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Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33-mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization-associated gene profile. Furthermore, miR-33-mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid-producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33-depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4(+) T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3(+) Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

Background: Patients undergoing cardiovascular procedures remain at increased risk for myocardial infarction, stroke, and cardiovascular death. Risk factor control in this patient population remains suboptimal and would likely benefit from strategies targeting education, lifestyle, and healthy behaviors., Design: The IMPACT trial is a 400-subject prospective randomized trial designed to compare different cardiovascular prevention strategies in subjects following a cardiovascular intervention. The trial began enrollment in the Spring of 2012 and is randomizing subjects in a 1:1:1 manner to usual care, a one-time cardiovascular prevention consult, or a one-time cardiovascular prevention consult plus behavioral intervention program (telephone-based motivational interviewing and tailored text messages) over a 6-month period. The primary end point is non-high-density lipoprotein cholesterol. Secondary end points include other plasma lipid values, metabolic risk, smoking cessation, physical activity, dietary intake, medication use and adherence, and quality of life., Conclusions: The IMPACT trial provides data on different management strategies for risk factor optimization in subjects following cardiovascular procedures. The results will provide a platform for the continued development of novel multidisciplinary interventions in this high-risk population., (Copyright © 2015 Elsevier Inc. All rights reserved.)

High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.

Unlabelled: Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)-facilitated TAM PET imaging in a breast cancer model., Methods: Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide (89)Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. (89)Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate (89)Zr-PL-HDL and (89)Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of (89)Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry., Results: The phospholipid- and apoA-I-labeled rHDL were produced at 79% ± 13% (n = 6) and 94% ± 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 ± 2.8 and 8.6 ± 1.3 percentage injected dose per gram for apoA-I- and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively., Conclusion: We have developed (89)Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our (89)Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr-/- mice in different metabolic states.

In mouse models of atherosclerosis, normalization of hyperlipidemia promotes macrophage emigration and regression of atherosclerotic plaques in part by liver X receptor (LXR)-mediated induction of the chemokine receptor CCR7. Here we report that LXRα serine 198 (S198) phosphorylation modulates CCR7 expression. Low levels of S198 phosphorylation are observed in plaque macrophages in the regression environment where high levels of CCR7 expression are observed. Consistent with these findings, CCR7 gene expression in human and mouse macrophages cell lines is induced when LXRα at S198 is nonphosphorylated. In bone marrow-derived macrophages (BMDMs), we also observed induction of CCR7 by ligands that promote nonphosphorylated LXRα S198, and this was lost in LXR-deficient BMDMs. LXRα occupancy at the CCR7 promoter is enhanced and histone modifications associated with gene repression are reduced in RAW264.7 cells expressing nonphosphorylated LXRα (RAW-LXRα S198A) compared to RAW264.7 cells expressing wild-type (WT) phosphorylated LXRα (RAW-LXRα WT). Expression profiling of ligand-treated RAW-LXRα S198A cells compared to RAW-LXRα WT cells revealed induction of cell migratory and anti-inflammatory genes and repression of proinflammatory genes. Modeling of LXRα S198 in the nonphosphorylated and phosphorylated states identified phosphorylation-dependent conformational changes in the hinge region commensurate with the presence of sites for protein interaction. Therefore, gene transcription is regulated by LXRα S198 phosphorylation, including that of antiatherogenic genes such as CCR7., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Inflammation drives atherosclerotic plaque progression and rupture, and is a compelling therapeutic target. Consequently, attenuating inflammation by reducing local macrophage accumulation is an appealing approach. This can potentially be accomplished by either blocking blood monocyte recruitment to the plaque or increasing macrophage apoptosis and emigration. Because macrophage proliferation was recently shown to dominate macrophage accumulation in advanced plaques, locally inhibiting macrophage proliferation may reduce plaque inflammation and produce long-term therapeutic benefits. To test this hypothesis, we used nanoparticle-based delivery of simvastatin to inhibit plaque macrophage proliferation in apolipoprotein E deficient mice ( Apoe -/- ) with advanced atherosclerotic plaques. This resulted in rapid reduction of plaque inflammation and favorable phenotype remodeling. We then combined this short-term nanoparticle intervention with an eight-week oral statin treatment, and this regimen rapidly reduced and continuously suppressed plaque inflammation. Our results demonstrate that pharmacologically inhibiting local macrophage proliferation can effectively treat inflammation in atherosclerosis.

High-density lipoprotein (HDL) is a natural nanoparticle that exhibits an intrinsic affinity for atherosclerotic plaque macrophages. Its natural targeting capability as well as the option to incorporate lipophilic payloads, e.g., imaging or therapeutic components, in both the hydrophobic core and the phospholipid corona make the HDL platform an attractive nanocarrier. To realize controlled release properties, we developed a hybrid polymer/HDL nanoparticle composed of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core. This novel HDL-like nanoparticle (PLGA-HDL) displayed natural HDL characteristics, including preferential uptake by macrophages and a good cholesterol efflux capacity, combined with a typical PLGA nanoparticle slow release profile. In vivo studies carried out with an ApoE knockout mouse model of atherosclerosis showed clear accumulation of PLGA-HDL nanoparticles in atherosclerotic plaques, which colocalized with plaque macrophages. This biomimetic platform integrates the targeting capacity of HDL biomimetic nanoparticles with the characteristic versatility of PLGA-based nanocarriers.

Objective: We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques, it has become appreciated that ≈40% of cells classified as macrophages by histological markers may be of VSMC origin. Therefore, we sought to gain insight into the molecular regulation of this clinically relevant process., Approach and Results: VSMC of mouse (or human) origin were incubated with cyclodextrin-cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins was reduced and of macrophage markers increased. Concurrent was downregulation of miR-143/145, which positively regulate the master VSMC differentiation transcription factor myocardin. Mechanisms were further probed in mouse VSMC. Maintaining the expression of myocardin or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core., Conclusions: Cholesterol loading of VSMC converts them to a macrophage-appearing state by downregulating the miR-143/145-myocardin axis. Although these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages., (© 2015 American Heart Association, Inc.)

Liver X receptor (LXR) signaling pathways regulate lipid metabolism and inflammation, which has generated widespread interest in developing synthetic LXR agonists as potential therapeutics for the management of atherosclerosis. In this study, it is demonstrated that nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (NP-LXR) exert anti-inflammatory effects and inhibit the development of atherosclerosis without causing hepatic steatosis. These NPs are engineered through self-assembly of a biodegradable diblock poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) copolymer. NP-LXR is significantly more effective than free GW3965 at inducing LXR-target gene expression and suppressing inflammatory factors in macrophages in vitro and in vivo. Additionally, the NPs elicit negligible lipogenic gene stimulation in the liver. Using the Ldlr (-/-) mouse model of atherosclerosis, abundant colocalization of fluorescently labeled NPs within plaque macrophages following systemic administration is seen. Notably, six intravenous injections of NP-LXR over 2 weeks markedly reduce the CD68-positive cell (macrophage) content of plaques (by 50%) without increasing total cholesterol or triglycerides in the liver and plasma. Together, these findings identify GW3965-encapsulated PLGA-b-PEG NPs as a promising nanotherapeutic approach to combat atherosclerosis, providing the benefits of LXR agonists without their adverse effects on hepatic and plasma lipid metabolism., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The atherosclerotic mouse aorta consists of a heterogeneous population of cells, including macrophages, endothelial cells, and smooth muscle cells that play critical roles in cardiovascular disease. Identification of these vascular cells in the vessel wall is important to understanding their function in pathological conditions. Immunohistochemistry is an invaluable technique used to detect the presence of cells in different tissues. Here, we describe immunohistochemical techniques commonly used for the detection of the vascular cells in the atherosclerotic mouse aorta using cell type-specific markers.

Objective: Diabetic hypertriglyceridemia is thought to be primarily driven by increased hepatic de novo lipogenesis. However, experiments in animal models indicated that insulin deficiency should decrease hepatic de novo lipogenesis and reduce plasma triglyceride levels., Approach and Results: To address the discrepancy between human data and genetically altered mouse models, we investigated whether insulin-deficient diabetic mice had triglyceride changes that resemble those in diabetic humans. Streptozotocin-induced insulin deficiency increased plasma triglyceride levels in mice. Contrary to the mouse models with impaired hepatic insulin receptor signaling, insulin deficiency did not reduce hepatic triglyceride secretion and de novo lipogenesis-related gene expression. Diabetic mice had a marked decrease in postprandial triglycerides clearance, which was associated with decreased lipoprotein lipase and peroxisome proliferator-activated receptor α mRNA levels in peripheral tissues and decreased lipoprotein lipase activity in skeletal muscle, heart, and brown adipose tissue. Diabetic heterozygous lipoprotein lipase knockout mice had markedly elevated fasting plasma triglyceride levels and prolonged postprandial triglycerides clearance., Conclusions: Insulin deficiency causes hypertriglyceridemia by decreasing peripheral lipolysis and not by an increase in hepatic triglycerides production and secretion., (© 2014 American Heart Association, Inc.)

We report the first systems biology investigation of regulators controlling arterial plaque macrophage transcriptional changes in response to lipid lowering in vivo in two distinct mouse models of atherosclerosis regression. Transcriptome measurements from plaque macrophages from the Reversa mouse were integrated with measurements from an aortic transplant-based mouse model of plaque regression. Functional relevance of the genes detected as differentially expressed in plaque macrophages in response to lipid lowering in vivo was assessed through analysis of gene functional annotations, overlap with in vitro foam cell studies, and overlap of associated eQTLs with human atherosclerosis/CAD risk SNPs. To identify transcription factors that control plaque macrophage responses to lipid lowering in vivo, we used an integrative strategy--leveraging macrophage epigenomic measurements--to detect enrichment of transcription factor binding sites upstream of genes that are differentially expressed in plaque macrophages during regression. The integrated analysis uncovered eight transcription factor binding site elements that were statistically overrepresented within the 5' regulatory regions of genes that were upregulated in plaque macrophages in the Reversa model under maximal regression conditions and within the 5' regulatory regions of genes that were upregulated in the aortic transplant model during regression. Of these, the TCF/LEF binding site was present in promoters of upregulated genes related to cell motility, suggesting that the canonical Wnt signaling pathway may be activated in plaque macrophages during regression. We validated this network-based prediction by demonstrating that β-catenin expression is higher in regressing (vs. control group) plaques in both regression models, and we further demonstrated that stimulation of canonical Wnt signaling increases macrophage migration in vitro. These results suggest involvement of canonical Wnt signaling in macrophage emigration from the plaque during lipid lowering-induced regression, and they illustrate the discovery potential of an epigenome-guided, systems approach to understanding atherosclerosis regression.

Objective: Advanced murine and human plaques are hypoxic, but it remains unclear whether plaque hypoxia is causally related to atherogenesis. Here, we test the hypothesis that reversal of hypoxia in atherosclerotic plaques by breathing hyperoxic carbogen gas will prevent atherosclerosis., Approach and Results: Low-density lipoprotein receptor-deficient mice (LDLR(-/-)) were fed a Western-type diet, exposed to carbogen (95% O2, 5% CO2) or air, and the effect on plaque hypoxia, size, and phenotype was studied. First, the hypoxic marker pimonidazole was detected in murine LDLR(-/-) plaque macrophages from plaque initiation onwards. Second, the efficacy of breathing carbogen (90 minutes, single exposure) was studied. Compared with air, carbogen increased arterial blood pO2 5-fold in LDLR(-/-) mice and reduced plaque hypoxia in advanced plaques of the aortic root (-32%) and arch (-84%). Finally, the effect of repeated carbogen exposure on progression of atherosclerosis was studied in LDLR(-/-) mice fed a Western-type diet for an initial 4 weeks, followed by 4 weeks of diet and carbogen or air (both 90 min/d). Carbogen reduced plaque hypoxia (-40%), necrotic core size (-37%), and TUNEL(+) (terminal uridine nick-end labeling positive) apoptotic cell content (-50%) and increased efferocytosis of apoptotic cells by cluster of differentiation 107b(+) (CD107b, MAC3) macrophages (+36%) in advanced plaques of the aortic root. Plaque size, plasma cholesterol, hematopoiesis, and systemic inflammation were unchanged. In vitro, hypoxia hampered efferocytosis by bone marrow-derived macrophages, which was dependent on the receptor Mer tyrosine kinase., Conclusions: Carbogen restored murine plaque oxygenation and prevented necrotic core expansion by enhancing efferocytosis, likely via Mer tyrosine kinase. Thus, plaque hypoxia is causally related to necrotic core expansion., (© 2014 American Heart Association, Inc.)

It is well recognized that macrophages in many contexts in vitro and in vivo display a spectrum of inflammatory features and functional properties. A convenient system to group together different subsets of macrophages has been the M1 (inflammatory)/M2 (anti-inflammatory) classification. In addition to other sites of inflammation, it is now established that atherosclerotic plaques contain both M1 and M2 macrophages. We review results made possible by a number of recent mouse models of atherosclerotic regression that, taken with other literature, have shown the M1/M2 balance in plaques to be dynamic, with M1 predominating in disease progression and M2 in regression. The regulation of the macrophage phenotype in plaques and the functional consequences of the M1 and M2 states in atherosclerosis will also be discussed.

Rationale: Diabetes mellitus increases cardiovascular disease risk in humans and remains elevated despite cholesterol-lowering therapy with statins. Consistent with this, in mouse models, diabetes mellitus impairs atherosclerosis plaque regression after aggressive cholesterol lowering. MicroRNA 33 (miR33) is a key negative regulator of the reverse cholesterol transport factors, ATP-binding cassette transporter A1 and high-density lipoprotein, which suggested that its inhibition may overcome this impairment., Objective: To assess the effects of miR33 inhibition on atherosclerosis regression in diabetic mice., Methods and Results: Reversa mice, which are deficient in the low-density lipoprotein receptor and in which hypercholesterolemia is reversed by conditional inactivation of the microsomal triglyceride transfer protein gene, were placed on an atherogenic diet for 16 weeks, then either made diabetic by streptozotocin injection or kept normoglycemic. Lipid-lowering was induced by microsomal triglyceride transfer protein gene inactivation, and mice were treated with anti-miR33 or control oligonucleotides. Although regression was impaired in diabetic mice treated with control oligonucleotides, anti-miR33 treatment decreased plaque macrophage content and inflammatory gene expression in these mice. The decreased macrophage content in anti-miR33 treated diabetic mice was associated with a blunting of hyperglycemia-induced monocytosis and reduced monocyte recruitment to the plaque, which was traced to an inhibition of the proliferation of bone marrow monocyte precursors associated with the upregulation of their Abca1., Conclusions: miR33 inhibition overcomes deleterious effects of diabetes mellitus in atherosclerosis regression in mice, which suggests a therapeutic strategy in diabetic patients, who remain at elevated cardiovascular disease risk, despite plasma cholesterol lowering., (© 2014 American Heart Association, Inc.)

Plasma high-density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR-33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short-term anti-miR-33 treatment has been previously studied, the long-term effect of miR-33 antagonism in vivo remains to be elucidated. Here, we show that long-term therapeutic silencing of miR-33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high-fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR-33 increases the expression of genes involved in fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR-33 antisense oligonucleotides. We also report that anti-miR-33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR-33 when mice are fed a high-fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR-33 in non-human primates before we can translate this therapy to humans., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

Liver X receptors (LXRs) attenuate inflammation by modulating the expression of key inflammatory genes, making LXRs and their ligands particularly attractive candidates for therapeutic intervention in cardiovascular, metabolic, and/or inflammatory diseases. Herein, enhanced proresolving activity of polymeric nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (LXR-NPs) is demonstrated, developed from a combinatorial library of more than 70 formulations with variations in critical physicochemical parameters. In vitro studies on peritoneal macrophages confirm that LXR-NPs are significantly more effective than the free agonist at downregulating pro-inflammatory mediators (MCP-1 and TNFα), as well as inducing the expression of LXR target genes (ABCA1 and SREBP1c). Through a zymosan-induced acute peritonitis in vivo model, LXR-NPs are found to be more efficient than free GW3965 at limiting the recruitment of polymononuclear neutrophils (50% vs 17%), suppressing the gene expression and secretion of pro-inflammatory factors MCP-1 and TNFα in peritoneal macrophages, and decreasing the resolution interval up to 4 h. Furthermore, LXR-NPs suppress the secretion of MCP-1 and TNFα by monocytes and macrophages more efficiently than the commercial drug dexamethasone. Overall, these findings demonstrate that LXR-NPs are capable of promoting resolution of inflammation and highlight the prospect of LXR-based nanotherapeutics for inflammatory diseases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inflammation contributes to many of the characteristics of plaques implicated in the pathogenesis of acute coronary syndromes. Moreover, inflammatory pathways not only regulate the properties of plaques that precipitate acute coronary syndromes but also modulate the clinical consequences of the thrombotic complications of atherosclerosis. This synthesis will provide an update on the fundamental mechanisms of inflammatory responses that govern acute coronary syndromes and also highlight the ongoing balance between proinflammatory mechanisms and endogenous pathways that can promote the resolution of inflammation. An appreciation of the countervailing mechanisms that modulate inflammation in relation to acute coronary syndromes enriches our fundamental understanding of the pathophysiology of this important manifestation of atherosclerosis. In addition, these insights provide glimpses into potential novel therapeutic interventions to forestall this ultimate complication of the disease., (© 2014 American Heart Association, Inc.)

Obesity is associated with infiltration of macrophages into adipose tissue (AT), contributing to insulin resistance and diabetes. However, relatively little is known regarding the origin of AT macrophages (ATMs). We discovered that murine models of obesity have prominent monocytosis and neutrophilia, associated with proliferation and expansion of bone marrow (BM) myeloid progenitors. AT transplantation conferred myeloid progenitor proliferation in lean recipients, while weight loss in both mice and humans (via gastric bypass) was associated with a reversal of monocytosis and neutrophilia. Adipose S100A8/A9 induced ATM TLR4/MyD88 and NLRP3 inflammasome-dependent IL-1β production. IL-1β interacted with the IL-1 receptor on BM myeloid progenitors to stimulate the production of monocytes and neutrophils. These studies uncover a positive feedback loop between ATMs and BM myeloid progenitors and suggest that inhibition of TLR4 ligands or the NLRP3-IL-1β signaling axis could reduce AT inflammation and insulin resistance in obesity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr(166)), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr(166) nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO2-Tyr(166)-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNACUA pair for functional studies. Studies with mAb 4G11.2 showed that NO2-Tyr(166)-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼ 8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO2-Tyr(166)-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063-1.21). NO2-Tyr(166)-apoA-I in plasma showed a similar distribution. Recovery of NO2-Tyr(166)-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr(166) is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.

Objective: Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques., Approach and Results: ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques., Conclusions: Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Macrophages in atherosclerotic plaques are activated, inflammatory cells that directly contribute to the disease process. De Nardo et al. (2013), now report that high-density lipoproteins (HDL) can reprogram macrophages to be less inflammatory through an ATF3-dependent pathway, providing another mechanistic basis for the atheroprotective properties of HDL., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi apparatus. Therefore, the transport of nascent VLDL from the ER to the Golgi represents a critical step during VLDL maturation and secretion and in regulating serum LDL cholesterol levels. Our previous studies showed that apoB100 exits the ER in coat complex II vesicles (COPII), but the cohort of related factors that control trafficking is poorly defined., Approach and Results: Expression levels of Kelch-like protein 12 (KLHL12), an adaptor protein known to assist COPII-dependent transport of procollagen, were manipulated by using a KLHL12-specific small interfering RNA and a KLHL12 expression plasmid in the rat hepatoma cell line, McArdle RH7777. KLHL12 knockdown decreased the secreted and intracellular pools of apoB100, an effect that was attenuated in the presence of an autophagy inhibitor. KLHL12 knockdown also significantly reduced secretion of the most lipidated apoB100-VLDL species and led to the accumulation of apoB100 in the ER. Consistent with these data, KLHL12 overexpression increased apoB100 recovery and apoB100-VLDL secretion. Images obtained from confocal microscopy revealed colocalization of apoB100 and KLHL12, further supporting a direct link between KLHL12 function and VLDL trafficking from the ER., Conclusions: KLHL12 plays a critical role in facilitating the ER exit and secretion of apoB100-VLDL particles, suggesting that KLHL12 modulation would influence plasma lipid levels.

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl(-) system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall.

High-density lipoprotein (HDL) particles transport (among other molecules) cholesterol (HDL-C). In epidemiological studies, plasma HDL-C levels have an inverse relationship to the risk of atherosclerotic cardiovascular disease. It has been assumed that this reflects the protective functions of HDL, which include their ability to promote cholesterol efflux. Yet, several recent pharmacological and genetic studies have failed to demonstrate that increased plasma levels of HDL-C resulted in decreased cardiovascular disease risk, giving rise to a controversy regarding whether plasma levels of HDL-C reflect HDL function, or that HDL is even as protective as assumed. The evidence from preclinical and (limited) clinical studies shows that HDL can promote the regression of atherosclerosis when the levels of functional particles are increased from endogenous or exogenous sources. The data show that regression results from a combination of reduced plaque lipid and macrophage contents, as well as from a reduction in its inflammatory state. Although more research will be needed regarding basic mechanisms and to establish that these changes translate clinically to reduced cardiovascular disease events, that HDL can regress plaques suggests that the recent trial failures do not eliminate HDL from consideration as an atheroprotective agent but rather emphasizes the important distinction between HDL function and plasma levels of HDL-C.

This article seeks to address how our understanding of the recovery process and resulting supports can be made more comprehensive: how can links from treatment to home to school to communities be made so that there are fewer and fewer recovery gaps for adolescents? Using the ecology of recovery model developed by White (2009) as the impetus for such a review, the article discusses the challenges inherent within adolescent substance abuse recovery, factors impacting successful recovery, and programs addressing this issue that have been empirically studied. Studied programs can be categorized as (1) formalized aftercare recovery and (2) recovery communities, and both examples will be described using existing literature. From the review of existing recovery support research and resources, the discussion highlights gaps and future research areas in order to address the complexity of recovery among young people.

Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show that this effect is mediated through the inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show that they accumulate in atherosclerotic lesions in which they directly affect plaque macrophages. Finally, we demonstrate that a 3-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a 1-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

One mechanism of the lipid-lowering effects of the fish oil n-3 fatty acids [e.g., docosahexaenoic acid (DHA)] in cell and animal models is induced hepatic apolipoprotein B100 (apoB) presecretory degradation. This degradation occurs post-endoplasmic reticulum, but whether DHA induces it before or after intracellular VLDL formation remains unanswered. We found in McA-RH7777 rat hepatic cells that DHA and oleic acid (OA) treatments allowed formation of pre-VLDL particles and their transport to the Golgi, but, in contrast to OA, with DHA pre-VLDL particles failed to quantitatively assemble into fully lipidated (mature) VLDL. This failure required lipid peroxidation and was accompanied by the formation of apoB aggregates (known to be degraded by autophagy). Preventing the exit of proteins from the Golgi blocked the aggregation of apoB but did not restore VLDL maturation, indicating that failure to fully lipidate apoB preceded its aggregation. ApoB autophagic degradation did not appear to require an intermediate step of cytosolic aggresome formation. Taken with other examples in the literature, the results of this study suggest that pre-VLDL particles that are competent to escape endoplasmic reticulum quality control mechanisms but fail to mature in the Golgi remain subject to quality control surveillance late in the secretory pathway.

High-density lipoprotein (HDL) is a natural nanoparticle that transports peripheral cholesterol to the liver. Reconstituted high-density lipoprotein (rHDL) exhibits antiatherothrombotic properties and is being considered as a natural treatment for cardiovascular diseases. Furthermore, HDL nanoparticle platforms have been created for targeted delivery of therapeutic and diagnostic agents. The current methods for HDL reconstitution involve lengthy procedures that are challenging to scale up. A central need in the synthesis of rHDL, and multifunctional nanomaterials in general, is to establish large-scale production of reproducible and homogeneous batches in a simple and efficient fashion. Here, we present a large-scale microfluidics-based manufacturing method for single-step synthesis of HDL-mimicking nanomaterials (μHDL). μHDL is shown to have the same properties (e.g., size, morphology, bioactivity) as conventionally reconstituted HDL and native HDL. In addition, we were able to incorporate simvastatin (a hydrophobic drug) into μHDL, as well as gold, iron oxide, quantum dot nanocrystals or fluorophores to enable its detection by computed tomography (CT), magnetic resonance imaging (MRI), or fluorescence microscopy, respectively. Our approach may contribute to effective development and optimization of lipoprotein-based nanomaterials for medical imaging and drug delivery.

In the current study we show the dissociation and tumor accumulation dynamics of dual-labeled near-infrared quantum dot core self-assembled lipidic nanoparticles (SALNPs) in a mouse model upon intravenous administration. Using advanced in vivo fluorescence energy transfer imaging techniques, we observed swift exchange with plasma protein components in the blood and progressive SALNP dissociation and subsequent trafficking of individual SALNP components following tumor accumulation. Our results suggest that upon intravenous administration SALNPs quickly transform, which may affect their functionality. The presented technology provides a modular in vivo tool to visualize SALNP behavior in real time and may contribute to improving the therapeutic outcome or molecular imaging signature of SALNPs.

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail.

Cardiovascular disease represents the most common cause of death in patients with nonalcoholic fatty liver disease (NAFLD). Patients with NAFLD exhibit an atherogenic dyslipidemia that is characterized by an increased plasma concentration of triglycerides, reduced concentration of high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) particles that are smaller and more dense than normal. The pathogenesis of NAFLD-associated atherogenic dyslipidemia is multifaceted, but many aspects are attributable to manifestations of insulin resistance. Here the authors review the structure, function, and metabolism of lipoproteins, which are macromolecular particles of lipids and proteins that transport otherwise insoluble triglyceride and cholesterol molecules within the plasma. They provide a current explanation of the metabolic perturbations that are observed in the setting of insulin resistance. An improved understanding of the pathophysiology of atherogenic dyslipidemia would be expected to guide therapies aimed at reducing morbidity and mortality in patients with NAFLD., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Prior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall., Methods and Results: A monoclonal antibody (10G1.5) was developed that equally recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination of homogenates of atherosclerotic plaque-laden aorta showed >100-fold enrichment of apoA1 compared with normal aorta (P<0.001). Surprisingly, buoyant density fractionation revealed that only a minority (<3% of total) of apoA1 recovered from either lesions or normal aorta resides within an HDL-like particle (1.063≤d≤1.21). In contrast, the majority (>90%) of apoA1 within aortic tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d>1.21). Moreover, both lesion and normal artery wall apoA1 are highly cross-linked (50% to 70% of total), and functional characterization of apoA1 quantitatively recovered from aorta with the use of monoclonal antibody 10G1.5 showed ≈80% lower cholesterol efflux activity and ≈90% lower lecithin-cholesterol acyltransferase activity relative to circulating apoA1., Conclusions: The function and distribution of apoA1 in human aorta are quite distinct from those found in plasma. The lipoprotein is markedly enriched within atherosclerotic plaque, predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and functionally impaired.

Atherosclerosis is a chronic inflammatory disease that arises from an imbalance in lipid metabolism and a maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Through the analysis of the progression and regression of atherosclerosis in animal models, there is a growing understanding that the balance of macrophages in the plaque is dynamic and that both macrophage numbers and the inflammatory phenotype influence plaque fate. In this Review, we summarize recently identified pro- and anti-inflammatory pathways that link lipid and inflammation biology with the retention of macrophages in plaques, as well as factors that have the potential to promote their egress from these sites.

Myeloperoxidase (MPO) and paraoxonase 1 (PON1) are high-density lipoprotein-associated (HDL-associated) proteins mechanistically linked to inflammation, oxidant stress, and atherosclerosis. MPO is a source of ROS during inflammation and can oxidize apolipoprotein A1 (APOA1) of HDL, impairing its atheroprotective functions. In contrast, PON1 fosters systemic antioxidant effects and promotes some of the atheroprotective properties attributed to HDL. Here, we demonstrate that MPO, PON1, and HDL bind to one another, forming a ternary complex, wherein PON1 partially inhibits MPO activity, while MPO inactivates PON1. MPO oxidizes PON1 on tyrosine 71 (Tyr71), a modified residue found in human atheroma that is critical for HDL binding and PON1 function. Acute inflammation model studies with transgenic and knockout mice for either PON1 or MPO confirmed that MPO and PON1 reciprocally modulate each other's function in vivo. Further structure and function studies identified critical contact sites between APOA1 within HDL, PON1, and MPO, and proteomics studies of HDL recovered from acute coronary syndrome (ACS) subjects revealed enhanced chlorotyrosine content, site-specific PON1 methionine oxidation, and reduced PON1 activity. HDL thus serves as a scaffold upon which MPO and PON1 interact during inflammation, whereupon PON1 binding partially inhibits MPO activity, and MPO promotes site-specific oxidative modification and impairment of PON1 and APOA1 function.

Our lab has previously shown in a mouse model that normalization of a low HDL level achieves atherosclerotic plaque regression. This included the shift from a pro ("M1") to an anti-inflammatory ("M2") phenotypic state of plaque macrophages. Whether HDL can directly cause this phenotypic change and, if so, what the signaling mechanism is, were explored in the present studies. Murine primary macrophages treated with HDL showed increased gene expression for the M2 markers Arginase-1 (Arg-1) and Fizz-1, which are classically induced by IL-4. HDL was able to potentiate the IL-4-induced changes in Arg-1, and tended to do the same for Fizz-1, while suppressing the expression of inflammatory genes in response to IFNγ. The effects of either IL-4 or HDL were suppressed when macrophages were from STAT6(-/-) mice, but inhibitor studies suggested differential utilization of JAK isoforms by IL-4 and HDL to activate STAT6 by phosphorylation. Overall, our results describe a new function of HDL, namely its ability to directly enrich macrophages in markers of the M2, anti-inflammatory, state in a process requiring STAT6.

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.

Objective: Diminished cholesterol efflux activity of apolipoprotein B (apoB)-depleted serum is associated with prevalent coronary artery disease, but its prognostic value for incident cardiovascular events is unclear. We investigated the relationship of cholesterol efflux activity with both prevalent coronary artery disease and incident development of major adverse cardiovascular events (death, myocardial infarction, or stroke)., Approach and Results: Cholesterol efflux activity from free cholesterol-enriched macrophages was measured in 2 case-control cohorts: (1) an angiographic cohort (n=1150) comprising stable subjects undergoing elective diagnostic coronary angiography and (2) an outpatient cohort (n=577). Analysis of media from cholesterol efflux assays revealed that the high-density lipoprotein fraction (1.063

We have previously reported enhancing the imaging of atherosclerotic plaques in mice using reconstituted high density lipoproteins (HDL) as nanocarriers for the MRI contrast agent gadolinium (Gd). This study focuses on the underlying mechanisms of Gd delivery to atherosclerotic plaques. HDL, LDL, and VLDL particles containing Gd chelated to phosphatidyl ethanolamine (DTPA-DMPE) and a lipidic fluorophore were used to demonstrate the transfer of Gd-phospholipids among plasma lipoproteins in vitro and in vivo. To determine the basis of this transfer, the roles of phospholipid transfer protein (PLTP) and lipoprotein lipase (LpL) in mediating the migration of Gd-DTPA-DMPE among lipoproteins were investigated. The results indicated that neither was an important factor, suggesting that spontaneous transfer of Gd-DTPA-DMPE was the most probable mechanism. Finally, two independent mouse models were used to quantify the relative contributions of HDL and LDL reconstituted with Gd-DTPA-DMPE to plaque imaging enhancement by MR. Both sets of results suggested that Gd-DTPA-DMPE originally associated with LDL was about twice as effective as that injected in the form of Gd-HDL, and that some of Gd-HDL's effectiveness in vivo is indirect through transfer of the imaging agent to LDL. In conclusion, the fate of Gd-DTPA-DMPE associated with a particular type of lipoprotein is complex, and includes its transfer to other lipoprotein species that are then cleared from the plasma into tissues.

Objective: Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 (Ntn1) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells., Approach and Results: We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro, Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1α, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1- and Unc5b-promoter activities are induced by oxidized low-density lipoprotein and require HIF-1α. Correspondingly, J774 macrophages overexpressing active HIF-1α show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1α-dependent manner., Conclusions: These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1α-induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.

Rationale: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking., Objective: To assess the role of macrophage cholesterol efflux pathways in atherogenesis., Methods and Results: We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC(DKO) mice) but not in hematopoietic stem or progenitor populations. MAC-ABC(DKO) bone marrow (BM) was transplanted into Ldlr(-/-) recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet-fed MAC-ABC(DKO) BM-transplanted Ldlr(-/-) mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production., Conclusions: These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Diabetes is a major risk factor for atherosclerosis. Although atherosclerosis is initiated by deposition of cholesterol-rich lipoproteins in the artery wall, the entry of inflammatory leukocytes into lesions fuels disease progression and impairs resolution. We show that diabetic mice have increased numbers of circulating neutrophils and Ly6-C(hi) monocytes, reflecting hyperglycemia-induced proliferation and expansion of bone marrow myeloid progenitors and release of monocytes into the circulation. Increased neutrophil production of S100A8/S100A9, and its subsequent interaction with the receptor for advanced glycation end products on common myeloid progenitor cells, leads to enhanced myelopoiesis. Treatment of hyperglycemia reduces monocytosis, entry of monocytes into atherosclerotic lesions, and promotes regression. In patients with type 1 diabetes, plasma S100A8/S100A9 levels correlate with leukocyte counts and coronary artery disease. Thus, hyperglycemia drives myelopoiesis and promotes atherogenesis in diabetes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: The persistence of myeloid-derived cells in the artery wall is a characteristic of advanced atherosclerotic plaques. However, the mechanisms by which these cells are retained are poorly understood. Semaphorins, a class of neuronal guidance molecules, play a critical role in vascular patterning and development, and recent studies suggest that they may also have immunomodulatory functions. The present study evaluates the expression of Semaphorin 3E (Sema3E) in settings relevant to atherosclerosis and its contribution to macrophage accumulation in plaques., Approach and Results: Immunofluorescence staining of Sema3E, and its receptor PlexinD1, demonstrated their expression in macrophages of advanced atherosclerotic lesions of Apoe(-/-) mice. Notably, in 2 different mouse models of atherosclerosis regression, Sema3E mRNA was highly downregulated in plaque macrophages, coincident with a reduction in plaque macrophage content and an enrichment in markers of reparative M2 macrophages. In vitro, Sema3E mRNA was highly expressed in inflammatory M1 macrophages and in macrophages treated with physiological drivers of plaque progression and inflammation, such as oxidized low-density lipoprotein and hypoxia. To explore mechanistically how Sema3E affects macrophage behavior, we treated macrophages with recombinant protein in the presence/absence of chemokines, including CCL19, a chemokine implicated in the egress of macrophages from atherosclerotic plaques. Sema3E blocked actin polymerization and macrophage migration stimulated by the chemokines, suggesting that it may immobilize these cells in the plaque., Conclusions: Sema3E is upregulated in macrophages of advanced plaques, is dynamically regulated by multiple atherosclerosis-relevant factors, and acts as a negative regulator of macrophage migration, which may promote macrophage retention and chronic inflammation in vivo.