Plaque Macrophages Research Articles

SIRT1-autophagy axis inhibits excess iron-induced ferroptosis of foam cells and subsequently increases IL-1Β and IL-18

ObjectiveFerroptosis is a new form of programmed cell death characterized by an iron-dependent increase in lipid ROS. It has recently been reported that elevated iron levels in macrophages in plaques are associated with atherosclerosis(AS). However, it is not clear whether iron induces ferroptosis and the mechanism of ferroptosis induced by iron in macrophages in plaque. MethodsTHP-1 macrophages were treated with ox-LDL and ferric ammonium citrate(FAC). Activate SIRT1 using SRT1720. Use of RAPA and CQ to promote and suppress autophagy. The expression of SIRT1, GPX4 was detected by Western Blot, and the cell activity and lipid ROS level were also performed. IL-1β and IL-18 levels were measured using qRT-PCR and ELISA. ResultsIn this study, we determined that FAC can induce a decrease in foam cell activity rather than macrophage activity, increase lipid ROS levels, decrease GPX4 expression and inhibit SIRT1 expression, and increase IL-1β and IL-18 levels. SRT1720 activated SIRT1 and reversed the above changes induced by FAC. CQ partially prevents the above changes caused by activating SIRT1. ConclusionActivation of SIRT1 can inhibit the ferroptosis and IL-1β and IL-18 levels of foam cells in excess iron by autophagy, providing a novel therapeutic target for AS.

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters.

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.

Macrophage Plasticity and Atherosclerosis Therapy.

Atherosclerosis is a chronic disease starting with the entry of monocytes into the subendothelium and the subsequent differentiation into macrophages. Macrophages are the major immune cells in atherosclerotic plaques and are involved in the dynamic progression of atherosclerotic plaques. The biological properties of atherosclerotic plaque macrophages determine lesion size, composition, and stability. The heterogenicity and plasticity of atherosclerotic macrophages have been a hotspot in recent years. Studies demonstrated that lipids, cytokines, chemokines, and other molecules in the atherosclerotic plaque microenvironment regulate macrophage phenotype, contributing to the switch of macrophages toward a pro- or anti-atherosclerosis state. Of note, M1/M2 classification is oversimplified and only represent two extreme states of macrophages. Moreover, M2 macrophages in atherosclerosis are not always protective. Understanding the phenotypic diversity and functions of macrophages can disclose their roles in atherosclerotic plaques. Given that lipid-lowering therapy cannot completely retard the progression of atherosclerosis, macrophages with high heterogeneity and plasticity raise the hope for atherosclerosis regression. This review will focus on the macrophage phenotypic diversity, its role in the progression of the dynamic atherosclerotic plaque, and finally discuss the possibility of treating atherosclerosis by targeting macrophage microenvironment.

Augmenting ATG14 alleviates atherosclerosis and inhibits inflammation via promotion of autophagosome-lysosome fusion in macrophages

ABSTRACT Dysfunction of macroautophagy/autophagy in macrophages contributes to atherosclerosis. Impaired autophagy-lysosomal degradation system leads to lipid accumulation, facilitating atherosclerotic plaque. ATG14 is an essential regulator for the fusion of autophagosomes with lysosomes. Whether ATG14 plays a role in macrophage autophagy dysfunction in atherosclerosis is unknown. To investigate the effects of ATG14 on macrophage autophagy, human atherosclerotic plaque, apoe−/- mice and cultured mouse macrophages were evaluated. Overexpression of ATG14 by adenovirus was used to reveal its function in autophagy, inflammation and atherosclerotic plaque formation. Results showed that impaired autophagy function with reduction of ATG14 expression existed in macrophages of human and mouse atherosclerotic plaques. Ox-LDL impaired autophagosome-lysosome fusion with reduction of ATG14 expression in macrophages. Overexpression of ATG14 in macrophages enhanced fusion of autophagosomes with lysosomes and promoted lipid degradation, decreasing Ox-LDL-induced apoptosis and inflammatory response. Augmenting ATG14 expression reversed the autophagy dysfunction in macrophages of apoe−/- mice plaque, blunted SQSTM1/p62 accumulation, inhibited inflammation, and upregulated the population of Treg cells, resulting in alleviating atherosclerotic lesions. Abbreviations: ABCC1: ATP-binding cassette, sub-family C (CFTR/MRP), member 1; ABCA1: ATP-binding cassette, sub-family A (ABC1), member 1; Ad-Atg14: adenovirus vector carrying the mouse Atg14 gene; Ad-LacZ: adenovirus vector carrying the gene for bacterial β-galactosidase; apoe−/- : apolipoprotein E knockout; ATG14: autophagy-related 14; CD68: CD68 antigen; DAPI: 4ʹ,6-diamidino-2-phenylindole; Dil-ox-LDL: Dil-oxidized low density lipoprotein; ELISA: enzyme-linked immunosorbent assay; HFD: high-fat diet (an atherogenic diet); IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LDL-C: low density lipoprotrein cholesterol; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; Ox-LDL: oxidized low density lipoprotein; PBMC: peripheral blood mononuclear cells; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; SREBF2/SREBP2: sterol regulatory element binding factor 2; STX17: syntaxin 17; TC: serum total cholesterol; TG: triglyceride; TNF: tumor necrosis factor; IFN: interferon; Treg cell: regulatory T cell

Macrophage Specific IGF‐1 Overexpression Decreases Atherosclerosis, CXCL12 Chemokine, And Increases Cholesterol Efflux In ApoE Deficient Mice

Increased bioavailability of Insulin Like Growth Factor (IGF-1) has been shown to decrease cardiovascular event risk. We have shown that systemic IGF-1 administration reduced atherosclerosis in ApoE deficient (Apoe-/-) mice fed a high fat diet and that those animals have reduced plaque macrophages. Thus, we have shown that IGF-1 is atheroprotective, but cell-specific mechanism(s) mediating IGF-1's effect need to be identified. IGF-1 interacts with major cell types in atherosclerotic plaque: macrophages, smooth muscle cells, and endothelial cells. Results of our previous in vitro experiments suggest that macrophages play a predominant role in mediating IGF-1 effects. We hypothesized that increasing IGF-1 levels strictly in macrophages will prevent atherosclerosis. Using a novel macrophage-specific IGF-1 overexpressing transgenic mouse bred to an Apoe-/- background (MF-IGF1 mice), we assessed atherosclerotic plaque burden, stability, and monocyte recruitment. We isolated plaque using laser capture microdissection and we analyzed monocyte recruitment via intravenous injection of fluorescent red beads among other traditional techniques. Macrophage IGF-1 overexpression downregulated plaque burden by 30% (P<0.01), reduced plaque macrophages by 47% (P<0.001) and promoted a stable plaque phenotype. Monocyte recruitment was reduced by 70% (P<0.05) in MF-IGF1 mice and was associated with a decrease in circulating levels of CXC Chemokine Ligand 12 (CXCL12) (27% reduction compared to control, P<0.05). CXCL12 protein levels were reduced in plaque and peritoneal macrophages in MF-IGF1 mice. IGF-1 completely blocked oxidized low-density lipoprotein (oxLDL)-dependent increase of CXCL12 mRNA transcription (98% reduction vs. control, P<0.01) and IGF-1 treatment reduced CXCL12 protein (56% decrease vs. control, P<0.001)in vitro. ATP-binding cassette transporter A1 (ABCA1) is the key cholesterol transporter mediating macrophage cholesterol efflux. We found that peritoneal macrophages isolated from MF-IGF1 mice have a 2-fold increase in ABCA1 protein levels. To study whether IGF-1 changes macrophage cholesterol efflux, we loaded THP-1 cells with oxLDL and then attached a fluorescent label to measure changes in efflux. We found a 27% increase in cholesterol efflux in IGF-1 (100ng/mL)-treated cells (P<0.01) when using Apolipoprotein A I as a cholesterol acceptor. Overall, our results indicate that macrophage IGF-1 reduces atherosclerosis and decreases CXCL12, a chemokine newly implicated in atheroprogression. CXCL12 downregulates ABCA1 expression. IGF-1 potentially exerts its atheroprotective effect via this reduction in CXCL12 by reducing monocyte recruitment and by increasing ABCA1 and thereby increasing cholesterol efflux capacity.

Deficiency of myeloid PHD proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signalling

AimsAtherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis.Methods and resultsMyeloid-specific PHD knockout (PHDko) mice were obtained via bone marrow transplantation (PHD1ko, PHD3ko) or conditional knockdown through lysozyme M-driven Cre recombinase (PHD2cko). Mice were fed high cholesterol diet for 6–12 weeks to induce atherosclerosis. Aortic root plaque size was significantly augmented 2.6-fold in PHD2cko, and 1.4-fold in PHD3ko compared to controls but was unchanged in PHD1ko mice. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the hypoxia-inducible factor (HIF) 1α/BNIP3 axis. Bulk and single-cell RNA data of PHD2cko bone marrow-derived macrophages (BMDMs) and plaque macrophages, respectively, showed enhanced HIF1α/BNIP3 signalling, which was validated in vitro by siRNA silencing. Human plaque BNIP3 mRNA was positively associated with plaque necrotic core size, suggesting similar pro-apoptotic effects in human. Furthermore, PHD2cko plaques displayed enhanced fibrosis, while macrophage collagen breakdown by matrix metalloproteinases, collagen production, and proliferation were unaltered. Instead, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. In silico analysis of macrophage-fibroblast communication predicted SPP1 (osteopontin) signalling as regulator, which was corroborated by enhanced plaque SPP1 protein in vivo. Increased SPP1 mRNA expression upon PHD2cko was preferentially observed in foamy plaque macrophages expressing ‘triggering receptor expressed on myeloid cells-2’ (TREM2hi) evidenced by single-cell RNA, but not in neutrophils. This confirmed enhanced fibrotic signalling by PHD2cko macrophages to fibroblasts, in vitro as well as in vivo.ConclusionMyeloid PHD2cko and PHD3ko enhanced atherosclerotic plaque growth and macrophage apoptosis, while PHD2cko macrophages further activated collagen secretion by fibroblasts in vitro, likely via paracrine SPP1 signalling through TREM2hi macrophages.

Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis.

Endothelial dysfunction, and the differentiation of smooth muscle cells (SMCs) into proliferative, secretory phenotypes, are two major pathophysiological processes in atherosclerosis. SMCs have the potential to recruit macrophages in atherosclerotic plaques, in which macrophages drive inflammatory responses. In this study, we found that microRNA-503-5p (miR-503-5p) was enriched in either extracellular vesicles (EVs), secreted by oxidized low-density lipoprotein-treated macrophages, or the EVs from peripheral blood mononuclear cells of atherosclerosis patients. miR-503-5p was transferred intercellularly from macrophages to the co-cultured human coronary artery endothelial cells (HCAECs) and HCASMCs via EVs, thus reducing the proliferative and angiogenic abilities of HCAECs and accelerating the proliferative and migrating abilities of HCASMCs. Smad family members 1, 2 and 7 were negatively regulated by miR-503-5p in HCAECs and HCASMCs. miR-503-5p was verified as an enhancer of inflammatory cytokines and adhesion molecules released by macrophages, in part via the down-regulation of smad family members 1, 2 and 7. The inhibition of miR-503-5p by lentivirus reduced atherosclerotic lesion formations in the aorta of atherosclerotic mice. Our work demonstrated a miR-503-5p- and EV-mediated mechanism for macrophage communication with HCAECs and HCASMCs in atherosclerosis. miR-503-5p is pro-atherosclerotic stimuli that may be a therapeutic target for atherosclerosis treatment.

7-Difluoromethoxy-5,4′-dimethoxy-genistein attenuates macrophages apoptosis to promote plaque stability via TIPE2/TLR4 axis in high fat diet-fed ApoE−/− mice

Promoting plaque stability is of great significance for prevention and treatment of cardiovascular diseases. 7-difluoromethoxy-5,4′-dimethoxygenistein (DFMG) is a novel active compound synthesized using genistein, which exerts anti-atherosclerotic effect. In this study, we evaluated effects of DFMG on plaque stability in ApoE−/− mice fed with high fat diet (HFD), and explored the molecular mechanism by using ApoE−/−TLR4−/− mice and RAW264.7 cells. Here, we found that DFMG significantly reduced plaque areas, macrophages infiltration and apoptosis, and TLR4 expression in HFD-fed ApoE−/− mice. Meanwhile, DFMG increased collagen fibers, smooth muscle cells and TIPE2 expression in plaques and media. Besides, TLR4 knockout promoted the protective effects of DFMG on plaques. In vitro, DFMG decreased lysophosphatidylcholine (LPC)-induced macrophages apoptosis and TLR4, while upregulated TIPE2. Moreover, TIPE2 reduced TLR4, MyD88, p-NF-κB p65Ser276, cleaved Caspase-3 overproduction, and enhanced effects of DFMG on LPC-induced macrophages. Overall, our study demonstrates that DFMG can promote plaque stability by reducing macrophage apoptosis through TIPE2/TLR4 signaling pathway, which suggests DFMG should be used to develop food additives or drugs for preventing atherosclerosis.

In This Issue

HomeCirculation ResearchVol. 128, No. 6In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published18 Mar 2021https://doi.org/10.1161/RES.0000000000000470Circulation Research. 2021;128:687is related toCaveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in CardiomyocytesAtherosclerosis Regression and Cholesterol Efflux in Hypertriglyceridemic MiceMolecular Mechanisms of Diaphragm Myopathy in Humans With Severe Heart FailureHypertriglyceridemia in Atherosclerosis Regression (p 690)High triglycerides do not impair atherosclerosis regression, report Josefs et al.Download figureDownload PowerPointThe balance of lipids in the blood can alter a person’s risk for developing cardiovascular disease, with low levels of high-density lipoproteins (HDLs) and high levels of both low-density lipoproteins (LDLs) and triglycerides (TGs) all being linked to increased risk. But, while these correlations are strong, untangling the effects of the individual fat types is tricky. LDL-lowering medications certainly reduce atherosclerosis burden, for example, but could high TGs and low HDLs in this context impair such regression? To find out, Josefs and colleagues engineered mice to have both high TGs and low HDLs (via lipoprotein lipase deficiency) and examined how plaque regression differed in these mice compared with controls. Both the test and control mice had normal LDL levels. The team used two models of plaque regression in the mice: surgical implantation of plaque-filled aortas, and blocking of the LDL receptor (to raise LDL levels) followed by release. In both models, the combination of high TG and low HDL did not affect plaque regression as measured by plaque size, macrophage content, and inflammation status. The findings support the role of lowering LDL levels to treat atherosclerosis, but suggest that having a combination of low HDLs and high TGs may not, in itself, be proatherogenic.Heart Failure Associated Diaphragm Dysfunction (p 706)Mangner et al examine the pathology of diaphragm weakness in heart failure patients.Download figureDownload PowerPointThe diaphragm—the primary muscle controlling a person’s breathing—can become weakened during heart failure (HF) exacerbating symptoms and increasing the risk of death. The pathological mechanisms underlying the diaphragm’s demise are largely unclear, however. Studies in animals have pointed to increased reactive oxygen species as a contributing factor, but human studies have been limited. For this reason, Mangner and colleagues have now evaluated the histological and molecular features of human diaphragm biopies from both HF patients and controls. The diaphragm samples were collected from 18 HF patients undergoing implantation of left ventricle assist devices, while the 21 control samples were from patients without HF having bypass graft surgery. Compared with the controls, the HF diaphragms showed significantly reduced thickness, severe muscle fiber atrophy, increased oxidative stress in the form of protein oxidation, increased proteolysis, impaired calcium handling and mitochondrial abnormalities and dysfunction. Pathological measures also correlated with clinical severity, the team showed. By providing the first insights into the pathology of HF-related diaphragm weakness, the work points to molecular players that could be targeted for novel treatments, say the authors.Identification of McT1 as Caveolin3 Interactor (p e102)Peper et al identify novel caveolin-associated proteins in heart cells.Download figureDownload PowerPointCaveolae are invaginations of the plasma membrane involved in endocytosis, signal transduction and other cell processes. Isoforms of caveolin (CAV)—a key component of caveolae—have been implicated in heart conditions. Mice lacking CAV1 develop heart failure, for example, and genome-wide association studies have linked human CAV1 variants with cardiac conduction disease and atrial fibrillation. Also, certain rare CAV3 variants are known to cause hypertrophic cardiomyopathy. However, little is known about the normal or pathological actions of CAV in heart cells where caveolae are plentiful. To learn more, Peper and colleagues performed mass spectrometry, immunoprecipitations and other analyses in cardiomyocytes to discover novel CAV-associated proteins, some of which turned out to be isoform-specific. For example, CAV1 interacted specifically with aquaporin, while CAV3 was associated specifically with the lactate transporting McT1 and the iron transporting TfR1. When the team knocked out the function of CAV3 in stem cell-derived human cardiomyocytes, they found that McT1 had reduced surface expression and function and that the cells exhibited abnormal depolarizations. Together the results set the stage for further studies of cardiomyocyte CAV biology including how CAV variants might contribute to disease. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesCaveolin3 Stabilizes McT1-Mediated Lactate/Proton Transport in CardiomyocytesJonas Peper, et al. Circulation Research. 2021;128:e102-e120Atherosclerosis Regression and Cholesterol Efflux in Hypertriglyceridemic MiceTatjana Josefs, et al. Circulation Research. 2021;128:690-705Molecular Mechanisms of Diaphragm Myopathy in Humans With Severe Heart FailureNorman Mangner, et al. Circulation Research. 2021;128:706-719 March 19, 2021Vol 128, Issue 6 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000470 Originally publishedMarch 18, 2021 PDF download Advertisement

A CROSS SECTIONAL STUDY ON C-REACTIVE PROTEIN LEVELAMONG OVERWEIGHT AND OBESE CHILDREN ATTENDING A TERTIARY CARE HOSPITAL OF DISTRICT KANPUR

Introduction- The global prevalence of overweight and obesity in children and adolescents has increased substantially over the past several decades. Accumulating evidence suggests that Hs-CRP, which is also found within macrophages of atheromatous plaques. However, there is no much data available that guarantee its utility as a marker of cardiovascular risk in obese children and adolescents. Hence, the present study was taken up to assess the metabolic abnormalities and its association with hsCRP in obese children and adolescents. Methodology-Present cross sectional study was conducted on 100participants attending the tertiary care hospital of District Kanpur and 5ml fasting sample had been collected after overnight fasting and data was entered in Microsoft excel and analysed in SPSS 15.0 using ANOVAand unpaired student t test.Result- among cases group there were 4 participants among the age group of 4-8yrs,10 in the age group of 9-13yrs,23 in the age group of &gt;13yrs while in control group there were 3 participants ampong 4-8yrs of age and 28 in 9-13 yrs ,32 participants among the age of &gt;13yrs. On applying independent sample T-test, we could not established correlation between age and Hs-CRP (p value &gt;0.05).Conclusion- obese children and adolescents have signicantly increased hsCRP compared with a normal weight group. Early intervention and prevention of obesity in children and adolescents decreases cardiovascular disease in later life

Hepatic Igf1-Deficiency Protects Against Atherosclerosis in Female Mice.

Atherosclerosis is the leading cause of cardiovascular disease (CVD), with distinct sex-specific pathogenic mechanisms that are poorly understood. Aging, a major independent risk factor for atherosclerosis, correlates with a decline in circulating insulin-like growth factor-1 (IGF-1). However, the precise effects of Igf1 on atherosclerosis remain unclear. In the present study, we assessed the essential role of hepatic Igf1, the major source of circulating IGF-1, in atherogenesis. We generated hepatic Igf1-deficient atherosclerosis-prone apolipoprotein E (ApoE)-null mice (L-Igf1-/-ApoE-/-) using the Cre-loxP system driven by the Albumin promoter. Starting at 6 weeks of age, these mice and their littermate controls, separated into male and female groups, were placed on an atherogenic diet for 18 to 19 weeks. We show that hepatic Igf1-deficiency led to atheroprotection with reduced plaque macrophages in females, without significant effects in males. This protection from atherosclerosis in females was associated with increased subcutaneous adiposity and with impaired lipolysis. Moreover, this impaired lipid homeostasis was associated with disrupted adipokine secretion with reduced circulating interleukin-6 (IL-6) levels. Together, our data show that endogenous hepatic Igf1 plays a sex-specific regulatory role in atherogenesis, potentially through athero-promoting effects of adipose tissue-derived IL-6 secretion. These data provide potential novel sex-specific mechanisms in the pathogenesis of atherosclerosis.

Dj1 deficiency protects against atherosclerosis with anti-inflammatory response in macrophages

Inflammation is a key contributor to atherosclerosis with macrophages playing a pivotal role through the induction of oxidative stress and cytokine/chemokine secretion. DJ1, an anti-oxidant protein, has shown to paradoxically protect against chronic and acute inflammation. However, the role of DJ1 in atherosclerosis remains elusive. To assess the role of Dj1 in atherogenesis, we generated whole-body Dj1-deficient atherosclerosis-prone Apoe null mice (Dj1−/−Apoe−/−). After 21 weeks of atherogenic diet, Dj1−/− Apoe−/−mice were protected against atherosclerosis with significantly reduced plaque macrophage content. To assess whether haematopoietic or parenchymal Dj1 contributed to atheroprotection in Dj1-deficient mice, we performed bone-marrow (BM) transplantation and show that Dj1-deficient BM contributed to their attenuation in atherosclerosis. To assess cell-autonomous role of macrophage Dj1 in atheroprotection, BM-derived macrophages from Dj1-deficient mice and Dj1-silenced macrophages were assessed in response to oxidized low-density lipoprotein (oxLDL). In both cases, there was an enhanced anti-inflammatory response which may have contributed to atheroprotection in Dj1-deficient mice. There was also an increased trend of plasma DJ-1 levels from individuals with ischemic heart disease compared to those without. Our findings indicate an atheropromoting role of Dj1 and suggests that targeting Dj1 may provide a novel therapeutic avenue for atherosclerosis treatment or prevention.

MiR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques.

[Figure: see text].

Analysis of MicroRNAs Associated With Carotid Atherosclerotic Plaque Rupture With Thrombosis.

Atherosclerosis is a progressive vascular wall inflammatory disease, and the rupture of atherosclerotic vulnerable plaques is the leading cause of morbidity and mortality worldwide. This study intended to explore the potential mechanisms behind plaque rupture and thrombosis in ApoE knockout mice. The spontaneous plaque rupture models were established, and left carotid artery tissues at different time points (1-, 2-, 4-, 6-, 8-, 12-, and 16-week post-surgery) were collected. By the extent of plaque rupture, plaque was defined as (1) control groups, (2) atherosclerotic plaque group, and (3) plaque rupture group. Macrophage (CD68), MMP-8, and MMP-13 activities were measured by immunofluorescence. Cytokines and inflammatory markers were measured by ELISA. The left carotid artery sample tissue was collected to evaluate the miRNAs expression level by miRNA-microarray. Bioinformatic analyses were conducted at three levels: (2) vs. (1), (3) vs. (2), and again in seven time series analysis. The plaque rupture with thrombus and intraplaque hemorrhage results peaked at 8 weeks and decreased thereafter. Similar trends were seen in the number of plaque macrophages and lipids, the expression of matrix metalloproteinase, and the atherosclerotic and plasma cytokine levels. MiRNA-microarray showed that miR-322-5p and miR-206-3p were specifically upregulated in the atherosclerotic plaque group compared with those in the control group. Meanwhile, miR-466h-5p was specifically upregulated in the plaque rupture group compared with the atherosclerotic plaque group. The highest incidence of plaque rupture and thrombosis occurred at 8 weeks post-surgery. miR-322-5p and miR-206-3p may be associated with the formation of atherosclerotic plaques. miR-466h-5p may promote atherosclerotic plaque rupture via apoptosis-related pathways.

Atherosclerosis Regression and Cholesterol Efflux in Hypertriglyceridemic Mice.

[Figure: see text].

Protective role of serpina3c as a novel thrombin inhibitor against atherosclerosis in mice.

Abnormal vascular smooth muscle cell (VSMC) proliferation is a critical step in the development of atherosclerosis. Serpina3c is a serine protease inhibitor (serpin) that plays a key role in metabolic diseases. The present study aimed to investigate the role of serpina3c in atherosclerosis and regulation of VSMC proliferation and possible mechanisms. Serpina3c is down-regulated during high-fat diet (HFD)-induced atherosclerosis. An Apoe-/-/serpina3c-/--double-knockout mouse model was used to determine the role of serpina3c in atherosclerosis after HFD for 12 weeks. Compared with Apoe-/- mice, the Apoe-/-/serpina3c-/- mice developed more severe atherosclerosis, and the number of VSMCs and macrophages in aortic plaques was significantly increased. The present study revealed serpina3c as a novel thrombin inhibitor that suppressed thrombin activity. In circulating plasma, thrombin activity was high in the Apoe-/-/serpina3c-/- mice, compared with Apoe-/- mice. Immunofluorescence staining showed thrombin and serpina3c colocalization in the liver and aortic cusp. In addition, inhibition of thrombin by dabigatran in serpina3c-/- mice reduced neointima lesion formation due to partial carotid artery ligation. Moreover, an in vitro study confirmed that thrombin activity was also decreased by serpina3c protein, supernatant and cell lysate that overexpressed serpina3c. The results of experiments showed that serpina3c negatively regulated VSMC proliferation in culture. The possible mechanism may involve serpina3c inhibition of ERK1/2 and JNK signaling in thrombin/PAR-1 system-mediated VSMC proliferation. Our results highlight a protective role for serpina3c as a novel thrombin inhibitor in the development of atherosclerosis, with serpina3c conferring protection through the thrombin/PAR-1 system to negatively regulate VSMC proliferation through ERK1/2 and JNK signaling.

Lactucopicrin Inhibits Cytoplasmic Dynein-Mediated NF-κB Activation in Inflammated Macrophages and Alleviates Atherogenesis in Apolipoprotein E-Deficient Mice.

Nuclear factor-κB (NF-κB) activation in macrophages aggravates atherosclerosis. Dietary plant secondary metabolites including sesquiterpene lactone lactucopicrin target multiple organs. This study is focused on the impact of lactucopicrin on NF-κB activation in inflammed macrophages and atherogenesis in a mouse model of atherosclerosis. In LPS-stimulated mouse bone marrow-derived macrophages, lactucopicrin inhibits NF-κB activation, and concomitantly represses the expression of IL-1β, IL-6, and tumor necrosis factor-alpha. This effect is not due to modulation of the inhibitor of NF-κB kinases (IKK) α/β/γ and NF-κB inhibitor α, and NF-κB/p65 DNA binding activity. Instead, the lactucopicrin effect is reliant on the inhibition of cytoplasmic dynein-mediated p65 transportation, a prerequisite step for p65 nuclear translocation. In high-fat diet-fed apolipoprotein E-deficient mice, lactucopicrin consumption dose-dependently reduces plaque area, inhibits plaque macrophage accumulation, attenuates plaque macrophage NF-κB activation, and reduces both plaque and serum inflammatory burden. However, lactucopicrin consumption does not affect the levels of serum lipids and anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor beta). Dietary lactucopicrin inhibits atherogenesis in mice likely by its anti-inflammatory property. These findings suggest that dietary supplementation with lactucopicrin is a promising strategy to inhibit atherosclerotic cardiovascular disease.

The hypoxia-sensor carbonic anhydrase IX affects macrophage metabolism, but is not a suitable biomarker for human cardiovascular disease

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.

Regulation of Macrophage Activation and Differentiation in Atherosclerosis.

Chronic inflammation is a hallmark of atherosclerosis and macrophages play a central role in controlling inflammation at all stages of atherosclerosis. In atherosclerosis, macrophages and monocyte-derived macrophages are continuously exposed to cholesterol, oxidized lipids, cell debris, cytokines, and chemokines. Not only do these stimuli induce a specific macrophage phenotype, but they also interact extensively, leading to macrophage heterogeneity in atherosclerotic plaques. Herein, we review the diverse phenotypes of macrophages, the mechanisms underlying macrophage activation, and the contributions of macrophages to atherosclerosis in this context. We also summarize recent studies on foamy macrophages and monocyte-derived macrophages in plaque during disease progression. We provide a comprehensive overview of transcriptional, epigenetic, and metabolic reprogramming of macrophages and discuss the emerging concepts of targeting cytokines and macrophages to modulate atherosclerosis.

In vivo MRI detection of intraplaque macrophages with biocompatible silica-coated iron oxide nanoparticles in murine atherosclerosis.

Identification of a vulnerable atherosclerotic plaque before rupture is an unmet clinical need. Integrating nanomedicine with multimodal imaging has the potential to precisely detect biological processes in atherosclerosis. We synthesized silica-coated iron oxide nanoparticles (SIONs) coated with rhodamine B isothiocyanate and polyethylene glycol and investigated their feasibility in the detection of macrophages in inflamed atherosclerotic plaques of apolipoprotein E-deficient (ApoE-/-) mice via magnetic resonance (MR) and fluorescence reflectance (FR) imaging. In vitro cellular uptake of SIONs was assessed in macrophages using confocal laser scanning microscopy (CLSM). In vivo MR imaging was performed 24 h after SION injection via the tail vein in 26-week-old ApoE-/- mice fed a high-cholesterol diet (HCD). We also performed FR imaging of the extracted aortas from four different mice: two normal-diet-fed C57BL/6 mice injected with saline or 10 mg/kg SIONs and two HCD-fed ApoE-/- mice injected with 5 or 10 mg/kg SIONs. The harvested aortas were cryosectioned and stained with immunohistochemical staining. The CLSM images at 24 h after incubation showed efficient uptake of SIONs by macrophages, with no evidence of cytotoxicity. The in vivo and ex vivo MR and FR images demonstrated SION deposition in the atheroma. Upon immunohistochemical staining of the aorta, CLSM images revealed colocalization of macrophages and SIONs in the atherosclerotic plaque. These results demonstrate that polyethylene glycosylated SIONs could be a highly effective method to identify macrophage activity in atherosclerotic plaques as a multimodal imaging agent.