Bcl-6 and NF-kappaB cistromes mediate opposing regulation of the innate immune response.

In the macrophage, toll-like receptors (TLRs) are key sensors that trigger signaling cascades to activate inflammatory programs via the NF-κB gene network. However, the genomic network targeted by TLR/NF-κB activation and the molecular basis by which it is restrained to terminate activation and re-establish quiescence is poorly understood. Here, using chromatin immunoprecipitation sequencing (ChIP-seq), we define the NF-κB cistrome, which is comprised of 31,070 cis-acting binding sites responsive to lipopolysaccharide (LPS)-induced signaling. In addition, we demonstrate that the transcriptional repressor B-cell lymphoma 6 (Bcl-6) regulates nearly a third of the Tlr4-regulated transcriptome, and that 90% of the Bcl-6 cistrome is collapsed following Tlr4 activation. Bcl-6-deficient macrophages are acutely hypersensitive to LPS and, using comparative ChIP-seq analyses, we found that the Bcl-6 and NF-κB cistromes intersect, within nucleosomal distance, at nearly half of Bcl-6-binding sites in stimulated macrophages to promote opposing epigenetic modifications of the local chromatin. These results reveal a genomic strategy for controlling the innate immune response in which repressive and inductive cistromes establish a dynamic balance between macrophage quiescence and activation via epigenetically marked cis-regulatory elements.

Macrophage polarization phenotype regulates adiponectin receptor expression and adiponectin anti-inflammatory response.

Adiponectin (APN), a pleiotropic adipokine that exerts anti-inflammatory, antidiabetic, and antiatherogenic effects through its receptors (AdipoRs), AdipoR1 and AdipoR2, is an important therapeutic target. Factors regulating AdipoR expression in monocyte/macrophages are poorly understood, and the significance of polarized macrophage activation in controlling AdipoR expression and the APN-mediated inflammatory response has not been investigated. The aim of this study was to investigate whether the macrophage polarization phenotype controls the AdipoR expression and APN-mediated inflammatory response. With the use of mouse bone marrow and peritoneal macrophages, we demonstrate that classical activation (M1) of macrophages suppressed (40-60% of control) AdipoR expression, whereas alternative activation (M2) preserved it. Remarkably, the macrophage polarization phenotypes produced contrasting inflammatory responses to APN (EC50 5 µg/ml). In M1 macrophages, APN induced proinflammatory cytokines, TNF-α, IL-6, and IL-12 (>10-fold of control) and AdipoR levels. In contrast, in M2 macrophages, APN induced the anti-inflammatory cytokine IL-10 without altering AdipoR expression. Furthermore, M1 macrophages adapt to a cytokine environment by reversing AdipoR expression. APN induced AdipoR mRNA and protein expression by up-regulating liver X receptor-α (LXRα) in macrophages. These results provide the first evidence that macrophage polarization is a key determinant regulating AdipoR expression and differential APN-mediated macrophage inflammatory responses, which can profoundly influence their pathogenic role in inflammatory and metabolic disorders.

The macrophage LBP gene is an LXR target that promotes macrophage survival and atherosclerosis.

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP(-/-) recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis.

Bone marrow NR4A expression is not a dominant factor in the development of atherosclerosis or macrophage polarization in mice.

The formation of the atherosclerotic lesion is a complex process influenced by an array of inflammatory and lipid metabolism pathways. We previously demonstrated that NR4A nuclear receptors are highly induced in macrophages in response to inflammatory stimuli and modulate the expression of genes linked to inflammation in vitro. Here we used mouse genetic models to assess the impact of NR4A expression on atherosclerosis development and macrophage polarization. Transplantation of wild-type, Nur77⁻/⁻, or Nor1⁻/⁻ null hematopoetic precursors into LDL receptor (LDLR)⁻/⁻ recipient mice led to comparable development of atherosclerotic lesions after high-cholesterol diet. We also observed comparable induction of genes linked to M1 and M2 responses in wild-type and Nur77-null macrophages in response to lipopolysaccharides and interleukin (IL)-4, respectively. In contrast, activation of the nuclear receptor liver X receptor (LXR) strongly suppressed M1 responses, and ablation of signal transductor and activator of transcription 6 (STAT6) strongly suppressed M2 responses. Recent studies have suggested that alterations in levels of Ly6C(lo) monocytes may be a contributor to inflammation and atherosclerosis. In our study, loss of Nur77, but not Nor1, was associated with decreased abundance of Ly6C(lo) monocytes, but this change was not correlated with atherosclerotic lesion development. Collectively, our results suggest that alterations in the Ly6C(lo) monocyte population and bone marrow NR4A expression do not play dominant roles in macrophage polarization or the development of atherosclerosis in mice.

Small RNA sequencing reveals microRNAs that modulate angiotensin II effects in vascular smooth muscle cells.

Angiotensin II (Ang II)-mediated vascular smooth muscle cell dysfunction plays a critical role in cardiovascular diseases. However, the role of microRNAs (miRNAs) in this process is unclear. We used small RNA deep sequencing to profile Ang II-regulated miRNAs in rat vascular smooth muscle cells (VSMC) and evaluated their role in VSMC dysfunction. Sequencing results revealed several Ang II-responsive miRNAs, and bioinformatics analysis showed that their predicted targets can modulate biological processes relevant to cardiovascular diseases. Further studies with the most highly induced miR-132 and miR-212 cluster (miR-132/212) showed time- and dose-dependent up-regulation of miR-132/212 by Ang II through the Ang II Type 1 receptor. We identified phosphatase and tensin homolog (PTEN) as a novel target of miR-132 and demonstrated that miR-132 induces monocyte chemoattractant protein-1 at least in part via PTEN repression in rat VSMC. Moreover, miR-132 overexpression enhanced cyclic AMP-response element-binding protein (CREB) phosphorylation via RASA1 (p120 Ras GTPase-activating protein 1) down-regulation, whereas miR-132 inhibition attenuated Ang II-induced CREB activation. Furthermore, miR-132 up-regulation by Ang II required CREB activation, demonstrating a positive feedback loop. Notably, aortas from Ang II-infused mice displayed similar up-regulation of miR-132/212 and monocyte chemoattractant protein-1, supporting in vivo relevance. In addition, microarray analysis and reverse transcriptase-quantitative PCR validation revealed additional novel miR-132 targets among Ang II-down-regulated genes implicated in cell cycle, motility, and cardiovascular functions. These results suggest that miR132/212 can serve as a novel cellular node to fine-tune and amplify Ang II actions in VSMC.

LXRα is uniquely required for maximal reverse cholesterol transport and atheroprotection in ApoE-deficient mice.

The liver X receptor (LXR) signaling pathway is an important modulator of atherosclerosis, but the relative importance of the two LXRs in atheroprotection is incompletely understood. We show here that LXRα, the dominant LXR isotype expressed in liver, plays a particularly important role in whole-body sterol homeostasis. In the context of the ApoE(-/-) background, deletion of LXRα, but not LXRß, led to prominent increases in atherosclerosis and peripheral cholesterol accumulation. However, combined loss of LXRα and LXRß on the ApoE(-/-) background led to an even more severe cholesterol accumulation phenotype compared to LXRα(-/-)ApoE(-/-) mice, indicating that LXRß does contribute to reverse cholesterol transport (RCT) but that this contribution is quantitatively less important than that of LXRα. Unexpectedly, macrophages did not appear to underlie the differential phenotype of LXRα(-/-)ApoE(-/-) and LXRß(-/-)ApoE(-/-) mice, as in vitro assays revealed no difference in the efficiency of cholesterol efflux from isolated macrophages. By contrast, in vivo assays of RCT using exogenously labeled macrophages revealed a marked defect in fecal sterol efflux in LXRα(-/-)ApoE(-/-) mice. Mechanistically, this defect was linked to a specific requirement for LXRα(-/-) in the expression of hepatic LXR target genes involved in sterol transport and metabolism. These studies reveal a previously unrecognized requirement for hepatic LXRα for optimal reverse cholesterol transport in mice.

Age-accelerated atherosclerosis correlates with failure to upregulate antioxidant genes.

Excess food intake leads to obesity and diabetes, both of which are well-known independent risk factors for atherosclerosis, and both of which are growing epidemics in an aging population. We hypothesized that aging enhances the metabolic and vascular effects of high fat diet (HFD) and therefore examined the effect of age on atherosclerosis and insulin resistance in lipoprotein receptor knockout (LDLR(-/-)) mice. We found that 12-month-old (middle-aged) LDLR(-/-) mice developed substantially worse metabolic syndrome, diabetes, and atherosclerosis than 3-month-old (young) LDLR(-/-) mice when both were fed HFD for 3 months, despite similar elevations in total cholesterol levels. Microarray analyses were performed to analyze the mechanism responsible for the marked acceleration of atherosclerosis in middle-aged mice. Chow-fed middle-aged mice had greater aortic expression of multiple antioxidant genes than chow-fed young mice, including glutathione peroxidase-1 and -4, catalase, superoxide dismutase-2, and uncoupling protein-2. Aortic expression of these enzymes markedly increased in young mice fed HFD but decreased or only modestly increased in middle-aged mice fed HFD, despite the fact that systemic oxidative stress and vascular reactive oxygen species generation, measured by plasma F2alpha isoprostane concentration (systemic) and dihydroethidium conversion and p47phox expression (vascular), were greater in middle-aged mice fed HFD. Thus, the mechanism for the accelerated vascular injury in older LDLR(-/-) mice was likely the profound inability to mount an antioxidant response. This effect was related to a decrease in vascular expression of 2 key transcriptional pathways regulating the antioxidant response, DJ-1 and forkhead box, subgroup O family (FOXOs). Treatment of middle-aged mice fed HFD with the antioxidant apocynin attenuated atherosclerosis, whereas treatment with the insulin sensitizer rosiglitazone attenuated both metabolic syndrome and atherosclerosis. Both treatments decreased oxidative stress. A novel effect of rosiglitazone was to increase expression of Nrf2 (nuclear factor [erythroid-derived 2]-like 2), a downstream target of DJ-1 contributing to enhanced expression of vascular antioxidant enzymes. This investigation underscores the role of oxidative stress when multiple atherosclerotic risk factors, particularly aging, converge on the vessel wall and emphasizes the need to develop effective strategies to inhibit oxidative stress to protect aging vasculature.

Impaired development of atherosclerosis in Abcg1-/- Apoe-/- mice: identification of specific oxysterols that both accumulate in Abcg1-/- Apoe-/- tissues and induce apoptosis.

OBJECTIVE: To generate Abcg1(-/-) Apoe(-/-) mice to understand the mechanism and cell types involved in changes in atherosclerosis after loss of ABCG1. METHODS AND RESULTS: ABCG1 is highly expressed in macrophages and endothelial cells, 2 cell types that play important roles in the development of atherosclerosis. Abcg1(-/-) Apoe(-/-) and Apoe(-/-) mice and recipient Apoe(-/-) mice that had undergone transplantation with bone marrow from Apoe(-/-) or Abcg1(-/-) Apoe(-/-) mice were fed a Western diet for 12 or 16 weeks before quantification of atherosclerotic lesions. These studies demonstrated that loss of ABCG1 from all tissues, or from only hematopoietic cells, was associated with significantly smaller lesions that contained increased numbers of TUNEL- and cleaved caspase 3-positive apoptotic Abcg1(-/-) macrophages. We also identified specific oxysterols that accumulate in the brains and macrophages of the Abcg1(-/-) Apoe(-/-) mice. These oxysterols promoted apoptosis and altered the expression of proapoptotic genes when added to macrophages in vitro. CONCLUSIONS: Loss of ABCG1 from all tissues or from only hematopoietic cells results in smaller atherosclerotic lesions populated with increased apoptotic macrophages, by processes independent of ApoE. Specific oxysterols identified in tissues of Abcg1(-/-) Apoe(-/-) mice may be critical because they induce macrophage apoptosis and the expression of proapoptotic genes.

PPARdelta-mediated antiinflammatory mechanisms inhibit angiotensin II-accelerated atherosclerosis.

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor delta (PPARdelta) has been shown to improve insulin resistance, adiposity, and plasma HDL levels. However, its antiatherogenic role remains controversial. Here we report atheroprotective effects of PPARdelta activation in a model of angiotensin II (AngII)-accelerated atherosclerosis, characterized by increased vascular inflammation related to repression of an antiinflammatory corepressor, B cell lymphoma-6 (Bcl-6), and the regulators of G protein-coupled signaling (RGS) proteins RGS4 and RGS5. In this model, administration of the PPARdelta agonist GW0742 (1 or 10 mg/kg) substantially attenuated AngII-accelerated atherosclerosis without altering blood pressure and increased vascular expression of Bcl-6, RGS4, and RGS5, which was associated with suppression of inflammatory and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in AngII-treated macrophages: PPARdelta activation increased both total and free Bcl-6 levels and inhibited AngII activation of MAP kinases, p38, and ERK1/2. These studies uncover crucial proinflammatory mechanisms of AngII and highlight actions of PPARdelta activation to inhibit AngII signaling, which is atheroprotective.

Ligand activation of LXR beta reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR alpha and apoE.

Liver X receptors (LXRs) alpha and beta are transcriptional regulators of cholesterol homeostasis and potential targets for the development of antiatherosclerosis drugs. However, the specific roles of individual LXR isotypes in atherosclerosis and the pharmacological effects of synthetic agonists remain unclear. Previous work has shown that mice lacking LXRalpha accumulate cholesterol in the liver but not in peripheral tissues. In striking contrast, we demonstrate here that LXRalpha(-/-)apoE(-/-) mice exhibit extreme cholesterol accumulation in peripheral tissues, a dramatic increase in whole-body cholesterol burden, and accelerated atherosclerosis. The phenotype of these mice suggests that the level of LXR pathway activation in macrophages achieved by LXRbeta and endogenous ligand is unable to maintain homeostasis in the setting of hypercholesterolemia. Surprisingly, however, a highly efficacious synthetic agonist was able to compensate for the loss of LXRalpha. Treatment of LXRalpha(-/-)apoE(-/-) mice with synthetic LXR ligand ameliorates the cholesterol overload phenotype and reduces atherosclerosis. These observations indicate that LXRalpha has an essential role in maintaining peripheral cholesterol homeostasis in the context of hypercholesterolemia and provide in vivo support for drug development strategies targeting LXRbeta.

A nuclear receptor corepressor-dependent pathway mediates suppression of cytokine-induced C-reactive protein gene expression by liver X receptor.

C-reactive protein (CRP), the prototypical human acute phase protein, is an independent risk predictor of future cardiovascular events, both in healthy individuals and in patients with known cardiovascular disease. In addition, previous studies indicate that CRP might have direct proatherogenic properties. Ligand activation of the liver X receptor (LXR), a member of the nuclear hormone receptor superfamily, inhibits inflammatory gene expression in macrophages and attenuates the development of atherosclerosis in various animal models. We demonstrate herein that 2 synthetic LXR ligands, T0901317 and GW3965, inhibit interleukin-1beta/interleukin-6-induced CRP mRNA and protein expression in human hepatocytes. Knockdown of LXRalpha/beta by short interfering RNAs completely abolished the inhibitory effect of the LXR agonist T0901317 on cytokine-induced CRP gene transcription. Transient transfection experiments with 5'-deletion CRP promoter constructs identified a region from -125 to -256 relative to the initiation site that mediated the inhibitory effect of LXR ligands on CRP gene transcription. Depletion of the nuclear receptor corepressor by specific short interfering RNA increased cytokine-inducible CRP mRNA expression and promoter activity and reversed LXR ligand-mediated repression of CRP gene transcription. Chromatin immunoprecipitation assays indicated that nuclear receptor corepressor is present on the endogenous CRP promoter under basal conditions. Cytokine-induced clearance of nuclear receptor corepressor complexes was inhibited by LXR ligand treatment, maintaining the CRP gene in a repressed state. Finally, treatment of C57Bl6/J mice with LXR ligands attenuated lipopolysaccharide-induced mouse CRP and serum amyloid P component gene expression in the liver, whereas no effect was observed in LXRalphabeta knockout mice. Our observations identify a novel mechanism of inflammatory gene regulation by LXR ligands. Thus, inhibition of CRP expression by LXR agonists may provide a promising approach to impact initiation and progression of atherosclerosis.

Transcriptional repression of ATP-binding cassette transporter A1 gene in macrophages: a novel atherosclerotic effect of angiotensin II.

Angiotensin II (Ang II) is a powerful accelerator of atherosclerosis. Herein, we describe a novel transcription mechanism through which Ang II inhibits macrophage expression of the ATP-binding cassette transporter A1 (ABCA1), a key regulator of reverse cholesterol transport. We demonstrate that chronic Ang II infusion substantially promotes macrophage infiltration, foam cell formation, and atherosclerosis in low-density lipoprotein receptor-deficient mice and significantly reduces ABCA1 expression in peripheral macrophages. Administration of the Ang II type 1 receptor blocker valsartan inhibited Ang II-induced ABCA1 mRNA repression, macrophage cholesterol accumulation, and atherosclerosis. Ang II treatment reduced ABCA1 promoter activity of in vitro cultured mouse peritoneal macrophages, inducing fos-related antigen 2 (Fra2) protein binding to an ABCA1 promoter E-box motif, a site known to negatively regulate macrophage ABCA1 transcription. Valsartan pretreatment blocked Fra2 binding to the ABCA1 promoter, and Fra2 small interfering RNA pretreatment attenuated Ang II-mediated ABCA1 transcriptional inhibition, confirming the role of Fra2 in this process. This new evidence suggests that Ang II, a well-known proinflammatory and pro-oxidative factor, alters macrophage cholesterol homeostasis by repressing ABCA1 to promote foam cell formation and atherosclerosis.

Impaired development of atherosclerosis in hyperlipidemic Ldlr-/- and ApoE-/- mice transplanted with Abcg1-/- bone marrow.

OBJECTIVE: The lungs of Abcg1-/- mice accumulate macrophage foam cells that contain high levels of unesterified and esterified cholesterol, consistent with a role for ABCG1 in facilitating the efflux of cholesterol from macrophages to high-density lipoprotein (HDL) and other exogenous sterol acceptors. Based on these observations, we investigated whether loss of ABCG1 affects foam cell deposition in the artery wall and the development of atherosclerosis. METHODS AND RESULTS: Bone marrow from wild-type or Abcg1-/- mice was transplanted into Ldlr-/- or ApoE-/- mice. After administration of a high-fat/high-cholesterol diet, plasma and tissue lipid levels and atherosclerotic lesion size were quantified and compared. Surprisingly, transplantation of Abcg1-/- bone marrow cells resulted in a significant reduction in lesion size in both mouse models, despite the fact that lipid levels increased in the lung, spleen, and kidney. Lesions of Ldlr-/- mice transplanted with Abcg1-/- cells contained increased numbers of apoptotic cells. Consistent with this observation, in vitro studies demonstrated that Abcg1-/- macrophages were more susceptible to oxidized low-density lipoprotein (ox-LDL)-dependent apoptosis than Abcg1+/+ cells. CONCLUSIONS: Diet-induced atherosclerosis is impaired when atherosclerotic-susceptible mice are transplanted with Abcg1-/- bone marrow. The demonstration that Abcg1-/- macrophages undergo accelerated apoptosis provides a mechanism to explain the decrease in the atherosclerotic lesions.

Macrophage liver X receptor is required for antiatherogenic activity of LXR agonists.

OBJECTIVE: Complications of atherosclerotic cardiovascular disease due to elevated blood cholesterol levels are the major cause of death in the Western world. The liver X receptors, LXRalpha and LXRbeta (LXRs), are ligand-dependent transcription factors that act as cholesterol sensors and coordinately control transcription of genes involved in cholesterol and lipid homeostasis as well as macrophage inflammatory gene expression. LXRs regulate cholesterol balance through activation of ATP-binding cassette transporters that promote cholesterol transport and excretion from the liver, intestine, and macrophage. Although LXR agonists are known to delay progression of atherosclerosis in mouse models, their ability to abrogate preexisting cardiovascular disease by inducing regression and stabilization of established atherosclerotic lesions has not been addressed. METHODS AND RESULTS: We demonstrate that LXR agonist treatment increases ATP-binding cassette transporter expression within preexisting atherosclerotic lesions, resulting in regression of these lesions as well as remodeling from vulnerable to stable lesions and a reduction in macrophage content. Further, using macrophage-selective LXR-deficient mice created by bone marrow transplantation, we provide the first evidence that macrophage LXR expression is necessary for the atheroprotective actions of an LXR agonist. CONCLUSIONS: These data substantiate that drugs targeting macrophage LXR activity may offer therapeutic benefit in the treatment of atherosclerotic cardiovascular disease.

Elevated Adiponectin Levels Suppress Perivascular and Aortic Inflammation and Prevent AngII-induced Advanced Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a degenerative disease characterized by aortic dilation and rupture leading to sudden death. Currently, no non-surgical treatments are available and novel therapeutic targets are needed to prevent AAA. We investigated whether increasing plasma levels of adiponectin (APN), a pleiotropic adipokine, provides therapeutic benefit to prevent AngII-induced advanced AAA in a well-established preclinical model. In the AngII-infused hyperlipidemic low-density lipoprotein receptor-deficient mouse (LDLR-/-) model, we induced plasma APN levels using a recombinant adenovirus expressing mouse APN (AdAPN) and as control, adenovirus expressing green florescent protein (AdGFP). APN expression produced sustained and significant elevation of total and high-molecular weight APN levels and enhanced APN localization in the artery wall. AngII infusion for 8 weeks induced advanced AAA development in AdGFP mice. Remarkably, APN inhibited the AAA development in AdAPN mice by suppressing aortic inflammatory cell infiltration, medial degeneration and elastin fragmentation. APN inhibited the angiotensin type-1 receptor (AT1R), inflammatory cytokine and mast cell protease expression, and induced lysyl oxidase (LOX) in the aortic wall, improved systemic cytokine profile and attenuated adipose inflammation. These studies strongly support APN therapeutic actions through multiple mechanisms inhibiting AngII-induced AAA and increasing plasma APN levels as a strategy to prevent advanced AAA.

Thermoneutral Housing Accelerates Metabolic Inflammation to Potentiate Atherosclerosis but Not Insulin Resistance.

Chronic, low-grade inflammation triggered by excess intake of dietary lipids has been proposed to contribute to the pathogenesis of metabolic disorders, such as obesity, insulin resistance, type 2 diabetes, and atherosclerosis. Although considerable evidence supports a causal association between inflammation and metabolic diseases, most tests of this link have been performed in cold-stressed mice that are housed below their thermoneutral zone. We report here that thermoneutral housing of mice has a profound effect on the development of metabolic inflammation, insulin resistance, and atherosclerosis. Mice housed at thermoneutrality develop metabolic inflammation in adipose tissue and in the vasculature at an accelerated rate. Unexpectedly, this increased inflammatory response contributes to the progression of atherosclerosis but not insulin resistance. These findings not only suggest that metabolic inflammation can be uncoupled from obesity-associated insulin resistance, but also point to how thermal stress might limit our ability to faithfully model human diseases in mice.

Adiponectin expression protects against angiotensin II-mediated inflammation and accelerated atherosclerosis.

Adiponectin (APN), an adipocytokine produced by adipose tissue, exerts pleiotropic actions regulating inflammation, metabolism and vascular homeostasis. APN levels are inversely correlated with obesity, type-2 diabetes, hypertension and cardiovascular disease. Although renin angiotensin system (RAS) activation in these interrelated metabolic syndrome components increases angiotensin II (AngII) levels leading to vascular damage, it is unknown whether APN under these conditions provides atheroprotection. We investigated whether increasing plasma APN provides atheroprotection in a hypertensive and accelerated atherosclerosis model. Using adenoviral gene transfer, sustained APN expression increased plasma levels of total and high-molecular weight APN, leading to a significant elevation of plasma HDL-cholesterol (HDL-C). Elevated APN levels were strongly atheroprotective, yet had no impact on blood pressure. Notably, gene expression analyses revealed that APN significantly inhibited the expression of pro-inflammatory and atherogenic genes while it increased the expression of the anti-inflammatory cytokine, IL-10 and the cholesterol efflux transporters, ABCA1 and ABCG1 in the artery wall. These findings suggest that increasing APN levels may be an effective therapeutic strategy to inhibit vascular inflammation and accelerated atherosclerosis associated with RAS activation in the metabolic syndrome.

The LXR-Idol axis differentially regulates plasma LDL levels in primates and mice.

The LXR-regulated E3 ubiquitin ligase IDOL controls LDLR receptor stability independent of SREBP and PCSK9, but its relevance to plasma lipid levels is unknown. Here we demonstrate that the effects of the LXR-IDOL axis are both tissue and species specific. In mice, LXR agonist induces Idol transcript levels in peripheral tissues but not in liver, and does not change plasma LDL levels. Accordingly, Idol-deficient mice exhibit elevated LDLR protein levels in peripheral tissues, but not in the liver. By contrast, LXR activation in cynomolgus monkeys induces hepatic IDOL expression, reduces LDLR protein levels, and raises plasma LDL levels. Knockdown of IDOL in monkeys with an antisense oligonucleotide blunts the effect of LXR agonist on LDL levels. These results implicate IDOL as a modulator of plasma lipid levels in primates and support further investigation into IDOL inhibition as a potential strategy for LDL lowering in humans.

The Bcl6-SMRT/NCoR cistrome represses inflammation to attenuate atherosclerosis.

Chronic inflammation is a hallmark of atherosclerosis, but its transcriptional underpinnings are poorly understood. We show that the transcriptional repressor Bcl6 is an anti-inflammatory regulator whose loss in bone marrow of Ldlr(-/-) mice results in severe atherosclerosis and xanthomatous tendonitis, a virtually pathognomonic complication in patients with familial hypercholesterolemia. Disruption of the interaction between Bcl6 and SMRT or NCoR with a peptide inhibitor in vitro recapitulated atherogenic gene changes in mice transplanted with Bcl6-deficient bone marrow, pointing to these cofactors as key mediators of Bcl6 inflammatory suppression. Using ChIP-seq, we reveal the SMRT and NCoR corepressor cistromes, each consisting of over 30,000 binding sites with a nearly 50% overlap. While the complete cistromes identify a diversity of signaling pathways, the Bcl6-bound subcistromes for each corepressor are highly enriched for NF-κB-driven inflammatory and tissue remodeling genes. These results reveal that Bcl6-SMRT/NCoR complexes constrain immune responses and contribute to the prevention of atherosclerosis.

Differential roles of cardiomyocyte and macrophage peroxisome proliferator-activated receptor gamma in cardiac fibrosis.

OBJECTIVE: Cardiac fibrosis is an important component of diabetic cardiomyopathy. Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands repress proinflammatory gene expression, including that of osteopontin, a known contributor to the development of myocardial fibrosis. We thus investigated the hypothesis that PPARgamma ligands could attenuate cardiac fibrosis. RESEARCH DESIGN AND METHODS: Wild-type cardiomyocyte- and macrophage-specific PPARgamma(-/-) mice were infused with angiotensin II (AngII) to promote cardiac fibrosis and treated with the PPARgamma ligand pioglitazone to determine the roles of cardiomyocyte and macrophage PPARgamma in cardiac fibrosis. RESULTS: Cardiomyocyte-specific PPARgamma(-/-) mice (cPPARgamma(-/-)) developed spontaneous cardiac hypertrophy with increased ventricular osteopontin expression and macrophage content, which were exacerbated by AngII infusion. Pioglitazone attenuated AngII-induced fibrosis, macrophage accumulation, and osteopontin expression in both wild-type and cPPARgamma(-/-) mice but induced hypertrophy in a PPARgamma-dependent manner. We pursued two mechanisms to explain the antifibrotic cardiomyocyte-PPARgamma-independent effects of pioglitazone: increased adiponectin expression and attenuation of proinflammatory macrophage activity. Adenovirus-expressed adiponectin had no effect on cardiac fibrosis and the PPARgamma ligand pioglitazone did not attenuate AngII-induced cardiac fibrosis, osteopontin expression, or macrophage accumulation in monocyte-specific PPARgamma(-/-) mice. CONCLUSIONS: We arrived at the following conclusions: 1) both cardiomyocyte-specific PPARgamma deficiency and activation promote cardiac hypertrophy, 2) both cardiomyocyte and monocyte PPARgamma regulate cardiac macrophage infiltration, 3) inflammation is a key mediator of AngII-induced cardiac fibrosis, 4) macrophage PPARgamma activation prevents myocardial macrophage accumulation, and 5) PPARgamma ligands attenuate AngII-induced cardiac fibrosis by inhibiting myocardial macrophage infiltration. These observations have important implications for potential interventions to prevent cardiac fibrosis.