is regulated by HDACs. The recruitment of HDACs to specific regions of genes leads to a decrease in acetylation, chromatin condensing, and gene silencing. HDACs exist in two different classes. Class I comprises HDACs 1–3 and 8, and Class II comprises HDACs 4–7, 9 and 10. Each class of HDACs interacts with specific chromatin factors and repressor molecules and thus they have slight differences in their actions [45].

Histone methylation

Histone methylation occurs on lysine or arginine residues and unlike acetylation and phosphorylation, it does not change the charge of the histones. The degree of methylation varies. Lysine residues can be mono-, di- and tri-methylated whereas arginine residues can be mono- or di-methylated symmetrically or asymmetrically. Histone methylation is associated with both transcriptional activation and repression. H3K4me2/3 are marks that are generally associated with gene activation [46] whereas H3K9me3 is a mark of inactivation, these marks can also be tissue dependent. For example, increased α-myosin heavy chain gene (α-MHC) expression requires H3K4me2 in the cardiac cells of the left ventricle in mice, however, this histone mark is not required in the cardiac cells of the right ventricle, demonstrating the specificity of the histone marks within tissues [47]. Smooth muscle cells (SMCs) cultured from db/db mice have an atherogenic and inflammatory phenotype associated with low levels of H3K9me3 at key inflammatory gene promoters [48].

Histone phosphorylation

Histone phosphorylation occurs on serine and threonine residues and like acetylation, the phosphorylation marks change the charge of the histone proteins from positive to negative. This mark is mostly associated with transcriptional repression however it can also be found on active chromatin alongside activation marks such as histone H3 acetylation/phosphorylation (H3K4ac/S10p).

microRNAs

Non-coding RNAs (ncRNAs) are functional RNAs that are not translated into proteins. ncRNAs has been implicated in the epigenetic regulation of gene expression. Micro RNAs (miRNA) are small trans-acting ncRNAs that are first transcribed from the genome by RNA polymerase II as pri-miRNAs. These pri-mRNAs are cleaved by an enzyme complex consisting of Drosha (RNase III endonuclease) and the dsRNA binding protein Pasha. This results in a 70–100 nucleotide long hair-pin shaped precursor miRNA which is transported to the cytoplasm from the nucleus. In the cytoplasm precursor miRNAs are then cleaved by the endoribonuclease III enzyme Dicer into around 18–22 nucleotide long fragments to form shorter transient, non-coding strands known as miRNAs. These small interfering RNAs (siRNA) then assemble with the RNA-induced silencing (RISC) complex. The RISC complex degrades specific mRNA resulting in the suppression of target genes [49]. A single miRNA is capable of regulating a large number of mRNAs simultaneously [50], mainly by destabilizing the target mRNAs and subsequently resulting in their destruction or by translation inhibition [51]. miRNAs control gene expression by directly targeting the gene promoters. A good example of this is miRNA-373 that was shown to be required for E-cadherin induction in cell culture [52].

Recent evidence suggests that miRNAs can also directly interact with chromatin and chromatin modifying complexes [53]. Thus, overlap and cross-talk exist between the various epigenetic pathways (Figure 1). For example, it has been shown that administration of a HDAC inhibitor (HDACi) not only increases histone acetylation but also effects histone methylation levels and miRNA pathways [5456]. miRNAs have been shown to regulate epigenetic factors such as methyl binding CpG protein 2 (MeCP2), which binds to methylated DNA and represses transcription of genes; dysregulation of this protein can lead to pathologies such as Rett syndrome [57]. Thus, it is well appreciated that the epigenetic system is a self-organizing network that regulates the context dependent expression of myriads of genes by the co-ordinated effects of the entirety of the epigenetic control mechanisms.

EVIDENCE FOR THE INVOLVEMENT OF EPIGENETICS IN THE PATHOLOGY OF CVD AND PAD

Atherosclerosis and laminar flow

Atherosclerotic occlusions usually occur in regions of disturbed blood flow near arterial branches. Endothelial cells (ECs) and SMCs are constantly exposed to haemodynamic forces, including blood flow-induced fluid shear stress and cyclic stretch. These forces modulate vascular cell gene expression as well as function and are implicated in vascular pathology [58]. Disturbed blood flow is associated with atherosclerosis development.

An important anti-inflammatory athero-protective transcription factor, Kruppel-Like Factor 4 (KLF4), was recently reported to be down regulated in regions of disturbed flow (Table 3). In vitro investigations suggested that disturbed flow leads to increased CpG island methylation in KLF4 leading to increased expression of downstream KLF4 transcription target genes such as endothelial nitric oxide (eNOS), thrombomodulin, and monocyte chemotactic protein-1 (MCP-1) [62]. Arterial tissue isolated from regions of disturbed flow in pigs had significantly lower expression of KLF4 and eNOS, and a hypermethylated myocyte enhancer factor-2 binding site in the KLF4 promoter [62]. Recently, it was also shown that disturbed flow regulates genome-wide DNA methylation patterns in a DNMT-dependent manner in studies using a partial carotid ligation murine model [19]. The reduction in DNMT1 activity by 5-azacytidine (5-Aza) or siRNA markedly attenuated endothelial inflammation. Furthermore, disturbed flow resulted in hypermethylation of several crucial mechano-sensitive genes, including HoxA5 and KLF3 whose expression was subsequently restored by 5-Aza administration [19]. These data suggest that disturbed flow controls the epigenetic phenotype of the vascular endothelium and thereby promotes atherosclerosis.

Table 3
Some evidence for the relevance of epigenetic changes to CVD and PAD
PathologyEpigenetic effectEffectCell type/model studiedGenes involvedReferences
Atherosclerosis Tricostatin A (HDAC inhibitor) increases histone acetylation Alters the expression of surface markers on macrophages that are involved in the uptake of oxidized low-density lipoprotein and can increase the progression of atherosclerosis Ldlr −/− mice CD36 [59
  Inhibits proliferation Smooth muscle cell p21(WAF1) [60
 Oxidized low-density lipoprotein induces acetylation of H4 and H3 acetylation/phosphorylation Statins reduced oxidized low-density lipoprotein-induced histone modifications and subsequent inflammation Endothelial cells Interleukin-8 and monocyte chemoattractant protein-1 [61
 Kruppel-Like Factor 4 (KLF4) is down-regulated in regions of disturbed blood flow Decreased transcription of endothelial-specific genes Swine aorta Endothelial nitric oxide, thrombomodulin and monocyte chemoattractant protein-1 [62
 Disturbed flow regulates genome-wide DNA methylation pattern Oscillatory stress enhanced DNMT1 expression and endothelial inflammation and hypermethylation of mechano sensitive genes Human aortic smooth muscle cells and swine aorta Kruppel-Like Factor 3 and HOXA5 [19
 Shear stress (atheroprone versus atheroprotective) atheroprone–resulted in globally decreased histone H4 acetylation Atheroprone decreased expression of genes associated with differentiated endothelial cells and smooth muscle cells and induced a pro-inflammatory phenotype. SMC genes that require serum response factor (SRF) binding for transcription are dependent on histone H4 acetylation during atheroprone stress Endothelial cell and smooth muscle cell (co-culture model) Vascular cell adhesion molecule-1, interleukin-8 and monocyte chemoattractant protein-1 [63
Hypoxia Suppressing HDAC2 expression can reduce ischaemic retinal injury Degeneration of inner retinal layers Hdac2+/− mice HDAC2 [64
 Reduced H3K9 methylation Expression of hypoxia inducible facor-1 is able to subsequently activate various gene promoters and induce more pro-inflammatory genes Human microvascular endothelial cell HIF-1, Jumonji domains 1A, 2A and 2B [65,66
 Reduced HDAC2 expression Decreased ischaemic retinal injury HDAC2+/− mice HDAC2 [64
PathologyEpigenetic effectEffectCell type/model studiedGenes involvedReferences
Atherosclerosis Tricostatin A (HDAC inhibitor) increases histone acetylation Alters the expression of surface markers on macrophages that are involved in the uptake of oxidized low-density lipoprotein and can increase the progression of atherosclerosis Ldlr −/− mice CD36 [59
  Inhibits proliferation Smooth muscle cell p21(WAF1) [60
 Oxidized low-density lipoprotein induces acetylation of H4 and H3 acetylation/phosphorylation Statins reduced oxidized low-density lipoprotein-induced histone modifications and subsequent inflammation Endothelial cells Interleukin-8 and monocyte chemoattractant protein-1 [61
 Kruppel-Like Factor 4 (KLF4) is down-regulated in regions of disturbed blood flow Decreased transcription of endothelial-specific genes Swine aorta Endothelial nitric oxide, thrombomodulin and monocyte chemoattractant protein-1 [62
 Disturbed flow regulates genome-wide DNA methylation pattern Oscillatory stress enhanced DNMT1 expression and endothelial inflammation and hypermethylation of mechano sensitive genes Human aortic smooth muscle cells and swine aorta Kruppel-Like Factor 3 and HOXA5 [19
 Shear stress (atheroprone versus atheroprotective) atheroprone–resulted in globally decreased histone H4 acetylation Atheroprone decreased expression of genes associated with differentiated endothelial cells and smooth muscle cells and induced a pro-inflammatory phenotype. SMC genes that require serum response factor (SRF) binding for transcription are dependent on histone H4 acetylation during atheroprone stress Endothelial cell and smooth muscle cell (co-culture model) Vascular cell adhesion molecule-1, interleukin-8 and monocyte chemoattractant protein-1 [63
Hypoxia Suppressing HDAC2 expression can reduce ischaemic retinal injury Degeneration of inner retinal layers Hdac2+/− mice HDAC2 [64
 Reduced H3K9 methylation Expression of hypoxia inducible facor-1 is able to subsequently activate various gene promoters and induce more pro-inflammatory genes Human microvascular endothelial cell HIF-1, Jumonji domains 1A, 2A and 2B [65,66
 Reduced HDAC2 expression Decreased ischaemic retinal injury HDAC2+/− mice HDAC2 [64

Research examining epigenetic mechanisms that may contribute to vascular remodelling in response to fluid shear stress is limited [67] (Table 3). Laminar shear stress is an important modulating factor of eNOS expression in ECs and stimulates activating histone (H3 and H4) acetylation marks [68]. The activation of these histone acetylation marks is mediated via the p300 HAT complex, at a shear stress reporter element in the human eNOS promoter in the EC [68]. The shear stress induced-epigenetic activation marks relax the chromatin complex keeping it open to allow constitutive expression of the eNOS gene. HDAC activity is believed to play a significant role in determining the severity of ischaemia/reperfusion damage, and tricostatin A (TSA)-mediated inhibition of HDAC activity in cultured embryonic stem cells was found to stimulate myogenesis and formation of collaterals [69].

These reports show that both elevated fluid shear stress and disturbed flow, which are associated with atherosclerosis development and progression, induce epigenetic changes in the vasculature.

Hypoxia

Hypoxia secondary to arterial occlusion plays a central role in initiating collateral formation by altering cell metabolism, changing cell growth, inducing angiogenesis and increasing cell motility. Hypoxia modulates an array of genes mainly through the activation of a transcriptional complex named hypoxia-inducible factor (HIF)-1α [70] (Table 3). Hypoxia also induces the expression of HDAC leading to down regulation of the expression of numerous genes [71]. Recent findings indicate that several types of HDACis (HDAC inhibitors) disrupt the function of HIF [72]. For example, hypoxia-induced histone modifications have been reported in both hypoxia-activated and hypoxia-repressed genes [73]. Certain histone demethylases and histone methyl transferases have been identified as HIF-1-regulated genes, including JMJD1A, JMJD2B, JARID1B and G9a histone methyltransferase [7479]. Additionally, it was found that hypoxia promotes removal of repressive histone marks such as H3K4 demethylation, H3K9 methylation, and H3K9 deacetylation, at the promoter region of vascular endothelial growth factor (VEGF) which is a crucial hypoxia-inducible gene [80]. Further studies using clinical samples from ischaemic tissues are necessary to assess the role of hypoxia in CVD and PAD. Recent evidence suggests a direct role of HIF-1α in the up-regulation of placental growth factor (PlGF) gene expression in cultured human and mouse ECs exposed to hypoxia through chromatin remodelling [81]. Brahma-related gene 1 (Brg1) is another gene implicated in the epigenetic response to hypoxia. Hypoxia induced Brg1 up-regulation promotes cell adhesion molecule expression, promoting leucocyte recruitment, endothelial dysfunction and inflammation in mice [82]. Brg1 has been suggested as a potential therapeutic target in CVD and PAD [83].

EVIDENCE FOR THE ROLE OF EPIGENETICS IN CVD

The main model of ischaemic stroke used in pre-clinical animal studies relies on middle cerebral artery occlusion (MCAO). MCAO can be achieved by inserting a silicon-coated microfilament into the common carotid artery and advancing it along the internal carotid artery into the circle of Willis. Within the circle of Willis the filament blocks the origin of the middle cerebral artery and the resulting change in the blood flow can be monitored using laser doppler. MCAO can be either temporary (e.g. 30 min–2 h) or permanent. Permanent MCAO can be achieved by leaving the microfilament in place. Studies in this animal model suggest that a number of epigenetic drugs have benefits when used before and after ischaemia injury. HDACis such as TSA, valproic acid (VPA) or sodium butyrate (SB) have been shown to reduce inflammation, apoptosis and blood–brain barrier (BBB) disruption, all of which are involved in the pathological progression of CVD. They have also been suggested to promote neurogenesis and angiogenesis (Table 4). It has been shown that ischaemia induces aberrant expression of genes [104] in part due to epigenetic changes. For example, changes in DNA methylation have been associated with stroke in experimental models. Mice undergoing MCAO demonstrate increased genome-wide DNA methylation within their brain tissue. This change is believed to render the tissue more vulnerable to injury after ischaemia [105]. Pharmacological inhibition of DNA methylation using the agent 5-Aza or a deficiency in DNMT1 gene expression significantly reduced ischaemic injury in post-mitotic neurons resulting in neuronal protection after MCAO in mice [105]. A later study supported this finding by showing that DNMT1 conditional knockdown mice which had decreased levels of DNMT1 in post-mitotic neurons were protected from cerebral infarction [106].

Table 4
Effect of drugs that modulate epigenetics in models of stroke
DrugTarget of the inhibitory actionClinical stagePre-clinical model usedAdministering condition (pre-/post-ischemia)EffectReferences
Tricostatin A (TSA) Direct Class I and II histone deacetylase 1–10 inhibitor Undergoing clinical trials Permanent middle cerebral artery occlusion Pre-ischaemia Reduced inflammation [8489
   Transient middle cerebral artery occlusion Post-ischaemia Increased neurogenesis  
   Hypoxic-ischaemic model  Increased stem cell migration  
     Reduced proliferation of vascular smooth muscle cells  
Valproic acid (2-propylpentanoic acid; VPA) Specific histone deacetylase I inhibitor Approved for clinical use in 1986 Permanent middle cerebral artery occlusion Pre-ischaemia Reduced inflammation [9095
   Transient middle cerebral artery occlusion Post-ischaemia Reduced blood–brain barrier disruption  
   Intracerebral haemorrhage  Increased angiogenesis  
     Increased neurogenesis  
     Reduced apoptosis  
     Increased mesenchymal stem cell migration  
Suberoylanilide hydroxamic acid (SAHA or vorinstat) Histone deacetylase Class I, II and IV inhibitor Food and Drug Administration approved in 2006 Permanent middle cerebral artery occlusion Post-ischaemia Reduced inflammation [96,97
     Increased neuroprotection  
     Increased angiogenesis  
Sodium butyrate (SB) Class I and II histone deacetylase 1–10 inhibitor Undergoing clinical trials Permanent middle cerebral artery occlusion Pre-ischaemia Reduced inflammation [84,98
    Post-ischaemia Increased neurogenesis  
Sodium 4-phenylbutyrate (buphenyl) Direct histone deacetylase inhibitor Food and Drug Administration approved in 1996 Hypoxia ischaemia Pre-ischaemia Reduced apoptosis [99101
     Reduced inflammation  
Depsipeptide (FK228 or romidepsin) Class histone deacetylase 1 and IV, V inhibitor Undergoing clinical trials Tumour-cell lines Not applicable Reduced angiogenesis [102,103
DrugTarget of the inhibitory actionClinical stagePre-clinical model usedAdministering condition (pre-/post-ischemia)EffectReferences
Tricostatin A (TSA) Direct Class I and II histone deacetylase 1–10 inhibitor Undergoing clinical trials Permanent middle cerebral artery occlusion Pre-ischaemia Reduced inflammation [8489
   Transient middle cerebral artery occlusion Post-ischaemia Increased neurogenesis  
   Hypoxic-ischaemic model  Increased stem cell migration  
     Reduced proliferation of vascular smooth muscle cells  
Valproic acid (2-propylpentanoic acid; VPA) Specific histone deacetylase I inhibitor Approved for clinical use in 1986 Permanent middle cerebral artery occlusion Pre-ischaemia Reduced inflammation [9095
   Transient middle cerebral artery occlusion Post-ischaemia Reduced blood–brain barrier disruption  
   Intracerebral haemorrhage  Increased angiogenesis  
     Increased neurogenesis  
     Reduced apoptosis  
     Increased mesenchymal stem cell migration  
Suberoylanilide hydroxamic acid (SAHA or vorinstat) Histone deacetylase Class I, II and IV inhibitor Food and Drug Administration approved in 2006 Permanent middle cerebral artery occlusion Post-ischaemia Reduced inflammation [96,97
     Increased neuroprotection  
     Increased angiogenesis  
Sodium butyrate (SB) Class I and II histone deacetylase 1–10 inhibitor Undergoing clinical trials Permanent middle cerebral artery occlusion Pre-ischaemia Reduced inflammation [84,98
    Post-ischaemia Increased neurogenesis  
Sodium 4-phenylbutyrate (buphenyl) Direct histone deacetylase inhibitor Food and Drug Administration approved in 1996 Hypoxia ischaemia Pre-ischaemia Reduced apoptosis [99101
     Reduced inflammation  
Depsipeptide (FK228 or romidepsin) Class histone deacetylase 1 and IV, V inhibitor Undergoing clinical trials Tumour-cell lines Not applicable Reduced angiogenesis [102,103

It has been shown that mouse forebrain ischaemia/reperfusion can lead to genetic instability leading to mutations in the brain thought to be due to oxidative stress. After DNA damage has occurred, the DNA is eventually repaired through the cells base-excision repair (BER) pathway and then re-methylated by DNMT1 [107]. These observations suggest that a balance of DNA methylation, controlled by DNMT1, is essential in limiting cell death during cerebral ischaemia.

Pharmacological manipulation of gene expression is a potential way to reduce post-ischaemic brain damage and inhibition of HDACs has been suggested as a potential neuroprotective approach [89,96]. A number of HDACis are currently being assessed as possible neuroprotective agents. The main suggested benefits of these drugs are reduction in inflammation, promotion of stem cell migration, reduction in neuron apoptosis, an increase in neurogenesis and angiogenesis, and preservation of the BBB. Table 4 shows a list of HDACis currently used in the clinical setting and their effects. For example, VPA is widely used for a variety of seizure disorders and in certain cases for depression and anxiety and is a potent HDACi. It has been shown that one of the major consequences of cerebral ischaemia is the reduction in histone H3 acetylation within the cortex and striatum in mice [93]. Administration of VPA after MCAO significantly reduced the infarct volume and resulted in an increase in H3K9 acetylation in a time-dependent manner. VPA administration also provided neuroprotection by inhibiting the expression of caspase-3, an enzyme that has been implicated in neuronal damage after focal cerebral ischaemia, and increasing the expression of heat shock protein 70 (HSP70) [93], a protein that has been associated with neuroprotection [108].

EVIDENCE FOR THE ROLE OF EPIGENETICS IN PAD

Studies exploring the role of epigenetic mechanisms in PAD are limited. There are many reports of successful revascularization in CLI by novel regenerative cell therapies [109111]. Pre-clinical studies using the most commonly used ischaemic mouse hind limb model (which requires unilateral ligation and excision of the femoral artery) showed increased vascularity and blood flow after local gene transfer of VEGF-A. Thus, altering gene expression through epigenetic manipulation was proposed as a novel approach for the treatment of CLI. It was previously shown that lentivirus-mediated delivery of short hairpin RNAs (shRNA) targeted to specific regions in the VEGF-A promoter induced or repressed gene expression via histone modification [112]. Recently, Palii et al. showed that transcription factor T-cell acute leukemia 1 (TAL1) is a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs) and that TAL1 up-regulates the expression of genes promoting migration and adhesion through recruitment of the HAT p300 [113]. Ex vivo priming of ECFCs with TSA improved the revascularization efficiency of ECFCs showing that modifying the epigenome of stem/progenitor cells using small molecule epigenetic drugs is a potential way of enhancing their therapeutic potential before transplantation. Recently, a novel protein, prothymosin α (ProTα), was shown to be released extracellularly upon ischaemia induced necrosis [114]. ProTα stimulates cell proliferation and differentiation through chromatin remodelling and was shown to be protective against ischaemic stress. This observation suggests the possibility of involvement of other unknown proteins in epigenetic control of ischaemic stress insults.

Even though HDACis are promising drugs for the treatment of several pathologies adverse side effects and cardiotoxicity have been reported in experimental models of PAD [115]. In a recent report, mice subjected to lower limb ischaemia were injected with selective Class I (DIs MS275) and IIa (MC1568) HDACis [115]. Between 14 and 21 days after ischaemia, the MC1568 compound increased the number of newly formed muscle fibres but delayed their terminal differentiation. On the other hand, MS275 abolished the early onset of the regeneration process resulting in atrophy and fibrosis [115]. Furthermore, the class selective HDACis had different effects. MC1568 promoted arteriogenesis whereas MS275 had an inhibitory effect. The HDACis had no effect on capillarogenesis, suggesting that class-selective HDACis interfere with normal mouse ischaemic hind limb regeneration and suggesting caution in the application of these drugs. The HDACis were also reported to have opposite effects on atherosclerosis and neointimal hyperplasia. For example, it was demonstrated previously that administration of TSA to low-density lipoprotein receptor deficient (Ldlr−/−) mice exacerbates atherosclerosis [59]. However, in a recent promising report, a novel HDACi, MCT-3, was shown to attenuate neointimal hyperplasia in male FVB/N mice [116]. These reports emphasize the need for better understanding of epigenetic pathways involved in PAD and more comprehensive studies on the actions of different HDACis.

EPIGENETIC EFFECTS OF RISK FACTORS FOR CVD AND PAD

A number of risk factors for both CVD and PAD have been identified to have epigenetic effects. Examples are shown in Table 5.

Table 5
Evidence for the epigenetic effects of some risk factors for CVD and PAD
Risk factorsMechanismEffectCell type/model studiedGenes involvedReferences
Diabetes HAT mediated regulation of vasoactive factors and extracellular matrix proteins Increased expression and binding of the HAT p300 to the gene promoters Human umbilical vein endothelial cells Endothelin, Fibronectin, Vascular endothelial growth factor [117
 Development of diabetic retinopathy and promoting metabolic memory Decrease in the activity of H3-specific acetyltransferase Retina and retinal endothelial cells from Streptozotocin-induced diabetic rats HDAC1, HDAC2, HDAC8 [118
 miRNA-expression profiling in diabetic retinopathy Upregulation of miRNAs responsive to crucial genes inducing diabetic retinopathy Retina and retinal endothelial cells of Streptozotocin-induced diabetic rats Nuclear factor kappa-light-chain-enhancer of activated B cells, Vascular endothelial growth factor, p53 [119
 HDAC2 is a key regulator of diabetes induced renal fibrosis Decreased expression of extracellular matrix components Streptozotocin-induced diabetic kidneys; Rat kidney tubular epithelial cells (NRK-52E) Transforming growth factor β1 [120
  Decreased epithelial to mesenchymal transition    
 miRNA-mediated gene regulation in diabetic retinopathy Decreased miR-200b and increased target gene expression Human umbilical vein endothelial cells; Bovine retinal endothelial cells treated with high glucose Vascular endothelial growth factor [121
Hypertension Hypermethylation of H3K79 mediated gene silencing Increased methylation leads to altered regulation of extracellular fluid volume and blood pressure mIMCD3 cells Epithelial sodium channel [122
 DNA methylation Decreased global DNA methylation levels Patient peripheral blood Global changes in genome [123
Hyper homocysteinemia DNA methylation Endothelial dysfunction, by inhibiting endothelial cell regeneration Endothelial cells p21ras [124
  Decreased intracellular Nitric oxide levels, altered gene expression and impaired endothelial function Human umbilical vein endothelial cells Endothelial nitric oxide, Vascular endothelial growth factor, Akt, Extracellular signal-regulated kinase 1/2 [125
  Altered S-Adenosyl methionine dependent methylation can contribute to vascular dysfunction and Hyper homocysteinemia CBS +/− mice S-Adenosyl methionine and S-adenosyl-L-homocysteine [126
Aging DNA methylation Decreased Estrogen receptor-α expression in aging cells was advantageous for proliferating cells Smooth muscle cells Estrogen receptor-α [127
 miRNA-200c Increased miRNA-200C induction by oxidative stress and in turn it induces senescence and apoptosis Human umbilical vein endothelial cells and p66ShcA−/− mice Zinc finger E-box binding homeobox-1 [128
 miRNA-146a/b Increased miRNA-146a/b expression in response to increasing cytokine levels and inhibits senescence associated secretory phenotype (SASP) and modulates inflammation by decreasing the secretion of IL-6 and IL-8 Fibroblasts Interleukin 1, 6, and 8 receptor-associated kinase-1 [129
 miRNA-146a Increased senescence in endothelial cells Endothelial cells Silent information regulator-1 [130
 miR-217 Increased senescence and inhibits angiogenesis in young Human umbilical vein endothelial cells Human umbilical vein endothelial cells and Endothelial cells Silent information regulator-1 [131
  Decreased senescence and increases angiogenesis of old endothelial cells    
Smoking DNA methylation Increased DNA methylation at a specific CpG site is associated with inflammation and intimal hyperplasia Patient peripheral blood Coagulation factor II thrombin receptor-like-3 [132
  Increased aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers Upper aerodigestive tract epithelium from heavy smokers Retinoic acid receptor beta-2, H-cadherin (, p16(INK4a), RAS association domain family I [133
  Tobacco-induced molecular damage Oral and bronchial brush specimens from 127 smokers p16(INK4a), and Fragile histidine triad [134
  Increased methylation of at least one gene present in 72% of cancer patients with no smoking history or those who smoked <20 pack-years Serum DNA Deleted in colorectal carcinoma, Kinesin family member 1A, Nischarin, Retinoic acid receptor β [135
  Patterns in the Long Interspersed Nucleotide Element-1 and in Alu elements Children exposed prenatally to tobacco smoke Global changes in genome; Glutathione S-transferase pi-1 [136
  Increased Long Interspersed Nucleotide Element-1 methylation and development of cardiovascular disease Serum Vascular cell adhesion molecule-1 [137
  Independent risk factor in CAD patients global DNA methylation Plasma Global changes in genome [138
Risk factorsMechanismEffectCell type/model studiedGenes involvedReferences
Diabetes HAT mediated regulation of vasoactive factors and extracellular matrix proteins Increased expression and binding of the HAT p300 to the gene promoters Human umbilical vein endothelial cells Endothelin, Fibronectin, Vascular endothelial growth factor [117
 Development of diabetic retinopathy and promoting metabolic memory Decrease in the activity of H3-specific acetyltransferase Retina and retinal endothelial cells from Streptozotocin-induced diabetic rats HDAC1, HDAC2, HDAC8 [118
 miRNA-expression profiling in diabetic retinopathy Upregulation of miRNAs responsive to crucial genes inducing diabetic retinopathy Retina and retinal endothelial cells of Streptozotocin-induced diabetic rats Nuclear factor kappa-light-chain-enhancer of activated B cells, Vascular endothelial growth factor, p53 [119
 HDAC2 is a key regulator of diabetes induced renal fibrosis Decreased expression of extracellular matrix components Streptozotocin-induced diabetic kidneys; Rat kidney tubular epithelial cells (NRK-52E) Transforming growth factor β1 [120
  Decreased epithelial to mesenchymal transition    
 miRNA-mediated gene regulation in diabetic retinopathy Decreased miR-200b and increased target gene expression Human umbilical vein endothelial cells; Bovine retinal endothelial cells treated with high glucose Vascular endothelial growth factor [121
Hypertension Hypermethylation of H3K79 mediated gene silencing Increased methylation leads to altered regulation of extracellular fluid volume and blood pressure mIMCD3 cells Epithelial sodium channel [122
 DNA methylation Decreased global DNA methylation levels Patient peripheral blood Global changes in genome [123
Hyper homocysteinemia DNA methylation Endothelial dysfunction, by inhibiting endothelial cell regeneration Endothelial cells p21ras [124
  Decreased intracellular Nitric oxide levels, altered gene expression and impaired endothelial function Human umbilical vein endothelial cells Endothelial nitric oxide, Vascular endothelial growth factor, Akt, Extracellular signal-regulated kinase 1/2 [125
  Altered S-Adenosyl methionine dependent methylation can contribute to vascular dysfunction and Hyper homocysteinemia CBS +/− mice S-Adenosyl methionine and S-adenosyl-L-homocysteine [126
Aging DNA methylation Decreased Estrogen receptor-α expression in aging cells was advantageous for proliferating cells Smooth muscle cells Estrogen receptor-α [127
 miRNA-200c Increased miRNA-200C induction by oxidative stress and in turn it induces senescence and apoptosis Human umbilical vein endothelial cells and p66ShcA−/− mice Zinc finger E-box binding homeobox-1 [128
 miRNA-146a/b Increased miRNA-146a/b expression in response to increasing cytokine levels and inhibits senescence associated secretory phenotype (SASP) and modulates inflammation by decreasing the secretion of IL-6 and IL-8 Fibroblasts Interleukin 1, 6, and 8 receptor-associated kinase-1 [129
 miRNA-146a Increased senescence in endothelial cells Endothelial cells Silent information regulator-1 [130
 miR-217 Increased senescence and inhibits angiogenesis in young Human umbilical vein endothelial cells Human umbilical vein endothelial cells and Endothelial cells Silent information regulator-1 [131
  Decreased senescence and increases angiogenesis of old endothelial cells    
Smoking DNA methylation Increased DNA methylation at a specific CpG site is associated with inflammation and intimal hyperplasia Patient peripheral blood Coagulation factor II thrombin receptor-like-3 [132
  Increased aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers Upper aerodigestive tract epithelium from heavy smokers Retinoic acid receptor beta-2, H-cadherin (, p16(INK4a), RAS association domain family I [133
  Tobacco-induced molecular damage Oral and bronchial brush specimens from 127 smokers p16(INK4a), and Fragile histidine triad [134
  Increased methylation of at least one gene present in 72% of cancer patients with no smoking history or those who smoked <20 pack-years Serum DNA Deleted in colorectal carcinoma, Kinesin family member 1A, Nischarin, Retinoic acid receptor β [135
  Patterns in the Long Interspersed Nucleotide Element-1 and in Alu elements Children exposed prenatally to tobacco smoke Global changes in genome; Glutathione S-transferase pi-1 [136
  Increased Long Interspersed Nucleotide Element-1 methylation and development of cardiovascular disease Serum Vascular cell adhesion molecule-1 [137
  Independent risk factor in CAD patients global DNA methylation Plasma Global changes in genome [138

Diabetes is an important risk factor for athero-thrombosis [139] and a number of studies suggest that dysglycaemia promotes epigenetic changes [140]. Elevated levels of homocysteine (Hcy) are also thought to cause epigenetic changes [141]. Hyperhomocysteinemia (HHcy) is associated with CVD and PAD [137,142144] and a deficiency of folic acid has been previously linked to endothelial dysfunction, atherosclerosis, stroke and CLI [145]. HHcy is associated with decreased levels of global genome-wide CpG methylation and promoter-specific gene methylation, promoting altered expression levels of a number of genes relevant to vascular damage [125,146149]. Furthermore, HHcy also suppresses angiogenesis, promotes SMC proliferation, promotes dyslipidaemia, stimulates vascular oxidative stress, and leads to impairment of EC regeneration and function [125,148]. Advanced age is a major risk factor for CVD and PAD and aging is known to cause altered gene expression patterns due to epigenetic changes [150,151]. It has been demonstrated that miRNAs play a role in cell senescence and that they can exert either positive or negative effects on cell growth depending on the genes they are regulating [128,129]. Advanced age and ischaemic injury have been related to aberrant miRNA transcription and altered expression of genes such as silent information regulator-1 (SiRT-1) [130,131] (Table 5).

Hypertension has a strong association with CVD and PAD. Overexpression of the connective tissue growth factor (CTGF) has been found in a number of pathologies such as atherosclerotic lesions and can be induced by angiotensin II in models of hypertensive nephropathy and vascular fibrosis [152]. It has also been shown that hypermethylation of H3K79 at the CTGF promoter by the enzyme disruptor of teleomeric silencing-1 (Dot-1) results in gene silencing in the collecting ducts of the nephron [153] and the inhibition of CTGF has been shown to reverse the process of fibrosis [154].

Smoking is a major risk factor for CVD and PAD and has been implicated in inducing a number of epigenetic modifications [132]. Smoking-induced epigenetic changes have been mainly associated with DNA methylation [133,135,155]. Prolonged smoking has been shown to increase gene promoter specific DNA methylation in the peripheral blood [156] as well as in the aerodigestive tract and oral epithelium [133,134] suggesting the profound impact of smoking on the epigenome. Tobacco abuse in the mother has also been shown to result in altered placental gene expression in addition to increased global DNA methylation levels [136,157] suggesting the transgenerational effects of smoking.

THE RELEVANCE OF EPIGENETICS TO THE EFFECTS OF ISCHAEMIA

The reduced blood flow associated with CVD and PAD can lead to progressive cell death and induce an inflammatory response which may exacerbate initial levels of tissue injury. Therapeutic revascularization aims to increase collateral capacity (arteriogenesis) and new capillary formation (neo-angiogenesis) [8].

Arteriogenesis

The formation of mature arteries from pre-existent interconnecting arterioles following an arterial occlusion is known as arteriogenesis. Even though arteriogenesis has many common features with angiogenesis, they follow different pathways. Arteriogenesis is potentially more able to compensate for an occluded artery than angiogenesis. Furthermore, arteriogenesis is induced by physical forces such as fluid shear stress and occurs in an environment of normoxia whereas angiogenesis is induced by hypoxia, leading to formation of new capillaries [158]. Elevated fluid shear stress activates ion channels, releasing nitric oxide (NO) and initiates two major signalling pathways. One pathway originating as a result of shear stress, attracts bone marrow-derived stem cells and endothelial progenitor cells (EPCs), which are essential for structural remodelling, and the other pathway results in the cell cycle entry of ECs and SMCs, leading to proliferation and differentiation [159].

Neoangiogenesis

The process of formation of new blood vessels is called neoangiogenesis. It consists of several successive steps of vascular destabilization, angiogenic sprouting, formation of lumen and vascular stabilization. A number of growth factors and signalling pathways orchestrate angiogenesis through the direct or indirect regulation of quiescence, migration and proliferation of ECs [160,161]. The majority of studies have focused on tumour angiogenesis, however additional insight is urgently required to improve our understanding about the role of genetic and epigenetic factors in angiogenesis in other diseases [162]. Gene or protein therapy delivering various growth factors such as VEGF, as well as cell therapy using EPCs, mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs), have been developed as pro-angiogenic therapeutics for ischaemic heart disease and PADs [161].

THE RELEVANCE OF EPIGENETICS TO THE CELLULAR RESPONSE TO ISCHAEMIA

Epigenetic modifiers can influence various aspects of the results and response to ischaemia by altering transcriptional regulation of cellular responses. The alteration in gene regulation may lead to a number of effects such as proliferation and differentiation of SMCs and ECs, attenuation of apoptosis and inflammation, improvement in blood flow and increased plasticity of newly formed vessels. Thus, identifying the epigenetic contribution to ischaemic injury and its resolution may influence drug development (Figure 2).

Relevance of epigenetic marks to the effect and response to ischaemic injury

Figure 2
Relevance of epigenetic marks to the effect and response to ischaemic injury

A simplified diagram highlighting the different types of epigenetic marks and the complexity of various epigenetic gene regulation mechanisms and the cell types involved in ischaemic injury development and resolution. ROS, reactive oxygen species; Hcy, homocysteine; VSM, vascular smooth muscle cells; Mir; microRNAs.

Figure 2
Relevance of epigenetic marks to the effect and response to ischaemic injury

A simplified diagram highlighting the different types of epigenetic marks and the complexity of various epigenetic gene regulation mechanisms and the cell types involved in ischaemic injury development and resolution. ROS, reactive oxygen species; Hcy, homocysteine; VSM, vascular smooth muscle cells; Mir; microRNAs.

SMC proliferation

SMC proliferation constitutes a central component of atherosclerosis formation and neointimal vascular remodelling. Histone acetylation constitutes a major epigenetic modification for the transcriptional cascade controlling proliferative gene expression in SMCs [163]. Pharmacological HDACis have been shown to arrest cell proliferation [164]. A novel non-selective HDACi, Scriptaid, has been shown to inhibit neointimal thickening in a mouse model [163]. Butyrate, a dietary HDACi, has been shown to stimulate PTMs of histone H3 and differently alters G1-specific cell cycle proteins leading to inhibition of SMC proliferation [165]. A recent study suggested the in vitro and in vivo potential of a novel HDACi, MCT-3, to modulate SMC migration, proliferation and neointimal hyperplasia [116]. MCT-3 administration induced histone H3 and H4 acetylation and regulation of expression of orphan nuclear receptor NUR77, plasminogen activator inhibitor type-1 (PAI-1) and cyclin dependent kinase inhibitors (CDKI) p21CIP1/WAF1 and p27KIP1. However, additional pre-clinical studies are necessary to determine the potential clinical utility of this novel compound [116] due to previous contradictory reports about the non-specific effects of HDACis. Previously, the specific HDACi TSA was shown to inhibit SMC proliferation via induction of p21CIP1/WAF1 [60]. However, in a subsequent study using Ldlr−/− mice, TSA exacerbated atherosclerosis and modulated expression of oxLDL receptors and some proatherogenic genes [59].

SMC differentiation

Many of the pathways involved in SMC differentiation leading to vascular remodelling in adults are similar to those involved in embryogenesis [166]. A recent study assessed the role of neural crest-derived SMC lineage in formation of the cardiac outflow tract in mice [167]. The HDAC3 (Class I HDAC) blocks SMC differentiation and is associated with down-regulation of the Notch ligand Jagged1, a key driver of SMC differentiation. In a new in vitro organotypic model system for rat limb bud development the effect of the epigenetic drug 5-Aza was assessed [168]. Initially, the limb buds were covered by immature epithelium and contained mesenchyme, and capillaries. However, following administration of 5-Aza, a decrease in cell proliferation was noted with the disappearance of structures typical for vasculogenesis. This negative effect of an epigenetic modifying drug suggests that specific drugs have to be identified for each cell phenotype involved in the disease process. Recently, a study compared the remodelling process in the femoral and internal carotid arteries within a novel mouse vascular injury model [169]. Compared with the remodelling in the femoral artery, intracranial internal carotid arteries showed a unique remodelling pattern. The formation of neointima was delayed and showed continuous macrophage accumulation, loss of SMCs followed by medial thinning and continuous expansion of the adventitia [169]. These findings suggest that pathophysiological differences exist in different peripheral arteries which are probably controlled by different genetic and epigenetic pathways.

EC differentiation

Angiogenesis involves complex and highly dynamic interactions between ECs and their environment that promote endothelial motility, filopodia extension, proliferation and the formation of new cell–cell junctions. Lysine acetylation and cytosine methylation are important transcriptional regulators of angiogenic genes in ECs such as VEGF and eNOS [170]. Angiopoietin-2 induces vessel destabilization leading to formation of new vessels or to regression of existing vessels. Angiopoietin-2 promoter methylation has been associated with decreased gene expression [171]. Studies suggest that laminar shear stress is an important modulator of eNOS expression in ECs and stimulates activating H3 and H4 acetylation marks via the HAT p300 complex [68]. The shear stress induced epigenetic activation marks result in chromatin remodelling promoting constitutive eNOS expression. Recent studies have shown that protein kinase D (PKD) stimulates phosphorylation and nuclear export of HDAC5 in ECs and regulates cell migration [172]. Studies in human umbilical vein ECs (HUVECs) showed that PKD also stimulates pro-angiogenic fibroblast growth factor (bFGF) expression through the HDAC5 signalling pathway. Age-related impairment of angiogenesis is likely plays a central role in the outcome of CVD and PAD. Aging-induced dysregulation of Dicer1 dependent miRNA expression impairs angiogenic capacity of rat cerebral microvascular ECs [173].

Fibroblasts

Studies of fibroblasts suggest that epigenetic factors including DNA methylation and histone acetylation regulate wound healing [174176]. MeCP2 plays a critical role in epithelial to myo-fibroblast transformation [174] and in many fibrotic disorders there is an imbalance in the histone acetylation and deacetylation [175]. Proliferation of fibroblasts induced by platelet-derived growth factor (PDGF) is reduced by TSA [176].

THE INFLUENCE OF EXERCISE ON EPIGENETICS AS RELEVANT TO CVD AND PAD

Increased physical activity is associated with reduced risk of developing CVD and PAD. Sedentary lifestyle has been associated with an unfavourable epigenome [177]. A study by Alibegovic et al. demonstrated that 9 days of bed rest led to increased peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α promoter methylation [178]. Physical activity can modify the genome in the peripheral vascular bed [179]. Exercise induces an increase in free radical production and concurrently improves antioxidative capacity, resulting in activation of signalling cascades that modulate the physiological adaptations of the peripheral vascular genome [10,180]. For example, apoptosis associated spec-like protein (ASC) modulates the expression of pro-inflammatory cytokines such as IL-1β and IL-18. Chronic moderate exercise was shown to attenuate the age-dependent decrease in ASC promoter methylation, and thereby decrease pro-inflammatory cytokine expression [181].

Exercise training has been associated with an increase in the levels of global DNA methylation in peripheral blood cells, furthermore the level of DNA methylation increases with increasing amounts of exercise [182,183]. A single intense exercise bout results in acute remodelling through promoter methylation of exercise-responsive genes within skeletal muscle. Muscle-specific miRNAs called myomirs are also up-regulated by a single bout of endurance exercise and reversibly down regulated by 12-week endurance training [184]. A study showed decreased whole genome methylation in skeletal muscle biopsies obtained from healthy sedentary men and women after acute exercise [185]. Exercise induced a dose-dependent expression with a marked hypomethylation on the promoter of several metabolic genes. Exercise training has also been shown to alter DNA methylation in a chronic way in the skeletal muscles biopsies collected after a 6 months training programme [186]. A previous study showed that the DNA methylation profiles of several genes involved in diverse metabolic pathways were differentially methylated in response to chronic exercise. Furthermore, chronic exercise also led to increased DNA methylation of a large number of genes in adipose tissue [186]. In contrast, an exercise intervention study in middle aged healthy, sedentary men showed that the majority of the genes showed decreased DNA methylation within skeletal muscle [187], supporting the fact that epigenetic modifications are probably tissue- and population-specific.

Exercise-induced epigenetic alterations relevant to CVD

Previous literature suggests a role for exercise in promoting stroke rehabilitation, and indicates that exercise training protects neural cells from inflammation, apoptosis and oxidative stress and can enhance choline acetyltransferase activity. Recent rodent animal model studies highlight the importance of exercise induced epigenetic modification in the brain [188190]. Previously, voluntary exercise in adult rats was shown to increase acetylated H3 within the hippocampus [188]. In adolescent male mice, voluntary wheel running for 1 week resulted in increased global H3 acetylation in both the hippocampus and cerebellum. In contrast, the expression pattern of DNMTs and HDAC were decreased in both regions following exercise [189]. Similarly, in another study, a single exercise session decreased both DNMT3b and DNMT1 levels in young adult rats [190]. The exercise protocol was shown to reduce H3-K9 methylation levels in young adult rats, whereas the single session reversed the changes in H3-K9 methylation levels induced by aging [190]. Furthermore, a forced exercise protocol ameliorated aging-related memory decline, decreased pro-inflammatory markers and increased histone H4 acetylation levels in the hippocampus of 20-month-old rats [191]. A study investigating the effects of antecedent exercise on functional recovery following MCAO in adult male rats showed that antecedent treadmill exercise prior to focal cerebral ischaemia had beneficial effects [192]. Antecedent exercise exerted neuroprotective effects against ischaemic brain injury by improving motor performance and decreasing the levels of calpain gene expression. Together, these results suggest that an imbalance in the key epigenetic modulators might be linked to the capacity of brain to recover after ischaemia induced by occlusion and that patient-specific exercise rehabilitation has to be carefully tailored.

Exercise-induced epigenetic alterations relevant to PAD

Supervised exercise programmes have been consistently demonstrated to improve walking time and walking distance in PAD patients [193,194]. Supervised exercise leads to alterations in skeletal muscle gene expression. In addition exercise-induced elevation of reactive oxygen species (ROS) is believed to be important in promoting angiogenesis [195]. Global lysine 36-H3 acetylation, a site associated with transcriptional elongation, was shown to be increased following 60 min of cycling [196]. Furthermore, during exercise HDAC4 and HDAC5 were exported from the nucleus, thereby removing their transcriptional repressive function. In addition, activation of two protein kinases that induce phosphorylation-dependent Class IIa HDAC nuclear export, namely the AMP-activated protein kinase (AMPK) and the calcium-calmodulin-dependent protein kinase II (CaMKII), was also induced in response to exercise [196]. These protein kinases phosphorylate HDACs, promoting transcription factor binding and subsequent initiation of transcriptional activity of exercise-responsive genes such as myogenin, muscle creatine kinase (MCK) and glucose transporter 4 (GLUT4) [197,198]. Furthermore, the effects of exercise training was blocked in transgenic mice showing overexpression of HDAC5 further suggesting that epigenetic transcriptomic regulation is important in controlling the effects of exercise [199].

Evidence for rapid and reversible DNA methylation in response to exercise is currently emerging [200]. The human ten eleven translocation (TET)-1 gene encodes an enzyme that catalyses the conversion of 5mC to 5-hydroxy methyl cytocine (5hmC), and plays a crucial role in active demethylation [201]. It has also been suggested that miRNA expression is differentially regulated in subjects that respond favourably and unfavourably to exercise, suggesting that taking the epigenetic profile into consideration will help the patients before subjecting them to exercise rehabilitation programmes [202].

FUTURE DIRECTIONS

Recently arginase 2 (Arg2), NO production and endothelial function were identified to be regulated by HDAC2 [203]. Thus, therapeutic activation of HDAC2 represents a novel method of correcting endothelial dysfunction and atherosclerosis. Lysine acetylation of eNOS is a PTM. HDAC3 antagonizes aspirin-stimulated endothelial NO production by reversing aspirin-induced lysine acetylation of eNOS [204]. This interesting observation suggests that selective pharmacological HDAC inhibition may be a way to improve the efficacy of drugs such as aspirin in the treatment of stroke and PADs.

Exploring epigenetic mechanisms in pathologies is gaining more momentum within scientific research. The fact that the epigenetic modifications can be reversed makes them an ideal target for drug treatment. In the oncology field, epigenetic drugs have already entered the clinical arena and methylation patterns are used as biomarkers to subtype and stage various cancers [205,206]. HDACis are being investigated in clinical trials for the treatment of solid and haematological malignancies [207,208]. There are potential limitations in applying these drugs to CVD and PAD however. These medications lack specificity, for example HDACis also influence non-histone proteins making their effects widespread. Furthermore, a lot of HDACs form co-repressor complexes in order to silence gene activity, which again amplifies the potential non-specific effect that HDACi technology can have within the cell.

The role of DNMTs, HDACs, HATs and miRNAs in transcriptional regulation is becoming increasingly clear. The complexity of the interactions, however, means applying findings in a safe way to patients is not straightforward. Thus, elucidating the cause and effect of these modifications depends on further pre-clinical and clinical studies. It should also be noted that translating pre-clinical findings to the clinical setting is also not simple, since the various components of the epigenetic machinery may be functionally different in animal models of CVD and PAD. Realistically, in order for epigenetic drugs to be applied to patients with CVD and PAD, there is a need for more specific interventions which can be applied in a safe and effective way to patients with a number of co-morbidities (Figure 3). Such developments likely require improved understanding of the epigenetic mechanisms involved in these diseases.

Epigenetic factors affecting occlusive cerebral and peripheral arterial diseases

Figure 3
Epigenetic factors affecting occlusive cerebral and peripheral arterial diseases

A number of risk factors common to both CVD and PAD have been identified to cause epigenetic changes. Epigenetic alterations influence common pathologic responses including inflammation, ischaemia, hypoxia and shear stress. Risk factors, such as diabetes, aging and life style habits such as smoking and physical inactivity, modulate the epigenome promoting maladaptive changes.

Figure 3
Epigenetic factors affecting occlusive cerebral and peripheral arterial diseases

A number of risk factors common to both CVD and PAD have been identified to cause epigenetic changes. Epigenetic alterations influence common pathologic responses including inflammation, ischaemia, hypoxia and shear stress. Risk factors, such as diabetes, aging and life style habits such as smoking and physical inactivity, modulate the epigenome promoting maladaptive changes.

Abbreviations

     
  • 5-Aza

    5-azacytidine

  •  
  • ASC

    apoptosis associated spec-like protein

  •  
  • Brg1

    Brahma-related gene 1

  •  
  • bFGF

    fibroblast growth factor

  •  
  • CAD

    coronary artery disease

  •  
  • CLI

    critical limb ischaemia

  •  
  • CTGF

    connective tissue growth factor

  •  
  • CVD

    cerebrovascular disease

  •  
  • DNMT

    DNA methyltransferase

  •  
  • ECFCs

    endothelial colony-forming cells

  •  
  • ECs

    Endothelial cells

  •  
  • eNOS

    endothelial nitric oxide

  •  
  • EPCs

    endothelial progenitor cells

  •  
  • HAT

    histone acetlytraferase

  •  
  • HDAC

    histone deacetylase

  •  
  • HDACis

    HDAC inhibitors

  •  
  • HHcy

    Hyperhomocysteinemia

  •  
  • HIF

    hypoxia-inducible factor

  •  
  • IC

    intermittent claudication

  •  
  • KLF4

    Kruppel-Like Factor 4

  •  
  • 5meC

    5-methylcytosine

  •  
  • MCAO

    middle cerebral artery occlusion

  •  
  • MeCP2

    methyl binding CpG protein 2

  •  
  • ncRNAs

    Non-coding RNAs

  •  
  • PAD

    peripheral arterial disease

  •  
  • PKD

    protein kinase D

  •  
  • PTM

    post translational modification

  •  
  • RISC

    RNA-induced silencing

  •  
  • SMCs

    Smooth muscle cells

  •  
  • TAL1

    T-cell acute leukemia 1

  •  
  • TIA

    transient ischaemic attack

  •  
  • TSA

    trichostatin A

  •  
  • VEGF

    vascular endothelial growth factor

  •  
  • VPA

    valproic acid

FUNDING

This work is funded in part by the National Health and Medical Research Council [grant numbers 1021416, 1020955, 1003707 and 1000967]; the Queensland Government and the Townsville Hospital Private Practice Trust; a Practitioner Fellowship from the National Health and Medical Research Council, Australia [grant number 1019921 (to J.G.)]; and a Senior Clinical Research Fellowship from the Queensland Government. The funding bodies played no role in generation of the data presented in this publication.

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Author notes

1

These authors contributed equally to this work.