Recent genome-wide association studies revealed that

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ecent genome-wide association studies revealed that a genetic variant in the loci corresponding to histone deacetylase 9 (HDAC9) is associated with large vessel stroke.¹ Moreover, HDAC9 is also a common genetic variant that share genetic susceptibility to ischemic stroke and coronary artery disease.² HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries.³ The molecular mechanisms how HDAC9 region might increase the risk of atherosclerosis is unknown.

See accompanying editorial on page 1798 There are 18 mammalian HDACs, which fall into 4 classes on the basis of their structural and biochemical characteristics.⁴ HDAC9 is expressed in heart, pancreatic islets, neuron, spi- nal cord, teeth, smooth and skeletal muscles, T lymphocytes, endothelium, and adipose tissues.⁴ One of the best character- ized mechanisms of action of HDAC9 is its ability to partner with MEF-2 (myocyte enhancer-binding factor) and repress MEF-2 activity.⁴ HDAC9 deacetylates Lys¹¹⁶ on ataxia telangiectasia group D-complementing (also called TRIM29) protein, prevents ataxia telangiectasia group D-complementing from binding p53, and consequently leads to activation of p53-inducible genes.⁵ Macrophages consist of ≥2 subgroups M1 and M2. Whereas M1 macrophages are proinflammatory and have a central role in atherosclerosis development and

plaque rupture, M2 macrophages are associated with response to anti-inflammatory reactions, tissue remodeling, fibrosis, and atherosclerosis regression.⁶ Recent studies demonstrate that among HDACs, HDAC3 and HDAC7 have been identified to play a key role in inflammatory gene expression program and alternative activation of macrophages.^7–9 However, the role of HDAC9 in these processes in macrophages is unknown.

Understanding whether HDAC9 plays a role in macrophage development and the pathogenesis of atherosclerosis will provide the rationale for the development of selective HDAC9 inhibitors that target macrophages for the treatment and pre- vention of atherosclerosis because of its significance with GWAS (Genome-Wide Association Study) study.

In the present study, we report that HDAC9 is expressed in macrophages and its role in cholesterol homeostasis and inflammation in macrophages and on atherosclerosis development. Our experiments revealed that systemic and bone marrow (BM) cell HDAC9 deficiency in LDLr^−/− (low density lipoprotein receptor) mice reduced atherosclerosis. HDAC9 deletion resulted upregulation of lipid homeostatic genes, downregulation of inflammatory genes, and polarization of macrophages toward M2-like phenotype via increased accumulation of acetylated H3 at the promoters of ABCA1 (ATP-binding cassette transporter), ABCG1, and PPAR-γ (peroxisome proliferator-activated receptor) in macrophages. We conclude that macrophage HDAC9 upregulation is

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.114.303393

Objective—Recent genome-wide association studies revealed that a genetic variant in the loci corresponding to histone deacetylase 9 (HDAC9) is associated with large vessel stroke. HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries. The molecular mechanisms how HDAC9 might increase atherosclerosis is not clear.

Approach and Results—In this study, we show that systemic and bone marrow cell deletion of HDAC9 decreased atherosclerosis in LDLr^−/− (low density lipoprotein receptor) mice with minimal effect on plasma lipid concentrations.

HDAC9 deletion resulted upregulation of lipid homeostatic genes, downregulation of inflammatory genes, and polarization toward an M2 phenotype via increased accumulation of total acetylated H3 and H3K9 at the promoters of ABCA1 (ATP-binding cassette transporter), ABCG1, and PPAR-γ (peroxisome proliferator-activated receptor) in macrophages.

Conclusions—We conclude that macrophage HDAC9 upregulation is atherogenic via suppression of cholesterol efflux and generation of alternatively activated macrophages in atherosclerosis. (Arterioscler Thromb Vasc Biol. 2014;34:1871-1879.)

Key Words: atherosclerosis ◼ epigenomics ◼ histones ◼ histone deacetylases ◼ macrophages

Received on: February 3, 2014; final version accepted on: June 25, 2014.

From the Departments of Internal Medicine/Rheumatology and Immunology (Q.C., N.M.), Pathology/Lipid Sciences (S.R., R.S.C., J.S.P.), and Biochemistry (J.S.P.), Wake Forest School of Medicine, Winston-Salem, NC; and Departments of Internal Medicine and Physiology, The University of Texas Southwestern Medical Center, Dallas (J.J.R.).

The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.303393/-/DC1.

Correspondence to Nilamadhab Mishra, MD, Department of Internal Medicine/Rheumatology and Immunology, Wake Forest School of Medicine, Medical Center Blvd, Winston Salem, NC 27157. E-mail nmishra@wakehealth.edu

Histone Deacetylase 9 Represses Cholesterol Efflux and Alternatively Activated Macrophages

in Atherosclerosis Development

Qiang Cao, Shunxing Rong, Joyce J. Repa, Richard St. Clair, John S. Parks, Nilamadhab Mishra

Basic Sciences

Downloaded from http://ahajournals.org by on September 23, 2021

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atherogenic via suppression of cholesterol efflux and generation of alternatively macrophages in atherosclerosis.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Systemic Deletion of HDAC9 Reduces

Atherosclerosis in Male and Female LDLr^−/− Mice Initially, we used animal model of atherosclerosis to under- stand the underlying molecular pathways of HDAC9 in atherogenesis. To examine whether systemic deficiency of HDAC9 inhibits atherogenesis, male and female LDLr^−/− (single knockout [SKO]) and LDLr^−/− HDAC9^−/− (double knockout [DKO]) mice were generated and fed an atherogenic diet (Table I in the online-only Data Supplement) for 16 weeks.

Female and male SKO and DKO mice had similar body weight and plasma lipids concentrations on a chow diet (Figure I in the online-only Data Supplement).

Plasma triglyceride and free cholesterol levels were decreased in female DKO mice compared with SKO female mice (Figure 1A); however, no difference was observed in male mice (Figure IIA in the online-only Data Supplement).

Total cholesterol, cholesterol ester, and phospholipid concentrations were similar for SKO versus DKO mice (Figure 1A; Figure IIA in the online-only Data Supplement). Very- low-density lipoprotein cholesterol was decreased, whereas low-density lipoprotein and high-density lipoprotein (HDL) were increased in DKO female but not male DKO mice compared with SKO (Figure 1B and 1C; Figure IIB and IIC in the online-only Data Supplement).

Compared with SKO mice, DKO mice had visibly decreased lesions in the unstained aortic arch in situ (Figure 1D; Figure IID in the online-only Data Supplement) by en face analysis (Figure 1E and 1F; Figure IIE and IIF in the online-only Data Supplement) and by morphometric analysis of Oil Red O–stained aortic root sections (Figure 1G and 1H; Figure IIG and IIH in the online-only Data Supplement).

Smooth muscle cells synthesize the interstitial collagens, which are responsible for resistance to rupture of atherosclerotic plaques. Pathological studies in human ruptured atherosclerotic plaques have shown a thin fibrous cap poor in smooth muscle cells and collagen.^10,11 To determine whether HDAC9 deficiency promotes plaque stability, we measured smooth muscle cell and collagen content in aortic root atherosclerotic lesions by immunohistochemistry. There was a significant increase in smooth muscle cells and an increased trend in collagen deposition in aortic atherosclerotic plaques in DKO compared with SKO mice (Figure IIIA–IIID in the online-only Data Supplement).

We determined the effect of HDAC9 deletion in genes expression levels in lesions that play key roles in atherogenesis in vivo. Real- time polymerase chain reaction analysis was done in pooled sam- ples of aortic roots from the SKO and DKO mice. Aortic roots of DKO mice had increased mRNA expression of ABCA1, ABCG1, and arginase-1, decreased expression of interleukin-1β and MCP-1 (monocyte chemoattractant protein), but no change in expression of CD36, scavenger receptor class A (SRA), tumor necrosis factor-α, and inducible nitric oxide synthase (iNOS) compared with SKO mice (Figure IV in the online-only Data Supplement).

Deletion of HDAC9 in BM Cells Inhibits the Formation of Atherosclerosis

HDAC9 is expressed in multiple tissues including endothelial cells, T lymphocytes, and adipose tissues.^4,12 To test the hypothesis whether HDAC9 expressed on hematopoietic cells (eg, monocytes/

MΦ, lymphocytes, and platelets) versus endothelial cells or all the cell types those promote the development of atherosclerosis lesions, we transplanted male SKO and DKO BM into female LDLr^−/−

recipient mice (Figure V in the online-only Data Supplement). Five weeks after transplantation, the recipient mice were fed an atherogenic diet for 16 weeks and euthanized. Plasma total cholesterol, free cholesterol, and cholesterol ester levels were similar, but triglyceride concentrations were reduced in LDLr^−/− mice reconsti- tuted with DKO versus SKO BM (Figure 2A). Very-low-density lipoprotein cholesterol was reduced, whereas HDL was increased in mice receiving DKO BM (Figure 2B and 2C). LDLr^−/− mice transplanted with DKO versus SKO BM had decreased visible atherosclerotic plaques in the aortic arch (Figure 2D), aortic surface lesion area (Figure 2E and 2F), and aorta root intimal area and lipid staining (Figure 2G–2I). Although BM transplantation experiments suggested that the possible deletion of macrophage HDAC9 was sufficient to decrease atherosclerosis, a beneficial role for other BM cell types in this process cannot be excluded.

HDAC9 Expression Is Increased During Macrophage Differentiation

To determine the role of HDAC9 biological and pathological functions in macrophages, we performed following experiments. We first determined HDAC9 expression in human THP-1 (acute monocytic leukemia cell) monocytes during in vitro differentiation into macrophages and mouse BM-derived macrophages. HDAC9 mRNA was most highly expressed in differentiated macrophages (Figure 3A and 3B). HDAC9 is alternatively spliced to generate different proteins. Among these, 2 major isoforms (HDAC9—a form containing HDAC domain and a truncated form HDRP (HDAC-related protein)/MITR (MEF-2 interacting transcription repressor protein) without HDAC domain but acquire deacetylase activity by recruitment of HDAC1 or HDAC3) expressed in tissue-specific manner.^13,14 These major isoforms were expressed in mouse and human macrophages (Figure 3C and 3D). HDAC9 mRNA expression is further increased in response to oxidized low-density lipoprotein, acetylated low-density lipoprotein, and toll-like receptor (TLR) (LTA [lipoteichoic acid], LPS [lipopolysaccharide], flagellin but not CpG [cytosine–phosphate–guanine] DNA) signals and thioglycollate-elicited peritoneal macrophages (TEPMs; Figure VI in the online-only Data Supplement).

Nonstandard Abbreviations and Acronyms BM bone marrow

HDAC9 histone deacetylase 9 HDL high-density lipoprotein

TEPM thioglycollate-elicited peritoneal macrophage

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HDAC9 Deficiency in Macrophages Results Increased Cholesterol Efflux via Increased Expression of ABCA1 and ABCG1 via Accumulation of Acetylated Histone at H3 and H4 at Promoters

Cholesterol efflux capacity from macrophages via ABCA1 and ABCG1 has a strong inverse relationship with atherosclerosis

development.¹⁵ To determine the role of macrophages HDAC9 in cholesterol efflux pathway, we performed following experiments. HDAC9-deficient macrophages demonstrated increased expression of the cholesterol efflux genes ABCA1 and ABCG1 with acetylated low-density lipoprotein and liver X receptor (LXR) ligand stimulation compared with wild-type mice (both HDAC9-deficient and wild-type mice were on C57/

Figure 1. Systemic histone deacetylase 9 (HDAC9) deficiency reduces atherosclerosis in female LDLr^−/− mice. Plasma lipid and atherosclerosis measurements were made after 16 weeks of atherogenic diet consumption by SKO (LDLr^−/−) and DKO (LDLr^−/−HDAC9 ^−/−) mice.

A, Plasma lipid concentrations. B and C, Cholesterol distribution in very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) by fast protein liquid chromatography analysis of pooled plasma from the terminal bleeds. D, Repre- sentative examples of light microscopic images taken of gross dissected aortas in situ. Atherosclerotic lesions are visualized as white areas, denoted by arrows, inside the arteries. Representative en face image (E) and surface lesion quantification (F) of aortas. G and H, Representative cross-sectional image and quantification of aortic root stained with Oil Red O. Data are shown as the mean±SD. *P<0.05;

**P<0.01.

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Bl6 back ground fed on chow diet) in vitro (Figure VIIA and VIIB in the online-only Data Supplement). Apolipoprotein A1– and HDL-mediated cholesterol efflux was increased in HDAC9-deficient versus wild-type macrophages (Figure VIIC and VIID in the online-only Data Supplement). ACAT

(acyl-coenzyme A:cholesterol acyltransferase) inhibition further increased apolipoprotein A1- but not HDL-mediated cholesterol efflux in HDAC9-deficient (Figure VIIE and VIIF in the online-only Data Supplement). To determine whether cholesterol efflux genes were upregulated in macrophages Figure 2. Histone deacetylase 9 deficiency in hematopoietic cells reduces atherosclerosis in LDLr^−/− mice. Plasma lipid and atherosclerosis measurements were made after 16 weeks of atherogenic diet consumption. Irradiated female LDLr^−/− mice were transplanted with bone marrow cells isolated from male SKO (white bar) and DKO (black bar) mice. A, Plasma lipid concentrations. B and C, Cholesterol distribution in very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) fractions by fast protein liquid chromatography analysis of pooled plasma from the terminal bleeds. D, Representative examples of light microscopic images taken of gross dissected aortas in situ. Atherosclerotic lesions are visualized as white areas, denoted by arrows. Representative en face image (E), quantification of en face surface lesion area (F), cross-sectional image of aortic root stained with Oil Red O (G), intimal area (H), and Oil Red O positive area (I). For (A) and (C), genotypes of the donor cells are indicated below the figures. Data are shown as the mean±SD. *P<0.05; **P<0.01.

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from atherogenic diet-fed mice, TEPMs were isolated from SKO and DKO mice fed an atherogenic diet for 16 weeks.

TEPMs from DKO mice had significantly less cholesterol ester compared with foam cells from SKO mice (Figure 4A), consistent with the finding of decreased atherosclerosis in DKO mice. Expression of ABCA1 and ABCG1 in DKO macrophages was increased compared with SKO macrophages (Figure 4B–4D). HDAC9 regulates gene expression via multiple mechanisms, including protein–protein interaction and histone and nonhistone protein acetylation, but these pathways are not necessarily mutually exclusive.^5,16,17 Western blot analyses revealed no global changes in histone acetylation in HDAC9-deficient macrophages (Figure VIII in the online- only Data Supplement). Control of inducible gene expression is dependent on signal-induced transcriptional elongation.

This process is facilitated by deposition of histone acetylation mark associated with transcriptional activation at the promoters.¹⁸ Using stable isotope labeling in combination with mass spectrometry analysis in splenocytes from knockout and MRL (Murphy Roths Large)/lprHDAC9^+/+ (wild-type) mice, we had previously demonstrated that lysine residues H3K9 and H3K18 were hyperacetylated (≈5-fold) in splenocytes from HDAC9-deficient mice.¹⁷ To investigate the consequences of HDAC9 deletion in macrophages and these histone acetylation marks, we performed chromatin immunoprecipitation (ChIP) assay using total H3, H4, site specific H3K9ac, and H3K18 ab. Quantitative ChIP assays demonstrated increased

levels of total H3, H4, and H3K9 acetylation but not H3K18 acetylation at the promoters of ABCA1 and ABCG1 in HDAC9-deficient macrophages compared with controls fed on atherogenic diet (Figure 4E and 4F). We also performed ChIP assays in BM-derived macrophages from these 2 groups of mice loaded with acetylated low-density lipoprotein in vitro. Similar results were observed in ChIP assay as above (Figure IXB and IXC in the online-only Data Supplement).

There was no increase in deposition of these acetylation marks in SRA promoter where transcription was unchanged between HDAC9 versus wild-type macrophages (Figure IXD in the online-only Data Supplement).

HDAC9 Deficiency in Macrophages Promotes M2 Polarization and Decreases M1 Inflammatory Genes via Upregulation of PPAR-γ via Chromatin Remodeling

To determine the role of HDAC9 in generation of M1 and M2 macrophages, we performed following experiments. Ex vivo stimulation with LPS demonstrated that DKO TEPMs produced significantly less proinflammatory cytokines (tumor necrosis factor-α, IFN-γ (interferon), MCP-1, interleukin-6, iNOS, and interleukin-1β) compared with SKO macrophages (Figure 5A and 5B). TEPMs from HDAC9-deficient mice had a significant increase in subset of genes characteristics of alternatively activated, M2-like, macrophages (MGL-1 [macrophage galactose N-acetyl-galactosamine specific lectin],

Figure 3. Histone deacetylase 9 (HDAC9) is overexpressed during macrophage differentiation. A, Real-time polymerase chain reaction (PCR) analysis of class IIa HDAC mRNAs in THP-1 monocyte cells and differentiated macrophages. THP-1 cells were cultured with PMA (phorbol 12-myristate 13-acetate; 20 ng/mL) for 4 days to differentiate to macrophages.

Data were normalized using the house- keeping gene GAPDH. mRNA expression was calculated relative to undifferentiated THP-1 cells (controls). B, HDAC9 mRNA level is increased during differentiation of mouse bone marrow–derived cells to macrophages. Bone marrow–derived cells from C57BL/6J mice were cultured with LCM (L929-cell–conditioned medium) media, and cells were isolated at given time points after differentiation. Data were normalized using the housekeep- ing gene GAPDH. mRNA expression was calculated relative to differentiated macrophages at day 1 (control).

C, Reverse transcription (RT) PCR analysis demonstrates 2 major HDAC9 isoforms (HDAC9 and HDRP) in mouse macrophages. 1 indicates mouse bone marrow cells; 2, macrophages differentiated from mouse bone marrow cells;

and 3, thioglycollate-elicited peritoneal macrophages from an LDLr^−/− mouse. D, RT PCR analysis demonstrates 2 major HDAC9 isoforms (HDAC9 and HDRP) in human macrophages. 1 indicates THP-1 cells; 2, macrophages differentiated from THP-1 cells; 3, human monocytes; and 4, macrophages differentiated from human monocytes.

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arginase-1, and interleukin-10) in the basal states compared with wild-type mice fed on atherogenic diet (Figure 5C).

Compared with wild-type mice macrophages, the macrophages from HDAC9-deficient mice fed on chow diet had a significant increase in basal, interleukin-4, and PPAR-γ agonists (rosiglitazone) stimulated expression of CD206, MGL-1, MGL-2, arginase-1, Chi3l3, and interleukin-10 mRNA (Figure X in the online-only Data Supplement). The upregulation of M2 and downregulation of M1 genes in HDAC9-deficient macrophages are probably through PPAR-γ pathway.¹⁹ Expression of PPAR-γ in DKO macrophages was increased compared with SKO macrophages (Figure 5D). Quantitative ChIP assays demonstrated increased levels of total H3, H4, H3K9 but not H3K18acetylation at the promoters of PPAR-γ (Figure 5E) but not at the arginase-1 promoter (Figure XI in the online-only Data Supplement).

These in vitro and ex vivo experiments in macrophages support the concept that increased HDAC9 expression in macrophages is atherogenic via suppression of cholesterol efflux and generation of alternatively activated macrophages via decreased accumulation of histone acetylation at ABCA1, ABCG1, and PPAR γ promoters. The consequences of HDAC9 deletion in macrophages on a genome-wide scale in response to different toll-like receptor agonists or infections need further investigations.

Discussion

In this study, we report several new findings. Among class IIa HDACs, HDAC9 is most abundantly expressed during macrophage differentiation. Systemic and macrophage HDAC9 deficiency reduces atherosclerosis development in different

sites in LDLr^−/− mice fed an atherogenic diet. The molecular mechanisms behind the decreased atherosclerosis in HDAC9- deficient mice are likely multifactorial, including increased macrophage cholesterol efflux by ABCA1 and ABCG1 and phenotypic switching of macrophages from a proinflammatory M1 to a less inflammatory M2 state via PPAR-γ.

We have not observed cardiac hypertrophy or polydactyly in DKO mice in contrast to previously reported studies in HDAC9 knockout mice.^20,21 The difference between the phenotypes observed between ours and others are most likely because of difference in background (C57Bl6 versus mixed 129 and C57Bl6) and age of the mice studied. Interestingly, in contrast to Chatterjee et al¹² study, the double knockout (LDLr^−/−HDAC9^−/−) mice have increased body, liver, and adipose tissue weight compared with single knockout (LDLr^−/−) mice on the atherogenic diet; these phenotypes were neither observed in chow-fed mice nor during atherogenic diet feeding of LDLr^−/− mice transplanted with HDAC9 knockout versus wild-type BM (data not shown).

The difference between the phenotypes observed between theirs and ours are most likely because of difference in background (C57Bl6 versus LDLr) and diet. The mechanisms behind the phenotypes observed with systemic deficiency of HDAC9 in atherogenic diet-fed LDLr^−/− mice are currently unknown.

Recent genome-wide association studies revealed that a genetic variant in the loci corresponding HDAC9 is associated with large vessel stroke.¹ Moreover, HDAC9 is also a common genetic variant that share genetic susceptibility to ischemic stroke and coronary artery disease. HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries.³ HDAC9 knockout mice are protected from adipose tissue dysfunction and systemic metabolic disease during high fat feeding.¹²

Figure 4. Histone deacetylase 9 regulates cholesterol homeostasis via alteration of ABCA1 and ABCG1 promoter architecture. A, Quantification of total cholesterol (TC), cholesterol ester (CE), and free cholesterol (FC) in thioglycollate-elicit peritoneal macrophages isolated from SKO and DKO mice fed with 16-week atherogenic diet. Thioglycollate-elicit peritoneal macrophages isolated from SKO and DKO mice fed with 16-week atherogenic diet.

ABCA1 and ABCG1 mRNA level (B), protein level (C), and Western blot quantification (D). E and F, Quantitative ChIP assays were performed on the promoter regions of ABCA1 and ABCG1 using anti-Ac-H3, Ac-H4, Ac-H3K9, Ac-H3K18, or IgG (control) for immunoprecipitation.

Nonimmunoprecipitated sample served as an input control. All assays performed in triplicate and are representative of 3 independent experiments. Data are pre- sented as mean±SD. *P<0.05; **P<0.01.

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Moreover, HDAC9 deficiency increases regulatory T cells and decreases effector T cells.^17,22 Finally, HDAC9 promotes angiogenesis by targeting the antiangiogenic microRNA-17 to -92 cluster in endothelial cells.²³ These studies consistent with our results to support the concept of inhibition of HDAC9 may have beneficial effect in atherosclerosis development.

Lund et al²⁴ demonstrated that epigenetic changes, such as DNA methylation polymorphisms, preceded any histological sign of atherosclerosis in apolipoprotein E–deficient mice and human THP-1 cells cultured with mixtures of different propor- tions of lipoproteins (very-low-density lipoprotein, low-density

lipoprotein, and HDL) decreased histone H4 acetylation.²⁴ Studies in endothelial cells have demonstrated opposing roles of HDACs in endothelial function.²⁵ HDAC3 expression protected endothelial integrity, and HDAC3 knockdown increased atherosclerosis in aortic isografts of apolipoprotein E mice.²⁶ Laminar blood flow inhibited HDAC5 expression, resulting in decreased binding of monocytes to endothelial cells.²⁷ HDAC2 and HDAC5 played an important role in smooth muscle cell differentiation.^27,28 Moreover, short interfering RNA-mediated knockdown of HDAC1, 2, or 3, and pharmacological inhibition of HDAC by scriptaid prevented smooth muscle cell Figure 5. Macrophage histone deacetylase 9 deficiency reduces LPS-mediated inflammatory cytokines secretion and increases M2 polarization through changing PPAR-γ1 promoter architecture. Thioglycollate-elicit SKO and DKO peritoneal macrophages were isolated from hypercholesterolemic mice and stimulated with LPS for 24 hours, M1 marks were detected at mRNA level (A) and protein level (B), and M2 markers were checked at mRNA level without LPS stimulation (C). D, PPAR-γ1 mRNA level in thioglycollate-elicit peritoneal macrophages isolated from SKO and DKO mice fed with 16-week atherogenic diet. E, Quantitative ChIP assays were performed on the promoter regions of PPAR-γ1 using anti-Ac-H3, Ac-H4, Ac-H3K9, Ac-H3K18 antibodies, or IgG (control) for immunoprecipitation. Nonim- munoprecipitated sample served as an input control. All assays performed in triplicate and are representative of 3 independent experi- ments. Data are presented as mean±SD. *P<0.05; **P<0.01.

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proliferation and neointima formation.²⁹ HDAC3 and HDAC7 promote TLR-4 dependent proinflammatory gene expression, whereas HDAC3 promotes macrophage alternate activation.^7-9 Finally, pan-HDAC inhibitors have both anti- and proinflammatory effects on macrophages and prevent smooth muscle cells migration in vitro.³⁰Importantly, endogenous HDAC inhibitor β-hydroxybutyrate protects against oxidative stress by upregulating oxidative stress resistance genes Foxo3a and MT2.³¹ Based on these opposing roles of HDACs in different cell types that are involved in atherosclerosis development, it is not surprising that trichostatin A, which inhibits both class I and class II HDACs, increased atherosclerosis in LDLr^−/−

mice.³² It is also possible that the high concentration of dietary cholesterol (1.25%) overwhelms a beneficial effect of trichostatin A.

The nuclear receptors (PPARs, LXRs, and RXRs [retinoid X receptors]) not only influence lipid metabolism at the systemic level but also regulate lipid homeostasis and inflammation in macrophages.³³ There is considerable interest in the development of selective PPAR and LXR agonists for the treatment of atherosclerosis.^33–35 Active repression of LXR target genes, such as ABCA1 and ABCG1, is mediated, in part, by interaction with corepressors nuclear receptor corepressor (NCoR) and SMART (silencing mediator of retinoic acid and thyroid hormone receptor) to repress basal expression of target promoters via recruitment of HDAC3.³⁶ Intriguingly, HDAC9 interacts and colocalizes in vivo with several transcriptional repressors or corepressor including NCoR.¹³ HDAC9, which represses these nuclear receptors by forming multiprotein complexes, antago- nizes the antiatherogenic effect of natural and synthetic nuclear receptor ligands. HDAC9 deletion relieves inhibition and results in increased transcription of ABCA1, ABCG1, and PPAR-γ in macrophages. Activation of the genes in macrophages decreased foam cell formation and inflammation via increased cholesterol efflux and phenotypic switching of macrophages from a proinflammatory M1 to anti-inflammatory M2 phenotype, ultimately with decreased atherosclerosis phenotype in LDLr^−/− mice. Our findings are consistent with recent study by Li etal³⁷ that macrophage specific deletion of NCoR results anti-inflammatory phenotype and generation of M2 macrophages via increase in histone acetylation at certain promoters.

Although, it is not clear from our study whether enzyme activity of HDAC9 or protein itself is required for atherogenesis, it is tempting to speculate that HDAC9 inhibition in macrophages may provide a therapeutic benefit in atherosclerosis in humans.HDAC9 is recently identified a common genetic variant that share genetic susceptibility to large artery stroke and coronary artery disease.¹ Moreover, HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries.³ Our study provides a mechanistic clue how HDAC9 gene variant may cause atherosclerosis. We envision that this study may provide the foundation for development of HDAC9 isoform–specific inhibitors that can selectively be delivered to macrophages via chemical conjugates technology may provide therapeutic benefit in individuals with coronary artery disease and stroke with genetically defined HDAC9 population.

Acknowledgments

We thank Eric N Olson and Rhonda Bassel-Dubey (UT Southwestern Medical Center, Dallas, TX) for providing the HDAC9^−/− mice. We acknowledge the help and assistance of Kailin Yan (decease), Lin Jia, Kristen Delany, Xuewei Zhu Xin Bi, and Karen Klein (Office of Research, Wake Forest School of Medicine).

Sources of Funding

This work was supported by National Institutes of Health grants RO1-HL 084592 (N. Mishra), RO1-HL084592 S1 (N. Mishra), P01 HL049373 (J.S. Parks), and R01 HL-094525 (J.S. Parks).

Disclosures

None.

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Recently, genome-wide association studies revealed that a genetic variant in the loci corresponding to histone deacetylase 9 (HDAC9) is associated with large vessel stroke. Moreover, HDAC9 expression was identified in human atherosclerotic plaques in different arteries. How- ever, the molecular mechanism of HDAC9 on atherogenesis is unknown. In this study, we demonstrate that HDAC9 in macrophage is atherogenic. Systemic and bone marrow cell deletion of HDAC9 decreases atherosclerosis in LDLr^−/− mice. Macrophages lacking HDAC9 suppress foam cell formation by increasing cholesterol efflux via increased expression of macrophage ABCA1 and ABCG1 gene expression. Moreover, macrophages lacking HDAC9 produce less inflammatory mediators and polarize toward M2-like macrophages. These important finding points toward development of epigenetic therapy for atherosclerosis by using small molecule inhibitors that targets HDAC9 isoform in macrophages.