Accepted Manuscript Histone Deacetylases and Atherosclerosis Xia-xia Zheng, Tian Zhou, Xin-An Wang, Xiaohong Tong, Jiawang Ding, PhD, Professor and Director PII:

S0021-9150(14)01670-0

DOI:

10.1016/j.atherosclerosis.2014.12.048

Reference:

ATH 13866

To appear in:

Atherosclerosis

Received Date: 10 July 2014 Revised Date:

17 December 2014

Accepted Date: 18 December 2014

Please cite this article as: Zheng X-x, Zhou T, Wang X-A, Tong X, Ding J, Histone Deacetylases and Atherosclerosis, Atherosclerosis (2015), doi: 10.1016/j.atherosclerosis.2014.12.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Histone Deacetylases and Atherosclerosis Xia-xia Zhenga,b,#, Tian Zhoua,b,#, Xin-An Wanga,b, Xiaohong Tonga,b, Jiawang Dinga,b,*

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Running Title: Histone Deacetylases and Atherosclerosis

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a Department of Cardiology, the First College of Clinical Medical Sciences, China Three Gorges University, Yichang 443000, Hubei Province, China. b Institute of Cardiovascular Diseases, China Three Gorges University, Yichang 443000, Hubei Province, China.

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#:co-first author

Correspondence to Jiawang Ding, PhD Professor and Director

Department of Cardiology, The First College of Clinical Medical Sciences

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China Three Gorges University

Yichang, 443000, Hubei, PR China. Tel: +8613972000609

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Fax: +8607176482302

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E-mail: [email protected]

Word Count: Abstract: 96; Text: 7944

ACCEPTED MANUSCRIPT 1 Introduction 2 Histone deacetylases and histone deacetylase inhibitions 2.1 Class I HDACs 2.2 Class II HDACs 2.3 Class III HDACs 2.5 Histone deacetylase inhibitions

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2.4 Class IV HDACs 3 HDACs have an impact on the atherosclerosis plaque formation 3.1 Role of HDACs in elevated blood glucose and plasma lipid

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3.1.1 HDACs have an effect on the blood glucose homeostasis 3.1.2 HDACs have an effect on the blood lipid homeostasis

3.2 Role of HDACs in endothelial cells activation and dyfunction as well as correlative cytokine

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secretion

3.2.1 HDACs partake in endothelium function regulation-mediated by shear stress 3.2.2 HDACs contribute to ECs dysfunction-induced by inflammation and oxidative stress 3.2.3The synthetic and secretory action of activated ECs are partially affected by HDACs

3.3Role of HDACs in monocyte accumulation migration and differentiation 3.4 HDACs regulate macrophage foam cell and VSMC foam cell formation

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3.4.1 Role of HDACs in LDL and cholesterol uptake and foam cell formation 3.4.2 HDACs regulate macrophage cholesterol reverse transport 3.5 HDACs contribute to VSMCs phenotype switch and fibrous cap formation synthetic state

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3.5.1 HDACSs influence phenotype switch of VSMCs to a proliferation, migration and 3.5.2 HDACs play a role in regulation synthetic function of VSMCs and fibrous cap formation

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3.5.3 HDACs have to do with VSMCs calcification, aging and apoptosis 3.6 Role of HDACs in plaque disruption and thrombosis 3.6.1 How can HDACs impact on atheromatous plaque disruption？ 3.6.2 HDACs and the thrombosis after plaque rupture 4 Summary

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Abstract Atherosclerosis is the most common pathological process that leads to cardiovascular diseases, a disease of large- and medium-sized arteries that is characterized by a formation of atherosclerotic plaques consisting of necrotic cores, calcified regions, accumulated modified lipids, smooth muscle cells (SMCs), endothelial cells, leukocytes, and foam cells. Recently, the question

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about how to suppress the occurrence of atherosclerosis and alleviate the progress of cardiovascular disease becomes the hot topic. Accumulating evidence suggests that histone deacetylases(HDACs) play crucial roles in arteriosclerosis. This review summarizes the effect of HDACs and HDAC

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inhibitors(HDACi) on the progress of atherosclerosis.

Keywords: HDACs; endothelial cells; macrophage cells; vascular smooth muscle cells; plaque;

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thrombosis

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1. Introduction Cardiovascular diseases (CVD) are associated with an increasing in the high morbility and mortality worldwide. Atherosclerosis(AS) is the leading pathophysiological process of CVD. HDACs are a family of enzymes that lead to the deacetylation of the lysine residues on histone and non-histone

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protein[1], which can regulate expression of multiple genes associated with the pathogenesis of diseases[2].Recently, a great number of studies have highlighted that HDACi plays a multiple role in CVD, such as hypertension, arrhythmia heart failure and so on[3]. In addition, HDACs have been shown to associate with atherosclerosis[4]. However, the function of HDACs in arteriosclerosis has

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not yet been elucidated. This review will discuss the impact of HDACs on the progress of

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atherosclerosis.

2. Histone deacetylases and histone deacetylase inhibitions HDACs are a group of enzymes that lead to the deacetylation of the lysine residues on histone and non-histone protein[1]. Until now there are at least 18 distinct mammalian genes have been identified,

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which can be categorized into four classes based on the structure, function and respective yeast orthologues termed class I HDACs (HDAC1, 2, 3, 8), class II HDACs (HDAC4, 5, 6, 7, 9, 10), class III HDACs, and class IV HDAC (HDAC11) [5]. The class II HDACs are further divided into the class IIa (HDAC4, 5, 7 and 9) and the class IIb (HDAC6 and 10) based on the function and

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localization[6]. Classes I, II and IV HDACs are also named classical HDACs for their requirement of zinc as one of the cofactors for enzymatic activity, while III class HDACs differ from classical

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HDACs and need NAD+ as a cofactor[6, 7].

2.1 Class I HDACs

Class I HDACs are significant homology to yeast HDAC retinoblastoma protein (Rpd3), which have ubiquitous tissue distribution. HDAC 1 and 2 are mostly exclusive localization in nucleus while HDAC 3 and 8 ubiquitously distributed throughout cytoplasm and nucleus[8]. They function as the catalytic core of the transcriptional repressor multiprotein complexes, such as silencing mediator of retinoid and thyroid receptors(SMRT), nuclear receptor co-repressor(NCoR), and CoREST (co-repressor for element-1-silencing transcription factor), which partake in transcriptional

ACCEPTED MANUSCRIPT repression[9]. Class I HDACs modulate the multiple steps involved in the progression of atherosclerosis.

HDAC2, also named the “canonical” HDAC, has strong enzymatic activity towards histones.

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HDAC2 increases the histone deacetylation of SM22α, which is one of the markers of differentiated SMCs, leading to the SMC phenotype switch from the “contractile” phenotype to a “synthetic” phenotype[10], which exhibits a pivotal role in the progression of atherosclerosis and the stability of plaque. In addition, HDAC2 also function as a regulator of non-histone proteins and modulate the

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deacetylation of transcription factors involved in cell proliferation, such as Krüppel-like factor 5 (Klf5), CREB binding protein (CBP), retinoic acid receptor (RAR) and so on[11], finally increase the

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proliferation of SMCs and aggravate the development of atherosclerosis. Similarly, HDAC1/2/3 promotes endothelial cells(ECs) proliferation via transcription factor p21 and maintains the integrity of vascular[12].

As mentioned above, HDAC3 interacts with transcriptional co-repressors complex (NCoR and

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SMRT), recruits coactivator, and accelerates the transcription of target genes[2, 13], such as peroxisome proliferators-activated receptors(PPARs), liver X receptors(LXR), Klf2 and forkhead box O (FoxO). They play an important role in regulating complex biological events, including

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glucose and lipid metabolism, ECs differentiation and survival[14, 15], which involving in the process of atherosclerosis.

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HDAC8 most similar to HDAC3 in the structure, with largely catalyze domain, and may have an important role on histone and non-histone proteins, such as estrogen-related receptor α (ERRα) and cAMP responsive element-binding protein (CREB)[16]. Traditionally, we deem that HDAC8 can catalyze deacetylation without the protein complex[7]. However, we now observe that HDAC8 can also effect on the function of cell and tissue via binding to associated protein complex[16].

2.2 Class II HDACs Class IIa HDACs shuttle between nucleus and cytoplasm depending on the serine residues phosphorylation which bind to 14-3-3 in the regulatory N-terminal domain, which is mediated by

ACCEPTED MANUSCRIPT numerous kinase families, consisting of Calcium/calmodulin-dependent protein kinase (CaMK)[17], specifically CaMKI and IV, and microtubule affinity-regulating kinase (MARK)/Par-1 family[18], subsequently abolishing the repression of HDAC target genes. The association of the Class IIa HDACs with MEF2 play an important role in regulating VSMCs proliferation [19] and ECs

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proliferation and migration to maintain the integrity of the vascular intima [20]. For example, HDAC3 interact with HDAC4, 5 and 7 through the formation of complex with NCoR and SMRT[21], which are involved in the proliferation of ECs, ultimately regulating the process of atherosclerosis.

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HDAC6 can deacetylate α-tubulin to regulate cell motility [22] , which may play a role, at least in part, in the progression of atherosclerosis. HDAC10 belongs to class IIb HDACs, which has a similar

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structure to HDAC6[23]. Recently, there is a series of evidence shows that HDAC10 involve in the occurrence of cancers[24], but we know little about the function of HDAC10 in cardiovascular diseases. Therefore, more experiments should be taken to expound the characters of HDAC10 as well as HDAC6.

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2.3 Class III HDACs

Class III HDACs comprise of sirtuins (SIRT1-7), which are considered to be the homologous of the yeast Sir2 family of proteins[25]. Sirtuin proteins contain a conserved catalytic core domain, which

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are characterized by NAD+/NADH binding proteins, as well as N- and C-terminal regions that are variable in length and sequence[26]. All of the seven sirtuins locate in different subcelluar[27]: SIRT1, SIRT6 and SIRT7 are mostly located in nucleus, SIRT2 are found in the cytoplasm, and

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SIRT3, SIRT4 and SIRT5 reside in the mitochondria. A body of studies has reported that human sirtuin proteins play an important role in the pathophysiological process in atherosclerosis.

2.4 Class IV HDACs

HDAC 11 is the exclusive HDAC in class IV HDACs and is similar to class I and II HDACs in the catalytic regions[28]. HDAC11 has been reported to play important roles in inflammatory, immunity tolerance and cancers[29-31]. But we find little information about how HDAC11 modulate the progression of atherosclerosis and needs to be further investigated in future.

ACCEPTED MANUSCRIPT 2.5 Histone deacetylase inhibitions A mass of evidences have reported that HDACs involve in the processes of various diseases, including metabolism disorder, cancers and cardiovascular diseases [32-34], and they maybe become the potential treatment targets for those diseases. Over the past decades, a body of HDACis have

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been identified and developed to target on the catalytic sites of HDACs, which can be divided into four groups based upon the chemical structures: short chain fatty acids, benzamides, hydroxamic acids and cyclic peptides[35]. HDACi was first used to treat cancers, but now, we also find that HDACi can exhibit non-oncological applications. In this review, we set out a series of HDACi

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related to atherosclerosis (as shown in the table1).

indicated HDACi

HDAC

Class

specificity

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Table1: The critical role of HDACi in atherosclerosis and metabolic diseases as previous reports

Function and result

reference

I

hydroxamate

decrease the expression of gluconeogenesis genes

[36]

TSA

I, II, IV

hydroxamic

decrease the cholesterol biosynthesis

[37]

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acids

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Ky-2

enhance MCP-1 secrection in ECs

[38]

reduce monocyte adhesion

[39]

inhibit VSMC proliferation

[40]

enhance VSMC proliferation

[41]

enhance the activity of VSMC and macrophage

[42]

stimulate t-PA expression in EC

[43]

increase the recruitment of monocyte to subintima

[44]

SAHA

I, II, IV

benzamides

mediate PDGF-Rα expression

[45]

MS275

I

hydroxamic

enhance oxidative metabolism in skeletal muscle

[46]

acids

and adipose, thus reducing glucose levels

VPA

I, IIa

short chain fatty acids

stimulate t-PA expression in EC

[47]

decrease BrdU uptake in ECs

[12]

ACCEPTED MANUSCRIPT butyrateB I, IIa

short chain

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fatty acids /

metacept-

inhibit VSMC proliferation

[40, 48, 49]

inhibit MMP-2 expression in VSMC

[50]

inhibit TF activity and protein level

[51]

apicidin

I

cyclic

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1

peptides

3. HDACs have an impact on the atherosclerosis plaque formation

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Over the past decades, researchers focused on the genetic and physiological mechanisms of AS and had made certain progress. A variety of risk factors initiate the atherosclerotic process via a chemical,

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mechanical or immunological mechanism, then injury and activate the ECs, contributing to endothelial dysfunction[52, 53]. Then the activated ECs in injury lesions produce adhesion molecules

(intercellular

adhesion

molecule(ICAM)

and

vascular

cell

adhesion

molecule-1(VCAM-1)), chemotactic proteins(monocyte chemotactic protein-1 (MCP-1)), E- and P-selectin, TNF-α and growth factors(macrophage colony-stimulating factor (M-CSF)). The

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inflammatory molecules recruit monocytes to the vessel wall and promote the monocytes transmigration across the endothelial monolayer into the subintima, where they proliferate, differentiate into macrophages and foam cells by taking up the lipoproteins[53-55]. With time, the

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foam cells and macrophages die and release lipid-filled contents and tissue factors, contributing to the formation of the lipid-rich necrotic core, which is a key component of unstable plaques[55].

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Meanwhile, SMCs migrate from the medial layer and accumulate to intima, where they synthesize and secrete interstitial collagen, elastin and form the fibrous cap over the lesion. Over time, macrophages and lymphocytes secret cytokines and matrix-degrading proteases, including collagenases, gelatinases, stromolysin and cathepsins, to inhibit the secretion of matrix as well as weaken and labilize the atheromatous plaque[56]. Ultimately, the thin fibrous caps rupture, then expose and release procoagulant materials into the blood, triggering the thrombosis that impedes blood flow and results in acute stenosis of the arteries, leading to clinical manifestations[57]. As mentioned above, HDACs may have an impact on atherosclerosis during the pathophysiological process of plaque formation, and we’ll review the correlated knowledge as follow.

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3.1 Role of HDACs in elevated blood glucose and plasma lipid During the past decades, we have found plenty of risk factors for atherosclerosis, such as metabolic syndrome, smoking, oxidant stress, disturbed flow induced by hypertension and infection, of which

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metabolic syndrome relative elevated levels of blood glucose and plasma lipid and insulin resistant appear to be the major contributors of atherosclerosis[53, 58, 59]. In physiological condition, glucolipid metabolism maintains the normal physiological needs. Once glucose and lipid metabolic disorder appeared, a series of pathophysiologic alterations occurred. Recently, accumulation of

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studies indicate that HDACs may affect the level of blood glucose and plasma lipid by regulating the expression of correlative rate-limiting enzyme genes involved in biosynthesis and oxidative

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metabolism through accommodating hormone-mediated signaling pathway and metabolic nuclear receptors(MNRs), thus affect the progression of AS.

Carbohydrates and fat belong to the three energy-yielding nutrients in human beings. Their metabolism is not independent of each other, but related through the common intermediate

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metabolites, the Krebs cycle and biological oxidation and so on. Glucose homeostasis is dependent on the balance between the production of hepatic glucose and peripheral glucose uptake by skeletal muscle as well as adipose tissue[36]. Similar to it, lipid homeostasis is related to the balance between

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lipid synthesis and lipid oxidation metabolism. Increased lipid synthesis and pentose phosphate pathway, and reduced lipid oxidation metabolism make the body plasma lipid rise. In the latter part of this section, we will review the specific mechanism about how HDACs regulate the level of blood

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glucose and plasma lipid.

3.1.1 HDACs have an effect on the blood glucose homeostasis In mammals, plasma glucose homeostasis depends on insulin, glucagon and glucocorticoids. In physiological state, glycogenolysis and gluconeogenesis increase the level of plasma glucose and maintain euglycemia. However, the uptake and oxidative metabolism of the peripheral glucose by skeletal and adipose tissues decrease the plasma glucose level. Hexokinase II(HKII) and glycogen phosphorylase in liver and sketetal muscle are the rate-limiting enzymes for glycogenolysis while the rate-limiting enzymes for gluconeogenesis are phosphoenolpyruvate carboxylase kinase (PEPCK),

ACCEPTED MANUSCRIPT 6-bisphosphatase (FBPase), fructose-1, and glucose-6-phosphatase (G6Pase). The synergy of transcription factors and transcriptional cofactors, including CREB, hepatocyte nuclear factor 4α (HNF4 α), FoxO1, glucocorticoids receptor(GR), CREB regulated transcription coactivator 2 (CRTC2) and PPARγ coactivator 1α (PGC-1α ), plays an important role in regulating the expression

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of the rate-limiting enzyme genes mentioned above[60].

Recently, accumulating of studies has focused on the relationship between glucose homeostasis and HDACs. By means of treating HepG2 cells with class I HDAC inhibitor, Ky-2, Oiso et al.[36] has

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found that class I HDAC, especially HDAC1 can increase the expression of PEPCK via suppressing the expression of HNF4α and the transcriptional activity of FoxO1. Moreover, the study has reported

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that HDAC2 suppress the insulin-mediated effects, including glycogen synthesis and glucose uptake[61], but the special mechanism is not clear. In addition, Sun et al has observed that deletion of HDAC3 in liver results in the obviously increasing of genes related to lipid synthesis and NADPH synthesis, while the blood glucose concentrations is decreased in mice, suggesting HDAC3 may promote gluconeogenesis through repressing lipid synthesis[62]. It indicates that class I HDACs can

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upregualte the expression of the gluconeogenic target genes and increase the level of blood glucose.

As mentioned above, class II HDAC enzymes can shuttle between the nucleus and cytoplasm and

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then have tissue-specific roles[7]. In liver, class IIa HDAC enzymes are dephosphorytated and shuttle into nucleus, then HDAC4/5 recruits HDAC3 and links to the promoter of the genes of gluconeogenesis, such as PEPCK and G6Pase, and promotes their transcription via promoting

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deacetylation and activation of FoxO1; Consistently, knockdown of the HDAC4/5 by siRNA leads to the hyperacetylation of FoxO1, and decreases the expression of FoxO1 target genes[34] leading to the reduction of hyperglycemia. Interestingly, the study has also found that HDAC6 deletion ameliorates glucocorticoid-induced hyperglycemia via decreasing the transcription of glucocorticoid receptor, and then reduces the expression of genes related to gluconeogenesis, such as PEPCK, FBP, and G6P[63]. It suggests that class IIa HDAC may facilitate the expression of the gluconeogenic target genes and increase the level of blood glucose.

In addition to the classical HDACs, sirtuins are able to maintain the plasma glucose homeostasis via

ACCEPTED MANUSCRIPT modulating the expression of gluconeogenic and glycolytic target genes[64, 65]. Recently, the new evidence has shown that SIRT6 plays an ambiguous role in glucose metabolism[66] which may due to the different environments. We can conclude that sirtuin family plays an important role in

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maintaining the level of plasma glucose, but the detail mechanism needs further investigation.

Additionally, HDACs can also maintain the glucose homestasis through decreasing the utilization of glucose in the peripheral tissues. Deletion of HDAC5 in human muscle cells increases the uptake of glucose via enhancing the transcription of glucose transporter 4 gene, and glycogen catabolism by

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amplifying the related genes including HK II and glycogen phosphorylase[67].

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In conclusion, plasma glucose homeostasis is regulated by the balance between the production in liver and utilization in skeletal muscle and tissues. HDACs modulate the enzymes involved in glucose synthesis and catabolism through modifing the transcription factor and cofactor. Classical HDACs increase the production and decrease the utilization of glucose, while SIRT6 plays an

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ambiguous role in plasma glucose homeostasis.

3.1.2 HDACs have an effect on the blood lipid homeostasis PPARs and LXRs are transcription factors which belong to the ligand-activated nuclear receptor (NR)

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superfamily. They bind to the PPARE and LXRE respectively and then regulate the expression of genes which are involved in regulating the synthesis/utilization of glucose and lipids[68, 69]. Otherwise, the transcription regulators sterol regulatory element binding protein(SREBP)-1c and

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carbohydrate responsive element binding protein (ChREBP) regulated by LXR also involve in the lipogenesis in liver[70]. In addition, FoxO, AMPK and PGC-1α which involved in insulin signaling pathway may modulate the lipid synthesis and metabolism. HDACs maintain the level of plasma lipid through deacetylating transcription factors and NR related to lipid and glucose metabolism.

Fatty acid mobilization is the first step of fatty acid oxidation. SIRT1 promotes fatty acid mobilization and decreases fatty acid accumulation in adipocytes through repressing PPARγ activity[71]. Consistently, Xu et al [72] has found that the expression of genes (PPARγ, LPL, FAS, and CD36) involved in lipogenesis was increased obviously, while the expression of fatty acid

ACCEPTED MANUSCRIPT oxidation-related gene was reduced in SIRT+/- mice, supporting that SIRT1 may contribute to the fatty acid mobilization and decrease the lipogenesis. Adipose triglyceride lipase (ATGL) is regarded as the rate-limiting enzyme of lipolysis. SIRT1 upregulats the transcription of ATGL by deacetylating FoxO1, promoting the lipolysis[73]. In addition, Walker[74] has demonstrated that SIRT1 inhibits

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the lipogenesis and promotes fat oxidation through deacetylation of SREBP and decreases the SREBP target genes expression involve in lipid synthesis, suggesting that SIRT1 regulate the lipogenesis and cholesterol synthesis. Otherwise, Sita et al has summaried the vital role of SIRT6 in lipid metabolism[66]. Moreover, higher expression of Hdac3 in E3 rat liver might suppress the

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expression of LXR-α, CYP7A1 and PPAR-γ [75]. On the contrary, deletion of Hdac3 by siRNA technology can upregulate LXR-α, CYP7A1 and PPAR-γ expression[75], suggesting that HDAC3

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can suppress triglyceride and cholesterol synthesis, inducing hepatic steatosis. Interestingly, HDAC1/HDAC3 may inhibit lipogenesis in HepG2 cells via forming the C/EBP-δ/HDAC1/HDAC3 complex and repressing the transcription of PPARG2[76]. Apart from regulating of the biosynthesis of lipid and cholesterol, HDACs also has a vital effect on the lipid utilization in the peripheral tissues. In contrast, class I HDACis contribute to the oxidative metabolism and enhance insulin sensitivity,

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through increasing the Pgc-1α action and PPARγ/PGC-1α signaling in peripheral tissues [46], leading to the downregulation of plasma glucose and lipid. In conclusion, class I HDACs and SIRT6 decreased plasma lipid through inhibiting lipogenesis, and class I HDACis downregulate plasma

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lipid via promoting oxidative metabolism. Otherwise, SIRT1 affects the lipid homeostasis through

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modulating the all progression of lipid metabolism.

3.2 Role of HDACs in endothelial cells activation and dyfunction as well as correlative cytokine secretion

The vascular ECs, forming a monolayer in the innermost part of the blood vessel wall, show pivotal roles in maintaining the homeostasis of vessels generally via regulating its antagonism effect on inflammation, shear stress and oxidative stress[54, 77]. Endothelial dysfunction is considered to be a trigger for atherosclerosis due to its enhancive vascular permeability and EC migration as well as proliferation.

3.2.1 HDACs partake in endothelium function regulation-mediated by shear stress

ACCEPTED MANUSCRIPT Blood flow in arteries exposes underlying vascular endothelium to multifarious types of hemodynamic forces consisting of fluid shear stress, tensile stretch forces and hydrostatic pressure forces[78]. The straight part of the artery is exposed to steady laminar blood flow(s-flow) which plays a protect role in the development of atherosclerosis, while the regions of curvature and

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bifurcations are exposed to the disturbed blood flow(d-flow) which accelerate the progression of atherosclerosis. Abundance of evidence show that s-flow up-regulates the expression of protective genes and proteins via activating mitogen extracellular-signal-regulated kinase 5/extracellular signal-regulated kinase-5/KLF2 (MEK5/ERK5/KLF2) signaling pathway while d-flow activates

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various signals, including activator protein 1, NF-κB and protein kinase c zeta(PKCζ), to promote inflammation and atherogenesis[78]. However, we need more studies to understand the specific

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mechanism.

A great deal of evidence shows that s-flow stimulates class II HDAC phosphorylation and nuclear export in ECs, whereafter p-HDACs mediate the anti-inflammation function via up-regulating the expression of KLF2 and endothelial nitric oxide synthase (eNOS) [12, 15, 79]. On the contrary, the

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mechanism can be reversed under disturbed flow conditions leading to pathology. Otherwise, SIRT1 is reported to decrease the EC proliferation to a normal level under s-flow, performing a protective role in vascular homeostasis [80]. In conclusion, under d-flow condition, class II HDAC plays a

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pro-atherosclerosis role while SIRT1 can prevent the progression of atherosclerosis.

3.2.2 HDACs contribute to ECs dysfunction-induced by inflammation and oxidative stress

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Apart from the physical factors described above, chemical factors, such as oxidative stress, inflammation and NO bioavailability, are potential candidates responsible for vascular dysfunction and atherosclerosis. HDAC, especially SIRT1, has been proved to maintain endothelial function and resistant to atherosclerosis through counteracting redox states, anti-inflammation, and activating eNOS. NF-κB, p53, p66shc as well as eNOS are considered critical target proteins of SIRT1 in the resistance to endothelial dysfunction.

Inflammation appears to be the common potential cellular mechanism for the development of endothelial dysfunction, generally mediated by NF-κB p65 signal. NF-κB can up-regualte the

ACCEPTED MANUSCRIPT expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1) and adhesion molecules (ICAM-1 and VCAM-1) in ECs, contributing to the dysfunction of ECs [81, 82]. SIRT1 are found to intervene the transcriptional activation of NF-κB through suppressing RelA/p65 binding to naked DNA thus reduce the endothelial activation [82]. New study has found that inhibition of SIRT6 can increase

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transcriptional activity of NF-κB and the expressions of NF-κB target genes. Subsequently the inflammation molecules, such as IL-1β, IL-6, COX-2, MMP-2, MMP-9, adhesion molecules and angiogenic growth factors partake in inflammation, ECs dysfunction, vascular remodeling and angiogenesis[83]. It seems that SIRT1 and SIRT6 play an opposite role in ECs activity in response to

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the inflammation.

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Oxidative stress, generally performing excessive production of reactive oxygen species(ROS), plays a crucial role in atherosclerosis through inducing ECs dysfunction and inflammation[81]. There are four enzymatic systems predominantly responsible for the sources of ROS in the human vasculature, namely NADPH oxidases(NOX), enzymes of the mitochondrial, uncoupled NOS (especially eNOS), respiratory and xanthine oxidase[84]. A great deal of study has confirmed that the regulatory effect of

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HDAC on ROS production is generally via its deacetylation function. Pan-HDAC inhibition or knockdown of HDAC3 in HUVEC leads to the reduction of NOX4 transcription through hyperacetylation and prevention transcription factors binding to the NOX4 promoter [85]. In contrast,

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inhibition of SIRT1 upregulates p22phox and NOX4, the NADPH oxidase subunits, contributing to an increased vascular O2·–production, thus leads to endothelial dysfunction in the rat aorta [86]. SIRT1 seems to play an opposite role in regulating NOXs transcription and the function of ECs

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compared to classical HDACs.

As a redox enzyme, P66shc plays a critical role in mitochondrial ROS production. P66shc deficiency has been reported to be more resistant to ROS-mediated ECs dysfunction by high glucose and aging in mice [87, 88]. SIRT1 inhibits hyperglycemia-induced p66shc expression via binding to and modifying the promoter of p66shc, antagonizing the endothelial dysfunction under high glucose [89]. Recently, the study has demonstrated that SIRT1 significantly inhibits the hyperglycemia induced expression of the senescence associated markers, including p53, p21 and tissue plasminogen activator-1 (PAI-1), in aortas of mice. They also found decreased p66shc expression in aortas of

ACCEPTED MANUSCRIPT SIRT1-Tg diabetic mice, suggesting that SIRT1 can protect against vascular cell senescence in hyperglycemia partially through reducing the oxidative stress[90]. Interestingly, a new study demonstrates that SIRT3 deletion unexpectedly do not affect the atherosclerosis burden and vulnerability of plaque in LDLR-/- mice, in spite of increasing the systemic levels of oxidative

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stress[91].

Dysregulation of NO by endothelial eNOS is also involved in the pathophysiology of endothelial dysfunction. Numerous researches show that SIRT1 may protect against ECs dysfunction induced by

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cigarette smoking, oxidative stress and aging presumably through deacetylating eNOS and subsequently enhancing availability of NO[92, 93]. In addition, resveratrol, SIRT1 activator, can

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increase the SIRT1 expression and NO secretion against the pro-atherosclerotic effects of high glucose [94], which can be impaired after knockdown SIRT1 in HUVECS [94].

3.2.3The synthetic and secretory action of activated ECs are partially affected by HDACs ECs activation accompanying endothelial dysfunction is represented by the production and secretion

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of inflammation factors, such as TNF-α, interleukins, platelet-derived growth factors (PDGFs), adhesion molecules, E- and P- selection and MCP-1. MCP-1 plays a pathogenic role in atherosclerosis chiefly through attracting monocytes/macrophages infiltration. HDACi can enhance

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liberation of pro-atherosclerotic cytokines MCP-1 and IL-8 induced by oxLDL in ECs [38]. The PDGFs are a family of potent chemoattractants as well as mitogens for cells, including VSMCs and fibroblasts. HDAC1 are reported to mediate PDGF-D gene silence induced by IL-1β through

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forming an inhibitory complex with p65 and IRF-1 at the PDGF-D promoter [95]. HDAC1 and HDAC2 can also potentiate IL-1β induced PDGF-Rα expression, which is one of the subunit of PDGF-R, through dissociating from the PDGF-Rα promoter [45]. Furthermore, HDAC7 can regulate PDGF-B/PDGFR-β gene expression, partly contributing to modulation of ECs migration and angiogenesis [20]. VCAM-1, which

expresses on the activated ECs, can exasperate vascular

endothelial injury through promoting the activation and recruitment of monocytes. These activated monocytes then transmigrate into the vascular intima stimulated by chemokine factors such as MCP-1 and MCP-4 [96]. SIRT1 and SIRT6 can decrease the expression of VCAM-1 through interfering NF-κB signal in ECs [82, 83, 97]. On the contrary, HDAC3 promotes the VCAM-1

ACCEPTED MANUSCRIPT expression induced by TNF-α in ECs [39]. E-selectin, a Ca2+-dependent EC surface glycoprotein, contributes to recruitment of leukocytes and participates in atherosclerosis[98]. The evidence shows that SIRT1 can hinder the expression of E-selectin induced by high glucose in ECs [94]. In conclusion, the pro-inflammation molecules mentioned above promote leukocyte activation,

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recruitment, adhesion and migration into vascular intima, triggering the early lesion of atherosclerosis. In this process, HDAC1, HDAC2, SIRT1, SIRT6 and HDACi perform a protective role in antioxidant, anti-inflammation and maintenance of ECs function. On the contrary, HDAC3

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and HDAC7 may play an opposite role in the pathological process of atherosclerosis.

3.3 Role of HDACs in monocyte accumulation, migration and differentiation

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Monocytes infiltration into the subintima is achieved through leukocyte adhesion cascade, including capture, rolling, activation, adhesion, and transendothelial migration [99]. Once entering into the subintima, monocytes differentiate into macrophages triggered by special transcription factor and inflammatory factors[100]. Then the macrophages are activated under the inflammation condition and produce amount of pro-inflammatory factors[100]. On the other hand, a part of macrophages

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uptake of modified lipid in the subintima and turn into the foam cells[101]. Hence, the accumulation of monocyte and macrophage promote the process of the formation of plaque and progressive

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atherosclerosis.

Capture may decelerate the speed of monocytes from the blood and make them to roll on the activated endothelium[102]. Subsequently, integrin expresses on monocyte, such as integrins very

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late antigen 4 (VLA-4) and lymphocyte function-associated antigen 1 (LFA), and binds to the VCAM-1 and ICAM-1 expressed on ECs respectively and mediates the activation of rolling monocytes and firm adhesion to the endothelium[55]. Inoue et al[39] has elaborated that TSA effectively decreases the expression of VCAM-1 in endothelium, and attenuated monocyte adhesion to ECs, suggesting that TSA may prevent monocyte adhesion to ECs via reducing the VCAM-1 expression. Studies have been showed that the arrest of monocyte during rolling is rapidly triggered by chemokines and other chemoattractants expressed on ECs, including CCL2 (MCP-1), CX3CL1 and CD31[103]. Chemokines and chemoattractants mentioned above, which bind to the corresponding receptors on monocyte, may mediate the transmigration of monocytes from

ACCEPTED MANUSCRIPT endothelium into plaques [55, 99]. In the presence of modified LDL and inflammatory molecules like IFN-γ, TGF-β, TNF-α and IL-10, monocyte differentiates into M1 macrophage, function as a type of pro-inflammatory cells. Recently, a study has showed that short-chain fatty acids can inhibit the expression of chemokines(MCP-2), adhere molecules(ICAM-1 and VCAM-1 on ECs and LFA-1

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on monocytes) and proinflammatory cytokines (TNF-α and NO) and enhance the release of IL-10[44]. Controversially, Choi et al has found that TSA treatment increased TNF-α and VCAM-1 expression[104]. Recently, studies show that deletion of HDAC9 in macrophages enhances macrophages phenotype switch from the proinflammatory M1 to anti-inflammatory M2 states via

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PPARγ, decreasing the progression of AS[105, 106]. Meanwhile, accumulation of studies show that Hdac3 deletion promotes phenotype switch of macrophages and increases anti-inflammatory

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cytokines as well as reduces the expression of pro-inflammatory cytokines[107, 108], indicating that HDAC3 promotes the progression of AS. Otherwise, VPA can also promote the phenotype switch of macrophage, reducing the development of AS[109]. The knowledge mentioned above shows that both HDAC and HDACi may modulate the recruitment and differentiation of monocytes and affect the progression of AS. However, the mechanism how HDACi regulate the differentiation of

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monocyte remains to be further investigated. Furthermore, selective HDACi inhibits HDAC enzymes activation as well as the expression and effects of proinflammatory cytokines[110, 111].

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3.4 HDACs regulate macrophage foam cell and VSMC foam cell formation The formation as well as accumulation of lipid-laden foam cells in the subendothelial space of the vascular wall is the key step during the development of atherosclerosis. In traditional, foam cells

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derives from macrophages through scavenger receptors(SRs)-dependent uptake of modified lipoproteins[112], especially oxidized LDL, and scavenger receptor-independent uptake of native and modified LDL including phagocytosis and pinocytisis[113]. Recently, it has been reported that foam cells also derives from SMCs[114]. The imbalance between focal LDL and cholesterol aggregation and cholesterol efflux in macrophages and SMCs leads to the formation of the foam cells. Recently, some studies suggest that HDACi regulates the process of foam cells formation, but the mechanism remains unclear. We will exhibit the possible mechanism below.

3.4.1 Role of HDACs in LDL and cholesterol uptake and foam cell formation

ACCEPTED MANUSCRIPT Both the modified LDL, such as oxidized LDL, acetylated LDL and chemically modified LDL, and native LDL can invert macrophage into foam cells, which play a proatherosclerotic role during the progression[115]. Macrophages ingest lipoproteins by SRs, including SR-A, CD36 and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)[116]. Inhibit the expression of SRs enable to

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reduce LDL uptake by macrophages and decrease the development of AS notably[117]. It has reported that TSA significantly increased the expression of CD36 and SR-A on the macrophage, resulting to the increasing uptake of LDL and accelerating AS[104, 118]. It is suggested that TSA may has the role of promoting the formation of foam cells, thereby contributing to the process of

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atherosclerosis. LOX-1, also known as OLR-1, is a class E scavenger receptor, which is detected on ECs, monocytes/macrophages and SMCs [119, 120]. Schaeffer et al [121]showed that

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pro-inflammatory cytokines promote the expression of LOX-1 in macrophages, which substantially increased ox-LDL uptake by nidus macrophages, providing the specificity of LOX-1 to modulate ox-LDL uptake in macrophages. SIRT1 suppresses the expression of LOX-1 in macrophages via suppressing of the NF-κB signalling pathway by deacetylation of RelA/p65, following diminishes the uptake of oxLDL and foam cells formation, finally decreases atherogenesis[65, 122]. Moreover,

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in addition to the traditional mechanisms that macrophages uptake of excess lipid by SRs in the subendothelial space, it has been shown that minimally oxidized LDL can induce macropinocytosis in macrophages via a TLR4/spleen tyrosine kinase dependent mechanism, thereby promoting to the

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uptake of both native and oxLDL, leading to the progression of atherosclerosis[123]. It suggests that TLR4 signaling may involve the formation of foam cells. Meanwhile, we know that the HDACi SAHA attenuates inflammation in macrophages through increasing acetylation of HSP90 in the

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TLR4 pathway [124]. We speculate that HDACi may have an effect on the element related to the TLR4 signaling to produce cytokines, and then regulate the uptake of ox-LDL and foam cells formation. In addition, Yamaki et al[125] has also showed that resveratrol can inhibit oxLDL uptake into macrophages, which has a protective effect against arteriosclerosis. Meanwhile, Schober et al [126] has showed that inhibition of macrophage macrophage migration inhibitory factor(MIF), which production by macrophage and T-cell and have a crucial role in immune-mediated disease, leads to the reducing macrophage/foam cell content. TSA inhibits MIF expression through specific deacetylation of MIF [127]. We infer that HDACi may reduce foam cell formation via inhibiting cytokines related to atherosclerosis. Otherwise, resveratrol also suppresses the formation of foam cell

ACCEPTED MANUSCRIPT induced by LPS throughting downregulating MCP-1 expression via AMP-SIRT1-PPAR signaling pathway[128]. In conclusion, SIRT1 and SAHA suppress the formation of foam cells and the progression of AS, while TSA play the controversial role in the formation of foam cells.

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SMCs can also invert into foam cells by taking up native LDL. LDL receptor is the primary receptor for uptake of plasma-derived LDL cholesterol, resulting in the formation of foam cells[129]. Lim et al [130]has showed that elevation of CD36 expression facilitates SMC derived foam cell formation. And we hypothesis that HDAC may regulate SMC derived foam cell formation through modulating

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the expression of CD36 on SMCs. However, the mechanism about how HDACs effect on SMC

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derived foam cell formation is complex and needs to be further investigated.

3.4.2 HDACs regulate macrophage cholesterol reverse transport

As it mentioned previously, imbalance between lipid uptake and efflux in macrophage results in the foam cells formation, which is the vital step in the development of atherosclerosis. Therefore, remove excess cholesterol from macrophage is regarded as the most important step to prevent against

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the development of atherosclerosis [131]. It has been shown that both ATP-binding cassette (ABC) transporters and SR-B are involved in the cholesterol efflux, which is the first procedure of reverse cholesterol transport[132, 133]. ABCA1 and ABCG1 as well as SR-B1 mediate macrophage

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cholesterol efflux to lipid-poor apolipoproteins (apoA-I and apoE) and mature HDL, respectively[132]. Studies indicated that decreasing the level of ABCA1, ABCG1 and SR-B in macrophages promotes cholesterol accumulation and accelerates AS[134, 135]. Therefore, it may

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prevent the development of AS through repressing the expression of all the three membrane transporters on macrophage. However, the question about how to regulate the expression of ABCA1, ABCG1 and SR-BI needs to be further investigated.

Ku et al observes that mRNA levels of ABCA1 and ABCG1 were significantly increased in macrophage while incubated with TSA[135], promoting the macrophage cholesterol reverse transportation. The study has shown that PPARs/LXR pathway involves in promoting the expression of ABC transporters[136]. Overexpression of HDAC3 decreased PPARγ, LXR and their target genes transcription in hepatocyte, while treatment with TSA results in LXR and target genes increasing,

ACCEPTED MANUSCRIPT which lead us to speculate that HDAC may downregulate the transcriptional factors ABCA1 and ABCG1 related to cholesterol efflux through PPARs/LXR pathway. Similarly, SIRT1 promotes ABCA1 related cholesterol efflux via regulating LXR activition[65], slowing the formation of foam cells. Otherwise, HDAC9-deficiency in macrophages increases the transcription of ABCA1, ABCG1

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and PPARγ, promoting the cholesterol efflux in macrophages and suppressing the progression of AS[105, 106]. Meanwhile, investigators show that resveratrol promotes expression of ABCA1, ABCG1 and SR-B1 relying on PPARγ and adenosine 2A receptor pathways in human THP-1 and monocyte-derived macrophages[134]. Otherwise, SR-BI is homologue to CD36 and CLA-1

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(lysosomal integral membrane protein-II analogous-1) in human[137]. SR-BI/CLA-1 has a pivotal role in cholesterol reverse transport by mediating selective uptake of cholesteryl ester from

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peripheral tissues to the liver. It has been reported that the HDACi, TSA, SAHA and sodium butyrate, can increase both mRNA and protein level of CLA-1/SR-BI in macrophages, which plays a role of antiatherosclerotic in atherosclerosis [118].

3.5 HDACs contribute to VSMCs phenotype switch and fibrous cap formation

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VSMCs play a critical role in process of atherosclerosis through proliferation and migration from the media to the intima and subsequently formation of fibrous cap. SMCs undergo switch from the “contractile” phenotype to a “synthetic” phenotype in response to multiple stimuli within the

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atherosclerotic plaque, such as shear stress, lipids, ROS, cytokines and ECM [138, 139]. Those medial SMCs exhibit decreased expression of “contractile” associated proteins such as α-smooth muscle actin(SMα-actin) and smooth muscle myosin heavy chain(SMMHC), showing a greater

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proliferating capacity and a stronger synthetic ability for cytokines, ECM and proteinase[19, 138, 139]. And a great deal of evidence shows that HDACs modulate the phenotype switch of SMCs in the progression of atherosclerosis.

3.5.1 HDACs influence phenotype switch of VSMCs to a proliferation, migration and synthetic state Phenotype switch induces higher proliferation index in VSMCs contributing to vascular remodel[138]. The study has shown that class II HDACs promote the phenotype switch of SMCs through Ca2+ signals[140] and the progression of AS. In addition, the increasing expression of SIRT2

ACCEPTED MANUSCRIPT can lead to the proliferation and phenotype switch of SMCs, resulting the formation of atherosclerosis[141]. Cell proliferation accompanied by cycle progression can be promoted by cyclins(cyclin D1, cyclin E and cyclin A)[142], while inhibited by cyclin-dependent kinases(CDKs) inhibitors, such as p21 and p27[143]. Interesting, Boda et al has summarized the ambiguous role of

expression of

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TSA in VSMCs proliferation [8]. Otherwise, although TSA and butyrate both can upregulate the proliferation-assosiated genes (cyclin D3, p21Cip1 and p15INK4B) and

downregulate the expression of cdk4, cdk6, and cdk2, resulting the histone H3 posttranslational modifications, butyrate display an opposite role compared with TSA for its contrary influence on

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cyclin D1 expression[40]. In addition, it has indicated that HDAC4 promote the PDGF-BB-induced VSMC proliferation and migration via activating the p38MAPK/HSP27 signal, ultimately may result

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in neointimal hyperplasia in vivo[144]. Those reports above indicate that classic HDACs may facilitate the proliferation of VSMC in atherosclerosis plaque. In contrast, SIRT1 and SIRT3 inhibit VSMCs proliferation, migration, neointimal formation and vascular remodeling[145, 146], repressing the development of AS. Above all, classical HDAC and SIRT2 may promote the atherosclerosis process through enhancing VSMCs proliferation and neointimal formation, while the

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SIRT1 and SIRT3 may have an anti-atherosclerosis function via the opposite effect on VSMCs.

Except proliferation, SMC phenotype switch can also increase migration in response to multiple

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stimuli. The study has demonstrated that strain inhibits VSMCs migration, which accompanied with decreasing HDAC3, 4 and increasing HDAC7, indicating a regulation effect of HDACs in VSMCs

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migration [147]. On the contrary, SIRT1 is found to inhibit the VSMCs migration [148].

3.5.2 HDACs play a role in regulation synthetic function of VSMCs and fibrous cap formation ECM mainly consists of collagen, fibrin, vitronectin, as well as proteoglycans and they play an important role in atherosclerosis and determine the integrity of plaque[149]. The synthetic phenotype VSMCs produce more ECM, resulting in the maturation of the atheromatous plaque. Accumulation of evidence has demonstrated that HDACs play an important role in the production of ECM. Yo shihide et al shows that HDAC1 overexpression promotes the inhibitory effect of Fli1 on COL1A2 via histone deacetylation, leading to the decreasing expression of collagen[150]. Consistently, HDAC2 forms a complex with regulatory factor for X-box (RFX5)-Sin3B-Gpa on the collagen site

ACCEPTED MANUSCRIPT to down-regulate collagen type I transcription in the COL1A2 repression induced by IFN-γ[151]. Recent study identified that HDAC3 deletion enhances collagen deposition in plaque which is not due to increasing number of VSMCs. Furthermore, increased TGF-β secreted by HDACdel macrophages

can

promote

collagen

production[107].

Otherwise,

HDACs

suppress

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proly1-4-hydroxylase (P4Hα1) transcription, which is the key enzyme associated with collagen modification, promoting the progression of AS[152]. On the contrary, SIRT1 promotes the production of collagen 1 partially through modulating RFX5 activity [153]. In conclusion, HDACs

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decrease the production of collagen while SIRT1 has the opposite role.

Apart of ECMs, VSMCs in atherosclerosis lesion can also perform a stronger capacity of synthetic

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pro-inflammation molecule such as adhesion molecules(VCAM-1 and ICAM-1), IL-1, IL-6, MCP-1, PDGF and TGF-β[138, 154], which can aggravate ECs dysfunction and activation as well as retain macrophage in plaque[138]. Treatment HDAC4 with RNA inhibitor can decrease the expression of VCAM-1 [155], suggesting that HDAC4 may promote the secretion of inflammation molecule,

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promoting the development of AS.

3.5.3 HDACs have to do with VSMCs calcification, aging and apoptosis Cellular senescence means a lower level of proliferation and apoptosis. VSMCs in the plaque may

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undergo senescence, which can interfer plaque repair and subsequently destabilize the plaque through their pro-inflammation effects and incapacity of proliferation[156]. SIRT1 can protect VSMCs from senescence induced by inorganic phosphate and aging through p53/p21 pathway and

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prevent senescence-related calcification induced by hyperphosphorylatemia[157, 158]. Calcification in AS is associated with plaque burden and plaque stability, in which, Wnt/β-catenin pathway play a vital role[159]. HDAC7 are reported to interact with β-catenin on a C-terminal site and retain it in the cytoplasm, which may influence the calcification process[160]. In cultured human aortic SMCs, TSA can promote Pi-induced calcification through increase the expression of ALP[161] in contrast with SIRT1.

VSMCs are the major source of ECM responsible for the stability of fibrous cap. Therefore, VSMCs apoptosis is a crucial process weakening plaque vulnerability and ultimately causing plaque rupture.

ACCEPTED MANUSCRIPT Boda et al summarized the promotion role of HDAC1 in the signal of oxidative-stress-induced SMC apoptosis[8]. VSMCs always undergo DNA damage and DNA repair in atherosclerosis. Isabelle et al found that SIRT1 deficiency cause reduced DNA repair, elevated DNA damage markers and correlative apoptosis in VSMCs[162]. In addition, overexpression of SIRT1 are reported to promote

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VSMCs express FasL in vitro and vivo, which can trigger apoptosis during injury[163]. It indicates that SIRT1 plays an equivocal role in atherosclerosis, while HDAC1 promotes SMCs apoptosis and the progression of AS. In conclusion, HDAC1 and HDAC7 can extensively participate in the proliferation, migration, differentiation, synthesis, calcification, aging and apoptosis progress of

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vascular smooth muscle cells, closely connecting with the development of atherosclerosis, while

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SIRT1 have a dual role during AS.

3.6 Role of HDACs in plaque disruption and thrombosis

Atherosclerosis is a continuous process, involving several pathological course: inflammation, endothelial activation and dysfunction, monocyte recruitment, foam cell formation, SMCs proliferation and migration and matrix deposition, vascular remodeling and consequently

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atherosclerosis plaque rupture, which is the main cause of occlusive thrombus resulting in myocardial infarction[164]. As we have discussed the widely role of HDACs in multiple stages of atherosclerosis process, here we will review the HDACs effects on the ultimate stage: plaque

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disruption and thrombosis.

3.6.1 How can HDACs impact on atheromatous plaque disruption?

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The stability of plaque depends on the integrity of the fibrous cap, which is influenced by inflammation within vessel wall [164]. The cytokines (IFN-γ, TNF-α and MCP-1) secreted by activated leukocytes decrease the matrix synthesis and increase the degradation of matrix[164, 165]. As mentioned above, HDACs involve in the process of VSMCs apoptosis which can cut down the quantity of VSMCs, demonstrating a partial role of HDACs influent the vulnerability of fibrous cap. Moreover, investigators demonstrate that HDACi, especially TSA, can potently suppress IFN-γ secreted by Th1[166], preventing the development of atherosclerosis. The major MHC II transactivator(CIITA) expressed on SMCs can be enhanced by IFN-γ[167], in return, represses the type I collagen expression and induce the class II MHC gene expression on SMCs[168]. Kong et al

ACCEPTED MANUSCRIPT has found that HDAC2 can down-regulate the expression of CIITA in a deacetylation-dependent manner in VSMCs as well as macrophages and promote the degradation of CIITA by proteasome [42]. Therefore, HDAC2 may weaken the IFN-γ mediated collagen repression and MHC II activation via downregulating the expression and function of CIITA [42]. These documents indicate that

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HDACs may have a complex role in stabilizing the atheromatous plaque through regulating the collagen synthesis.

The depletion of matrix components in the fibrous cap through increasing matrix breakdown

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primarily mediated by zinc-required neutral MMPs [165] can weaken the fibrous cap, predisposing it to rupture. MMPs are expressed in plaques mainly by macrophages and foam cells[165]. They can be

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activated by IFN-γ, CD-40L and other cytokines(IL-1, TNF-α and MCP-1)[164]. HDACs are involved in the inflammation process, such as expression of IFN-γ[166] and CD-40[169] , and cytokines expression(mentioned before). Also, HDACs may partially influence the activation and expression of MMPs on macrophages. Megan et al reported that HDAC1 can be recruited by IFN-β to the proximal AP-1 site and repress MMP-9 transcription[170]. Metacept-1, a novel HDAC

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inhibitor, inhibits the expression of MMP-2 in human VSMCs in vitro [50]. Similarly, SIRT1 can suppress the expression of MMP-9 on monocytes/macrophages[171] and VSMCs[148]. In addition, investigators have reported that SIRT1 [172] and HDAC3 [173] can upregulate the tissue inhibitor of

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MMP3 and 1 respectively, preventing the degradation of matrix.

Above all, HDACs seem to influence the synthesis and breakdown of matrix in fibrous cap, the

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imbalance of which can induce matrix depletion in vulnerable plaque, ultimately leading to plaque rupture under extrinsic trigger, such as mechanical and hemodynamic forces[174].

3.6.2 HDACs and the thrombosis after plaque rupture The rupture of plaque often causes thrombosis, leading to a series of serious clinical syndrome. The balance between anti-thrombotic molecules (NO and t-PA) and pro-coagulant molecules(TF and PAI-1) is a vital factor in the development of atheromatous plaque[175]. NO release from the endothelium related to platelet activation and thrombin formation can delay the thrombosis production[176]. As mentioned before, HDACs has found to influence NO synthesis in ECs,

ACCEPTED MANUSCRIPT suggesting that HDAC may partially affect the thrombotic process. Fibrin clot formation in early stage is dissolved by plasminogen activated by t-PA, while PAI-1 can block this process[176]. With platelet activation, the balance between t-PA and PAI-1 will be broken, then accelerating the thrombosis[175]. Researches have shown that the non-specific HDACi (TSA, VPA and butyrate)[43,

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49, 177] and MS275(class I HDAC inhibitor)[47] can stimulate expression of t-PA in ECs through regulating the acetylation state of t-PA histones[47]. In addition, PAI-1 can inhibit ECM degradation through restraining the effect of plasminogen activators. A recent study found that the HDACi CBHA may decrease the expression of PAI-1 in human pleural mesothelial cell line[178]. Consistently,

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on PAI-1 expression in ECs needs further investigation.

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SIRT6 inhibition can increase the expression of PAI-1 in HUVECs[83]. However, the role of HDACs

Lipid-rich plaques are more prone to thrombosis due to their high content of TF mainly coming from macrophages. Exposure of TFs to the blood flow can boost the thrombotic response[165]. TSA and four other HDACis (apicidin, MS275, sodium butyrate and VPA) are all found to inhibit TF activity and protein level in HUVECs stimulated by TNF-α and LPS [51]. Inhibition of SIRT1 is also

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reported to enhance TF expression and activity through activating NF-κB/p65 signal in ECs [179]. It seems that the classical HDAC and SIRT1 play an opposite role in regulating TF expression in ECs.

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Above all, HDACs may take part in the production of thrombosis after plaque rupture partially through influence the expression and activity of molecules related to thrombosis such as NO, t-PA,

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PAI-1 and TF.

4. Summary

The formation of atherosclerotic plaque is indeed a complex biological process, including the constant stimulation of risk factors, endothelial cell injury and activation, the migration and activation of monocyte cells, the formation of foam cells and fibrous plaque. Once plaque rupture and thrombogenesis, a variety of cardiovascular events occurred. In this review, we summarized the role of HDACs in the occurrence of atherosclerosis. Firstly, i) HDACs may regulate the most of risk factors for atherosclerosis. HDACs regulate the progress of AS via modulating the limiting-enzymes

ACCEPTED MANUSCRIPT activity involved in the production and utilization of glucose and lipid. In addition, ii) HDACs have an influence on the ECs activation and dyfunction. Classical HDACs and SIRT6 promote the secretion of pro-inflammation molecules such as MCP-1, PDGF, IL-1 and selectin, and all of them participate in the activation, recruitment, adhesion and transendothelial migration of monocytes and

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lymphocytes as well as the migration of VSMCs. On the contrary, SIRT1 protects the ECs against those risk factors and inhibit the secretion of correlative proflammatory cytokines. Moreover, iii) the formation of foam cells derived from macrophages and VSMCs decreased due to the expression of CD36, SR-A and LOX-1, which is related to the uptake of nature and modified LDL by macrophages

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and VSMCs, was suppressed by HDACi (TSA, SAHA and sodium butyrate) and SIRT1. Meanwhile, the molecules including PPARγ, LXR, ABCA1, ABCG1 and SR-BI/CLA-1 involved in cholesterol

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reverse transport was decreased by HDAC 3 and HDAC9 but increased by classical HDACi and SIRT1. We can conclude that classical HDACi and SIRT1 may decrease foam cells formation via inhibiting the uptake of lipid and enhancing the transport of cholesterol in foam cells. Subsequently, iv) HDAC1 and HDAC7 promote VSMCs phonotype switch to a proliferation, migration and synthetic states and promote VSMCs calcification, apoptosis, leading to facilitate the formation of

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fibroatheroma while SIRT1 plays a dual role in VSMCs. Over time, according to the studies, we know that v) TSA, HDAC2, HDAC3, and SIRT1 promote the stability of plaque via increasing the matrix synthesis and decreasing the degradation of matrix, while HDAC2 play the opposite role.

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Along with the rupture of plaque, HDACis and SIRT6 inhibit the thrombosis through modulating the balance between anti-thrombotic and pro-coagulant. On the contrary, SIRT1 may promote the thrombosis via the opposite role. In general, classical HDACs and class III HDACs have a pivotal

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role in the process of atherosclerosis, and both of them will be the new and promising target for the treatment of atherosclerosis in the future.

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ACCEPTED MANUSCRIPT Highlight 1、HDACs regulate the progress of AS via modulating the limiting-enzymes activity involved in the production and utilization of glucose and lipid.

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2、Classical HDACs and SIRT6 promote the secretion of pro-inflammation molecules such as MCP-1, PDGF, IL-1 and selectin, and all of them participate in the activation, recruitment, adhesion and transendothelial migration of monocytes and lymphocytes as well as the migration of VSMCs. On the contrary, SIRT1 protects the ECs against

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those risk factors and inhibit the secretion of correlative proflammatory cytokines.

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3、Classical HDACi and SIRT1 may decrease foam cells formation via inhibiting the uptake of lipid and enhancing the transport of cholesterol in foam cells.

4、HDAC1 and HDAC7 promote VSMCs phonotype switch to a proliferation, migration and synthetic states and promote VSMCs calcification, apoptosis, leading to

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facilitate the formation of fibroatheroma, while SIRT1 plays a dual role in VSMCs.

5、TSA, HDAC2, HDAC3, and SIRT1 promote the stability of plaque via increasing

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the matrix synthesis and decreasing the degradation of matrix, while HDAC2 play the opposite role. Along with the rupture of plaque, HDACis and SIRT6 inhibit the through

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thrombosis

modulating

the

balance

between

anti-thrombotic

and

pro-coagulant. On the contrary, SIRT1 may promote the thrombosis via the opposite role.