Chromatin is a nucleoprotein complex consisting of repeating units of nucleosomal core particles. It offers a dynamic platform for all DNA-mediated processes within the nucleus. The nucleosomal core particles are the basic units of chromatin consisting of 147 base pair (bp) of DNA that wraps almost twice around two copies of each of the four core histone proteins, H3, H4, H2A and H2B. Each nucleosome is separated by 10-16 bp long linkers DNA, which gives an appearance of a bead on a string structure that constitutes the chromatin fiber of ~10 nm in diameter. The linker DNA assists further compaction of chromatin structure into higher-order chromatin structure, which is essential for packaging of remarkable lengths of DNA into the cell nucleus. Furthermore, this compact chromatin structure limits accessibility of DNA to DNA-mediated processes like transcription, DNA replication, and DNA repair (Kouzarides, 2007). Evidence accumulated during the past 15 years reveals that three interrelated epigenetic mechanisms alter the highly compacted chromatin structure and facilitate accessibility of DNA for gene transcription. The interrelated epigenetic mechanisms that include DNA methylation, histone modification and ncRNA expression contribute to the epigenome making the epigenome dynamic rather than static like genome and thus, being predisposed to and influenced by environmental factors and extracellular stimuli. Deregulation of epigenetic mechanisms is observed in many different cancers and other human diseases. Thus, understanding of how epigenetic mechanisms contribute to gene

epigenetics in the regulation of VSMC proliferation.

regulation will provide insight into the disease process.

**3.1 VSMC epigenetics**

disease.

**3.2 Chromatin structure**

oxLDL offers protection against atherosclerosis, which appears to operate both through cellular and humoral immunity (Zhou, 2001). The increased titers of T cell-dependent IgG antibodies to oxLDL (Habets et al., 2010; Zhou, 2001) and natural IgM antibodies to phosphocholine (Binder et al., 2004) are also in agreement with the atheroprotection. Furthermore, two recent studies report promising immunotherapeutic approach for the prevention of atherosclerosis. In one study, LDL-receptor deficient mice were vaccinated with oxLDL-pulsed mature dendritic cells to determine the effect on atherosclerosis (Habets et al., 2010). In the second immunotherapy study, tolerogenic apo-B100-loaded dendritic cells in combination with immunosuppressive cytokine interleukin-10 were injected intravenously to hypercholesterolemic mice. This immunotherapy significantly prevented atherosclerosis by reducing autoimmune response against LDL ( Hermansson et al., 2011) Although, these studies are encouraging and promising from a clinical perspective to translate these promising outcomes to the clinics, antigens that can be easily manufactured under good manufacturing practice conditions and that have a reproducible quality are necessary. However, several clinical studies are currently underway to evaluate the therapeutic implications of immunotherapy.

#### **3. Epigenetics and vascular proliferative diseases**

Advancement in technological innovations during the past 25 years has resulted in farreaching in-depth comprehension of the biology and the etiology of vascular diseases, and thus influencing the perception of the pathophysiology of vascular proliferative diseases like atherosclerosis and restenosis. In spite of the substantial understanding of the etiology and the clinical management of these vascular proliferative diseases, they are still life threatening diseases and reasons are not fully evident. Based on the recent finding of the role of epigenetics in human diseases, it is proper to expect that epigenetic mechanisms enforces an additional layer of gene regulation that alters chromatin structure, and dynamics in the pathogenesis of vascular proliferative diseases (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Epigenetic mechanisms are essential for the functioning of genomes to regulate normal development and maintenance of organisms, and to facilitate their interaction with surrounding environment. Compilation of the past 10 to 20 years of studies has resulted in the identification of three highly interrelated epigenetic mechanisms that alter the chromatin structure and accessibility. These include, DNA methylation, histone posttranslational modifications and non-coding RNA (ncRNA) expression based mechanisms, each of these mechanisms is essential for regulation of gene expression. Therefore, it is anticipated that the genetic and environmental factors that are relevant to the development of vascular proliferative diseases by their effect on inflammation, VSMC proliferation and vessel remodeling, is regulated by epigenetic mechanisms through the modification of chromatin structure, dynamics and accessibility (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Although there is an outbreak of interest and enthusiasm in linking altered epigenetic mechanisms to human pathologies particularly cancers, it is relatively unexplored area regarding cardiovascular diseases. Moreover, deregulation of epigenetic processes are linked to changes in many aspects of cell biology including cell growth, cell cycle control, and cell death by altering the expression and in turn functions of target genes without changing their primary gene structure. Because VSMC proliferation is the hallmark of vascular proliferative diseases, understanding the epigenetics of VSMC proliferation and in particular their susceptibility to perturbation by the epigenetic modifiers may offer novel insights into disease pathogenesis and epigenetic therapeutic approaches. Therefore, it is appropriate to review the current knowledge of epigenetics in the regulation of VSMC proliferation.

#### **3.1 VSMC epigenetics**

86 Atherogenesis

oxLDL offers protection against atherosclerosis, which appears to operate both through cellular and humoral immunity (Zhou, 2001). The increased titers of T cell-dependent IgG antibodies to oxLDL (Habets et al., 2010; Zhou, 2001) and natural IgM antibodies to phosphocholine (Binder et al., 2004) are also in agreement with the atheroprotection. Furthermore, two recent studies report promising immunotherapeutic approach for the prevention of atherosclerosis. In one study, LDL-receptor deficient mice were vaccinated with oxLDL-pulsed mature dendritic cells to determine the effect on atherosclerosis (Habets et al., 2010). In the second immunotherapy study, tolerogenic apo-B100-loaded dendritic cells in combination with immunosuppressive cytokine interleukin-10 were injected intravenously to hypercholesterolemic mice. This immunotherapy significantly prevented atherosclerosis by reducing autoimmune response against LDL ( Hermansson et al., 2011) Although, these studies are encouraging and promising from a clinical perspective to translate these promising outcomes to the clinics, antigens that can be easily manufactured under good manufacturing practice conditions and that have a reproducible quality are necessary. However, several clinical studies are currently underway to evaluate the

Advancement in technological innovations during the past 25 years has resulted in farreaching in-depth comprehension of the biology and the etiology of vascular diseases, and thus influencing the perception of the pathophysiology of vascular proliferative diseases like atherosclerosis and restenosis. In spite of the substantial understanding of the etiology and the clinical management of these vascular proliferative diseases, they are still life threatening diseases and reasons are not fully evident. Based on the recent finding of the role of epigenetics in human diseases, it is proper to expect that epigenetic mechanisms enforces an additional layer of gene regulation that alters chromatin structure, and dynamics in the pathogenesis of vascular proliferative diseases (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Epigenetic mechanisms are essential for the functioning of genomes to regulate normal development and maintenance of organisms, and to facilitate their interaction with surrounding environment. Compilation of the past 10 to 20 years of studies has resulted in the identification of three highly interrelated epigenetic mechanisms that alter the chromatin structure and accessibility. These include, DNA methylation, histone posttranslational modifications and non-coding RNA (ncRNA) expression based mechanisms, each of these mechanisms is essential for regulation of gene expression. Therefore, it is anticipated that the genetic and environmental factors that are relevant to the development of vascular proliferative diseases by their effect on inflammation, VSMC proliferation and vessel remodeling, is regulated by epigenetic mechanisms through the modification of chromatin structure, dynamics and accessibility (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Although there is an outbreak of interest and enthusiasm in linking altered epigenetic mechanisms to human pathologies particularly cancers, it is relatively unexplored area regarding cardiovascular diseases. Moreover, deregulation of epigenetic processes are linked to changes in many aspects of cell biology including cell growth, cell cycle control, and cell death by altering the expression and in turn functions of target genes without changing their primary gene structure. Because VSMC proliferation is the hallmark of vascular proliferative diseases, understanding the

therapeutic implications of immunotherapy.

**3. Epigenetics and vascular proliferative diseases**

Curiosity in epigenetics has surged during past decade even though the principle question it aims to address has been there for decades. That is, how a multicellular organism maintain drastically different gene expression profile in different cell types of the organism, while all the different cell types of the organism have exactly the same DNA. This is where epigenetics come into picture. Epigenetics refers to the inheritance of gene function/activity/expression that may be stable over long periods, last through several cell divisions or inherit through several generations, all without any change in their primary DNA (Ng & Gurdon, 2008; Probst, 2009). The three interrelated epigenetic mechanisms, which involve: methylation of DNA at CpG dinucleotides at specific position in the DNA molecule suppress expression of nearby genes (Esteller, 2008); posttranslational modifications of histones alters chromatin structure and changes promoter accessibility (Kouzarides, 2007); and small RNA molecules generated from noncoding RNAs (ncRNAs) inhibit gene expression (Mattick et al., 2009). All these mechanisms involved in epigenetic regulation contribute to epigenome. This review focuses on the role of posttranslational modifications of histones in the regulation of VSMC proliferation, and on the epigenetic regulators of histone modifications as potential candidates for drug targeting in the treatment and management of vascular proliferative disease.

#### **3.2 Chromatin structure**

Chromatin is a nucleoprotein complex consisting of repeating units of nucleosomal core particles. It offers a dynamic platform for all DNA-mediated processes within the nucleus. The nucleosomal core particles are the basic units of chromatin consisting of 147 base pair (bp) of DNA that wraps almost twice around two copies of each of the four core histone proteins, H3, H4, H2A and H2B. Each nucleosome is separated by 10-16 bp long linkers DNA, which gives an appearance of a bead on a string structure that constitutes the chromatin fiber of ~10 nm in diameter. The linker DNA assists further compaction of chromatin structure into higher-order chromatin structure, which is essential for packaging of remarkable lengths of DNA into the cell nucleus. Furthermore, this compact chromatin structure limits accessibility of DNA to DNA-mediated processes like transcription, DNA replication, and DNA repair (Kouzarides, 2007). Evidence accumulated during the past 15 years reveals that three interrelated epigenetic mechanisms alter the highly compacted chromatin structure and facilitate accessibility of DNA for gene transcription. The interrelated epigenetic mechanisms that include DNA methylation, histone modification and ncRNA expression contribute to the epigenome making the epigenome dynamic rather than static like genome and thus, being predisposed to and influenced by environmental factors and extracellular stimuli. Deregulation of epigenetic mechanisms is observed in many different cancers and other human diseases. Thus, understanding of how epigenetic mechanisms contribute to gene regulation will provide insight into the disease process.

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 89

Consistent with the variety of posttranslational modifications of histones, a disparate family of enzymes, which are referred as histone modification writers, catalyzes addition of specific functional groups to histones. These modifications of histones alter the chromatin structure and function by two distinct manner: First, by directly altering the charges of histone proteins, certain posttranslational modifications cause localized relaxation of chromatin structure and second, by serving as recognition and binding sites for various classes of effector proteins that participate in chromatin remodeling, certain histone modifications indirectly alter the chromatin structure. However, most histone modification are reversible and diverse families of proteins, which include histone acetyltransferases (HATs) /histone deacetylases (HDACs), histone methyltransferases (HMTs)/demethylases, histone kinases/phosphatases, and ubiquitin ligases, catalyze addition and removal of the modifications from histones. One of the highlights of epigenetics is that it offers new therapeutic targets for diseases including cardiovascular diseases. The epigenetic regulators, HATs/HDACs and HMTs/demethylases, which exhibit counterbalancing activities are essential for the regulation of gene expression, which are required for the basic cellular processes such as cell proliferation and differentiation. This essential role of epigenetic regulators in basic cellular processes identifies potential therapeutic targets for diseases including cardiovascular diseases. Moreover, identification of those HATs and HDACs that plays a role in the transcriptional regulation of genes, products of which contributes to the processes of neointima formation like inflammation, VSMC proliferation, and matrix formation is also important in designing potential epigenetic therapy to target vascular proliferative diseases. Thus, pharmacological inhibition of enzyme activities involved in epigenetic DNA and histone modifications designed to induce or silence the transcription of disease-relevant genes offers an amenable therapeutic intervention for atherosclerosis and restenosis. In addition to modifying the effects of diseased genes, it is possible to change the effects of environmental risk factors by targeting epigenetic mechanisms. Here we will focus on HATs/HDACs, the principal epigenetic regulators that control histone acetylation, a major epigenetic modification for transcriptional control of gene expression. Because HATs/HDACs are essential for the regulation of gene expression, in all probability, they play crucial role in the development of multigene and multifactorial diseases such as

**4. Histone acetyltransferases (HATs) and histone deacetylases (HDACs)**

One of the best- and most-studied posttranslational histone modifications is lysine acetylation catalyzed by HATs, the modification that is generally associated with gene activation (Ekstrom, 2009; Kouzarides, 2007; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Hyperacetylation of histones causes decondensation of chromatin allowing a more relaxed or open and active chromatin structure, which allows accessibility of DNA to basal transcription initiation machinery (Kouzarides, 2007; Roth, 2001). In contrast, gene repression is mediated by HDACs, and other co-repressors, which cause deacetylation of hyperacetylated histones and offset the activity of HATs resulting in a closed conformation of chromatin structure. Thus, the acetylation status of the chromatin associated with particular genes is dictated by the balance between the activities of HATs and HDACs. These enzymes are shown to regulate expression of genes associated with various cellular processes like inflammation, proliferation and matrix modulation (Cao et al., 2005; Pons et

**3.4 Chromatin modifying enzymes**

atherosclerosis and restenosis.

#### **3.3 Histone modifications**

Histones are highly conserved basic proteins that undergo an amazing number and types of posttranslational modifications, which contributes to the active or inactive chromatin **(**Ekstrom, 2009; Kouzarides, 2007; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Each of the four core histones are composed of a conserved globular domain that forms the nucleosome core, and a highly dynamic amino-terminal tail of 20-35 residues rich in basic amino acids. Additionally, H2A histone has an extended tail of about 35 residues at the carboxy-terminal end. Both amino- and carboxy-terminal tails protrude from nucleosome into the nucleoplasm. Histones tails are the targets of an array of site-specific posttranslational modifications including lysine acetylation/deacetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitylation and sumoylation, and glutamic acid ADPribosylation (Fischle et al., 2003; Ito, 2007; Jenuwein & Allis, 2001; Turner, 2003).

Histone modifications are dynamic and reversible, and their 'off' and 'on" modification states are influenced by different physiological and environmental factors like developmental state, stress condition and environmental cues. Many of histone modifications are associated with transcriptionally active euchromatin regions, while other histone modifications are localized to transcriptionally inactive heterochromatin regions. Even though issues such as how the process of modification is regulated, and how many modifications are required for their biological effect are still elusive, recognition of specific histone modifications by various effector proteins is suggested to mediate specific biological processes like gene activation or gene suppression by altering the chromatin structure and gene accessibility. Generally, conformationally relaxed and decondensed chromatin structure that is associated with histone acetylation and DNA hypomethylation is the feature of transcriptionally active chromatin. On the other hand, compact and condensed chromatin structure that is associated with deacetylation of histones and hypermethylation of DNA is transcriptionally silent. Condensed chromatin structure is essential during cell cycle, mitosis and meiosis, whereas decondensed chromatin structure is required for gene expression, replication, repair and recombination. The combinatorial pattern of the histone modifications indicates the state of the chromatin structure, and thus, regulates the accessibility of the DNA to the transcription-regulatory complexes, through it controls gene expression. The collection of various covalent histone modifications serve as epigenetic marks for the recruitment of different proteins or protein complexes to regulate disparate chromatin functions such as gene expression, gene suppression, mitosis, repair, replication and chromosome segregation (Taverna et al., 2007). Furthermore, there is also crosstalk between different histone modifications like acetylation, methylation and phosphorylation at independent sites forming a "histone code" that is translated to a specific biological event through the mediation of various effector proteins. For example, phosphorylation of serine 10 of histone H3 facilitates acetylation of lysine 14 and methylation of lysine 4, which create an open or relaxed chromatin conformation associated with an active gene. Serine 10 phosphorylation also facilitates the acetylation of lysine 9, thus preventing the repressive lysine 9-methylation associated with an inactive gene (Jenuwein & Allis., 2001; Lund & van Lohuizen, 2004; Mathew et al., 2010; Schreiber & Bernstein, 2002; Turner, 2003).

#### **3.4 Chromatin modifying enzymes**

88 Atherogenesis

Histones are highly conserved basic proteins that undergo an amazing number and types of posttranslational modifications, which contributes to the active or inactive chromatin **(**Ekstrom, 2009; Kouzarides, 2007; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Each of the four core histones are composed of a conserved globular domain that forms the nucleosome core, and a highly dynamic amino-terminal tail of 20-35 residues rich in basic amino acids. Additionally, H2A histone has an extended tail of about 35 residues at the carboxy-terminal end. Both amino- and carboxy-terminal tails protrude from nucleosome into the nucleoplasm. Histones tails are the targets of an array of site-specific posttranslational modifications including lysine acetylation/deacetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitylation and sumoylation, and glutamic acid ADPribosylation (Fischle et al., 2003; Ito, 2007; Jenuwein &

Histone modifications are dynamic and reversible, and their 'off' and 'on" modification states are influenced by different physiological and environmental factors like developmental state, stress condition and environmental cues. Many of histone modifications are associated with transcriptionally active euchromatin regions, while other histone modifications are localized to transcriptionally inactive heterochromatin regions. Even though issues such as how the process of modification is regulated, and how many modifications are required for their biological effect are still elusive, recognition of specific histone modifications by various effector proteins is suggested to mediate specific biological processes like gene activation or gene suppression by altering the chromatin structure and gene accessibility. Generally, conformationally relaxed and decondensed chromatin structure that is associated with histone acetylation and DNA hypomethylation is the feature of transcriptionally active chromatin. On the other hand, compact and condensed chromatin structure that is associated with deacetylation of histones and hypermethylation of DNA is transcriptionally silent. Condensed chromatin structure is essential during cell cycle, mitosis and meiosis, whereas decondensed chromatin structure is required for gene expression, replication, repair and recombination. The combinatorial pattern of the histone modifications indicates the state of the chromatin structure, and thus, regulates the accessibility of the DNA to the transcription-regulatory complexes, through it controls gene expression. The collection of various covalent histone modifications serve as epigenetic marks for the recruitment of different proteins or protein complexes to regulate disparate chromatin functions such as gene expression, gene suppression, mitosis, repair, replication and chromosome segregation (Taverna et al., 2007). Furthermore, there is also crosstalk between different histone modifications like acetylation, methylation and phosphorylation at independent sites forming a "histone code" that is translated to a specific biological event through the mediation of various effector proteins. For example, phosphorylation of serine 10 of histone H3 facilitates acetylation of lysine 14 and methylation of lysine 4, which create an open or relaxed chromatin conformation associated with an active gene. Serine 10 phosphorylation also facilitates the acetylation of lysine 9, thus preventing the repressive lysine 9-methylation associated with an inactive gene (Jenuwein & Allis., 2001; Lund & van Lohuizen, 2004; Mathew et al., 2010; Schreiber & Bernstein, 2002;

**3.3 Histone modifications**

Allis, 2001; Turner, 2003).

Turner, 2003).

Consistent with the variety of posttranslational modifications of histones, a disparate family of enzymes, which are referred as histone modification writers, catalyzes addition of specific functional groups to histones. These modifications of histones alter the chromatin structure and function by two distinct manner: First, by directly altering the charges of histone proteins, certain posttranslational modifications cause localized relaxation of chromatin structure and second, by serving as recognition and binding sites for various classes of effector proteins that participate in chromatin remodeling, certain histone modifications indirectly alter the chromatin structure. However, most histone modification are reversible and diverse families of proteins, which include histone acetyltransferases (HATs) /histone deacetylases (HDACs), histone methyltransferases (HMTs)/demethylases, histone kinases/phosphatases, and ubiquitin ligases, catalyze addition and removal of the modifications from histones. One of the highlights of epigenetics is that it offers new therapeutic targets for diseases including cardiovascular diseases. The epigenetic regulators, HATs/HDACs and HMTs/demethylases, which exhibit counterbalancing activities are essential for the regulation of gene expression, which are required for the basic cellular processes such as cell proliferation and differentiation. This essential role of epigenetic regulators in basic cellular processes identifies potential therapeutic targets for diseases including cardiovascular diseases. Moreover, identification of those HATs and HDACs that plays a role in the transcriptional regulation of genes, products of which contributes to the processes of neointima formation like inflammation, VSMC proliferation, and matrix formation is also important in designing potential epigenetic therapy to target vascular proliferative diseases. Thus, pharmacological inhibition of enzyme activities involved in epigenetic DNA and histone modifications designed to induce or silence the transcription of disease-relevant genes offers an amenable therapeutic intervention for atherosclerosis and restenosis. In addition to modifying the effects of diseased genes, it is possible to change the effects of environmental risk factors by targeting epigenetic mechanisms. Here we will focus on HATs/HDACs, the principal epigenetic regulators that control histone acetylation, a major epigenetic modification for transcriptional control of gene expression. Because HATs/HDACs are essential for the regulation of gene expression, in all probability, they play crucial role in the development of multigene and multifactorial diseases such as atherosclerosis and restenosis.

#### **4. Histone acetyltransferases (HATs) and histone deacetylases (HDACs)**

One of the best- and most-studied posttranslational histone modifications is lysine acetylation catalyzed by HATs, the modification that is generally associated with gene activation (Ekstrom, 2009; Kouzarides, 2007; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). Hyperacetylation of histones causes decondensation of chromatin allowing a more relaxed or open and active chromatin structure, which allows accessibility of DNA to basal transcription initiation machinery (Kouzarides, 2007; Roth, 2001). In contrast, gene repression is mediated by HDACs, and other co-repressors, which cause deacetylation of hyperacetylated histones and offset the activity of HATs resulting in a closed conformation of chromatin structure. Thus, the acetylation status of the chromatin associated with particular genes is dictated by the balance between the activities of HATs and HDACs. These enzymes are shown to regulate expression of genes associated with various cellular processes like inflammation, proliferation and matrix modulation (Cao et al., 2005; Pons et

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 91

anticancer agents that are under clinical trials for different cancer treatment. Some of the early clinical studies have demonstrated that certain HDACi exhibit promising activity against several neoplasms (Bhalla, 2005). Naturally, it has stimulated great interest to determine how HATs/HDACs regulate transcriptome of different processes that are linked to the development of atherosclerosis and restenosis like inflammation, VSMC proliferation and matrix modification and to assess therapeutic potential of HDACi in these vascular proliferative diseases (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). The following sections will focus on the role of HATs and HDACs in the transcriptional regulation of genes in the context of their contribution to VSMC proliferation and its disorders as well as on the potential applicability of HDACi in vascular disease

In the past few years, great effort has been focused on seeking and designing most effective HDACi because of their potential roles in reversing the silenced genes in tumor cells by modulating transcriptional processes. The balance between the acetylated/deacetylated states of histones, which is mediated by the counterbalancing activities of HATs and HDACs, contributes to the transcriptional states of chromatin structure. The structural modification of histones by acetylation/deacetylation of their N-terminal tails is crucial in modulating gene expression, because it affects the accessibility of DNA for the transcriptionregulatory protein complexes. HATs preferentially acetylate specific lysine residues of histones, which relaxes the DNA conformation, thus allowing its access to transcription machinery to turn on gene expression. On the contrary, HDACs restore the positive charge on lysine residues by removing acetyl groups, which promotes condensed chromatin structure. This promotes silencing of gene expression by blocking the access of transcription machinery to DNA. Inappropriate silencing of critical genes such as tumor suppressor genes can result in cancer based on the recent understanding of the cancer cell cycle (Kristeleit et al., 2004). This provides a rationale for using inhibition of HDAC activity to release transcriptional repression. As result, a flurry of HDACi has been recognized for their ability

Structurally diverse classes of naturally occurring and synthetic compounds have been recognized for their ability to bind to the catalytic pocket of HDACs and chelate the zinc ion

management.

to inhibit HDACs activity.

**4.1.1 Histone deacetylase inhibitors (HDACi)**

Fig. 1.  HATs and HDACs are also recruited to gene promoters by multiprotein transcriptional complexes, where they regulate transcription through chromatin modification without directly binding the DNA.

A number of different HATs are identified and organized as families based on the presence of highly conserved structural motifs, which include PCAF/Gcn5, p300/CBP, MYST, SRC, and TAFII250 families. While they all differ in their HAT domains and substrate specificity, they all require the assembly of multiprotein complexes for acetylation of nucleosomes (Marmorstein, 2001). Likewise a number of HDACs are identified and are classified into three different classes based on cellular localization, substrates and binding site features (Lindemann et al., 2004; Santini et al., 2007). Class I and class II include zinc-dependent HDACs, and class III includes NAD-dependent HDACs, which are also called as sirtuins. Class I HDACs are widely expressed and include HDACs 1-3 and 8 that are exclusively localized to nucleus. They are known to modulate cell proliferation and survival. Class II HDACs are HDACs 4-7, 9, and 10, which shuttle between the nucleus and cytoplasm in response to certain cellular signals. They may be involved in cell differentiation (Pons et al., 2009; Santini et al., 2007). Class II HDACs are further divided into Class IIa and Class IIb , which include HDACs 4, 5 ,7, and 9, and, HDACs 6 and 10, respectively. While Class IIa members have an extended N-terminal regulatory domain, Class IIb exhibit an extra catalytic domain (Lindemann et al., 2004; Pons et al., 2009; Santini et al., 2007). Class III HDACs are sirtuins (SIRT), which include NAD+-dependent enzymes (SIRT 1-7) potentially involved in apoptosis (Pons et al., 2009).

HATs and HDACs are also recruited to gene promoters by multiprotein transcriptional complexes. There they regulate transcription through chromatin modification without directly binding the DNA (Johnstone & Licht, 2003; Pons et al., 2009). Moreover, HATs and HDACs are also involved in the acetylation status of lysine residues of transcription factors such as p53, E2F1, GATA1, RelA, YY1 and hormone receptors. Acetylation status of these transcription factors affects their DNA binding and transcriptional activity (Glozak et al., 2005; Johnstone & Licht., 2003; Marks, 2001; Pons et al., 2009). Besides histones and transcription factors, several other non-histone proteins like α-tubulin, nuclear import protein importin-α7, and signal transduction protein β-catenin are also modified by HATs and HDACs, but their effects on gene expression is not dependent on chromatin remodeling (Johnstone & Licht., 2003; Marks, 2001).

#### **4.1 Epigenetic therapy targeting VSMC proliferation**

Because HATs/HDACs are involved in dynamic reversible epigenetic processes that contribute to modulation of gene expression profiles specific to cellular processes like cell proliferation, they probably play important role in cardiovascular pathologies such as atherosclerosis and restenosis. Moreover, epigenetic deregulation affects several aspects of cell biology, including cell growth, cell cycle control, differentiation, DNA repair, and cell death. This elevates the strong possibility that reversing deregulated epigenetic mechanisms may be an effective treatment strategy for proliferative diseases. Incidentally, the property of HDACs, suppression of gene expression by epigenetic mechanism, has been exploited in the field of cancer to reactivate transcriptionally silent tumor suppressor gene to arrest proliferation of cancer cells and growth (Pons et al., 2009; Ranganna, et al., 2005, 2007; Sharma et al., 2010). Moreover, HDAC inhibitors (HDACi) are emerging as a new class of

al., 2009; Sahar et al., 2007; Vinh et al., 2008; Waltregny et al., 2005; Xu et al., 2007; Yan et al., 2009). HATs and HDACs are also recruited to gene promoters by multiprotein transcriptional complexes, where they regulate transcription through chromatin

A number of different HATs are identified and organized as families based on the presence of highly conserved structural motifs, which include PCAF/Gcn5, p300/CBP, MYST, SRC, and TAFII250 families. While they all differ in their HAT domains and substrate specificity, they all require the assembly of multiprotein complexes for acetylation of nucleosomes (Marmorstein, 2001). Likewise a number of HDACs are identified and are classified into three different classes based on cellular localization, substrates and binding site features (Lindemann et al., 2004; Santini et al., 2007). Class I and class II include zinc-dependent HDACs, and class III includes NAD-dependent HDACs, which are also called as sirtuins. Class I HDACs are widely expressed and include HDACs 1-3 and 8 that are exclusively localized to nucleus. They are known to modulate cell proliferation and survival. Class II HDACs are HDACs 4-7, 9, and 10, which shuttle between the nucleus and cytoplasm in response to certain cellular signals. They may be involved in cell differentiation (Pons et al., 2009; Santini et al., 2007). Class II HDACs are further divided into Class IIa and Class IIb , which include HDACs 4, 5 ,7, and 9, and, HDACs 6 and 10, respectively. While Class IIa members have an extended N-terminal regulatory domain, Class IIb exhibit an extra catalytic domain (Lindemann et al., 2004; Pons et al., 2009; Santini et al., 2007). Class III HDACs are sirtuins (SIRT), which include NAD+-dependent enzymes (SIRT 1-7) potentially

HATs and HDACs are also recruited to gene promoters by multiprotein transcriptional complexes. There they regulate transcription through chromatin modification without directly binding the DNA (Johnstone & Licht, 2003; Pons et al., 2009). Moreover, HATs and HDACs are also involved in the acetylation status of lysine residues of transcription factors such as p53, E2F1, GATA1, RelA, YY1 and hormone receptors. Acetylation status of these transcription factors affects their DNA binding and transcriptional activity (Glozak et al., 2005; Johnstone & Licht., 2003; Marks, 2001; Pons et al., 2009). Besides histones and transcription factors, several other non-histone proteins like α-tubulin, nuclear import protein importin-α7, and signal transduction protein β-catenin are also modified by HATs and HDACs, but their effects on gene expression is not dependent on chromatin remodeling

Because HATs/HDACs are involved in dynamic reversible epigenetic processes that contribute to modulation of gene expression profiles specific to cellular processes like cell proliferation, they probably play important role in cardiovascular pathologies such as atherosclerosis and restenosis. Moreover, epigenetic deregulation affects several aspects of cell biology, including cell growth, cell cycle control, differentiation, DNA repair, and cell death. This elevates the strong possibility that reversing deregulated epigenetic mechanisms may be an effective treatment strategy for proliferative diseases. Incidentally, the property of HDACs, suppression of gene expression by epigenetic mechanism, has been exploited in the field of cancer to reactivate transcriptionally silent tumor suppressor gene to arrest proliferation of cancer cells and growth (Pons et al., 2009; Ranganna, et al., 2005, 2007; Sharma et al., 2010). Moreover, HDAC inhibitors (HDACi) are emerging as a new class of

modification without directly binding the DNA.

involved in apoptosis (Pons et al., 2009).

(Johnstone & Licht., 2003; Marks, 2001).

**4.1 Epigenetic therapy targeting VSMC proliferation**

anticancer agents that are under clinical trials for different cancer treatment. Some of the early clinical studies have demonstrated that certain HDACi exhibit promising activity against several neoplasms (Bhalla, 2005). Naturally, it has stimulated great interest to determine how HATs/HDACs regulate transcriptome of different processes that are linked to the development of atherosclerosis and restenosis like inflammation, VSMC proliferation and matrix modification and to assess therapeutic potential of HDACi in these vascular proliferative diseases (Ekstrom, 2009; Pons et al., 2009; Ranganna et al., 2006; Turunen, 2009). The following sections will focus on the role of HATs and HDACs in the transcriptional regulation of genes in the context of their contribution to VSMC proliferation and its disorders as well as on the potential applicability of HDACi in vascular disease management.

#### **4.1.1 Histone deacetylase inhibitors (HDACi)**

In the past few years, great effort has been focused on seeking and designing most effective HDACi because of their potential roles in reversing the silenced genes in tumor cells by modulating transcriptional processes. The balance between the acetylated/deacetylated states of histones, which is mediated by the counterbalancing activities of HATs and HDACs, contributes to the transcriptional states of chromatin structure. The structural modification of histones by acetylation/deacetylation of their N-terminal tails is crucial in modulating gene expression, because it affects the accessibility of DNA for the transcriptionregulatory protein complexes. HATs preferentially acetylate specific lysine residues of histones, which relaxes the DNA conformation, thus allowing its access to transcription machinery to turn on gene expression. On the contrary, HDACs restore the positive charge on lysine residues by removing acetyl groups, which promotes condensed chromatin structure. This promotes silencing of gene expression by blocking the access of transcription machinery to DNA. Inappropriate silencing of critical genes such as tumor suppressor genes can result in cancer based on the recent understanding of the cancer cell cycle (Kristeleit et al., 2004). This provides a rationale for using inhibition of HDAC activity to release transcriptional repression. As result, a flurry of HDACi has been recognized for their ability to inhibit HDACs activity.

Fig. 1. Structural class of histone deacetylase inhibitors

Structurally diverse classes of naturally occurring and synthetic compounds have been recognized for their ability to bind to the catalytic pocket of HDACs and chelate the zinc ion

2005).

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 93

processing and activation of caspases-9 and-3 (Bhalla, 2005). HDACi appear to promote apoptotic cell death not only by upregulating several proteins that participate in apoptotic cell death including Bak, Bax, Bim, DR4, DR5, and TRAIL, but also by attenuating the levels of a number of antiapoptotic proteins such as Bcl-xL, Bcl-2, XIAP, and survivin (Bhalla,

Fig. 3. Cell cycle regulatory proteins that are altered by the HDACi-induced acetylation of

Fig. 2. Display of cell cycle targets of HDAC inhibition.

histones and transcription factors.

at its base, thereby inhibiting HDAC activity (Marks et al., 2000). A wide range of structures inhibits activity of class I/II HDAC enzymes with a few exceptions [Figure 1]. The HDACi are classified into structural classes including 1) short-chain fatty acids (carboxylates), 2) hydroxamic acids, 3) benzamides, and 4) cyclic and non-cyclic peptides. The various HDACi studied so far have been shown to inhibit class I (HDACs 1, 2, 3, and 8) and II (HDACs 4, 5, 6, 9, and 10) HDACs. Their activities have been tested in cell lines and preclinical murine models, and appropriate drugs that are selected for clinical trials, demonstrated good tolerance and clinical activity against different human neoplasms (Santini et al., 2007). However, Class III HDACs (SIRT 1, 2, 3, 4, 5, 6, and 7), also known as sirtuins, require NAD rather than zinc as a co-factor for their activity, and are not inhibited by the HDACi. Instead, they are inhibited by Nicotinamide (Luo et al., 2001).

#### **4.1.2 HDACi effects on cellular processes**

HDACi exhibit multiple cellular effects, which are linked to chromatin-mediated altered transcriptional activity. In general, most HDACi exhibit inhibition of cell proliferation, stimulation of cell differentiation and/or induction of cell death by selectively modulating gene expression (Bhalla, 2005; Mathew et al., 2010; Ranganna, 2005). HDACi arrest cells at the G1 or G2/M, and promote cell differentiation mainly by stimulating cyclin-dependent kinase inhibitor (cdkI) p21Cip1 expression (Bhalla, 2005; Mathew et al., 2010; Ranganna et al., 2005). HDACi also cause cell cycle blockade through the modulation of mechanisms that involve repression of cyclin D and cyclin A and upregulation of other cdkI like p27Kip1, p16INK4A and p15INK4B, which blocks pRb/E2F pathway, thus preventing the cell cycle progression (Mathew et al., 2010; Bhalla, 2005) [Figure 2]. Now with the array technologies, it is recognized that HDACi selectively modulate about 2% to 10% of all genes, with as many genes upregulated as are downregulated genes in different cell types (Bhalla, 2005; Ranganna et al., 2003). One of the genes that are universally upregulated is the cdkI p21Cip1, in a p53-independent manner, which is necessary for HDACi-induced G1 arrest. Induction of GADD45α and β and upregulation of transforming growth factor beta, which inhibits c-*myc* expression may also contribute to the cell cycle arrest in G1 or G2 (Bhalla, 2005.). HDACi treatment is also shown to transcriptionally downregulate the expression of CTP synthetase and thymidylate synthetase, which are required for DNA synthesis, thus, causing inhibition of S phase progression [Figure 3].

HDACi also stimulate differentiation of several cancer cells by inhibiting cell proliferation (Bhalla, 2005). Again, upregulation of p21Cip1 appears to be essential for differentiation because cells lacking p21Cip1 fail to respond to HDACi treatment (Bhalla, 2005). Furthermore, acute promyelocytic leukemic cells and primary leukemia blasts, expressed differentiated phenotype in response to a combination of ATRA, a retinoid-based chemotherapeutic drug and HDACi (Bhalla, 2005). HDACi stimulated gelsolin, an actinbinding protein required for morphological and cytostructural changes associated with differentiation (Bhalla, 2005).

It is interesting that HDACi induce growth arrest and cell differentiation in some cell, and in others, they cause apoptosis (Bhalla, 2005; Johnstone & Licht, 2003). HDACi-induced apoptosis triggers both the intrinsic and extrinsic pathways of apoptosis. Several types of HDACi, particularly hydroxamic acid analogs are shown to induce mitochondrial permeability transition, which releases prodeath molecules such as cytochrome c, Smac and Omi into cytosol (Bhalla, 2005). This triggers activation of Apaf-1, which leads to the

at its base, thereby inhibiting HDAC activity (Marks et al., 2000). A wide range of structures inhibits activity of class I/II HDAC enzymes with a few exceptions [Figure 1]. The HDACi are classified into structural classes including 1) short-chain fatty acids (carboxylates), 2) hydroxamic acids, 3) benzamides, and 4) cyclic and non-cyclic peptides. The various HDACi studied so far have been shown to inhibit class I (HDACs 1, 2, 3, and 8) and II (HDACs 4, 5, 6, 9, and 10) HDACs. Their activities have been tested in cell lines and preclinical murine models, and appropriate drugs that are selected for clinical trials, demonstrated good tolerance and clinical activity against different human neoplasms (Santini et al., 2007). However, Class III HDACs (SIRT 1, 2, 3, 4, 5, 6, and 7), also known as sirtuins, require NAD rather than zinc as a co-factor for their activity, and are not inhibited by the HDACi. Instead,

HDACi exhibit multiple cellular effects, which are linked to chromatin-mediated altered transcriptional activity. In general, most HDACi exhibit inhibition of cell proliferation, stimulation of cell differentiation and/or induction of cell death by selectively modulating gene expression (Bhalla, 2005; Mathew et al., 2010; Ranganna, 2005). HDACi arrest cells at the G1 or G2/M, and promote cell differentiation mainly by stimulating cyclin-dependent kinase inhibitor (cdkI) p21Cip1 expression (Bhalla, 2005; Mathew et al., 2010; Ranganna et al., 2005). HDACi also cause cell cycle blockade through the modulation of mechanisms that involve repression of cyclin D and cyclin A and upregulation of other cdkI like p27Kip1, p16INK4A and p15INK4B, which blocks pRb/E2F pathway, thus preventing the cell cycle progression (Mathew et al., 2010; Bhalla, 2005) [Figure 2]. Now with the array technologies, it is recognized that HDACi selectively modulate about 2% to 10% of all genes, with as many genes upregulated as are downregulated genes in different cell types (Bhalla, 2005; Ranganna et al., 2003). One of the genes that are universally upregulated is the cdkI p21Cip1, in a p53-independent manner, which is necessary for HDACi-induced G1 arrest. Induction of GADD45α and β and upregulation of transforming growth factor beta, which inhibits c-*myc* expression may also contribute to the cell cycle arrest in G1 or G2 (Bhalla, 2005.). HDACi treatment is also shown to transcriptionally downregulate the expression of CTP synthetase and thymidylate synthetase, which are required for DNA synthesis, thus,

HDACi also stimulate differentiation of several cancer cells by inhibiting cell proliferation (Bhalla, 2005). Again, upregulation of p21Cip1 appears to be essential for differentiation because cells lacking p21Cip1 fail to respond to HDACi treatment (Bhalla, 2005). Furthermore, acute promyelocytic leukemic cells and primary leukemia blasts, expressed differentiated phenotype in response to a combination of ATRA, a retinoid-based chemotherapeutic drug and HDACi (Bhalla, 2005). HDACi stimulated gelsolin, an actinbinding protein required for morphological and cytostructural changes associated with

It is interesting that HDACi induce growth arrest and cell differentiation in some cell, and in others, they cause apoptosis (Bhalla, 2005; Johnstone & Licht, 2003). HDACi-induced apoptosis triggers both the intrinsic and extrinsic pathways of apoptosis. Several types of HDACi, particularly hydroxamic acid analogs are shown to induce mitochondrial permeability transition, which releases prodeath molecules such as cytochrome c, Smac and Omi into cytosol (Bhalla, 2005). This triggers activation of Apaf-1, which leads to the

they are inhibited by Nicotinamide (Luo et al., 2001).

causing inhibition of S phase progression [Figure 3].

differentiation (Bhalla, 2005).

**4.1.2 HDACi effects on cellular processes**

processing and activation of caspases-9 and-3 (Bhalla, 2005). HDACi appear to promote apoptotic cell death not only by upregulating several proteins that participate in apoptotic cell death including Bak, Bax, Bim, DR4, DR5, and TRAIL, but also by attenuating the levels of a number of antiapoptotic proteins such as Bcl-xL, Bcl-2, XIAP, and survivin (Bhalla, 2005).

Fig. 2. Display of cell cycle targets of HDAC inhibition.

Fig. 3. Cell cycle regulatory proteins that are altered by the HDACi-induced acetylation of histones and transcription factors.

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 95

modulating certain gene expressions, but the mechanistic basis for these actions are far from clear. Butyrate has been known to alter chromatin structure and organization via hyperacetylation of histone amino-terminal tails, modulate gene expression and play a protective role in the prevention of cancer and inflammatory diseases of colon for a long time (Ranganna et al., 2005, 2006). However, its importance in the prevention of cancer of other tissues and different diseases has been recognized during the past ten years (Anderson, 2003; Dashwood et al., 2006; Kim, 2000; Ranganna et al., 2005, 2006). On the other hand, no similar studies are performed to indicate the protective role of butyrate in

During last few years, significant interest is focused on potential utility of butyrate and its stable derivatives in the intervention of vascular proliferative diseases, besides their therapeutic applications in other diseases including cancers. Butyrate and its more stable in vivo analogue tributyrin, arrested proliferation and inhibited DNA synthesis of smooth muscle cells in a cAMP-independent manner. Butyrate also abolished serum-induced c-fos, c-myc, and Ki-Ras expression that are important for early G1 events initiated by serum growth factors, but stimulated the expression of PS4 and thromospondin (Feng et al., 1996). Moreover, studies performed in our own lab further supports the efficacy of butyrate and its stable derivatives in vascular proliferative diseases. Treatment of VSMC with butyrate inhibited serum and PDGF-induced proliferation and abolished expression of proliferation markers such as c-myc and proliferating cell nuclear antigen [PCNA] (Ranganna et al., 1995, 2000). Furthermore, our analysis of profiles of VSMC transcriptome by array technology disclosed that butyrate-arrested VSMC proliferation is a multigene and multipathwaymediated process. Our array data identified differential expression of several genes in butyrate arrested VSMC proliferation, which are mainly belonging to four different functional classes: cell proliferation and differentiation; stress response; vascular function; and genes normally present in neuronal cells (Ranganna, et al., 2003). Extension of this study reveals that an upper level regulatory mechanism mediated through epigenetic modification of chromatin structure controls the expression of both positive and negative cell cycle regulatory genes linked to VSMC proliferation arrest by butyrate (Mathew et al., 2010). To establish the mechanistic link between chromatin remodeling and antiproliferation action of butyrate, influence of butyrate on posttranslational modifications of histone H3 and its consequence on G1-specific cell cycle regulators were investigated [Figure 2]. Outcomes of the study indicate interplay between different site-specific posttranslational modifications of histone H3 in butyrate treated VSMCs that seem to alter chromatin structure and organization that supports downregulation of cdk2, cdk4/6, and PCNA, and upregulation of cdkI, p21Cip1 and p15INK4B. This causes inhibition of Rb phosphorylation resulting in arrest of VSMC proliferation [Figure 2 and Figure 3]. The effects of HDACi on cell cyclerelated gene expressions appear to be highly selective, leading to transcriptional activation of certain genes such as the cdkIs but repression of others like cdks to efficiently block cell

Hypernuclear acetylation (HNA) also plays a role in proliferation (Kawahara et al., 2003). Presence of increased histone acetylation is observed in VSMC of atherosclerotic lesions unlike in normal arteries. Thrombin, a humoral factor that is known to activate and stimulate VSMC proliferation, strongly induced HNA in cultures of VSMC. MAP kinase

cardiovascular diseases due to atherosclerosis and restenosis.

proliferation.

**4.2.2 Histone acetylation**

Besides affecting cell proliferation, differentiation and apoptosis, HDACi also alter the function of some of the non-histone transcription factors like p53, RelA, GATA1 and FoxO3A because HDACi enhance their acetylation, which may affect their DNA binding and transcriptional activity (Marks et al, 2001; Lindemann et al., 2004). Similarly, stimulating acetylation status of other non-histone protein such as nuclear import protein importin-α7, signaling protein β-catenin, DNA repair enzyme Ku70, and the cytoskeletal protein βtubulin, HDACi alter their activity (Bhalla, 2005). Taken together, by inducing acetylation of histones and non-histones, HDACi alter the levels of proteins that control cell cycle progression, differentiation and apoptosis appropriately by transcriptional and posttranscriptional mechanisms, which implicate their potential use in disease treatment.

#### **4.2 VSMC proliferation and histone acetylation**

Although the common mechanism of pathogenesis shared by the atherosclerosis and cancer are linked to abnormal cell proliferation, very limited information is available with reference to anti-atherogenic potential of HDACi. Because HDACi not only alter gene expressions, but also cause inhibition of cell proliferation and induction of differentiation and/or apoptosis, a number of studies are initiated in the past few years to test the effects of HDACi as potential antiatherogenic agents. Even though both in vitro and in vivo studies have been done with the intention of targeting VSMC proliferation for the intervention and management of vascular proliferative diseases, most of the information that is available currently is from in vitro cell culture studies. There is limited in vivo data supporting the protective role of HDACi but needs further evaluation in models of VSMC injury.

#### **4.2.1 VSMC proliferation**

In general, HDACi exhibit almost same effects in VSMC as they do in cancer cells. They arrest cell proliferation, induce differentiation and/or apoptosis, and modulate expression of cell cycle regulators. Several studies have shown that trichostatin A (TSA), a well-known HDACi, arrests VSMC proliferation via upregulation of p21Cip1 and subsequent reduction of the phosphorylation of Rb protein at the G1-S phase (Okamoto et al., 2006; Pons et al., 2009), the effects consistently observed in cancer cell (Bhalla, 2005). In contrast, in one of the studies TSA unexpectedly exhibited paradoxical pro-atherogenic effect on VSMCs via the reduction of thioredoxin 1 instead of antiatherogenic properties (Song et al., 2010).

Besides TSA, butyrate, a well-known dietary HDACi, which has been used in different human cancer and other disease treatments, appears to exhibit potential antiatherogenic effect by arresting VSMC proliferation and appropriately altering both negative and positive cell cycle regulators (Davie, 2003; Mathew et al., 2010; Ranganna et al, 2005). Butyrate belongs to the class of short-chain fatty acids and is a derived from the intestinal microbial fermentation of dietary fiber. A number of epidemiological, animal and interventional studies suggest an inverse relationship between dietary fiber and chronic diseases such as bowel disorders and colorectal cancer, cancer of other tissues, cardiovascular disease, diabetes, obesity and hypertension (Anderson, 2003; Dashwood et al., 2006; Kim, 2000; Ranganna et al., 2005, 2006). Some of the studies suggest that the protective effect of dietary fibers in chronic diseases is linked to bioactivity of butyrate (Anderson, 2003; Dashwood et al., 2006; Kim, 2000; Ranganna et al., 2005, 2006). Butyrate elicits many cytoprotective, chemopreventive and chemotherapeutic activities mainly through inhibition of cell proliferation, stimulation of cell differentiation and/or induction of cell death by selectively

Besides affecting cell proliferation, differentiation and apoptosis, HDACi also alter the function of some of the non-histone transcription factors like p53, RelA, GATA1 and FoxO3A because HDACi enhance their acetylation, which may affect their DNA binding and transcriptional activity (Marks et al, 2001; Lindemann et al., 2004). Similarly, stimulating acetylation status of other non-histone protein such as nuclear import protein importin-α7, signaling protein β-catenin, DNA repair enzyme Ku70, and the cytoskeletal protein βtubulin, HDACi alter their activity (Bhalla, 2005). Taken together, by inducing acetylation of histones and non-histones, HDACi alter the levels of proteins that control cell cycle progression, differentiation and apoptosis appropriately by transcriptional and posttranscriptional mechanisms, which implicate their potential use in disease treatment.

Although the common mechanism of pathogenesis shared by the atherosclerosis and cancer are linked to abnormal cell proliferation, very limited information is available with reference to anti-atherogenic potential of HDACi. Because HDACi not only alter gene expressions, but also cause inhibition of cell proliferation and induction of differentiation and/or apoptosis, a number of studies are initiated in the past few years to test the effects of HDACi as potential antiatherogenic agents. Even though both in vitro and in vivo studies have been done with the intention of targeting VSMC proliferation for the intervention and management of vascular proliferative diseases, most of the information that is available currently is from in vitro cell culture studies. There is limited in vivo data supporting the

In general, HDACi exhibit almost same effects in VSMC as they do in cancer cells. They arrest cell proliferation, induce differentiation and/or apoptosis, and modulate expression of cell cycle regulators. Several studies have shown that trichostatin A (TSA), a well-known HDACi, arrests VSMC proliferation via upregulation of p21Cip1 and subsequent reduction of the phosphorylation of Rb protein at the G1-S phase (Okamoto et al., 2006; Pons et al., 2009), the effects consistently observed in cancer cell (Bhalla, 2005). In contrast, in one of the studies TSA unexpectedly exhibited paradoxical pro-atherogenic effect on VSMCs via the

Besides TSA, butyrate, a well-known dietary HDACi, which has been used in different human cancer and other disease treatments, appears to exhibit potential antiatherogenic effect by arresting VSMC proliferation and appropriately altering both negative and positive cell cycle regulators (Davie, 2003; Mathew et al., 2010; Ranganna et al, 2005). Butyrate belongs to the class of short-chain fatty acids and is a derived from the intestinal microbial fermentation of dietary fiber. A number of epidemiological, animal and interventional studies suggest an inverse relationship between dietary fiber and chronic diseases such as bowel disorders and colorectal cancer, cancer of other tissues, cardiovascular disease, diabetes, obesity and hypertension (Anderson, 2003; Dashwood et al., 2006; Kim, 2000; Ranganna et al., 2005, 2006). Some of the studies suggest that the protective effect of dietary fibers in chronic diseases is linked to bioactivity of butyrate (Anderson, 2003; Dashwood et al., 2006; Kim, 2000; Ranganna et al., 2005, 2006). Butyrate elicits many cytoprotective, chemopreventive and chemotherapeutic activities mainly through inhibition of cell proliferation, stimulation of cell differentiation and/or induction of cell death by selectively

protective role of HDACi but needs further evaluation in models of VSMC injury.

reduction of thioredoxin 1 instead of antiatherogenic properties (Song et al., 2010).

**4.2 VSMC proliferation and histone acetylation**

**4.2.1 VSMC proliferation**

modulating certain gene expressions, but the mechanistic basis for these actions are far from clear. Butyrate has been known to alter chromatin structure and organization via hyperacetylation of histone amino-terminal tails, modulate gene expression and play a protective role in the prevention of cancer and inflammatory diseases of colon for a long time (Ranganna et al., 2005, 2006). However, its importance in the prevention of cancer of other tissues and different diseases has been recognized during the past ten years (Anderson, 2003; Dashwood et al., 2006; Kim, 2000; Ranganna et al., 2005, 2006). On the other hand, no similar studies are performed to indicate the protective role of butyrate in cardiovascular diseases due to atherosclerosis and restenosis.

During last few years, significant interest is focused on potential utility of butyrate and its stable derivatives in the intervention of vascular proliferative diseases, besides their therapeutic applications in other diseases including cancers. Butyrate and its more stable in vivo analogue tributyrin, arrested proliferation and inhibited DNA synthesis of smooth muscle cells in a cAMP-independent manner. Butyrate also abolished serum-induced c-fos, c-myc, and Ki-Ras expression that are important for early G1 events initiated by serum growth factors, but stimulated the expression of PS4 and thromospondin (Feng et al., 1996). Moreover, studies performed in our own lab further supports the efficacy of butyrate and its stable derivatives in vascular proliferative diseases. Treatment of VSMC with butyrate inhibited serum and PDGF-induced proliferation and abolished expression of proliferation markers such as c-myc and proliferating cell nuclear antigen [PCNA] (Ranganna et al., 1995, 2000). Furthermore, our analysis of profiles of VSMC transcriptome by array technology disclosed that butyrate-arrested VSMC proliferation is a multigene and multipathwaymediated process. Our array data identified differential expression of several genes in butyrate arrested VSMC proliferation, which are mainly belonging to four different functional classes: cell proliferation and differentiation; stress response; vascular function; and genes normally present in neuronal cells (Ranganna, et al., 2003). Extension of this study reveals that an upper level regulatory mechanism mediated through epigenetic modification of chromatin structure controls the expression of both positive and negative cell cycle regulatory genes linked to VSMC proliferation arrest by butyrate (Mathew et al., 2010). To establish the mechanistic link between chromatin remodeling and antiproliferation action of butyrate, influence of butyrate on posttranslational modifications of histone H3 and its consequence on G1-specific cell cycle regulators were investigated [Figure 2]. Outcomes of the study indicate interplay between different site-specific posttranslational modifications of histone H3 in butyrate treated VSMCs that seem to alter chromatin structure and organization that supports downregulation of cdk2, cdk4/6, and PCNA, and upregulation of cdkI, p21Cip1 and p15INK4B. This causes inhibition of Rb phosphorylation resulting in arrest of VSMC proliferation [Figure 2 and Figure 3]. The effects of HDACi on cell cyclerelated gene expressions appear to be highly selective, leading to transcriptional activation of certain genes such as the cdkIs but repression of others like cdks to efficiently block cell proliferation.

#### **4.2.2 Histone acetylation**

Hypernuclear acetylation (HNA) also plays a role in proliferation (Kawahara et al., 2003). Presence of increased histone acetylation is observed in VSMC of atherosclerotic lesions unlike in normal arteries. Thrombin, a humoral factor that is known to activate and stimulate VSMC proliferation, strongly induced HNA in cultures of VSMC. MAP kinase

Emerging Epigenetic Therapy for Vascular Proliferative Diseases 97

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pathway and CBP are implicated in thrombin-induced HNA suggesting that coactivators cooperating with signaling-dependent transcription activators play a role in atherosclerosis through HNA (Kawahara et al., 1999).

#### **5. Conclusions and perspectives**

Over the past few years, it has become abundantly evident that several interdependent epigenetic changes collaborate with genetic changes in the development of human diseases including cardiovascular diseases such as atherosclerosis and restenosis. Since the genetic foundations of diseases are generally immutable, but their epigenetic and chromatin changes are reversible, they are suitable for epigenetic therapy with epigenetic and chromatin modifiers. Therefore, thorough understanding on the roles of epigenetic processes in the etiology of atherosclerosis and restenosis is essential to launch an epigenetic therapy designed to target the epigenetic processes. Although a number of different therapeutic approaches have been investigated in the treatment of atherosclerosis and restenosis such as brachytherapy, pharmacotherapy, gene therapy, and immunotherapy, the therapeutic efficacy of these treatment modalities for atherosclerosis and restenosis is not adequate for a number of patients. Possible reasons are multiple factors, genes, pathways are involved in the disease pathogenesis, and targeting one or two genes or pathways are not sufficient to treat complex vascular pathologies. In these scenarios, epigenetic therapy, which is reversible, appears to be appropriate because HDACi exhibit multiple cellular effects that play major roles in vascular pathogenesis. HDACi exhibit antiproliferative, antioxidant and antiinflammatory effects and cause inhibition of cell proliferation and stimulation of differentiation or apoptosis by modulating expression of multiple genes (Natarajan, 2011; Ranganna, et al.,2005; 2006; 2007). For example, HDACi inhibit cell proliferation by appropriately altering both positive and negative regulators of cell cycle. While cell cycle inhibitors such as p21Cip1, p27kip1, p16INK4a and p15INK4b are upregulated, expressions of cyclin D, cyclin A, cdk2, cdk4/6, PCNA and pRb that promote cell cycle progression are downregulated (Figure 2 and Figure 3). With one single HDACi, multiple genes are altered that control cell cycle progression unlike gene therapy, where a cocktail of genes is required to bring about inhibition of cell proliferation. Furthermore, stents are ideal platform for the localized delivery of HDACi to the vascular wall because of their widespread use and safety in the treatment of restenosis. It is recognized that many of the processes that play critical role in atherosclerosis and restenosis such as VSMC proliferation, migration, inflammation, cellular redox state and matrix protein synthesis (Natarajan, 2011) are regulated by epigenetic mechanisms. As such, they present an exciting opportunity for therapeutic intervention, particularly to refractory or recurrent vascular pathologies such as restenosis and in-stent restenosis and vein graft failure. A number of natural and synthetic HDACi are already in the pipeline for the treatment of cancer either stand alone, or in combination with other anticancer drugs and several clinical trials are in progress. Exploring these particulars will speed the necessary epigenetic treatment strategies for the management of atherosclerosis and restenosis.

#### **6. Acknowledgements**

The work from our laboratory described in this review is supported by G12RR0345 and C06RR012537-01 grants from National Institutes of Health/National Center for Research Resources.

#### **7. References**

96 Atherogenesis

pathway and CBP are implicated in thrombin-induced HNA suggesting that coactivators cooperating with signaling-dependent transcription activators play a role in atherosclerosis

Over the past few years, it has become abundantly evident that several interdependent epigenetic changes collaborate with genetic changes in the development of human diseases including cardiovascular diseases such as atherosclerosis and restenosis. Since the genetic foundations of diseases are generally immutable, but their epigenetic and chromatin changes are reversible, they are suitable for epigenetic therapy with epigenetic and chromatin modifiers. Therefore, thorough understanding on the roles of epigenetic processes in the etiology of atherosclerosis and restenosis is essential to launch an epigenetic therapy designed to target the epigenetic processes. Although a number of different therapeutic approaches have been investigated in the treatment of atherosclerosis and restenosis such as brachytherapy, pharmacotherapy, gene therapy, and immunotherapy, the therapeutic efficacy of these treatment modalities for atherosclerosis and restenosis is not adequate for a number of patients. Possible reasons are multiple factors, genes, pathways are involved in the disease pathogenesis, and targeting one or two genes or pathways are not sufficient to treat complex vascular pathologies. In these scenarios, epigenetic therapy, which is reversible, appears to be appropriate because HDACi exhibit multiple cellular effects that play major roles in vascular pathogenesis. HDACi exhibit antiproliferative, antioxidant and antiinflammatory effects and cause inhibition of cell proliferation and stimulation of differentiation or apoptosis by modulating expression of multiple genes (Natarajan, 2011; Ranganna, et al.,2005; 2006; 2007). For example, HDACi inhibit cell proliferation by appropriately altering both positive and negative regulators of cell cycle. While cell cycle inhibitors such as p21Cip1, p27kip1, p16INK4a and p15INK4b are upregulated, expressions of cyclin D, cyclin A, cdk2, cdk4/6, PCNA and pRb that promote cell cycle progression are downregulated (Figure 2 and Figure 3). With one single HDACi, multiple genes are altered that control cell cycle progression unlike gene therapy, where a cocktail of genes is required to bring about inhibition of cell proliferation. Furthermore, stents are ideal platform for the localized delivery of HDACi to the vascular wall because of their widespread use and safety in the treatment of restenosis. It is recognized that many of the processes that play critical role in atherosclerosis and restenosis such as VSMC proliferation, migration, inflammation, cellular redox state and matrix protein synthesis (Natarajan, 2011) are regulated by epigenetic mechanisms. As such, they present an exciting opportunity for therapeutic intervention, particularly to refractory or recurrent vascular pathologies such as restenosis and in-stent restenosis and vein graft failure. A number of natural and synthetic HDACi are already in the pipeline for the treatment of cancer either stand alone, or in combination with other anticancer drugs and several clinical trials are in progress. Exploring these particulars will speed the necessary epigenetic treatment strategies

The work from our laboratory described in this review is supported by G12RR0345 and C06RR012537-01 grants from National Institutes of Health/National Center for Research

through HNA (Kawahara et al., 1999).

**5. Conclusions and perspectives**

for the management of atherosclerosis and restenosis.

**6. Acknowledgements**

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**6**

*Italy*

**Endothelial and Vascular Smooth Cell**

*Anatomy Section, Department of Biomedical Sciences and Biotechnology,*

Luigi Fabrizio Rodella and Rita Rezzani

*University of Brescia,*

**Dysfunctions: A Comprehensive Appraisal**

Cardiovascular disease (CvDs) such as coronary artery disease, hypertension, congestive heart failure and stroke are the leading causes of death and disability in the Western World (Madamanchi et al., 2005; Thom, 1989). The majority of CvDs results from complication of atherosclerosis. Prevention of cardiovascular events is therefore urgently needed and is one of the major recent challenges of medicine. New molecular imaging approaches featuring the assessment of inflammatory processes in the vascular wall (on top of existing anatomic and functional vessel imaging procedures) could emerge as decisive tools for the understanding and prevention of cardiovascular events (Schafers et

Atherosclerosis is a progressive disease, affecting medium and large-sized arteries, characterized by patchy intramural thickening of the subintimal that encroaches on the arterial lumen (Bonomini et al., 2008). The atherosclerosis plaque is characterized by an accumulation of lipid in the artery wall, together with infiltration of macrophages, T cells and mast cells, and the formation by vascular smooth muscle cells (VSMCs) of a fibrous cap composed mostly of collagen. Early lesions called "fatty streaks" consist of subendothelial deposition of lipid, macrophage foam cells loaded with cholesterol and T cells. Over time, a more complex lesion develops, with apoptotic as well as necrotic cells, cell debris and cholesterol crystals forming a necrotic core in the lesion. This structure is covered by a fibrous cap of variable thickness, and its "shoulder" regions are infiltrated by activated T cells, macrophages and mast cells, which produce proinflammatory mediators and enzymes (Hansson et al., 2006). Plaque growth can cause stenosis (narrowing of the lumen) that can contribute to ischemia in the surrounding tissue

Although the pathophysiological mechanisms underlying atherosclerosis are not completely understood, it is widely recognized that both inflammation and oxidative stress play important roles in all of the phases of atherosclerosis evolution (Cipollone et

**1. Introduction**

al., 2010).

al., 2007).

**2. Atherosclerosis**

(Hansson & Hermansson, 2011).

### **Endothelial and Vascular Smooth Cell Dysfunctions: A Comprehensive Appraisal**

Luigi Fabrizio Rodella and Rita Rezzani

*Anatomy Section, Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy*

#### **1. Introduction**

104 Atherogenesis

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> Cardiovascular disease (CvDs) such as coronary artery disease, hypertension, congestive heart failure and stroke are the leading causes of death and disability in the Western World (Madamanchi et al., 2005; Thom, 1989). The majority of CvDs results from complication of atherosclerosis. Prevention of cardiovascular events is therefore urgently needed and is one of the major recent challenges of medicine. New molecular imaging approaches featuring the assessment of inflammatory processes in the vascular wall (on top of existing anatomic and functional vessel imaging procedures) could emerge as decisive tools for the understanding and prevention of cardiovascular events (Schafers et al., 2010).

#### **2. Atherosclerosis**

Atherosclerosis is a progressive disease, affecting medium and large-sized arteries, characterized by patchy intramural thickening of the subintimal that encroaches on the arterial lumen (Bonomini et al., 2008). The atherosclerosis plaque is characterized by an accumulation of lipid in the artery wall, together with infiltration of macrophages, T cells and mast cells, and the formation by vascular smooth muscle cells (VSMCs) of a fibrous cap composed mostly of collagen. Early lesions called "fatty streaks" consist of subendothelial deposition of lipid, macrophage foam cells loaded with cholesterol and T cells. Over time, a more complex lesion develops, with apoptotic as well as necrotic cells, cell debris and cholesterol crystals forming a necrotic core in the lesion. This structure is covered by a fibrous cap of variable thickness, and its "shoulder" regions are infiltrated by activated T cells, macrophages and mast cells, which produce proinflammatory mediators and enzymes (Hansson et al., 2006). Plaque growth can cause stenosis (narrowing of the lumen) that can contribute to ischemia in the surrounding tissue (Hansson & Hermansson, 2011).

Although the pathophysiological mechanisms underlying atherosclerosis are not completely understood, it is widely recognized that both inflammation and oxidative stress play important roles in all of the phases of atherosclerosis evolution (Cipollone et al., 2007).

Endothelial and Vascular Smooth Cell Dysfunctions: A Comprehensive Appraisal 107

context. Numerous studies have shown that various physiological stimuli that contribute to pathogenesis of vascular disease can induce the formation of ROS (Lakshmi et al., 2009). ROS have detrimental effects on vascular function through several mechanisms. First, ROS, especially hydroxyl radicals, directly injure cell membranes and nuclei. Second, by interacting with endogenous vasoactive mediators formed in ECs, ROS modulate vasomotion and the atherogenic process. Third, ROS peroxidize lipid components, leading to the formation of oxidized lipoproteins (LDL), one of the key mediators of atherosclerosis

Fig. 2. Potential sources of ROS production in atherosclerosis progression.

acid (HOCl), phenoxyl radical intermediates or ONOO-

inflammation and atherosclerosis (Hansson & Hermansson, 2011).

Cholesterol is transported in the blood by LDL. These particles contain esterified cholesterol and triglycerides surrounded by a shell of phospholipids, free cholesterol and apolipoprotein B100 (ApoB100). Circulating LDL particles can accumulate in the intimal, the innermost layer of the artery. Here ApoB100 binds to proteoglycans of the extracellular matrix (ECM) through ionic interactions (Tabas et al., 2007). This is an important initiating factor in early atherogenesis (Skålen et al., 2002; Steinberg, 2009; Witztum & Steinberg, 2001). As a consequence of this subendothelial retention, LDL particles are trapped in the tunica intima, where they are prone to oxidative modifications caused by enzymatic attack of myeloperoxidase (Heinecke, 2007) and lipoxygenases, or by ROS such as hypoclorous

Oxidized LDL (Ox-LDL) has several biological effects (Madamanchi et al., 2005); it is proinflammatory; it causes inhibition of endothelial NOS (eNOS); it promotes vasoconstriction and adhesion; it stimulates cytokines such as interleukins (ILs) and increases platelet aggregation. Ox-LDL-derived products are cytotoxic and induce apoptosis. Ox-LDL can adversely affect coagulation by stimulating tissue factor and plasminogen activator

generated in the intimal during

(Bonomini et al., 2008).

Fig. 1. Steps involved in atherosclerosis progression from endothelial dysfunction to cardiovascular complication.

#### **2.1 Atherosclerosis and oxidative stress**

Oxidative stress can be defined as an "imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage" (Sies, 1991). Age, gender, obesity, cigarette smoking, hypertension, diabetes mellitus and dyslipidemia are known atherogenic risk factors that promote the impairment of endothelial function, smooth muscle function and vessel wall metabolism. These risk factors are associated with an increased production of reactive oxygen species (ROS) (Antoniades et al., 2003). ROS play a physiological role in the vessel wall and participate as second messengers in endothelium-dependent function, in smooth muscle cells and endothelial cells (ECs) growth and survival, and in remodelling of the vessel wall. Each of these responses, when uncontrolled, contributes to vascular diseases (Fortuño et al., 2005; Griendling & Harrison, 1999; Irani, 2000; Taniyama & Griendling, 2003).

In the vasculature wall, ROS are produced by all the layers, including tunica intima, media and adventitia. ROS include superoxide anion radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH), nitric oxide (NO), and peroxynitrite (ONOO- ) (Lakshmi et al., 2009). The major vascular ROS is O2−, which inactivates NO, the main vascular relaxing factor, thus impairing relaxation (Cai & Harrison, 2000; Kojda & Harrison, 1999). Dismutation of O2<sup>−</sup> by superoxide dismutase (SOD) produces H2O2, a more stable ROS, which, in turn, is converted to water by catalase and glutathione peroxidase. H2O2 and other peroxides appear to be important in the regulation of growth-related signalling in VSMCs and inflammatory responses in vascular lesions (Irani, 2000; Li, P.F. et al., 1997). High levels of O2−, the consequent accumulation of H2O2 and diminished NO bioavailability play a critical role in the modulation of vascular remodelling. Finally, ONOO- , resulting from the reaction between O2<sup>−</sup> and NO, constitutes a strong oxidant molecule, which is able to oxidize proteins, lipids and nucleic acids and then causes cell damage (Beckman & Koppenol, 1996; Fortuño et al., 2005).

There are several potential sources of ROS production. In cardiovascular disease the sources include xanthine oxidase, cyclooxygenase, lipooxygenase, mitochondrial respiration, cytochrome P450, uncoupled nitric oxide synthase (NOS) and NAD(P)H oxidase. They have been identified as sources of ROS generation in all type of vasculature. These sources may contribute to ROS formation, depending on cell type, cellular activation site and disease

Fig. 1. Steps involved in atherosclerosis progression from endothelial dysfunction to

Oxidative stress can be defined as an "imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage" (Sies, 1991). Age, gender, obesity, cigarette smoking, hypertension, diabetes mellitus and dyslipidemia are known atherogenic risk factors that promote the impairment of endothelial function, smooth muscle function and vessel wall metabolism. These risk factors are associated with an increased production of reactive oxygen species (ROS) (Antoniades et al., 2003). ROS play a physiological role in the vessel wall and participate as second messengers in endothelium-dependent function, in smooth muscle cells and endothelial cells (ECs) growth and survival, and in remodelling of the vessel wall. Each of these responses, when uncontrolled, contributes to vascular diseases (Fortuño et al., 2005; Griendling & Harrison, 1999; Irani, 2000; Taniyama &

In the vasculature wall, ROS are produced by all the layers, including tunica intima, media and adventitia. ROS include superoxide anion radical (O2-), hydrogen peroxide (H2O2),

The major vascular ROS is O2−, which inactivates NO, the main vascular relaxing factor, thus impairing relaxation (Cai & Harrison, 2000; Kojda & Harrison, 1999). Dismutation of O2<sup>−</sup> by superoxide dismutase (SOD) produces H2O2, a more stable ROS, which, in turn, is converted to water by catalase and glutathione peroxidase. H2O2 and other peroxides appear to be important in the regulation of growth-related signalling in VSMCs and inflammatory responses in vascular lesions (Irani, 2000; Li, P.F. et al., 1997). High levels of O2−, the consequent accumulation of H2O2 and diminished NO bioavailability play a critical role in the modulation of vascular remodelling. Finally, ONOO-, resulting from the reaction between O2<sup>−</sup> and NO, constitutes a strong oxidant molecule, which is able to oxidize proteins, lipids and nucleic acids and then causes cell damage (Beckman & Koppenol, 1996;

There are several potential sources of ROS production. In cardiovascular disease the sources include xanthine oxidase, cyclooxygenase, lipooxygenase, mitochondrial respiration, cytochrome P450, uncoupled nitric oxide synthase (NOS) and NAD(P)H oxidase. They have been identified as sources of ROS generation in all type of vasculature. These sources may contribute to ROS formation, depending on cell type, cellular activation site and disease

) (Lakshmi et al., 2009).

hydroxyl radical (OH), nitric oxide (NO), and peroxynitrite (ONOO-

cardiovascular complication.

Griendling, 2003).

Fortuño et al., 2005).

**2.1 Atherosclerosis and oxidative stress**

context. Numerous studies have shown that various physiological stimuli that contribute to pathogenesis of vascular disease can induce the formation of ROS (Lakshmi et al., 2009). ROS have detrimental effects on vascular function through several mechanisms. First, ROS, especially hydroxyl radicals, directly injure cell membranes and nuclei. Second, by interacting with endogenous vasoactive mediators formed in ECs, ROS modulate vasomotion and the atherogenic process. Third, ROS peroxidize lipid components, leading to the formation of oxidized lipoproteins (LDL), one of the key mediators of atherosclerosis (Bonomini et al., 2008).

Fig. 2. Potential sources of ROS production in atherosclerosis progression.

Cholesterol is transported in the blood by LDL. These particles contain esterified cholesterol and triglycerides surrounded by a shell of phospholipids, free cholesterol and apolipoprotein B100 (ApoB100). Circulating LDL particles can accumulate in the intimal, the innermost layer of the artery. Here ApoB100 binds to proteoglycans of the extracellular matrix (ECM) through ionic interactions (Tabas et al., 2007). This is an important initiating factor in early atherogenesis (Skålen et al., 2002; Steinberg, 2009; Witztum & Steinberg, 2001). As a consequence of this subendothelial retention, LDL particles are trapped in the tunica intima, where they are prone to oxidative modifications caused by enzymatic attack of myeloperoxidase (Heinecke, 2007) and lipoxygenases, or by ROS such as hypoclorous acid (HOCl), phenoxyl radical intermediates or ONOO generated in the intimal during inflammation and atherosclerosis (Hansson & Hermansson, 2011).

Oxidized LDL (Ox-LDL) has several biological effects (Madamanchi et al., 2005); it is proinflammatory; it causes inhibition of endothelial NOS (eNOS); it promotes vasoconstriction and adhesion; it stimulates cytokines such as interleukins (ILs) and increases platelet aggregation. Ox-LDL-derived products are cytotoxic and induce apoptosis. Ox-LDL can adversely affect coagulation by stimulating tissue factor and plasminogen activator

Endothelial and Vascular Smooth Cell Dysfunctions: A Comprehensive Appraisal 109

evidence supports selective recruitment of a proinflammatory subset of monocytes to

A number of proinflammatory cytokines have been shown to participate in atherosclerotic plaque development, growth and rupture (Dabek, 2010; Libby et al., 2002). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) seems to be a crucial transcription factor in the cross-talk among cytokines, adhesion molecules and growth factors. On one hand, NF-kB is a major transcription factor leading to cytokine synthesis, and on the other hand, the above mentioned factors keep NF-kB persistently activated in acute coronary syndromes (Dabek, 2010). In atherogenesis, NF-kB before regulates the expression of cyclooxygenases, lipooxygenases, cytokines, chemokines (i.e., MCP-1) and adhesion molecules (Dabek, 2010; Kutuk & Basaga, 2003). Later in the progression of the atherosclerotic lesion, NF-kB regulates gene expression of M-CSF, a factor stimulating infiltrating monocyte differentiation and transformation into "foamy cells", and other genes participating in the transformation (Brach et al., 1991; Dabek, 2010). As stated, atherosclerosis is an inflammatory reaction of the arterial wall. The factors IL-1β, TNF-α, IL-6, IL-12 and interferon γ (IFNγ) are involved in this reaction and their expression is

Intracellular matrix degradation is an important process in both plaque development and rupture. The vital factors involved include MMPs, particularly those that are able to break down the vascular base membrane. It has been shown that NF-kB is an essential regulator of MMP gene expression, especially MMP-2 and MMP-9, which are critical in plaque rupture (Bond et al., 1998; Dabek, 2010). Thus, NF-kB regulates the expression of a wide spectrum of atherosclerosis mediating factors. On the other hand, most of these factors also up-regulate NF-kB activity. Increased NF-kB activity was found in unstable regions of atherosclerotic plaques (Brand et al., 1997; Dabek, 2010). The significance of NF-kB activity has been confirmed in some clinical studies as well. Li and colleagues reported significantly increased NF-kB activity in white blood cells from unstable angina patients *vs.* stable angina

patients and *vs.* control patients (the lowest activity in the latter) (Li, J.J. et al., 2004).

Fig. 3. Role of oxidative stress and inflammation in the early atherosclerosis.

nascent atheroma in mice (Packard & Libby, 2008).

coregulated by NF-kB.

inhibitor-1 (PAI-1) synthesis. Another atherogenic property of Ox-LDL is its immunogenicity and its ability to promote retention of macrophages in the arterial wall by inhibiting macrophage motility (Singh & Jialal, 2006). In addiction, Ox-LDL stimulates VSMCs proliferation (Stocker & Keaney, 2004). Thus, intimal thickening further reduces the lumen of blood vessels, leading to further potentation of hypertension and atherosclerosis (Singh & Jialal, 2006). With ongoing oxidation, the physicochemical properties gradually change, including alterations in charge, particle size, lipid content and other features. The precise nature of each of these alterations obviously depends on the oxidizing agent. For all these reasons, Ox-LDL is not a defined molecular species but is instead a spectrum of LDL particles that have undergone a variety of physicochemical changes (Hansson & Hermansson, 2011).

#### **2.2 Atherosclerosis and inflammation**

Inflammation participates in atherosclerosis from its inception onwards. Fatty streaks do not cause symptoms, and may either progress to more complex lesions or involute. Fatty streaks have focal increases in the content of lipoproteins within regions of the intimal, where they associate with components of the ECM such as proteoglycans, slowing their egress. This retention sequesters lipoproteins within the intimal, isolating them from plasma antioxidants, thus favoring their oxidative modification (Kruth, 2002; Packard & Libby, 2008; Skålen et al., 2002). Oxidatively modified LDL particles comprise an incompletely defined mixture, because both the lipid and protein moieties can undergo oxidative modification. Constituents of such modified lipoprotein particles can induce a local inflammatory response (Miller et al., 2003; Packard & Libby, 2008).

Vascular ECs function to prevent clotting of blood and adhesion of blood cells to the endothelial cells, in addition to playing the role of a barrier, as a cell monolayer, to prevent blood constituents from invading the vascular wall. When ECs are injured or activated by various coronary risk factors, infections or physical stimuli, adhesion molecules become expressed in ECs, and peripheral monocytes adhere to the endothelial cell surface. Adhesion molecules are broadly divided into three molecular families: integrin family, immunoglobulin family, and selectin family (L-selectin, Eselectin, P-selectin) (Yamada, 2001).

Chemoattractant factors, which include monocyte chemoattractant protein-1 (MCP-1) produced by vascular wall cells in response to modified lipoproteins, direct the migration and diapedesis of adherent monocytes (Boring et al., 1998; Packard & Libby, 2008). Monocytic cells, directly interacting with human ECs, increase several fold monocyte matrix metalloproteinase (MMP) 9 production, allowing for the subsequent infiltration of leukocytes through the endothelial layer and its associated basement membrane (Amorino & Hoover, 1998; Packard & Libby, 2008) Within the intima, monocytes mature into macrophages under the influence of macrophage colony stimulating factor (M-CSF), which is overexpressed in the inflamed intima. M-CSF stimulation also increases macrophage expression of scavenger receptors, members of the pattern-recognition receptor superfamily, which engulf modified lipoproteins through receptor-mediated endocytosis. Accumulation of cholesteryl esters in the cytoplasm converts macrophages into foam cells, i.e., lipid-laden macrophages characteristic of early-stage atherosclerosis. In parallel, macrophages proliferate and amplify the inflammatory response through the secretion of numerous growth factors and cytokines, including tumor necrosis factor α (TNFα) and IL-1β. Recent

inhibitor-1 (PAI-1) synthesis. Another atherogenic property of Ox-LDL is its immunogenicity and its ability to promote retention of macrophages in the arterial wall by inhibiting macrophage motility (Singh & Jialal, 2006). In addiction, Ox-LDL stimulates VSMCs proliferation (Stocker & Keaney, 2004). Thus, intimal thickening further reduces the lumen of blood vessels, leading to further potentation of hypertension and atherosclerosis (Singh & Jialal, 2006). With ongoing oxidation, the physicochemical properties gradually change, including alterations in charge, particle size, lipid content and other features. The precise nature of each of these alterations obviously depends on the oxidizing agent. For all these reasons, Ox-LDL is not a defined molecular species but is instead a spectrum of LDL particles that have undergone a variety of physicochemical changes (Hansson &

Inflammation participates in atherosclerosis from its inception onwards. Fatty streaks do not cause symptoms, and may either progress to more complex lesions or involute. Fatty streaks have focal increases in the content of lipoproteins within regions of the intimal, where they associate with components of the ECM such as proteoglycans, slowing their egress. This retention sequesters lipoproteins within the intimal, isolating them from plasma antioxidants, thus favoring their oxidative modification (Kruth, 2002; Packard & Libby, 2008; Skålen et al., 2002). Oxidatively modified LDL particles comprise an incompletely defined mixture, because both the lipid and protein moieties can undergo oxidative modification. Constituents of such modified lipoprotein particles can induce a

Vascular ECs function to prevent clotting of blood and adhesion of blood cells to the endothelial cells, in addition to playing the role of a barrier, as a cell monolayer, to prevent blood constituents from invading the vascular wall. When ECs are injured or activated by various coronary risk factors, infections or physical stimuli, adhesion molecules become expressed in ECs, and peripheral monocytes adhere to the endothelial cell surface. Adhesion molecules are broadly divided into three molecular families: integrin family, immunoglobulin family, and selectin family (L-selectin, Eselectin, P-selectin) (Yamada,

Chemoattractant factors, which include monocyte chemoattractant protein-1 (MCP-1) produced by vascular wall cells in response to modified lipoproteins, direct the migration and diapedesis of adherent monocytes (Boring et al., 1998; Packard & Libby, 2008). Monocytic cells, directly interacting with human ECs, increase several fold monocyte matrix metalloproteinase (MMP) 9 production, allowing for the subsequent infiltration of leukocytes through the endothelial layer and its associated basement membrane (Amorino & Hoover, 1998; Packard & Libby, 2008) Within the intima, monocytes mature into macrophages under the influence of macrophage colony stimulating factor (M-CSF), which is overexpressed in the inflamed intima. M-CSF stimulation also increases macrophage expression of scavenger receptors, members of the pattern-recognition receptor superfamily, which engulf modified lipoproteins through receptor-mediated endocytosis. Accumulation of cholesteryl esters in the cytoplasm converts macrophages into foam cells, i.e., lipid-laden macrophages characteristic of early-stage atherosclerosis. In parallel, macrophages proliferate and amplify the inflammatory response through the secretion of numerous growth factors and cytokines, including tumor necrosis factor α (TNFα) and IL-1β. Recent

local inflammatory response (Miller et al., 2003; Packard & Libby, 2008).

Hermansson, 2011).

2001).

**2.2 Atherosclerosis and inflammation**

evidence supports selective recruitment of a proinflammatory subset of monocytes to nascent atheroma in mice (Packard & Libby, 2008).

A number of proinflammatory cytokines have been shown to participate in atherosclerotic plaque development, growth and rupture (Dabek, 2010; Libby et al., 2002). Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) seems to be a crucial transcription factor in the cross-talk among cytokines, adhesion molecules and growth factors. On one hand, NF-kB is a major transcription factor leading to cytokine synthesis, and on the other hand, the above mentioned factors keep NF-kB persistently activated in acute coronary syndromes (Dabek, 2010). In atherogenesis, NF-kB before regulates the expression of cyclooxygenases, lipooxygenases, cytokines, chemokines (i.e., MCP-1) and adhesion molecules (Dabek, 2010; Kutuk & Basaga, 2003). Later in the progression of the atherosclerotic lesion, NF-kB regulates gene expression of M-CSF, a factor stimulating infiltrating monocyte differentiation and transformation into "foamy cells", and other genes participating in the transformation (Brach et al., 1991; Dabek, 2010). As stated, atherosclerosis is an inflammatory reaction of the arterial wall. The factors IL-1β, TNF-α, IL-6, IL-12 and interferon γ (IFNγ) are involved in this reaction and their expression is coregulated by NF-kB.

Intracellular matrix degradation is an important process in both plaque development and rupture. The vital factors involved include MMPs, particularly those that are able to break down the vascular base membrane. It has been shown that NF-kB is an essential regulator of MMP gene expression, especially MMP-2 and MMP-9, which are critical in plaque rupture (Bond et al., 1998; Dabek, 2010). Thus, NF-kB regulates the expression of a wide spectrum of atherosclerosis mediating factors. On the other hand, most of these factors also up-regulate NF-kB activity. Increased NF-kB activity was found in unstable regions of atherosclerotic plaques (Brand et al., 1997; Dabek, 2010). The significance of NF-kB activity has been confirmed in some clinical studies as well. Li and colleagues reported significantly increased NF-kB activity in white blood cells from unstable angina patients *vs.* stable angina patients and *vs.* control patients (the lowest activity in the latter) (Li, J.J. et al., 2004).

Fig. 3. Role of oxidative stress and inflammation in the early atherosclerosis.

2009).

**3.1 Endothelial cell-factors**

Endothelial and Vascular Smooth Cell Dysfunctions: A Comprehensive Appraisal 111

apoptosis and inflammation through activation of various signaling cascades, redoxsensitive transcriptional factors and expression of pro-inflammatory phenotype (Higashi et al., 2009). ECs dysfunction has been shown to be associated with an increase of ROS in atherosclerotic animal models and in human subjects with atherosclerosis (Dai, D.Z. & Dai, Y., 2010; Davies et al., 2010; Higashi et al., 2009). Moreover, in APOE-deficient mice, a widely used animal model of atherosclerosis (Xu, 2009; Zhang, S.H. et al., 1992), studies have demonstrated that aged-ECs are more sensitive to apoptosis than younger ones. ECs in the areas of the artery resistant to atherosclerosis have a life span of about 12 months, whereas cells at lesion-prone sites live for few weeks and even shorter in aged animals (Xu,

The vascular endothelium is nowadays considered to be a paracrine organ responsible for the secretion of several substances exerting atherogenic effects. The reduced bioavailability of NO as an indirect result of the effects of those factors, leads to atherosclerosis and its clinical manifestations (Muller & Morawietz, 2009; Tousoulis et al., 2010). Under normal conditions, ECs constantly produce a number of vasoactive and trophic substances that control inflammation, VSMC growth, vasomotion, platelet function and plasmatic

Normal vascular activity is essential for maintaining normal function of organs, dependent on a balance of vasoconstrictive and vasodilative substances derived from the endothelium, which mainly include NO to dilate and endothelin-1 (ET-1) to constrict the cells of tunica media. Furthermore, ECs activated by ROS can regulate vascular function via the release of inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), MCP-1, ILs, angiotensin-II (A-II), TNFα, NF-kB and E- and Pselectin, or the release of haemostatic regulators, such as von Willebrand factor, tissue factor inhibitor and plasminogen activator, fibrinogen and NO (Sima et al., 2009; Vanhoutte, 2009).

Fig. 5. Regulatory functions of the endothelium maintaining the equilibrium between

antiatherogenic and atherogenic properties.

coagulation (Barton & Haudenschild, 2001; Traupe et al., 2003).

#### **3. Endothelial cells dysfunction in atherosclerosis**

The endothelium is responsible for the regulation of vascular tone, the exchange of plasma and cell biomolecules, inflammation, lipid metabolism and modulation of fibrinolysis and coagulation (Andrews et al., 2010). Aging affects many pathways involved in cardiovascular functions and particularly of ECs (Barton, 2010; Virdis et al., 2010). In fact, endothelial-aging is associated with anatomical disruption, morphological abnormalities in ECs size and shape (Haudenschild et al., 1981), susceptibility to apoptosis and abnormal release of EC-derived factors (Barton, 2010). These factors, which are synthetized not only by ECs, but also by VSMCs, are now known to contribute to pathogenetic mechanisms of CVDs (Higashi et al., 2009).

Fig. 4. Central role of ROS in inducing endothelial dysfunction in vascular diseases.

ECs dysfunction, inflammation, oxidative stress and dyslipidaemia are known to play prominent and vital roles not only in the development of atherosclerotic lesions, but also in their progression. (Andrews et al., 2010; Bai et al., 2010; Higashi et al., 2009 ; Virdis et al., 2010). Number of factors and modalities are available to interfere with age related changes in EC function (Barton, 2010; Jensen-Urstad et al., 1999). When endothelial damage compromises the normal vascular function, the intracellular dynamic balance probably leans on an athero-prone phenotype.