Cell‑specific histone modifications in atherosclerosis (Review).

During the progression of atherosclerosis, oxidized low-density lipoprotein (oxLDL) initiates endothelial dysfunction and increases the expression of vascular cell adhesion molecule-1 (VCAM-1), P-selectin, and chemokine monocyte chemoattractant protein-1 (MCP-1) that recruits monocytes, T-lymphocytes and platelets. Monocytes are stimulated by monocyte-colony stimulating factor (M-CSF), and pro-inflammatory chemokines and cytokines, which differentiate them into macrophages. Macrophages then engulf oxLDL by receptor-mediated phagocytosis and transform themselves into lipid-laden foam cells. Accumulated foam cells and immune cells in the vessel wall develop an identifiable pathological condition called, the ‘fatty streak’. This first symptom represents asymptomatic and non-stenotic plaque, and is the early sign for the development of atherosclerosis. Subsequently, the smooth muscle cells (SMCs) triggered by cytokines, chemokines, and growth factors, gain the ability to proliferate and migrate to develop a fibrous cap covering the atherosclerotic core. In this advanced pathological stage of atherosclerosis, matrix degradation, cytotoxic T-cells and cholesterol crystals accumulate to form a lipid-rich necrotic core. In the very late stage of atherosclerosis, apoptosis in the SMCs of the fibrous cap occurs which causes plaque instability and thrombosis. As a result, distal embolism occurs which leads to blockage in the cerebral arteries. Thus, atherosclerosis in coronary arteries and in carotid arteries results in coronary heart disease and ischemia in the brain which in severe conditions may cause significant morbidity and mortality ( 1 ). During the progression of atherosclerosis from ‘fatty streak’ to atheroma development, changes in the gene expression in different cell types have been reported. Below, we describe the de novo epigenetic changes, specifically histone modifications which were reported in different cell types that are involved in the development of atherosclerosis.

Several histone modifications regulating the off-target drug effects have been reported which indicates their additional benefits. Both Hydralazine and Osalazine which are used to treat hypertension can be repurposed for treating neoplasia as their treatment can restore the functions of tumor suppressor genes. As a histone-deacetylase inhibitor, Romidepsin when used in endometriosis, efficiently induced apoptosis in the epithelial cells of endometrium. However, off-targets effects on histone modification can potentially exert negative effects in the cells. Similarly, the histone deacetylase inhibitor, valproic acid which is prescribed for epilepsy treatment, was identified as a causative factor for spina bifida in the growing embryos ( 59 ). Thus, for future considerations, specific mechanisms of histone modifications should be determined in patients for prognosis to avoid adverse drug effects. During the early developmental stages of the embryo, unmethylated CpG islands were found to induce the expression of DNMT3A and DNMT3B. At the same time, DNMT3L interacts with the chromatin and targets H3K4. G9a was found to methylate H3K9 and recruit heterochromatin protein 1, DNMT3A and DNMT3B during stem cell production ( 60 ). Existence of such epigenetic cross connections may also lead to de novo gene regulations which can be critically involved in the disease progression. Another key epigenetic mechanism that was found to intercept histone modifications is the expression of long non-coding RNAs. Under hypoxic conditions, upregulated expression of HDAC3 was reported to inhibit the expression of long non-coding RNA ‘lncRNA-LET’ ( 61 ). During the phenotype switch in VSMCs, the long non-coding RNA ‘taurine up-regulated gene-1’ (TUG1), was found to form a hetero-complex with EZH2 to methylate α-actin. As a result, the levels of α-actin were found decreased, and simultaneously F-actin was elevated. This imbalance was presumed to be a causative factor for the phenotype switch in the VSMCs which change from contractile to synthetic phenotype ( 62 ). In lung cancer, the long non-coding RNA, MALAT1, was reported to upregulate the expression of histone demethylase JMJD1A, which was known to promote cell migration and invasion ( 63 ). Several factors such as hyperlipidemia, hypertension, diabetes and also diet play an important role in the development and progression of atherosclerosis. The onset of de novo molecular mechanisms which are discussed above indicates epigenetic mechanisms to be critically involved in the pathogenesis. It was reported that high-fat diet decreases ATP citrate-lyase levels as well as acetyl-CoA and/or the ratio of acetyl-CoA:CoA in white adipose tissue and pancreas, leading to inhibition of H3K23 acetylation in mice fed with high-fat diet ( 64 ). Dyslipidemia can cause monocyte priming such as increase in the adhesion of monocytes. Chemotaxis in the monocytes was promoted due to reduction in the H3K27 acetylation in non-human primates ( 65 ). H3K4 methylation of lysine-specific demethylase-1 (LSD1), H3K6 methylation of dehydrogenase 2 (HSD11B2), H3K79 methylation on epithelial sodium channel subunit α (SCNN1A) were also shown to be associated with arterial hypertension ( 66 ). Panobinostat is a non-specific HDAC inhibitor that has been shown to have nanomolar potency, and can effectively inhibit Class I, II and IV HDACs. It induces apoptosis primarily by activating caspases and inducing PARP cleavage. In a phaseI/II clinical trial on 15 HIV positive patients, treatment with an HDAC inhibitor, panobinostat, was found to lower cardiovascular biomarkers such as CRP, LDL receptor, MCP1, E-Selectin, and HMGB1 in addition to lowering several inflammatory markers ( 67 ). Trichostatin A, is a nonspecific inhibitor of Class I and Class II HDACs. Although it has been identified to prevent inflammatory markers such as IL-1b and IL-6, and have significant anti-tumor properties, it appears to have a more deleterious role in atherosclerosis. Treatment with Trichostatin A was found to promote proatherogenic markers such as TNF-α, SRA, CD36, eNOS and VCAM-1 ( 68 ). However, Trichostatin A was found to inhibit p21 mediated proliferation of VSMCs ( 69 ). These reports suggest that Trichostatin A may have a role in preventing neointimal hyperplasia or maintaining plaque stability. Trichostatin A treatment upregulates the expression of Arginase 2 enzyme which promotes eNOS expression, inflammation and cell proliferation during progression of atherosclerosis. Earlier, it was shown that HDAC2 directly binds to the Arginase 2 promoter and inhibits its expression. In the same study, treatment with the HDAC2 specific inhibitor ‘mocetinostat’, was shown to enhance oxLDL induced endothelial dysfunction ( 70 ). Though certain HDAC inhibitors have shown promising results in treating cancer and other diseases, the same had a different role in atherosclerosis as identified above. However, there is no detailed information available yet on the clinical trials using the HDAC inhibitors for the treatment of atherosclerosis in humans. Additional studies emphasizing the specific role of each HDAC and their effects on different cell types during the progression of atherosclerosis are needed. The ultimate outcome of such studies should substantiate the need for extensive evaluation of HDAC inhibitors through clinical trials which are currently lacking. Improvements in sequencing strategies, mass spectrometry, Chromatin immunoprecipitation, microarray and other novel techniques have contributed to substantial progress in health science research. Increasing applications of these technological advancements would have positive effects in the development of treatment strategies that target de novo epigenetic changes in different disease conditions including atherosclerosis. Time-resolved NMR spectroscopy was innovatively developed to characterize asymmetric histone PTMs ( 71 ). Chromatin immunoprecipitation in conjunction with sequencing methodologies are typically used for mapping histone modifications. Through inclusion of micrococcal nuclease (MNase) digestion and barcoding, disease specific signatures can be identified at the single-cell level. Moreover, single-cell DamID can also be performed for genome-wide analysis of histone modifications using a cell line expressing Escherichia coli deoxyadenosine methylase (Dam) and combining it with specific histone readers or modifiers ( 72 ).

Histone acetylation is regulated by two key enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDAC). By transferring an acetyl group to lysine side chains of histone proteins, HATs weaken the binding between histone and DNA. On the contrary, HDACs remove the acetyl group on the histones to increase the interaction between histone and DNA. HATs are further categorized into two groups, type-A and type-B ( 38 ). HDACs regulate transcription and cell cycle progression by deacetylating lysine residues located on the N-terminal of the core histone proteins (H2A, H2B, H3 and H4). HDACs are further classified into four Classes I, II, III and IV. Classes I, II and IV represent ‘classical’ HDACs which include 11 genes, and in Class III, 7 genes are included which are named as sirtuins ( 39 ). Based on the type of modification occurring on the histones, gene expression is either favored or inhibited. This characteristic feature of the PTMs on histones and their role in transcriptional regulation is developmental and tissue specific. However, such histone modifications, to regulate gene expression, are frequently observed in many pathological conditions. Such modifications occurring in atherosclerosis are discussed below. In the heart tissues of New Zealand white rabbits that were treated with high cholesterol diet and atorvastatin, the mRNA levels of ACE2 was reported to be elevated when compared with control high cholesterol diet. Notably, atorvastatin was identified to promote histone H3 acetylation (H3-Ac) at the promoter region of ACE2 ( 40 ). High plasma levels of Hcy was identified not only to increase abnormal DNA methylation in the cells but also elevates the levels of N-methyl-d-aspartate receptor-1 (NMDAR1), DNA (cytosine-5)-methyltransferase-1 (DNMT1) and MMP-9, via acetylation on H3K9 and by inhibiting the expression of HDAC1. These regulatory proteins were identified as possible targets that have high potential to cause heart failure as reported in cardiomyocytes ( 41 ). The ‘Silent mating type information regulator two homologue one’ (SIRT1), which belongs to class III HDAC, requires Nicotinamide adenine dinucleotide (NAD+) as a cofactor for its functional activity. The role of SIRT1 has been implicated in many cellular processes such as in cell cycle progression, inflammation, DNA damage, apoptosis, autophagy, aging as well as in metabolic disease ( 42 ). SIRT1 mediates transfer of the acetyl group from proteins to the targeting co-substrate by removing the nicotinamide ribosyl bond of NAD+. In the peripheral blood mononuclear cells (PBMCs) of patients with T2DM, increased transcription of HDAC3 was reported. SIRT1, which was identified as a protective marker of cardiovascular diseases, was found reduced in PBMCS of these T2DM patients. In addition, the pro-inflammatory markers TNF-α, IL-6, MCP-1, IL1-β, NFκB, TLR2, and TLR4 were also reported to be upregulated in the plasma of the T2DM patients. Further, DBC1 (deleted in breast cancer 1), which represses HDAC3, was also reported to be decreased in the PBMCs of patients with T2DM ( 43 ). Myeloid HDAC3 deficiency was reported to have plaque stabilization effect by increasing the deposition of collagen, HDAC3 expression can potentially induce macrophage polarization, favoring the formation M2 phenotypes. These M2 macrophages by secreting TGFβ1 were shown to initiate VSMCs to secrete excess collagen ( 44 , 45 ) Under hypercholesterolemic conditions, the orphan nuclear receptor NR4A1, which can effectively reduce IL-6 and MCP-1 expression, was found increased due to the recruitment of p300 acetyltransferase. As a result, HDAC7 (histone deacetylase 7) was found repressed which contributes to the expression of NR4A1 in human monocytes, THP-1 and U937 ( 46 ). The histone deacetylase 9, HDAC9, was reported to be associated with MMP12 expression in the pro-inflammatory macrophages M2 and M4, in the advanced human plaques. However, the presence of both these types of macrophages could not be correlated with genes regulating plaque stabilization or thrombosis ( 47 ). HDAC9 deficient cells were identified to express increased levels of ABCA1, ABCG1, and PPAR-γ. This was identified to be mediated through upregulation of H3 and H4 acetylation, preventing the cholesterol efflux. Moreover, HDAC9 knockout macrophages were identified to be secreting lower levels of pro-inflammatory cytokines and showed prominent expression of M2 phenotype markers, and at the same time, it simultaneously decreased the expression of M1 markers ( 48 , 49 ). In Table IV , we summarized acetylation events on different residues of histones in the above monocytes and macrophages occurring during atherosclerosis. The same was represented in Fig. 2 . The protein ‘General Control Non-repressible 5’ (GCN5)-related N-acetyltransferase (GNAT), mediates transfer of the acyl group from acyl coenzyme A (acyl-CoA) to different proteins substrates which are involved in transcription, cell proliferation, antioxidant functions, antibiotic resistance and detoxification ( 50 ). The p300/CREB-binding protein (CBP) which is a HAT, acetylates non-histone proteins and controls transcription and DNA repair mechanisms in the cells ( 51 ). When compared between the cardiac mesenchymal stem cells from normoglycemic subjects, cardiac mesenchymal stem cells from type 2 diabetic patients had decreased levels of H3K9Ac and H3K14Ac acetylations, which are caused primarily due to the reduced activity of GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor. Also its isoform, GCN5a, decreased proliferation and differentiation of MSCs. Moreover treatment with the HAT activator, pentadecylidenemalonate 1b (SPV106) not only recovered the levels of H3K9Ac and H3K14Ac acetylation, but also decreased CpG hypermethylation in the genomic DNA, suggesting the involvement of two different epigenetic mechanisms regulating at the same time in these cells ( 52 ).

As cell specific histone modifications have been identified during atherosclerosis, targeting the enzymes which regulate these de novo changes can be a promising strategy for the treatment of atherosclerosis. This strategy is favored by two important features. Frist, like all epigenetic therapies, targeting histone modification alone do not affect the genetic component in the cells. Second, advances in the structural biology greatly assists the researchers to develop unique compounds that can specifically target the desired histone modification, such as the DNMT inhibitor (2′-deoxy-5-azacytidine, DAC) which targets H3K9 dimethylation, and the HDAC inhibitor TSA, which targets H3K27 trimethylation ( 53 ). Since any given histone modification is not unique for a specific disease and as they can be observed in different pathologies, drugs targeting a specific histone modification can be repurposed for other disease conditions. In addition, use of these specific histone targeting drugs as combination therapy with other therapeutic drugs appears to be more promising. The selective inhibitor, 3-deazaneplanocin A (DZNep) prevents methylation at lysine 27 on histone H3 (H3K27me3) and lysine 20 on histone H4 (H4K20me3) is currently used in the treatment of cancer ( 54 ). Upregulation of Lysine methyltransferases (KMTs) plays an important role in the differentiation of monocytes into immature dendritic cells (iDCs). Two of the know drugs that inhibit histone methylation, BIX-01294 which is KMT1c inhibitor, and DZNep, prevents global KMT activity were found to inhibit monocytes differentiating into iDCs ( 55 ). HDAC1 expression was identified to decrease acetylation of H3K9ac which induces accumulation of total cholesterol, free cholesterol, and triglycerides in foam cells, as seen in the aorta of ApoE-/-mice, which was abrogated by upregulation of the microRNA miR-34a ( 56 ). The compound 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE) isolated from the royal jelly was found to acetylate histone H3 and H4 at the proximal promoter region of EC-SOD (extracellular superoxide dismutase) in THP-1 cells, which is considered as a potential target for the treatment of atherosclerosis ( 57 ). Treatment with histone deacetylase inhibitor, valproic acid, was shown to promote the release of endogenous t-PA in males with vascular disease ( 58 ). Several HDAC inhibitors are used in the current standard of care. The known synthetic HDAC inhibitors which inhibit Class III HDACs include; dihydrocoumarin, naphthopyranone, 2-hydroxynaphaldehydes and other sirtuin-inhibiting agents. Class I, II, and IV HDAC inhibitors were grouped as hydroxamates such as trichostatin A (TSA), vorinostat (suberoylanilide hydroxamic acid; SAHA), belinostat (PXD101), panobinostat (LBH589), LAQ824, peptide inhibitors such as cyclic tetrapeptides (trapoxin B) and depsipeptides, benzamides (entinostat (MS-275), mocetinostat (MGCD0103), CI994, aliphatic acid components (phenylbutyrate, valproic acid), and electrophilic ketones ( 51 ). Some of the plant polyphenols such as curcumin (diferuloylmethane) and resveratrol were identified as naturally available HDAC inhibitors.