**4. Epigenetics**

The nucleosome is the fundamental subunit of chromatin in eukaryotes. Each nucleosome consists of a 146-bp DNA segment wrapped around an octamer of core histone proteins that includes two molecules of histones H2A, H2B, H3 and H4 associated with a single copy of histone H1. Epigenetics is defined as the study of stable alterations of gene expression without alterations of DNA itself. These alterations include the post-translational addition or removal of methyl groups to DNA as well as methyl, acetyl, sumoyl and phospho groups to histones and other kind of proteins. These changes participate in remodelling chromatin and modifying its accessibility to transcription factors and cofactors [17]. Epigenetic control is one of the main regulatory systems contributing to phenotypic differences between cell types in multicellular organisms. Epigenetic changes may explain why subjects with similar genetic backgrounds and risk factors for particular diseases can differ greatly in clinical manifestation and therapeutic response [18]. It has been reported that epigenetic mechanisms play a critical role in regulating endothelial gene expression [19]. Among these epigenetic changes are the methylation of DNA, RNA-based mechanisms and the posttranslational modification of histone proteins.

## **DNA methylation**

that the process can also take place in adults, which opens up a new avenue for clinical

– Angiogenesis is the formation of new blood vessels by sprouting from pre-existing small vessels in embryonic and adult tissue or by intravascular subdivision process [11]. This process is believed to be induced by angiogenic factors including fibroblast growth factor

– Arteriogenesis results from the hypertrophy and luminal distention of pre-existing collateral vessels, which involves specific remodelling of existing nascent EC tubules for greater size, elasticity and stability through the recruitment of and enclosure by SMCs and pericytes that secrete specific extracellular matrices. Therefore, these vessels have fully devel-

Angiogenesis is a very complex process that can be simplified into three categories: mechani-

– **Cellular factors**: There are many molecules that can modulate angiogenesis. The most essential angiogenic growth factors are as follows: FGF, VEGF, placenta growth factor, angiopoietin-1 and angiopoietin-2. Several pathological conditions can also initiate angiogenesis. For example, hypoglycaemia increases the expression of critical angiogenic inductor VEGF [13]. It has also been extensively demonstrated that the presence of inflammatory

– **Environmental factors**: Angiogenesis can be induced by hypoxia and through increased EC production of NO. Hypoxia stimulates the release of several angiogenic factors including platelet-derived growth factor and FGF-1 and FGF-2 by macrophages. Hypoxia also upregulates VEGF production, which is known to induce the production and secretion of NO from ECs, while eNOS production is amplified during VEGF-induced angiogenesis [15]. – **Mechanical factors**: There are two main factors: haemodynamic and shear stress. Haemodynamic changes trigger an augmentation of blood flow and might therefore stimulate vascular sprouting, maintain patency of the newly formed collateral vessels and provide blood flow to the ischemic area [16]. Shear stress has an important influence on the

The nucleosome is the fundamental subunit of chromatin in eukaryotes. Each nucleosome consists of a 146-bp DNA segment wrapped around an octamer of core histone proteins that includes two molecules of histones H2A, H2B, H3 and H4 associated with a single copy of histone H1. Epigenetics is defined as the study of stable alterations of gene expression without alterations of DNA itself. These alterations include the post-translational addition or removal of methyl groups to DNA as well as methyl, acetyl, sumoyl and phospho groups to histones and other kind of proteins. These changes participate in remodelling chromatin and modifying its accessibility to transcription factors and cofactors [17]. Epigenetic control is one of the main

cells, like macrophages and neutrophils, is sufficient to induce angiogenesis [14].

cal, chemical and molecular factors (see Ref. [12] for a more extensive review).

development of collateral vessel networks in the ischaemic tissues.

(FGF) and vascular endothelial growth factor (VEGF).

152 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

oped tunica media and tunica adventitia [11].

applications [10].

**4. Epigenetics**

The methylation of DNA involves the covalent modification of the 5-position of cytosine to define the 'fifth base of DNA', 5-methyl-cytosine [20]. In mammals, DNA methylation is almost exclusively restricted to CpG dinucleotides. DNA methylation is catalysed by DNA methyltransferases and regulates biological processes underlying CVD, such as atherosclerosis, inflammation, hypertension, and diabetes [21].

## **RNA-based mechanisms**

**1.** miRNA therapeutics

MicroRNAs (miRNA or miR) are short (20–22 nucleotides) non-coding RNAs modulating gene expression further by down-regulating the translation of target mRNAs through the inhibition of post-transcriptional events, through transcript degradation or through direct translational repression.

**2.** Long non-coding RNAs (lncRNAs)

Long non-coding RNAs (lncRNAs) are gaining more prominence as regulators of gene expression. The central role that lncRNAs play in heart development is only slowly being recognised [18]. Besides, understanding the function of these molecules in CVD is even further away.

#### **Histone modification**

It is well established that histone residues can undergo a wide array of modifications. At least eight different types of modification have been characterised with a range of enzymes identified for each: acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADPribosylation, deimination, and proline isomerisation (**Table 1**).

Histone methylation is modulated by two enzymes: histone methyltransferases and histone demethylases. The acetylation status of histone is fine-tuned by histone acetyltransferases (HATs) and HDACs. HDACs are enzymes that remove acetyl groups from histone lysine residues thereby increasing their negative charges, which lead to chromatin condensation and gene repression [17].

## **4.1. The HDAC family**

Deacetylation of histones in nucleosomes induces chromatin compaction, which represses transcription by preventing the binding of transcription factors and other components of the


**Table 1.** Types of histone modifications and the enzymes responsible (modified from Ref. [22]).

transcriptional machinery onto the gene promoter and enhancer regions. HDACs are enzymes that remove acetyl groups from hyperacetylated histones, and modification by HDACs leads to a closed chromatin structure and suppression of genes. HDACs are recruited to gene promoters by DNA-binding proteins that recognise certain DNA sequences and in this way provide specific modulation on gene expression.

There are 18 characterised members of the HDAC family in mammals, which can be grouped into four classes depending on their functional similarities and their homology with yeast HDACs. The class I and class II HDACs are considered as the 'classical' HDACs [23].

Class I HDACs comprise nuclear, ubiquitously expressed HDACs 1, 2, 3, and 8. HDAC1, 2, and 8 reside nearly exclusively in the nucleus. HDAC3 is found to shuttle between nucleus and cytoplasm. Because these are ubiquitously expressed and involved in cell proliferation and survival, aberrations in their gene expression have been implicated in a wide range of cancers [24, 25].

Class II HDACs shuttle between the cytoplasm and the nucleus depending on specific cellular signals; they share a tissue-specific expression pattern and are divided into two subgroups: class IIa (HDACs 4, 5, 7, and 9) and class IIb (HDACs 6 and 10). Class IIa HDACs distinguish themselves with their extended N-terminal regulatory domain, whereas class IIb HDACs contain two catalytic domains. Class IIa HDACs appear to have tissue-specific roles and can shuttle between the cytosol and the nucleus. In fact, the phosphorylation status is a critical event to determine their localisation in the nucleus or cytoplasm and the ability to act as transcriptional co-repressors in the nuclear region. Conversely, class IIb is mostly found in the cytosol [26].

Class III HDACs regroup the ubiquitously expressed silent information regulator 2 (Sir2) family of nicotinamide adenine dinucleotide (NAD+)-dependent HDACs (SIRT1–7), which share structural and functional similarities with the yeast Sir2 protein. Interestingly, these have a critical role in a wide range of cellular processes such as ageing, transcription, cell survival, DNA repair, apoptosis, and inflammation. Sirtuins appear to have contradictory roles in disease. On the one hand, they control many vital functions involved in cellular protection, while on the other hand, they are also involved in several disease pathologies such as metabolic diseases, neurodegenerative disorders, and cancer [27].

Finally, class IV HDAC is the newly discovered HDAC11. HDAC11 is most closely related to class I HDACs. However, since the overall sequence similarities are low, it cannot be grouped into any of the three existing classes. HDAC11 is primarily expressed in heart, smooth muscle, kidney, and brain tissues.

Recent reports suggest that HDACs can deacetylate non-histone proteins as additional functions of HDACs (**Figure 1**). The roles of HDACs in cancer and neurological diseases have

transcriptional machinery onto the gene promoter and enhancer regions. HDACs are enzymes that remove acetyl groups from hyperacetylated histones, and modification by HDACs leads to a closed chromatin structure and suppression of genes. HDACs are recruited to gene promoters by DNA-binding proteins that recognise certain DNA sequences and in this way pro-

There are 18 characterised members of the HDAC family in mammals, which can be grouped into four classes depending on their functional similarities and their homology with yeast

Class I HDACs comprise nuclear, ubiquitously expressed HDACs 1, 2, 3, and 8. HDAC1, 2, and 8 reside nearly exclusively in the nucleus. HDAC3 is found to shuttle between nucleus and cytoplasm. Because these are ubiquitously expressed and involved in cell proliferation and survival, aberrations in their gene expression have been implicated in a wide range of cancers [24, 25].

Class II HDACs shuttle between the cytoplasm and the nucleus depending on specific cellular signals; they share a tissue-specific expression pattern and are divided into two subgroups:

HDACs. The class I and class II HDACs are considered as the 'classical' HDACs [23].

vide specific modulation on gene expression.

**Modification type Amino acid modification Examples of modifying** 

154 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**Acetylation** Lysine Histone acetyl transferases

**Ubiquitination** Lysine Ubiquinases (ubiquitin

**SUMOylation** Lysine Small ubiquitin-like modifier

**Methylation** Lysine Lysine methyltransferases Transcription

**Phosphorylation** Serine Serine/threonine kinases Transcription

**ADP ribosylation** Glutamate ADP-ribosyltransferases Transcription **Deimination** Arginine (to Citrulline) Peptidylarginine deiminases Transcription **Proline isomerisation** Proline Proline isomerases Transcription

**Table 1.** Types of histone modifications and the enzymes responsible (modified from Ref. [22]).

Threonine Dephosphorylated by

**enzymes**

(HATs)

(HDACs)

Arginine Arginine methyltransferases Transcription

phosphatases

ligases)

(SUMO)

Histone deacetylases

Arginine demethylases

Lysine demethylases Repair

Deubiquinating enzymes Repair

De-SUMOylating enzymes: sentrin-specific proteases

**Role**

Transcription Repair

Repair Condensation

Transcription

Transcription

Replication Condensation

**Figure 1.** Schematic illustration of HDACi downstream effects. Inhibition of HDACs by HDACi induces acetylation of histone proteins as well as non-histone proteins, which leads to the alteration in various physiological and pathological processes (modified from Refs. [28–30]).

been extensively examined. However, the functions of HDACs in cardiovascular diseases and arteriosclerosis are less explored [23].

#### **4.2. HDAC inhibitors**

There has been a breakthrough in the development of HDACi. These HDACi induce acetylation of histone proteins, as well as non-histone proteins, which leads to the alteration and regulation of biological events including angiogenesis, apoptosis/autophagy, cell cycle, fibrogenesis, immune response, inflammation, and metabolism (**Figure 1**). As a result, HDAC inhibitor-based therapies have gained substantial attention as treatments for cardiovascular diseases and cancer.

In the following sections, we will describe the different exerted functions of HDACi in different physiological and pathological conditions.
