Lipid Accumulation In Macrophages Research Articles

2,2',4,4'-Tetrabromodiphenyl Ether (PBDE 47) Selectively Stimulates Proatherogenic PPARγ Signatures in Human THP-1 Macrophages to Contribute to Foam Cell Formation.

2,2',4,4'-Tetrabromodiphenyl ether (PBDE 47) is one of the most prominent PBDE congeners detected in the human body, suggesting that the potential health risks of PBDE 47 should be thoroughly considered. However, the cardiovascular toxicity of PBDE 47 remains poorly understood. Here, toxic outcomes of PBDE 47 in human THP-1 macrophages concerning foam cell formation, which play crucial roles in the occurrence and development of atherosclerosis, were elucidated. First, our results indicated that PBDE 47 affected the PPARγ pathway most efficiently in THP-1 macrophages by transcriptomic analysis. Second, the PPARγ target genes CD36 and FABP4, responsible for lipid uptake and accumulation in macrophages, were consistently upregulated both at transcriptional and translational levels in THP-1 macrophages upon PBDE 47. Unexpectedly, PBDE 47 failed to activate the PPARγ target gene LXRα and PPARγ-LXRα-ABCA1/G1 cascade, which is activated by the PPARγ full agonist rosiglitazone and enables cholesterol efflux in macrophages. Thus, coincident with the selective upregulation of the PPARγ target genes CD36 and FABP4, PBDE 47, distinct from rosiglitazone, functionally resulted in more lipid accumulation and oxLDL uptake in THP-1 macrophages through high-content analysis (HCA). Moreover, these effects were markedly abrogated by the addition of the PPARγ antagonist T0070907. Mechanistically, the structural basis of selective activation of PPARγ by PBDE 47 was explored by molecular docking and dynamics simulation, which indicated that PBDE 47 interacted with the PPARγ ligand binding domain (PPARγ-LBD) distinctively from that of rosiglitazone. PBDE 47 was revealed to interact with helix 3 and helix 5 but not helix 12 in the PPARγ-LBD. Collectively, these results unraveled the potential cardiovascular toxicity of PBDE 47 by selective activation of PPARγ to facilitate foam cell formation for the first time.

Candida albicans Induces Foaming and Inflammation in Macrophages through FABP4: Its Implication for Atherosclerosis

Abstract Atherosclerosis is a chronic degenerative disorder characterized by lipid-dense plaques and low-grade inflammation affecting arterial walls. Foamy macrophages are important in the formation of atherosclerotic plaques and the induction of low-grade inflammation. Candida albicans is the major cause of candidiasis in immunocompromised patients, including those with diabetes mellitus. However, the role played by C. albicans in macrophage foaming and the associated inflammation is poorly understood. We investigated whether C. albicans induces foaming along with inflammation in macrophages and, if so, by which mechanism(s). We incubated THP-1 macrophages with heat-killed C. albicans (HKCA). HKCA-induced lipid accumulation in macrophages along with increased expression of inflammatory markers, including CD11b and CD11c or expression and secretion of IL-1β. HKCA also increased the expression of PPAR, CD36, and FABP4 in macrophages. Mechanistically, we found that the foamy and inflammatory macrophage phenotype induced by HKCA requires FABP4 because disruption of FABP4 in macrophages either by chemical inhibitor BMS309404 or small interfering RNA (siRNA) abrogated foam cell formation and expression of inflammatory markers CD11b, CD11c, and IL-1β. Furthermore, HKCA-treated macrophages displayed high expression and secretion of MMP-9. Inhibition of FABP4 resulted in suppression of HCKA-induced MMP-9 production. Overall, our results demonstrate that C. albicans induces foam cell formation, inflammation, and MMP-9 expression in macrophages via the upregulation of FABP4, which may constitute a novel therapeutic target for treating C. albicans-induced atherosclerosis. Supported by Kuwait Foundation for the Advancement of Sciences (KFAS) (RA CB-2019-002)

Novel Roles of FFAR4 in Macrophage Foam Cell Formation

Free fatty acid receptor 4 (FFAR4), also known as G‐protein coupled receptor 120 (GPR120), is a long‐chain unsaturated fatty acid receptor expressed in adipocytes, endothelial cells, and macrophages. Activation of FFAR4 helps maintain metabolic homeostasis by regulating adipogenesis, insulin sensitivity, and inflammation. While FFAR4 is best known for its role its role in preventing obesity and diabetes, recent studies have demonstrated that FFAR4 may also play an important role in the development of atherosclerosis and cardiovascular disease (CVD). Given FFAR4’s importance in anti‐inflammatory signaling and high expression levels in macrophages, we designed experiments to test the hypothesis that FFAR4 prevents the development of atherosclerosis by reversing macrophage foam cell formation, a hallmark of early atherogenesis. In these studies, we isolated peritoneal macrophages from wild‐type C57/BL6J mice and incubated them with oxidized low‐density lipoprotein (oxLDL) to generate foam cells. We then investigated the effects of FFAR4 activation by GW9508 (a synthetic agonist) on lipid accumulation, cytokine secretion, and cholesterol efflux. Activation of FFAR4 by GW9508 decreased macrophage secretion of pro‐inflammatory cytokines. We also found that activation of FFAR4 with GW9508 reduced lipid accumulation in macrophages as observed by decreased Oil Red O staining and reduced cellular cholesterol content. Additionally, activation of FFAR4 by GW9508 increased [3H]‐cholesterol efflux to high‐density lipoprotein (HDL). Interestingly, the increased efflux was accompanied by decreased scavenger receptor CD36 expression (that mediates oxLDL uptake) and increased expression of ATP binding cassette transporters, ABCA1 and ABCG1 (that mediate cholesterol efflux). Taken together, our results support an exciting and novel role for FFAR4 in the reversal of foam cell formation and could emerge this receptor as a new target for treating CVD by preventing accumulation of atherosclerotic plaque.

Integrated metabolomic and transcriptomic analysis identifies benzo[a]pyrene-induced characteristic metabolic reprogramming during accumulation of lipids and reactive oxygen species in macrophages

Polycyclic aromatic hydrocarbon exposure is a major risk factor for cardiovascular diseases. Macrophage lipid accumulation is a characteristic molecular event in the pathophysiology of cardiovascular diseases. Metabolic reprogramming is an intervention target for diseases and toxic effects of environmental pollutants. However, comprehensive metabolic reprogramming related to BaP-induced macrophage lipid accumulation is currently unexplored. Therefore, metabolomics and transcriptomics were conducted to unveil relevant metabolic reprogramming in BaP-exposed macrophages, and to discover potential intervention targets. Metabolomics revealed that most amino acids, nucleotides, monosaccharides, and organic acids were significantly decreased, while most fatty acids and steroids accumulated in BaP-exposed macrophages. Transcriptomics showed that fatty acid synthesis and oxidation, and steroid synthesis and export were decreased, while import of fatty acids and steroids was increased, indicating potential roles of lipid transport in macrophage lipid accumulation following BaP exposure. Meanwhile, alanine, aspartate and glutamate metabolism, branched-chain amino acid degradation, nucleotide synthesis, monosaccharide import, pentose phosphate pathway, citrate synthesis, and glycolysis were decreased, while nucleotide degradation was increased, thus inducing decreases in most amino acids, nucleotides, monosaccharides, and organic acids in BaP-exposed macrophages. Additionally, increases in oxidative stress and the activation of antioxidant systems were observed in BaP-exposed macrophages, which was evinced by increases in reactive oxygen species, and the activation of Fenton reaction, Vdac2/3, Sod2, and Nrf2. Moreover, BaP-induced accumulation of reactive oxygen species and lipids in macrophages could be abolished by epigallocatechin-3-gallate. Quantitative PCR showed that BaP exposure activated aryl hydrocarbon receptor signaling and promoted the proinflammatory phenotype in macrophages, and these effects were inhibited or even abolished by the separate treatment with epigallocatechin-3-gallate or CH-223191, suggesting the regulatory role of aryl hydrocarbon receptor signaling in BaP-induced toxic effects. This study provides novel insights into the toxic effects of polycyclic aromatic hydrocarbons on macrophage metabolism and potential intervention targets.

Triglyceride breakdown from lipid droplets regulates the inflammatory response in macrophages

In response to inflammatory activation by pathogens, macrophages accumulate triglycerides in intracellular lipid droplets. The mechanisms underlying triglyceride accumulation and its exact role in the inflammatory response of macrophages are not fully understood. Here, we aim to further elucidate the mechanism and function of triglyceride accumulation in the inflammatory response of activated macrophages. Lipopolysaccharide (LPS)-mediated activation markedly increased triglyceride accumulation in macrophages. This increase could be attributed to up-regulation of the hypoxia-inducible lipid droplet–associated (HILPDA) protein, which down-regulated adipose triglyceride lipase (ATGL) protein levels, in turn leading to decreased ATGL-mediated triglyceride hydrolysis. The reduction in ATGL-mediated lipolysis attenuated the inflammatory response in macrophages after ex vivo and in vitro activation, and was accompanied by decreased production of prostaglandin-E2 (PGE2) and interleukin-6 (IL-6). Overall, we provide evidence that LPS-mediated activation of macrophages suppresses lipolysis via induction of HILPDA, thereby reducing the availability of proinflammatory lipid precursors and suppressing the production of PGE2 and IL-6.

Attenuating oxidized low density lipoprotein (ox-LDL)-induced macrophages damage via inhibiting C-type lectin domain family 2 (CLEC2) expression through janus kinase 1 (JAK1)/ signal transducers and activators of transcription-1 (STAT1) pathway

ABSTRACT Our study aimed to explore the effect of C-type lectin-like receptor 2 (CLEC2) expression level on oxidized low-density lipoprotein (ox-LDL)-induced macrophage damage and the regulatory mechanism of macrophage foaming. Foam cells were derived from RAW264.7 by ox-LDL, and the cell viability was detected by cell counting kit-8 (CCK-8) assay. Enzyme-linked immunosorbent assay (ELISA) was applied to detect the levels of inflammatory cytokines tumor necrosis factor (TNF-α), Interleukin-6 (IL-6), and Interleulin-1β (IL-1β). Small interfering CLEC2 (si-CLEC2) was synthesized and transfected into RAW264.7, and the apoptosis rate was analyzed by flow cytometry. Western blotting was employed to detect the protein expressions of Janus kinase 1 (JAK1), Signaltransducersandactivators oftranscription-1 (STAT1), phosphorylation-JAK1 (p-JAK1), phosphorylation-STAT1 (p-STAT1), CLEC2, and the apoptosis-related proteins. The levels of total cholesterol (TC) and free cholesterol (FC) were measured using colorimetric kits. Results showed that ox-LDL could activate the JAK1/STAT1 pathway of macrophages and up-regulate the expression of CLEC2. CLEC2 knockdown could reduce macrophage inflammation and lipid accumulation. Inactivating JAK1/STAT1 pathway with JAK1 inhibitor can significantly reduce the phosphorylation of STAT1 and alleviate the ox-LDL-induced damage in macrophages by regulating the expression of CLEC2. In conclusion, targeting JAK1/STAT1 to inhibit CLEC2 can attenuate ox-LDL-induced macrophage damage. This study enriched the pathogenesis of atherosclerosis and provided the possible treatment targets.

Excess iron promotes emergence of foamy macrophages that overexpress ferritin in the lungs of silicosis patients.

Background and objectiveInhalation of high concentrations of respirable crystalline silica (RCS) can lead to silicosis. RCS contains varying levels of iron, which can cause oxidative stress and stimulate ferritin production. This study evaluated iron‐related and inflammatory markers in control and silicosis patients.MethodsA cohort of stone benchtop industry workers (n = 18) were radiologically classified by disease severity into simple or complicated silicosis. Peripheral blood and bronchoalveolar lavage (BAL) were collected to measure iron, ferritin, C‐reactive protein, serum amyloid A and serum silicon levels. Ferritin subunit expression in BAL and transbronchial biopsies was analysed by reverse transcription quantitative PCR. Lipid accumulation in BAL macrophages was assessed by Oil Red O staining.ResultsSerum iron levels were significantly elevated in patients with silicosis, with a strong positive association with serum ferritin levels. In contrast, markers of systemic inflammation were not increased in silicosis patients. Serum silicon levels were significantly elevated in complicated disease. BAL macrophages from silicosis patients were morphologically consistent with lipid‐laden foamy macrophages. Ferritin light chain (FTL) mRNA expression in BAL macrophages was also significantly elevated in simple silicosis patients and correlated with systemic ferritin.ConclusionOur findings suggest that elevated iron levels during the early phases of silicosis increase FTL expression in BAL macrophages, which drives elevated BAL and serum ferritin levels. Excess iron and ferritin were also associated with the emergence of a foamy BAL macrophage phenotype. Ferritin may represent an early disease marker for silicosis, where increased levels are independent of inflammation and may contribute to fibrotic lung remodelling.

The elevation of miR-185-5p alleviates high-fat diet-induced atherosclerosis and lipid accumulation in vivo and in vitro via SREBP2 activation.

Objective: SREBP2, a member of the SREBP family, is a primary regulator of lipid metabolism. In recent years, an increasing number of studies have suggested that miRNAs regulate lipid metabolism-related genes. It was speculated in this study that miRNAs may be implicated in the regulation of lipid accumulation in macrophages by SREBP2 protein.Methods and results: GSE34812, GSE132651 and GSE28829 datasets comprised of atherosclerosis samples were downloaded to explore the gene expression profiles related to the miRNAs and SREBP2, and miR-185-5p was predicted to be a target of SREBP2. The GO annotations and KEGG pathway analysis were adopted for functional classification of differentially expressed genes, and lipid metabolic process was an enriched pathway in atherosclerosis. Besides, the effects of SREBP2 on increasing lipid accumulation were investigated in vivo using miR-185-5p mimic/apoE−/− mice and miR-185-5p NC/apoE−/− mice. All mice fed with a HFD suffered from atherosclerosis. Moreover, the effects of miR-185-5p on atherosclerotic plaque formation in mice were analyzed. An in vitro assay was also performed to determine the effect of miR-185-5p on ox-LDL-stimulated RAW 264.7 macrophages. Finally, miR-185-5p mimic was transfected into cultured macrophages. The results showed that the miR-185-5p elevation might regulate lipid accumulation in mice by targeting SREBP2. Furthermore, miR-185-5p mimic repressed the activation of SREBP1, SREBP2, LDLR, SCD-1, HMGCR as well as NLRP3, IL-1β, TNF-α in HFD fed mice or ox-LDL-stimulated macrophages.Conclusions: our study demonstrated that miR-185-5p effectively alleviates atherosclerosis and lipid accumulation by regulating the miR-185-5p/SREBP2 axis.

Monocyte and Macrophage Lipid Accumulation Results in Down-Regulated Type-I Interferon Responses.

Macrophages are critical components of atherosclerotic lesions and their pro- and anti-inflammatory responses influence atherogenesis. Type-I interferons (IFNs) are cytokines that play an essential role in antiviral responses and inflammatory activation and have been shown to promote atherosclerosis. Although the impact of type-I IFNs on macrophage foam cell formation is well-documented, the effect of lipid accumulation in monocytes and macrophages on type-I IFN responses remains unknown. Here we examined IFN stimulated (ISG) and non-ISG inflammatory gene expression in mouse and human macrophages that were loaded with acetylated LDL (acLDL), as a model for foam cell formation. We found that acLDL loading in mouse and human macrophages specifically suppressed expression of ISGs and IFN-β secretion, but not other pro-inflammatory genes. The down regulation of ISGs could be rescued by exogenous IFN-β supplementation. Activation of the cholesterol-sensing nuclear liver X receptor (LXR) recapitulated the cholesterol-initiated type-I IFN suppression. Additional analyses of murine in vitro and in vivo generated foam cells confirmed the suppressed IFN signaling pathways and suggest that this phenotype is mediated via down regulation of interferon regulatory factor binding at gene promoters. Finally, RNA-seq analysis of monocytes of familial hypercholesterolemia (FH) patients also showed type-I IFN suppression which was restored by lipid-lowering therapy and not present in monocytes of healthy donors. Taken together, we define type-I IFN suppression as an athero-protective characteristic of foamy macrophages. These data provide new insights into the mechanisms that control inflammatory responses in hyperlipidaemic settings and can support future therapeutic approaches focusing on reprogramming of macrophages to reduce atherosclerotic plaque progression and improve stability.

Perivascular adipose-derived exosomes reduce macrophage foam cell formation through miR-382-5p and the BMP4-PPARγ-ABCA1/ABCG1 pathways

BackgroundPerivascular adipose tissue (PVAT) releases exosomes (EXOs) to regulate vascular homeostasis. PVAT-derived EXOs reduce macrophage foam cell formation, but the underlying molecular mechanism has yet to be fully elucidated. We hypothesize that PVAT release miRNA through EXOs and regulate the expression of cholesterol transporter of macrophages, thereby reducing foam cell formation.Methods and resultsThrough RT-qPCR, we identified that miR-382-5p, which was expressed at lower levels in PVAT-EXOs from coronary atherosclerotic heart disease patients than healthy individuals, was expressed at higher levels in wild-type C57BL/6 J mouse aortic PVAT-EXOs than in subcutaneous adipose tissue-derived EXOs. We explored macrophage lipid accumulation through oil red O staining, assessed cholesterol uptake and efflux, and verified cholesterol transporter expression. We found that transfection with a miR-382-5p inhibitor offset PVAT-EXO-related reductions in macrophage foam cell formation and increases in cholesterol efflux mediated by ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1). In addition, bone morphogenetic protein 4 (BMP4) pretreatment and si-peroxisome proliferator-activated receptor γ (PPARγ) transfection showed that BMP4-PPARγ participated in PVAT-EXO-mediated upregulation of the cholesterol efflux transporters ABCA1 and ABCG1.ConclusionsPVAT-EXOs reduce macrophage foam cell formation through miR-382-5p- and BMP4-PPARγ-mediated upregulation of the cholesterol efflux transporters ABCA1 and ABCG1. This finding suggests a promising strategy for the prevention and treatment of atherosclerosis.

Forkhead box protein 1 transcriptionally activates sestrin1 to alleviate oxidized low-density lipoprotein-induced inflammation and lipid accumulation in macrophages

ABSTRACT Transcription factor forkhead box protein 1 (FOXP1) has been shown cardiovascular protection. We aimed to analyze the role of FOXP1 in oxidized low-density lipoprotein (ox-LDL)-induced macrophages and its possible regulatory effect on sestrin1 (SESN1) expression. After stimulation with ox-LDL, FOXP1 expression in RAW264.7 cells was evaluated with RT-qPCR and Western blotting. Then, FOXP1 was overexpressed, followed by detection of inflammatory mediator levels using ELISA kits and RT-qPCR. Lipid accumulation was detected with oil red O staining. Additionally, the JASPAR database was used to predict the potential genes that could be transcriptionally regulated by FOXP1. ChIP and luciferase reporter assays were used to verify this combination. To further clarify the regulatory effects of FOXP1 on SESN1 in damage of macrophages triggered by ox-LDL, SESN1 was silenced to determine the inflammation and lipid accumulation under the condition of FOXP1 overexpression. Results indicated that ox-LDL stimulation led to a significant decrease in FOXP1 expression. FOXP1 overexpression notably reduced the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, accompanied by a decreased in phosphorylated NF-κB p65 expression. Besides, FOXP1-upregulation inhibited lipid accumulation and reduced CD36 expression level in RAW264.7 cells upon ox-LDL stimulation. Moreover, results of ChIP and luciferase reporter assays suggested that FOXP1 could transcriptionally regulate SESN1 expression. Further experiments supported that SESN1 silencing restored the inhibitory effects of FOXP1 overexpression on the inflammation and lipid accumulation in RAW264.7 cells exposed to ox-LDL. Collectively, FOXP1 transcriptionally activates SESN1 for the alleviation of ox-LDL-induced inflammation and lipid accumulation in macrophages.

Myelin and non-myelin debris contribute to foamy macrophage formation after spinal cord injury

Tissue damage after spinal cord injury (SCI) elicits a robust inflammatory cascade that fails to resolve in a timely manner, resulting in impaired wound healing and cellular regeneration. This inflammatory response is partly mediated by infiltrating immune cells, including macrophages. As professional phagocytes, macrophages initially play an important role in debris clearance at the injury site, which would be necessary for proper tissue regeneration. After SCI, most macrophages become filled with lipid droplets due to excessive uptake of lipid debris, assuming a “foamy” phenotype that is associated with a proinflammatory state. Myelin has been assumed to be the main source of lipid that induces foamy macrophage formation after injury given its abundance in the spinal cord. This assumption has led to the widespread use of purified myelin treatment to model foamy macrophage formation in vitro. However, the assumption that myelin is necessary for foamy macrophage formation remains untested. To this end, we developed a novel foamy macrophage assay utilizing total spinal cord homogenate to include all sources of lipid present at the injury site. Using the myelin basic protein knockout (MBP KO, i.e., Shiverer) mice that lack myelin, we investigated lipid accumulation in foamy macrophages. Primary macrophages treated with myelin-deficient spinal cord homogenate still formed large lipid droplets typically observed in foamy macrophages, although to a lesser degree than cells treated with normal homogenate. Similarly, MBP KO mice subjected to contusive spinal cord injury also formed foamy macrophages that exhibited reduced lipid content and associated with improved histological outcomes and reduced immune cell infiltration. Therefore, the absence of myelin does not preclude foamy macrophage formation, indicating that myelin is not the only major source of lipid that contributes this pathology, even though myelin may alter certain aspects of its inflammatory profile.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease

Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and exvivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. Invitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Abnormal histidine metabolism promotes macrophage lipid accumulation under Ox-LDL condition

Distinct macrophage populations exert highly heterogeneity and perform various functions, among which, a crucial function of lipid metabolism is highlighted. However, the role of histidine metabolism disorder in macrophage lipid metabolism remains elusive. Addressed this question, we sorted and cultured the bone marrow-derived macrophages (BMDMs) of histidine decarboxylase (Hdc) knockout (Hdc−/-) mice with an in vitro oxidized low-density lipoprotein (ox-LDL) model, and detected the intracellular lipids by Oil Red O staining as well as lipid probe staining. Astemizole, a canonical and long-acting histamine H1 receptor (H1R) antagonist, was applied to elucidate the impact of antagonizing the H1R-dependent signaling pathway on macrophage lipid metabolism. Subsequently, the differential expressed genes were screened and analyzed in the bone marrow-derived CD11b+ immature myeloid cells of Hdc−/- and Hdc+/+ mice with a high fat diet by the microarray study. The expression levels of cholesterol metabolism-related genes were examined by qRT-PCR to explore underlying mechanisms. Lastly, we used a high-sensitivity histidine probe to detect the intracellular histidine in the BMDMs after oxidative stress. The results revealed that histidine metabolism disorder and histamine deficiency aggravated lipid accumulation in the ox-LDL-treated BMDMs. The expression level of H1R gene in the BMDMs was down-regulated after ox-LDL stimulation. The disruption of the H1R-dependent signaling pathway by astemizole further exacerbated ox-LDL-induced lipid deposition in the BMDMs partly by up-regulating scavenger receptor class A (SR-A) for lipid intake, down-regulating neutral cholesteryl ester hydrolase (nCEH) for cholesterol esterification and down-regulating ATP-binding cassette transporters A1 (ABCA1) and ABCG1 for reverse cholesterol transport. The intracellular histidine increased under ox-LDL condition, which was further increased by Hdc knockout. Collectively, these results partially reveal the relationship between histidine metabolism and lipid metabolism in the BMDMs and offer a novel strategy for lipid metabolism disorder-associated diseases.

Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155

Background:Foam cells are markers of atherosclerosis and characterise advanced atherosclerotic plaque, stimulated by inflammation caused by high lipid levels in macrophages. The combination of decaffeinated coffee and green tea extract (DCGTE) has been suggested to have a role in foam cell inhibition.Objective: we investigated the inhibiting role of DCGTE against foam cell formation, through modulation of the inflammation process and cholesterol metabolism in macrophage colony stimulating factor- (M-CSF) and oxidized low-density lipoprotein (oxLDL)-exposed macrophages.Methods: Coffee and green tea were extracted by filtration and infusion respectively, and underwent decaffeination using active carbon and blanching methods, respectively. Cells were administered 160/160 and 320/320μg/ml of DCGTE. Foam cell formation was observed using a light microscope after staining with Oil Red O (ORO), and the accumulation of lipids in macrophages with ELISA. Observations of lipid influx and efflux were determined through semiquantitative cluster differentiation 36 (CD36) and ATP binding cassette transporter A1 (ABCA1) expression through immunofluorescence. The inflammation process was quantified using inflammatory/anti-inflammatory markers, e.g., tumor necrosis factor α (TNFα) and interleukin 10 (IL10) with ELISA. Peroxisome proliferator activated response γ (PPARγ) expression and activity were assessed with PCR and ELISA, respectively. The expression of microRNA 155 (miR-155) was examined using qPCR.Results:DCGTE at the above concentrations tended to reduce foam cell numbers, significantly inhibited lipid accumulation (p=0.000), reduced CD36 expression (p=0.000) and TNFα secretion (p=0.000) in Raw264.7 exposed to M-CSF 50ng/ml and oxLDL 50μg/ml. PPARγ expression (p=0.00) and activity (p=0.001), miR-155 relative expression (p=0.000), and IL10 production (p=0.000) also increased.Conclusion:DCGTE lowered foam cell numbers, possibly through attenuation of the inflammatory process and improvement of lipid/efflux mechanisms in M-CSF and oxLDL-stimulated Raw264.7 cells, via upregulation of PPARγ and miR-155. Our results suggest DCGTE may help prevent atherosclerosis-based diseases.

Abstract 11102: Ginsenoside Rb2 Alleviated Atherosclerosis Progression by Inhibiting M1 Macrophages Polarization Induced by Microrna-216a

Background: It has been reported that microRNA-216a (miR-216a) advanced atherosclerosis progression by promoting macrophage polarization to M1 pro-inflammation phenotype. Ginsenoside Rb2 (Rb2) is the major pharmacologically active compound extracted from ginseng. Bioinformatics predicted that Rb2 had a high affinity with miR-216a. In the present study, we aimed to investigate the effect of Rb2 on macrophages in atherosclerosis progression induced by miR-216a. Methods: ApoE -/- mice with miR-216a adenovirus infection and Rb2 intraperitoneal injection were used to explore the effect of Rb2 on miR-216a-mediated atherosclerosis development in vivo. Oil red O, Hematoxylin-Eosin (HE) and Masson's trichrome staining were performed to assess the thoracic aortic plaque progression and stability. Immunohistochemical staining was used to detect the M1 macrophages in plaque. Furthermore, macrophages transfected with miR-216a and treatment with Rb2 were used to explore the mechanism of Rb2 on M1 polarization in vitro. Neutral red phagocytic experiment and cholesterol efflux assay were performed to assess the role of Rb2 in lipid accumulation of M1 macrophages. Results: The results in ApoE -/- mice showed that Rb2 alleviated miR-216a-mediated atherosclerosis lesion area by 33%, and promoted plaque stability with a lipid accumulation decrease by 55% and a collage content increase by 250%. We further observed that Rb2 inhibited M1 macrophages in plaque by 76%. Mechanistically, the data from macrophages revealed that Rb2 reserved miR-216a-mediated Smad3 and IκBα protein expression to inhibit NF-κB-responsive genes, including M1 maker monocyte chemotactic protein-1 (MCP1) and tumor necrosis factor alpha (TNFα), and Rb2 attenuated miR-216a-mediated lipid deposit in M1 macrophages by decreasing lipid uptake and promoting cholesterol efflux. Conclusion: Our results first time showed that Rb2 alleviated atherosclerosis progression and stability induced by miR-216a. By inhibiting M1 macrophage polarization through Smad3/IκBα, Rb2 decreases lipid accumulation and increases collage content increase in plaque. The Rb2 could serve as a potential small molecule drug for atherosclerosis associated M1 polarization.

Angiotensin II induces the expression of periostin to promote foam cell formation in oxLDL-treated macrophages

A matricellular protein periostin has been documented to promote macrophage recruitment in atherosclerotic lesions. However, the role of periostin in macrophage foam cell formation is still unknown. In this study, we examined the expression and function of periostin in cholesterol homeostasis in macrophages. The role of periostin in mediating Ang II-induced foam cell formation was also investigated. The mechanism by which Ang II induced the expression of periostin was explored. It was found that oxLDL treatment significantly increased the expression and secretion of periostin in THP-1 macrophages. Knockdown of periostin blocked oxLDL-induced lipid accumulation and enhanced cholesterol efflux. In contrast, treatment with recombinant periostin protein enhanced oxLDL-induced macrophage foam cell formation. Ang II caused a time-dependent induction of periostin in THP-1 macrophages, which was ascribed to Twist2-mediated transactivation of periostin. Ang II treatment significantly augmented lipid accumulation in THP-1 macrophages, and knockdown of periostin blocked the effect of Ang II on foam cell formation. Moreover, periostin depletion restored cholesterol efflux in Ang II-treated THP-1 macrophages. Clinically, there was a significant positive correlation between serum periostin and Ang II levels in patients with atherosclerosis. Collectively, we show that periostin is essential for Ang II-induced enhancement of macrophage foam cell formation via suppression of cholesterol efflux.

MicroRNA-22 inhibition promotes the development of atherosclerosis via targeting interferon regulator factor 5

Atherosclerosis is generally accepted as a chronic inflammatory disease and is the most important pathological process underlying the cardiovascular diseases. MiR-22 exerts an important role in tumorgenesis, obesity and NAFLD development, as well as cardiovascular diseases. However, a certain role of miR-22 in the pathogenesis of atherosclerosis remains undetermined. Here, we showed that miR-22 exhibited a negative association with the deteriorated atherosclerotic plaque and showed significant downregulated expression in macrophages. Next, treatment of ApoE deficiency (ApoE−/-) mice with miR-22 inhibitors which were then subjected to high fat diet (HFD) for 12 weeks were performed to investigate the function of miR-22 on atherogenesis. The results exhibited that miR-22 inhibition dramatically promoted atherosclerotic plaques but attenuated plaque stabilization which were accompanied by decreased smooth muscle cell and collagen content, but increased macrophage infiltration and lipid accumulation. More importantly, the in vivo and in vitro experiments suggested that miR-22 inhibition accelerated inflammatory response and foam cell formation. Mechanistically, we demonstrated interferon regulator factor 5 (IRF5) was an important target of miR-22 and it was required for the regulation of inflammation mediated by miR-22 inhibition. Collectively, these evidences revealed that miR-22 inhibition promoted the atherosclerosis progression through activation of IRF5.

The Pathogenic Role of Long Non-coding RNA H19 in Atherosclerosis via the miR-146a-5p/ANGPTL4 Pathway.

The abnormally expressed long non-coding RNA (lncRNA) H19 has a crucial function in the development and progression of cardiovascular disease; however, its role in atherosclerosis is yet to be known. We aimed to examine the impacts of lncRNA H19 on atherogenesis as well as the involved mechanism. The outcomes from this research illustrated that the expression of lncRNA H19 was elevated in mouse blood and aorta with lipid-loaded macrophages and atherosclerosis. Adeno-associated virus (AAV)-mediated lncRNA H19 overexpression significantly increased the atherosclerotic plaque area in apoE−/− mice supplied with a Western diet. The upregulation of lncRNA H19 decreased the miR-146a-5p expression but increased the levels of ANGPTL4 in mouse blood and aorta and THP-1 cells. Furthermore, lncRNA H19 overexpression promoted lipid accumulation in oxidized low-density lipoprotein (ox-LDL)-induced THP-1 macrophages. However, the knockdown of lncRNA H19 served as a protection against atherosclerosis in apoE−/− mice and lowered the accumulation of lipids in ox-LDL-induced THP-1 macrophages. lncRNA H19 promoted the expression of ANGPTL4 via competitively binding to miR-146a-5p, thus promoting lipid accumulation in atherosclerosis. These findings altogether demonstrated that lncRNA H19 facilitated the accumulation of lipid in macrophages and aggravated the progression of atherosclerosis through the miR-146a-5p/ANGPTL4 pathway. Targeting lncRNA H19 might be an auspicious therapeutic approach for preventing and treating atherosclerotic disease.

Candida albicans Induces Foaming and Inflammation in Macrophages through FABP4: Its Implication for Atherosclerosis.

Atherosclerosis is a chronic degenerative disorder characterized by lipid-dense plaques and low-grade inflammation affecting arterial walls. Foamy macrophages are important in the formation of atherosclerotic plaques and the induction of low-grade inflammation. The presence of lipid-laden macrophages has occurred in infections caused by opportunistic pathogens. Candida albicans is the major cause of candidiasis in immunocompromised patients, including those with diabetes mellitus. However, the role played by C. albicans in macrophage foaming and the associated inflammation is poorly understood. We investigated whether C. albicans induces foaming along with inflammation in macrophages and, if so, by which mechanism(s). We incubated THP-1 macrophages with heat-killed C. albicans (HKCA). HKCA-induced lipid accumulation in macrophages along with increased expression of inflammatory markers, including CD11b and CD11c or expression and secretion of IL-1β. HKCA also increased the expression of PPARγ, CD36, and FABP4 in macrophages. Mechanistically, we found that the foamy and inflammatory macrophage phenotype induced by HKCA requires FABP4 because disruption of FABP4 in macrophages either by chemical inhibitor BMS309404 or small interfering RNA (siRNA) abrogated foam cell formation and expression of inflammatory markers CD11b, CD11c, and IL-1β. Furthermore, HKCA-treated macrophages displayed high expression and secretion of MMP-9. Inhibition of FABP4 resulted in suppression of HCKA-induced MMP-9 production. Overall, our results demonstrate that C. albicans induces foam cell formation, inflammation, and MMP-9 expression in macrophages via the upregulation of FABP4, which may constitute a novel therapeutic target for treating C. albicans-induced atherosclerosis.