RFX1 regulates foam cell formation and atherosclerosis by mediating CD36 expression.

BACKGROUND AND AIMS: Atherosclerosis (AS) is a continuously low-grade inflammatory disease, and monocyte-derived macrophages play a vital role in AS pathogenesis. Regulatory factor X1 (RFX1) has been reported to participate in differentiation of various cells. Our previous report showed that RFX1 expression in CD14+ monocytes from AS patients was decreased and closely related to AS development. Macrophages mostly derive from monocytes and play an important role in AS plaque formation and stability. However, the functions of RFX1 in the formation of macrophage-derived foam cells and consequent AS development are unclear. METHODS: We explored the effects of RFX1 on oxidation low lipoprotein (ox-LDL)-stimulated foam cell formation and CD36 expression by increasing or silencing Rfx1 expression in mouse peritoneal macrophages (PMAs). The ApoE-/-Rfx1f/f or ApoE-/-Rfx1f/f Lyz2-Cre mice fed a high-fat diet for 24 weeks were used to further examine the effect of RFX1 on AS pathogenesis. We then performed dual luciferase reporter assays to study the regulation of RFX1 for CD36 transcription. RESULTS: Our results demonstrate that RFX1 expression was significantly reduced in ox-LDL induced foam cells and negatively correlated with lipid uptake in macrophages. Besides, Rfx1 deficiency in myeloid cells aggravated atherosclerotic lesions in ApoE-/- mice. Mechanistically, RFX1 inhibited CD36 expression by directly regulating CD36 transcription in macrophages. CONCLUSIONS: The reduction of RFX1 expression in macrophages is a vital determinant for foam cell formation and the initiation of AS, proving a potential novel approach for the treatment of AS disease.

AtheroSpectrum Reveals Novel Macrophage Foam Cell Gene Signatures Associated With Atherosclerotic Cardiovascular Disease Risk.

BACKGROUND: Whereas several interventions can effectively lower lipid levels in people at risk for atherosclerotic cardiovascular disease (ASCVD), cardiovascular event risks remain, suggesting an unmet medical need to identify factors contributing to cardiovascular event risk. Monocytes and macrophages play central roles in atherosclerosis, but studies have yet to provide a detailed view of macrophage populations involved in increased ASCVD risk. METHODS: A novel macrophage foaming analytics tool, AtheroSpectrum, was developed using 2 quantitative indices depicting lipid metabolism and the inflammatory status of macrophages. A machine learning algorithm was developed to analyze gene expression patterns in the peripheral monocyte transcriptome of MESA participants (Multi-Ethnic Study of Atherosclerosis; set 1; n=911). A list of 30 genes was generated and integrated with traditional risk factors to create an ASCVD risk prediction model (30-gene cardiovascular disease risk score [CR-30]), which was subsequently validated in the remaining MESA participants (set 2; n=228); performance of CR-30 was also tested in 2 independent human atherosclerotic tissue transcriptome data sets (GTEx [Genotype-Tissue Expression] and GSE43292). RESULTS: Using single-cell transcriptomic profiles (GSE97310, GSE116240, GSE97941, and FR-FCM-Z23S), AtheroSpectrum detected 2 distinct programs in plaque macrophages-homeostatic foaming and inflammatory pathogenic foaming-the latter of which was positively associated with severity of atherosclerosis in multiple studies. A pool of 2209 pathogenic foaming genes was extracted and screened to select a subset of 30 genes correlated with cardiovascular event in MESA set 1. A cardiovascular disease risk score model (CR-30) was then developed by incorporating this gene set with traditional variables sensitive to cardiovascular event in MESA set 1 after cross-validation generalizability analysis. The performance of CR-30 was then tested in MESA set 2 (P=2.60×10-4; area under the receiver operating characteristic curve, 0.742) and 2 independent data sets (GTEx: P=7.32×10-17; area under the receiver operating characteristic curve, 0.664; GSE43292: P=7.04×10-2; area under the receiver operating characteristic curve, 0.633). Model sensitivity tests confirmed the contribution of the 30-gene panel to the prediction model (likelihood ratio test; df=31, P=0.03). CONCLUSIONS: Our novel computational program (AtheroSpectrum) identified a specific gene expression profile associated with inflammatory macrophage foam cells. A subset of 30 genes expressed in circulating monocytes jointly contributed to prediction of symptomatic atherosclerotic vascular disease. Incorporating a pathogenic foaming gene set with known risk factors can significantly strengthen the power to predict ASCVD risk. Our programs may facilitate both mechanistic investigations and development of therapeutic and prognostic strategies for ASCVD risk.

PAK1 Silencing Attenuated Proinflammatory Macrophage Activation and Foam Cell Formation by Increasing PPARγ Expression.

Macrophage polarization in response to environmental cues has emerged as an important event in the development of atherosclerosis. Compelling evidences suggest that P21-activated kinases 1 (PAK1) is involved in a wide variety of diseases. However, the potential role and mechanism of PAK1 in regulation of macrophage polarization remains to be elucidated. Here, we observed that PAK1 showed a dramatically increased expression in M1 macrophages but decreased expression in M2 macrophages by using a well-established in vitro model to study heterogeneity of macrophage polarization. Adenovirus-mediated loss-of-function approach demonstrated that PAK1 silencing induced an M2 macrophage phenotype-associated gene profiles but repressed the phenotypic markers related to M1 macrophage polarization. Additionally, dramatically decreased foam cell formation was found in PAK1 silencing-induced M2 macrophage activation which was accompanied with alternation of marker account for cholesterol efflux or influx from macrophage foam cells. Moderate results in lipid metabolism and foam cell formation were found in M1 macrophage activation mediated by AdshPAK1. Importantly, we presented mechanistic evidence that PAK1 knockdown promoted the expression of PPARγ, and the effect of macrophage activation regulated by PAK1 silencing was largely reversed when a PPARγ antagonist was utilized. Collectively, these findings reveal that PAK1 is an independent effector of macrophage polarization at least partially attributed to regulation of PPARγ expression, which suggested PAK1-PPARγ axis as a novel therapeutic strategy in atherosclerosis management.

Liposome interaction with macrophages and foam cells for atherosclerosis treatment: effects of size, surface charge and lipid composition.

Liposomes are potential drug carriers for atherosclerosis therapy due to low immunogenicity and ease of surface modifications that allow them to have prolonged circulation half-life and specifically target atherosclerotic sites to increase uptake efficiency. However, the effects of their size, charge, and lipid compositions on macrophage and foam cell behaviour are not fully understood. In this study, liposomes of different sizes (60 nm, 100 nm and 180 nm), charges (-40 mV, -20 mV, neutral, +15 mV and +30 mV) and lipid compositions (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, L-a-phosphatidylcholine, and egg sphingomyelin) were synthesized, characterized and exposed to macrophages and foam cells. Compared to 100 nm neutral 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes, flow cytometry and confocal imaging indicated that cationic liposomes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPC) liposomes were internalized more by both macrophages and foam cells. Through endocytosis inhibition, phagocytosis and clathrin-mediated endocytosis were identified as the dominant mechanisms of uptake. Anionic and DSPC liposomes induced more cholesterol efflux capacity in foam cells. These results provide a guide for the optimal size, charge, and lipid composition of liposomes as drug carriers for atherosclerosis treatment.

Inhibition of NFAT suppresses foam cell formation and the development of diet-induced atherosclerosis.

Deciphering the molecular and cellular processes involved in foam cell formation is critical for us to understand the pathogenesis of atherosclerosis. Nuclear factor of activated T cells (NFAT) is a transcription factor originally identified as a key player in the differentiation of T cells and maturation of immune system. Nowadays it has been brought into attention that NFAT also regulates multiple pathophysiological processes and targeted intervention in NFAT may be effective in the treatment of some cardiovascular diseases. However, whether NFAT is involved in foam cell formation remains elusive. NFAT in human monocyte-derived macrophage was activated by ox-LDL and translocated from the cytoplasm to the nucleus. NFAT then directly bound to peroxisome proliferator-activated receptor γ (PPARγ) in the nucleus and negatively regulated its transcriptional activity. NFATc2 knockdown or NFAT inhibitor 11R-VIVIT increased cholesterol efflux (by activating PPARγ-LXRα-ABCA1 cascade) and reduced the uptake of modified lipoprotein (in a PPARγ-independent way) in macrophage, thus prevented foam cell formation. Besides, 11R-VIVIT also exerted a protective role in the development of atherosclerosis in western diet-fed ApoE-/- mice. These results suggest NFAT inhibition as a potential therapeutic strategy in atherosclerosis.

Syzygium cumini leaf extract protects macrophages against the oxidized LDL-induced toxicity: A promising atheroprotective effect.

Oxidized LDL (oxLDL) plays a pivotal role on atherosclerosis development, mainly in the formation of lipid-laden macrophage "foam cells". As a consequence, substances that can modulate LDL oxidation have a pharmacological and therapeutic relevance. Based in previous findings showing the ability of Syzigium cumini leaf extract (ScExt) in preventing LDL oxidation in vitro, this study was aimed to assess the effects of ScExt on oxLDL-mediated toxicity in murine J774 macrophages-like cells. For biochemical analyses, LDL isolated from fresh human plasma and oxidized with CuSO4 was incubated with ScExt pre-treated macrophages. Our results demonstrated that ScExt was efficient in preventing the overproduction of reactive oxygen/nitrogen species (ROS/RNS), the loss of macrophage's viability and the foam cells formation induced by oxLDL. These protective effects of ScExt make it a promising antioxidant for future trials toward atherogenesis.

Identification and functional analysis of a biflavone as a novel inhibitor of transient receptor potential vanilloid 4-dependent atherogenic processes.

Atherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Herein, using a fluorometric imaging plate reader-supported Ca2+ influx assay, we report semi high-throughput screening-based identification of ginkgetin, a biflavone, as a novel inhibitor of transient receptor potential vanilloid 4 (TRPV4)-dependent proatherogenic and inflammatory processes in macrophages. We found that ginkgetin (1) blocks TRPV4-elicited Ca2+ influx into macrophages, (2) inhibits oxidized low-density lipoprotein (oxLDL)-induced foam cell formation by suppressing the uptake but not the binding of oxLDL in macrophages, and (3) attenuates oxLDL-induced phosphorylation of JNK2, expression of TRPV4 proteins, and induction of inflammatory mRNAs. Considered all together, the results of this study show that ginkgetin inhibits proatherogenic/inflammatory macrophage function in a TRPV4-dependent manner, thus strengthening the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.

The benzoate plant metabolite ethyl gallate prevents cellular- and vascular-lipid accumulation in experimental models of atherosclerosis.

Ethyl gallate (EG) is a well-known constituent of medicinal plants, but its effects on atherosclerosis development are not clear. In the present study, the anti-atherosclerosis effects of EG and the underlying mechanisms were explored using macrophage cultures, zebrafish and apolipoprotein (apo) E deficient mice. Treatment of macrophages with EG (20 µM) enhanced cellular cholesterol efflux to HDL, and reduced net lipid accumulation in response to oxidized LDL. Secretion of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from activated macrophages was also blunted by EG. Fluorescence imaging techniques revealed EG feeding of zebrafish reduced vascular lipid accumulation and inflammatory responses in vivo. Similar results were obtained in apoE-/- mice 6.5 months of age, where plaque lesions and monocyte infiltration into the artery wall were reduced by 70% and 42%, respectively, after just 6 weeks of injections with EG (20 mg/kg). HDL-cholesterol increased 2-fold, serum cholesterol efflux capacity increased by ∼30%, and the levels of MCP-1 and IL-6 were reduced with EG treatment of mice. These results suggest EG impedes early atherosclerosis development by reducing the lipid and macrophage-content of plaque. Underlying mechanisms appeared to involve HDL cholesterol efflux mechanisms and suppression of pro-inflammatory cytokine secretion.

MicroRNA-328-5p Alleviates Macrophage Lipid Accumulation through the Histone Deacetylase 3/ATP-binding cassette transporter A1 pathway.

MicroRNA-328 (miR-328) was reported to protect against atherosclerosis, but its role in foam cell formation remains unknown. The aim of this study was to investigate the effect of miR-328-5p on macrophage lipid accumulation and the underlying mechanisms. The results showed that miR-328-5p expression was robustly decreased in oxidized low-density lipoprotein (ox-LDL)-treated macrophages. Treatment of human acute monocytic leukemia cel (THP-1) macrophage-derived foam cells with a miR-328-5p mimic markedly increased [3 H]-cholesterol efflux, inhibited lipid droplet accumulation, and decreased intracellular total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) contents. Upregulation of miR-328-5p also reduced the expression of histone deacetylase 3 (HDAC3) but increased the levels of ATP-binding cassette transporter A1 (ABCA1) in THP-1 macrophage-derived foam cells. Mechanistically, miR-328-5p inhibited HDAC3 expression by directly targeting its 3'UTR, thereby promoting ABCA1 expression and the subsequent cholesterol efflux. Furthermore, miR-328-5p mimic treatment did not affect the uptake of Dil-ox-LDL or the expression of scavenger receptor-A (SR-A), thrombospondin receptor (CD36) and ABCG1. Taken together, these findings suggest that miR-328-5p alleviates macrophage lipid accumulation through the HDAC3/ABCA1 pathway.

The vitamin E long-chain metabolite α-13'-COOH affects macrophage foam cell formation via modulation of the lipoprotein lipase system.

The α-tocopherol-derived long-chain metabolite (α-LCM) α-13'-carboxychromanol (α-13'-COOH) is formed via enzymatic degradation of α-tocopherol (α-TOH) in the liver. In the last decade, α-13'-COOH has emerged as a new regulatory metabolite revealing more potent or even different effects compared with its vitamin precursor α-TOH. The detection of α-13'-COOH in human serum has further strengthened the concept of its physiological relevance as a potential regulatory molecule. Here, we present a new facet on the interaction of α-13'-COOH with macrophage foam cell formation. We found that α-13'-COOH (5 µM) increases angiopoietin-like 4 (ANGPTL4) mRNA expression in human THP-1 macrophages in a time- and dose-dependent manner, while α-TOH (100 µM) showed no effects. Interestingly, the mRNA level of lipoprotein lipase (LPL) was not influenced by α-13'-COOH, but α-TOH treatment led to a reduction of LPL mRNA expression. Both compounds also revealed different effects on protein level: while α-13'-COOH reduced the secreted amount of LPL protein via induction of ANGPTL4 cleavage, i.e. activation, the secreted amount of LPL in the α-TOH-treated samples was diminished due to the inhibition of mRNA expression. In line with this, both compounds reduced the catalytic activity of LPL. However, α-13'-COOH but not α-TOH attenuated VLDL-induced lipid accumulation by 35%. In conclusion, only α-13'-COOH revealed possible antiatherogenic effects due to the reduction of VLDL-induced foam cell formation in THP-1 macrophages. Our results provide further evidence for the role of α-13'-COOH as a functional metabolite of its vitamin E precursor.

Aggregated LDL turn human macrophages into foam cells and induce mitochondrial dysfunction without triggering oxidative or endoplasmic reticulum stress.

Uptake of modified lipoproteins by macrophages turns them into foam cells, the hallmark of the atherosclerotic plaque. The initiation and progression of atherosclerosis have been associated with mitochondrial dysfunction. It is known that aggregated low-density lipoproteins (agLDL) induce massive cholesterol accumulation in macrophages in contrast with native LDL (nLDL) and oxidized LDL (oxLDL). In the present study we aimed to assess the effect of agLDL on the mitochondria and ER function in macrophage-derived foam cells, in an attempt to estimate the potential of these cells, known constituents of early fatty streaks, to generate atheroma in the absence of oxidative stress. Results show that agLDL induce excessive accumulation of free (FC) and esterified cholesterol in THP-1 macrophages and determine mitochondrial dysfunction expressed as decreased mitochondrial membrane potential and diminished intracellular ATP levels, without generating mitochondrial reactive oxygen species (ROS) production. AgLDL did not stimulate intracellular ROS (superoxide anion or hydrogen peroxide) production, and did not trigger endoplasmic reticulum stress (ERS) or apoptosis. In contrast to agLDL, oxLDL did not modify FC levels, but stimulated the accumulation of 7-ketocholesterol in the cells, generating oxidative stress which is associated with an increased mitochondrial dysfunction, ERS and apoptosis. Taken together, our results reveal that agLDL induce foam cells formation and mild mitochondrial dysfunction in human macrophages without triggering oxidative or ERS. These data could partially explain the early formation of fatty streaks in the intima of human arteries by interaction of monocyte-derived macrophages with non-oxidatively aggregated LDL generating foam cells, which cannot evolve into atherosclerotic plaques in the absence of the oxidative stress.

Endocytic recycling as cellular trafficking fate of simvastatin-loaded discoidal reconstituted high-density lipoprotein to coordinate cholesterol efflux and drug influx.

Reconstituted high-density lipoproteins (rHDLs) hold promise as nanocarriers for atherosclerosis-targeted delivery, with biofunctions typified by mediating cholesterol efflux. The paradox is how rHDL offloads the delivered drugs into atherosclerotic foam cells, while simultaneously transferring cholesterol out of cells. Herein, simvastatin-loaded discoidal rHDL (ST-d-rHDL), constructed based on established paradigms, was employed to investigate its basic trafficking mechanism in foam cells. As proved, ST-d-rHDL was resecreted via lysosomal and Golgi apparatus-recycling endosome-mediated pathways following clathrin-mediated endocytosis. And the resecretion ratio reached 60% within 6-h chase with excessive ST-d-rHDLs. During the rHDL resecretion, 39% of cellular cholesterol efflux was detected, accompanied by 85% of the encapsulated cargo released intracellularly. Furthermore, the recycling rate was demonstrated to be promoted by smaller rHDL size and higher cellular lipid contents. Collectively, endocytic recycling confers the synergism in ST-d-rHDL to coordinate cholesterol efflux and intracellular drug release, providing new insights into design of biofunctional rHDL.

An advanced method for quantitative measurements of cholesterol crystallization.

BACKGROUND: Cholesterol crystallization within an atherosclerotic plaque significantly contributes to the acceleration of plaque rupture - a problematic event due to the current lack of specific treatments to prevent such formations. Modelling this pathogenic process is also difficult due to the lack of suitable experimental models that enable quantitative analysis of crystal formation and bioactivity screening of potential therapeutic compounds. AIM: To develop an in vitro human cell model of cholesterol crystallization combined with an imaging system that incorporates both quantitative analysis and real-time continuous imaging of cholesterol crystal formation. METHODS AND RESULTS: An enhanced in vitro model of cholesterol crystallization was developed through the use of acetylated low-density lipoprotein (AcLDL) and 7-ketocholesterol as agents of foam cell induction within a human THP-1 monocytic cell line. Advanced confocal and polarizing microscopies were incorporated into the model so as to allow for quantitation of cholesterol crystallization, with the lipid-loaded group producing significantly greater numbers of cholesterol crystals than the untreated group. The utility of this system was also demonstrated by investigating the effects of the cholesterol-lowering drug lovastatin and therapeutic bile compound ursodeoxycholic acid (UDCA), showing that these drugs influence different aspects of cholesterol crystal formation. CONCLUSIONS: The in vitro human THP-1 monocyte model of cholesterol crystallization provides an effective and efficient means of quantitating cholesterol crystallization in the pre-clinical stage of research. The model also allows for the screening of potentially therapeutic compounds that may be used in attenuating or preventing cholesterol crystallization.

CTRP9 alleviates foam cells apoptosis by enhancing cholesterol efflux.

The apoptosis of foam cells leads to instability of atherosclerotic plaques. This study was designed to explore the protective role of CTRP9 in foam cell apoptosis. In our experiment, CTRP9 alleviated foam cell apoptosis. Meanwhile, CTRP9 upregulated the expression of proteins important for cholesterol efflux, such as LXRα, CYP27A1, ABCG1 and ABCA1, and improved cholesterol efflux in foam cells. Moreover, CTRP9 inhibited Wnt3a and ß-catenin expression and ß-catenin nuclear translocation in foam cells. In addition, adenovirus overexpression of Wnt3a abolished the effect of CTRP9 on macrophage apoptosis. Mechanistically, the AMPK inhibitor abolished the effect of CTRP9 on foam cell apoptosis, and downregulation of AdipoR1 by siRNA abrogated the activation of AMPK and the effect of CTRP9 on foam cell apoptosis. We concluded that CTRP9 achieved these protective effects on foam cells through the AdipoR1/AMPK pathway.

Monocyte Recruitment, Specification, and Function in Atherosclerosis.

Atherosclerotic lesions progress through the continued recruitment of circulating blood monocytes that differentiate into macrophages within plaque. Lesion-associated macrophages are the primary immune cells present in plaque, where they take up cholesterol and store lipids in the form of small droplets resulting in a unique morphology termed foam cell. Recent scientific advances have used single-cell gene expression profiling, live-cell imaging, and fate mapping approaches to describe macrophage and monocyte contributions to pro- or anti-inflammatory mechanisms, in addition to functions of motility and proliferation within lesions. Yet, many questions regarding tissue-specific regulation of monocyte-to-macrophage differentiation and the contribution of recruited monocytes at stages of atherosclerotic disease progression remain unknown. In this review, we highlight recent advances regarding the role of monocyte and macrophage dynamics in atherosclerotic disease and identify gaps in knowledge that we hope will allow for advancing therapeutic treatment or prevention strategies for cardiovascular disease.

Modeling early stage atherosclerosis in a primary human vascular microphysiological system.

Novel atherosclerosis models are needed to guide clinical therapy. Here, we report an in vitro model of early atherosclerosis by fabricating and perfusing multi-layer arteriole-scale human tissue-engineered blood vessels (TEBVs) by plastic compression. TEBVs maintain mechanical strength, vasoactivity, and nitric oxide (NO) production for at least 4 weeks. Perfusion of TEBVs at a physiological shear stress with enzyme-modified low-density-lipoprotein (eLDL) with or without TNFα promotes monocyte accumulation, reduces vasoactivity, alters NO production, which leads to endothelial cell activation, monocyte accumulation, foam cell formation and expression of pro-inflammatory cytokines. Removing eLDL leads to recovery of vasoactivity, but not loss of foam cells or recovery of permeability, while pretreatment with lovastatin or the P2Y11 inhibitor NF157 reduces monocyte accumulation and blocks foam cell formation. Perfusion with blood leads to increased monocyte adhesion. This atherosclerosis model can identify the role of drugs on specific vascular functions that cannot be assessed in vivo.

Macrophage polarisation associated with atherosclerosis differentially affects their capacity to handle lipids.

BACKGROUND AND AIMS: Lipid-rich foam cell macrophages drive atherosclerosis via several mechanisms, including inflammation, lipid uptake, lipid deposition and plaque vulnerability. The atheroma environment shapes macrophage function and phenotype; anti-inflammatory macrophages improve plaque stability while pro-inflammatory macrophages promote rupture. Current evidence suggests a variety of macrophage phenotypes occur in atherosclerotic plaques with local lipids, cytokines, oxidised phospholipids and pathogenic stimuli altering their phenotype. In this study, we addressed differential functioning of macrophage phenotypes via a systematic analysis of in vitro polarised, human monocyte-derived macrophage phenotypes, focussing on molecular events that regulate foam-cell formation. METHODS: We examined transcriptomes, protein levels and functionally determined lipid handling and foam cell formation capacity in macrophages polarised with IFNγ+LPS, IL-4, IL-10, oxPAPC and CXCL4. RESULTS: RNA sequencing of differentially polarised macrophages revealed distinct gene expression changes, with enrichment in atherosclerosis and lipid-associated pathways. Analysis of lipid processing activity showed IL-4 and IL-10 macrophages have higher lipid uptake and foam cell formation activities, while inflammatory and oxPAPC macrophages displayed lower foam cell formation. Inflammatory macrophages showed low lipid uptake, while higher lipid uptake in oxPAPC macrophages was matched by increased lipid efflux capacity. CONCLUSIONS: Atherosclerosis-associated macrophage polarisation dramatically affects lipid handling capacity underpinned by major transcriptomic changes and altered protein levels in lipid-handling gene expression. This leads to phenotype-specific differences in LDL uptake, cellular cholesterol levels and cholesterol efflux, informing how the plaque environment influences atherosclerosis progression by influencing macrophage phenotypes.

Sorting Nexin 10 Mediates Metabolic Reprogramming of Macrophages in Atherosclerosis Through the Lyn-Dependent TFEB Signaling Pathway.

RATIONALE: SNX10 (sorting nexin 10) has been reported to play a critical role in regulating macrophage function and lipid metabolism. OBJECTIVE: To investigate the precise role of SNX10 in atherosclerotic diseases and the underlying mechanisms. METHODS AND RESULTS: SNX10 expression was compared between human healthy vessels and carotid atherosclerotic plaques. Myeloid cell-specific SNX10 knockdown mice were crossed onto the APOE-/- (apolipoprotein E) background and atherogenesis (high-cholesterol diet-induced) was monitored for 16 weeks. We found that SNX10 expression was increased in atherosclerotic lesions of aortic specimens from humans and APOE-/- mice. Myeloid cell-specific SNX10 deficiency (Δ knockout [KO]) attenuated atherosclerosis progression in APOE-/- mice. The population of anti-inflammatory monocytes/macrophages was increased in the peripheral blood and atherosclerotic lesions of ΔKO mice. In vitro experiments showed that SNX10 deficiency-inhibited foam cell formation through interrupting the internalization of CD36, which requires the interaction of SNX10 and Lyn-AKT (protein kinase B). The reduced Lyn-AKT activation by SNX10 deficiency promoted the nuclear translocation of TFEB (transcription factor EB), thereby enhanced lysosomal biogenesis and LAL (lysosomal acid lipase) activity, resulting in an increase of free fatty acids to fuel mitochondrial fatty acid oxidation. This further promoted the reprogramming of macrophages and shifted toward the anti-inflammatory phenotype. CONCLUSIONS: Our data demonstrate for the first time that SNX10 plays a crucial role in diet-induced atherogenesis via the previously unknown link between the Lyn-Akt-TFEB signaling pathway and macrophage reprogramming, suggest that SNX10 may be a potentially promising therapeutic target for atherosclerosis treatment.

Myeloid-specific deficiency of pregnane X receptor decreases atherosclerosis in LDL receptor-deficient mice.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXRΔMyeLDLR-/-) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXRΔMyeLDLR-/- mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXRΔMyeLDLR-/- mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Isoborneol Attenuates Low-Density Lipoprotein Accumulation and Foam Cell Formation in Macrophages.

PURPOSE: Isoborneol has been used in the treatment of cardiovascular disease for several years in China. However, the mechanism is still unclear. The aim of this study was to identify the novel mechanism of isoborneol for its application in atherosclerotic disease. MATERIALS AND METHODS: The whole-genome gene expression profiles of MCF-7 cells treated with/or without isoborneol were detected by mRNA microarray analysis. The degree of similarity between the gene expression profiles was compared with the Connectivity Map (CMAP) database. An MTT assay was used to assess the toxicity of isoborneol on RAW 264.7 cells. Oil red O staining and a Dil-ox-LDL uptake assay in RAW 264.7 cells were also used to detect the accumulation of lipids in the macrophages and the uptake of oxidized low-density lipoprotein (ox-LDL). RESULTS: Isoborneol was proved to have mRNA expression profiles similar to that of ikarugamycin which can inhibit the uptake of ox-LDL. This process has proved to be an important cause of foam cell formation and early atherosclerotic lesions. It is speculated, therefore, that isoborneol may show similar activity to that shown by ikarugamycin. Subsequently, it was shown that RAW 264.7 cells reduced the absorption of ox-LDL and the accumulation of intracellular lipids after treatment with different concentrations of isoborneol. CONCLUSION: The results indicate that isoborneol inhibits macrophage consumption of ox-LDL, thereby preventing the accumulation of lipids in the macrophages. These results provide evidence for the application of isoborneol in atherosclerotic disease.