Vitamin A mediates conversion of monocyte-derived macrophages into tissue-resident macrophages during alternative activation.

It remains unclear whether activated inflammatory macrophages can adopt features of tissue-resident macrophages, or what mechanisms might mediate such a phenotypic conversion. Here we show that vitamin A is required for the phenotypic conversion of interleukin 4 (IL-4)-activated monocyte-derived F4/80intCD206+PD-L2+MHCII+ macrophages into macrophages with a tissue-resident F4/80hiCD206-PD-L2-MHCII-UCP1+ phenotype in the peritoneal cavity of mice and during the formation of liver granulomas in mice infected with Schistosoma mansoni. The phenotypic conversion of F4/80intCD206+ macrophages into F4/80hiCD206- macrophages was associated with almost complete remodeling of the chromatin landscape, as well as alteration of the transcriptional profiles. Vitamin A-deficient mice infected with S. mansoni had disrupted liver granuloma architecture and increased mortality, which indicates that failure to convert macrophages from the F4/80intCD206+ phenotype to F4/80hiCD206- may lead to dysregulated inflammation during helminth infection.

Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism.

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

Autophagy Is Differentially Regulated in Leukocyte and Nonleukocyte Foam Cells During Atherosclerosis.

RATIONALE: Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. OBJECTIVE: We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts. METHODS AND RESULTS: Atherosclerosis was induced in GFP-LC3 (microtubule-associated proteins 1A/1B light chain 3) transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMCs and macrophages, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) was used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells. CONCLUSIONS: These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.

miR-223 Exerts Translational Control of Proatherogenic Genes in Macrophages.

BACKGROUND: A significant burden of atherosclerotic disease is driven by inflammation. Recently, microRNAs (miRNAs) have emerged as important factors driving and protecting from atherosclerosis. miR-223 regulates cholesterol metabolism and inflammation via targeting both cholesterol biosynthesis pathway and NFkB signaling pathways; however, its role in atherosclerosis has not been investigated. We hypothesize that miR-223 globally regulates core inflammatory pathways in macrophages in response to inflammatory and atherogenic stimuli thus limiting the progression of atherosclerosis. METHODS AND RESULTS: Loss of miR-223 in macrophages decreases Abca1 gene and protein expression as well as cholesterol efflux to apoA1 (Apolipoprotein A1) and enhances proinflammatory gene expression. In contrast, overexpression of miR-223 promotes the efflux of cholesterol and macrophage polarization toward an anti-inflammatory phenotype. These beneficial effects of miR-223 are dependent on its target gene, the transcription factor Sp3. Consistent with the antiatherogenic effects of miR-223 in vitro, mice receiving miR223-/- bone marrow exhibit increased plaque size, lipid content, and circulating inflammatory cytokines (ie, IL-1ß). Deficiency of miR-223 in bone marrow-derived cells also results in an increase in circulating pro-atherogenic cells (total monocytes and neutrophils) compared with control mice. Furthermore, the expression of miR-223 target gene (Sp3) and pro-inflammatory marker (Il-6) are enhanced whereas the expression of Abca1 and anti-inflammatory marker (Retnla) are reduced in aortic arches from mice lacking miR-223 in bone marrow-derived cells. In mice fed a high-cholesterol diet and in humans with unstable carotid atherosclerosis, the expression of miR-223 is increased. To further understand the molecular mechanisms underlying the effect of miR-223 on atherosclerosis in vivo, we characterized global RNA translation profile of macrophages isolated from mice receiving wild-type or miR223-/- bone marrow. Using ribosome profiling, we reveal a notable upregulation of inflammatory signaling and lipid metabolism at the translation level but less significant at the transcription level. Analysis of upregulated genes at the translation level reveal an enrichment of miR-223-binding sites, confirming that miR-223 exerts significant changes in target genes in atherogenic macrophages via altering their translation. CONCLUSIONS: Our study demonstrates that miR-223 can protect against atherosclerosis by acting as a global regulator of RNA translation of cholesterol efflux and inflammation pathways.

Lipophagy pathways in yeast are controlled by their distinct modes of induction.

Lipid droplet (LD) autophagy (lipophagy) is a recently discovered selective form of autophagy and is a pathway for LD catabolism. This ubiquitous process has been an ongoing area of research within the budding yeast, Saccharomyces cerevisiae. Yeast lipophagy phenotypically resembles microautophagy, although it has a distinct set of genetic requirements depending on the mode of induction. This review highlights the similarities and differences between different forms of yeast lipophagy and offers perspectives on how our knowledge of lipophagy in yeast may guide our understanding of this process within mammalian cells to ultimately inform future applications of lipophagy.

Building Your Mentoring Network in Your Early Career.

The pursuit of knowledge, curiosity about the natural world, and a drive to better the human condition are several of the many motivations that encourage someone to further their education in the biological sciences. However noble the intentions, success in an academic graduate program, and perhaps more importantly, in the career options that follow, is not guaranteed. While it is often said that a trainee needs support from their mentors and network to succeed, the Arteriosclerosis, Thrombosis and Vascular Biology Early Career Committee has observed, through our many interactions, both face to face and in the virtual space, that many trainees do not appreciate that building their mentoring network is an active process, and the trainee has more agency in the relationship than perhaps they perceive. In the article below, we discuss our views on building relationships and identifying mentors at different levels and for different purposes. We also highlight events hosted by the Arteriosclerosis, Thrombosis and Vascular Biology Early Career Committee at Vascular Discoveries, Scientific Sessions, and in the virtual space that can help you at the critical career stage.

What Makes a Great Mentor: Interviews With Recipients of the ATVB Mentor of Women Award.

[Figure: see text].

HDL and Reverse Cholesterol Transport.

Cardiovascular disease, with atherosclerosis as the major underlying factor, remains the leading cause of death worldwide. It is well established that cholesterol ester-enriched foam cells are the hallmark of atherosclerotic plaques. Multiple lines of evidence support that enhancing foam cell cholesterol efflux by HDL (high-density lipoprotein) particles, the first step of reverse cholesterol transport (RCT), is a promising antiatherogenic strategy. Yet, excitement towards the therapeutic potential of manipulating RCT for the treatment of cardiovascular disease has faded because of the lack of the association between cardiovascular disease risk and what was typically measured in intervention trials, namely HDL cholesterol, which has an inconsistent relationship to HDL function and RCT. In this review, we will summarize some of the potential reasons for this inconsistency, update the mechanisms of RCT, and highlight conditions in which impaired HDL function or RCT contributes to vascular disease. On balance, the evidence still argues for further research to better understand how HDL functionality contributes to RCT to develop prevention and treatment strategies to reduce the risk of cardiovascular disease.

Loss of MLKL (Mixed Lineage Kinase Domain-Like Protein) Decreases Necrotic Core but Increases Macrophage Lipid Accumulation in Atherosclerosis.

OBJECTIVES: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe-knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. CONCLUSIONS: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.

Foam Cell Induction Activates AMPK But Uncouples Its Regulation of Autophagy and Lysosomal Homeostasis.

The dysregulation of macrophage lipid metabolism drives atherosclerosis. AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics and plays essential roles regulating macrophage lipid dynamics. Here, we investigated the consequences of atherogenic lipoprotein-induced foam cell formation on downstream immunometabolic signaling in primary mouse macrophages. A variety of atherogenic low-density lipoproteins (acetylated, oxidized, and aggregated forms) activated AMPK signaling in a manner that was in part due to CD36 and calcium-related signaling. In quiescent macrophages, basal AMPK signaling was crucial for maintaining markers of lysosomal homeostasis as well as levels of key components in the lysosomal expression and regulation network. Moreover, AMPK activation resulted in targeted upregulation of members of this network via transcription factor EB. However, in lipid-induced macrophage foam cells, neither basal AMPK signaling nor its activation affected lysosomal-associated programs. These results suggest that while the sum of AMPK signaling in cultured macrophages may be anti-atherogenic, atherosclerotic input dampens the regulatory capacity of AMPK signaling.

Cholesterol Efflux Pathways Suppress Inflammasome Activation, NETosis, and Atherogenesis.

BACKGROUND: The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) showed that antagonism of interleukin (IL)-1ß reduces coronary heart disease in patients with a previous myocardial infarction and evidence of systemic inflammation, indicating that pathways required for IL-1ß secretion increase cardiovascular risk. IL-1ß and IL-18 are produced via the NLRP3 inflammasome in myeloid cells in response to cholesterol accumulation, but mechanisms linking NLRP3 inflammasome activation to atherogenesis are unclear. The cholesterol transporters ATP binding cassette A1 and G1 (ABCA1/G1) mediate cholesterol efflux to high-density lipoprotein, and Abca1/g1 deficiency in myeloid cells leads to cholesterol accumulation. METHODS: To interrogate mechanisms connecting inflammasome activation with atherogenesis, we used mice with myeloid Abca1/g1 deficiency and concomitant deficiency of the inflammasome components Nlrp3 or Caspase-1/11. Bone marrow from these mice was transplanted into Ldlr-/- recipients, which were fed a Western-type diet. RESULTS: Myeloid Abca1/g1 deficiency increased plasma IL-18 levels in Ldlr-/- mice and induced IL-1ß and IL-18 secretion in splenocytes, which was reversed by Nlrp3 or Caspase-1/11 deficiency, indicating activation of the NLRP3 inflammasome. Nlrp3 or Caspase-1/11 deficiency decreased atherosclerotic lesion size in myeloid Abca1/g1-deficient Ldlr-/- mice. Myeloid Abca1/g1 deficiency enhanced caspase-1 cleavage not only in splenic monocytes and macrophages, but also in neutrophils, and dramatically enhanced neutrophil accumulation and neutrophil extracellular trap formation in atherosclerotic plaques, with reversal by Nlrp3 or Caspase-1/11 deficiency, suggesting that inflammasome activation promotes neutrophil recruitment and neutrophil extracellular trap formation in atherosclerotic plaques. These effects appeared to be indirectly mediated by systemic inflammation leading to activation and accumulation of neutrophils in plaques. Myeloid Abca1/g1 deficiency also activated the noncanonical inflammasome, causing increased susceptibility to lipopolysaccharide-induced mortality. Patients with Tangier disease, who carry loss-of-function mutations in ABCA1 and have increased myeloid cholesterol content, showed a marked increase in plasma IL-1ß and IL-18 levels. CONCLUSIONS: Cholesterol accumulation in myeloid cells activates the NLRP3 inflammasome, which enhances neutrophil accumulation and neutrophil extracellular trap formation in atherosclerotic plaques. Patients with Tangier disease, who have increased myeloid cholesterol content, showed markers of inflammasome activation, suggesting human relevance.

Immune cell screening of a nanoparticle library improves atherosclerosis therapy.

Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. We here propose a radically different approach: implementing and evaluating in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library's nanoparticles are based on endogenous high-density lipoprotein, which can preferentially deliver therapeutic compounds to pathological macrophages in atherosclerosis. Using the apolipoprotein E-deficient (Apoe-/-) mouse model of atherosclerosis, we quantitatively evaluated the library's immune cell specificity by combining immunological techniques and in vivo positron emission tomography imaging. Based on this screen, we formulated a liver X receptor agonist (GW3965) and abolished its liver toxicity while still preserving its therapeutic function. Screening the immune cell specificity of nanoparticles can be used to develop tailored therapies for atherosclerosis and other inflammatory diseases.

microRNA-33 Regulates Macrophage Autophagy in Atherosclerosis.

OBJECTIVE: Defective autophagy in macrophages leads to pathological processes that contribute to atherosclerosis, including impaired cholesterol metabolism and defective efferocytosis. Autophagy promotes the degradation of cytoplasmic components in lysosomes and plays a key role in the catabolism of stored lipids to maintain cellular homeostasis. microRNA-33 (miR-33) is a post-transcriptional regulator of genes involved in cholesterol homeostasis, yet the complete mechanisms by which miR-33 controls lipid metabolism are unknown. We investigated whether miR-33 targeting of autophagy contributes to its regulation of cholesterol homeostasis and atherogenesis. APPROACH AND RESULTS: Using coherent anti-Stokes Raman scattering microscopy, we show that miR-33 drives lipid droplet accumulation in macrophages, suggesting decreased lipolysis. Inhibition of neutral and lysosomal hydrolysis pathways revealed that miR-33 reduced cholesterol mobilization by a lysosomal-dependent mechanism, implicating repression of autophagy. Indeed, we show that miR-33 targets key autophagy regulators and effectors in macrophages to reduce lipid droplet catabolism, an essential process to generate free cholesterol for efflux. Notably, miR-33 regulation of autophagy lies upstream of its known effects on ABCA1 (ATP-binding cassette transporter A1)-dependent cholesterol efflux, as miR-33 inhibitors fail to increase efflux upon genetic or chemical inhibition of autophagy. Furthermore, we find that miR-33 inhibits apoptotic cell clearance via an autophagy-dependent mechanism. Macrophages treated with anti-miR-33 show increased efferocytosis, lysosomal biogenesis, and degradation of apoptotic material. Finally, we show that treating atherosclerotic Ldlr-/- mice with anti-miR-33 restores defective autophagy in macrophage foam cells and plaques and promotes apoptotic cell clearance to reduce plaque necrosis. CONCLUSIONS: Collectively, these data provide insight into the mechanisms by which miR-33 regulates cellular cholesterol homeostasis and atherosclerosis.

Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages.

Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRß. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.

IL-19 Halts Progression of Atherosclerotic Plaque, Polarizes, and Increases Cholesterol Uptake and Efflux in Macrophages.

Atherosclerosis regression is an important clinical goal, and treatments that can reverse atherosclerotic plaque formation are actively being sought. Our aim was to determine whether administration of exogenous IL-19, a Th2 cytokine, could attenuate progression of preformed atherosclerotic plaque and to identify molecular mechanisms. LDLR(-/-) mice were fed a Western diet for 12 weeks, then administered rIL-19 or phosphate-buffered saline concomitant with Western diet for an additional 8 weeks. Analysis of atherosclerosis burden showed that IL-19-treated mice were similar to baseline, in contrast to control mice which showed a 54% increase in plaque, suggesting that IL-19 halted the progression of atherosclerosis. Plaque characterization showed that IL-19-treated mice had key features of atherosclerosis regression, including a reduction in macrophage content and an enrichment in markers of M2 macrophages. Mechanistic studies revealed that IL-19 promotes the activation of key pathways leading to M2 macrophage polarization, including STAT3, STAT6, Kruppel-like factor 4, and peroxisome proliferator-activated receptor γ, and can reduce cytokine-induced inflammation in vivo. We identified a novel role for IL-19 in regulating macrophage lipid metabolism through peroxisome proliferator-activated receptor γ-dependent regulation of scavenger receptor-mediated cholesterol uptake and ABCA1-mediated cholesterol efflux. These data show that IL-19 can halt progression of preformed atherosclerotic plaques by regulating both macrophage inflammation and cholesterol homeostasis and implicate IL-19 as a link between inflammation and macrophage cholesterol metabolism.

Macrophage Mitochondrial Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis.

RATIONALE: Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis. Macrophage energy metabolism can significantly influence macrophage phenotype, but how this is controlled in foam cells is not known. Bioinformatic pathway analysis predicts that miR-33 represses a cluster of genes controlling cellular energy metabolism that may be important in macrophage cholesterol efflux. OBJECTIVE: We hypothesized that cellular energy status can influence cholesterol efflux from macrophages, and that miR-33 reduces cholesterol efflux via repression of mitochondrial energy metabolism pathways. METHODS AND RESULTS: In this study, we demonstrated that macrophage cholesterol efflux is regulated by mitochondrial ATP production, and that miR-33 controls a network of genes that synchronize mitochondrial function. Inhibition of mitochondrial ATP synthase markedly reduces macrophage cholesterol efflux capacity, and anti-miR33 required fully functional mitochondria to enhance ABCA1-mediated cholesterol efflux. Specifically, anti-miR33 derepressed the novel target genes PGC-1α, PDK4, and SLC25A25 and boosted mitochondrial respiration and production of ATP. Treatment of atherosclerotic Apoe(-/-) mice with anti-miR33 oligonucleotides reduced aortic sinus lesion area compared with controls, despite no changes in high-density lipoprotein cholesterol or other circulating lipids. Expression of miR-33a/b was markedly increased in human carotid atherosclerotic plaques compared with normal arteries, and there was a concomitant decrease in mitochondrial regulatory genes PGC-1α, SLC25A25, NRF1, and TFAM, suggesting these genes are associated with advanced atherosclerosis in humans. CONCLUSIONS: This study demonstrates that anti-miR33 therapy derepresses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, works to promote macrophage cholesterol efflux and reduce atherosclerosis.

miRNA Targeting of Oxysterol-Binding Protein-Like 6 Regulates Cholesterol Trafficking and Efflux.

OBJECTIVE: Cholesterol homeostasis is fundamental to human health and is, thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. APPROACH AND RESULTS: Here, we identify oxysterol-binding protein-like 6 (OSBPL6) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by liver X receptor and in response to cholesterol loading and in mice and nonhuman primates by Western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains dual membrane- and endoplasmic reticulum-targeting motifs. Subcellular localization studies showed that ORP6 is associated with the endolysosomal network and endoplasmic reticulum, suggesting a role for ORP6 in cholesterol trafficking between these compartments. Accordingly, knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures, resulting in reduced cholesterol esterification at the endoplasmic reticulum. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of high-density lipoprotein cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. CONCLUSIONS: These studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

Cholesterol loading reprograms the microRNA-143/145-myocardin axis to convert aortic smooth muscle cells to a dysfunctional macrophage-like phenotype.

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.

HDL-mimetic PLGA nanoparticle to target atherosclerosis plaque macrophages.

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.