THP-1 Macrophage-derived Foam Cells Research Articles

Biochanin A Mitigates Atherosclerosis by Inhibiting Lipid Accumulation and Inflammatory Response.

Biochanin A (BCA), a dietary isoflavone extracted from red clover and cabbage, has been shown to antagonize hypertension and myocardial ischemia/reperfusion injury. However, very little is known about its role in atherogenesis. The aim of this study was to observe the effects of BCA on atherosclerosis and explore the underlying mechanisms. Our results showed that administration of BCA promoted reverse cholesterol transport (RCT), improved plasma lipid profile, and decreased serum proinflammatory cytokine levels and atherosclerotic lesion area in apoE−/− mice fed a Western diet. In THP-1 macrophage-derived foam cells, treatment with BCA upregulated ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG1 expression and facilitated subsequent cholesterol efflux and diminished intracellular cholesterol contents by activating the peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) and PPARγ/heme oxygenase 1 (HO-1) pathways. BCA also activated these two signaling pathways to inhibit the secretion of proinflammatory cytokines. Taken together, these findings suggest that BCA is protective against atherosclerosis by inhibiting lipid accumulation and inflammatory response through the PPARγ/LXRα and PPARγ/HO-1 pathways. BCA may be an attractive drug for the prevention and treatment of atherosclerotic cardiovascular disease.

DPP-4 Inhibitor Linagliptin Ameliorates Oxidized LDL-Induced THP-1 Macrophage Foam Cell Formation and Inflammation.

IntroductionAtherosclerosis is one of the major causes of cardiovascular diseases. Lipid uptake and accumulation in macrophages play a major role in atherosclerotic plaque formation from its initiation to advanced atheroma formation. The dipeptidyl peptidase-4 (DPP-4) inhibitor Linagliptin is commonly used to lower blood glucose in type 2 diabetes patients. Recent studies report that Linagliptin has cardiovascular protective and anti-inflammatory effects.MethodsTHP-1 macrophage cells were treated with 100 nM PMA for 72 hour to induce foam cell formation. The differentiated cells were exposed to 100 μg/mL ox-LDL in the presence or absence of the DPP-4 inhibitor Linagliptin. The expression levels of DPP-4 and inflammatory cytokines were detected by RT-PCR, ELISA, and Western blot experiments. The cellular ROS level was measured by staining the cells with the fluorescent probe DCFH-DA. The separation of lipoprotein fractions was achieved by high-performance liquid chromatography (HPLC). The cells were labeled with fluorescent-labeled cholesterol to measure cholesterol efflux, and lipid droplets were revealed by Nile red staining.ResultsThe presence of Linagliptin significantly reduced ox-LDL-induced cytokine production (IL-1β and IL-6) and ROS production. Linagliptin ameliorated ox-LDL-induced lipid accumulation and impaired cholesterol efflux in macrophages. Mechanistically, this study showed that Linagliptin mitigated ox-LDL-induced expression of the scavenger receptors CD36 and LOX-1, but not SRA. Furthermore, Linagliptin increased the expression of the cholesterol transporter ABCG1, but not ABCA1.ConclusionLinagliptin possesses a potent inhibitory effect on THP-1 macrophage-derived foam cell formation in response to ox-LDL. This effect could be mediated through a decrease in the expression of CD36 and LOX-1 on macrophages and an increase in the expression of the cholesterol transporter ABCG1. This study indicates that the DPP-4 inhibitor Linagliptin plays a critical role in preventing foam cell formation in vitro. However, future research using an atherosclerotic animal model is necessary to determine its effectiveness and to prove its potential implication in the prevention and treatment of atherosclerosis.

RETRACTED: Long non-coding RNA CDKN2B-AS1 contributes to atherosclerotic plaque formation by forming RNA-DNA triplex in the CDKN2B promoter

BackgroundAtherosclerosis involves a slow process of plaque formation on the walls of arteries, and comprises a leading cause of cardiovascular disease. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. In this study, we aim to explore the possible involvement of lncRNA ‘cyclin-dependent kinase inhibitor 2B antisense noncoding RNA’ (CDKN2B-AS1) and CDKN2B in the progression of atherosclerosis. MethodsInitially, we quantified the expression of CDKN2B-AS1 in atherosclerotic plaque tissues and, in THP-1 macrophage-derived, and human primary macrophage (HPM)-derived foam cells. Next, we established a mouse model of atherosclerosis using apolipoprotein E knockout (ApoE−/−) mice, where lipid uptake, lipid accumulation, and macrophage reverse cholesterol transport (mRCT) were assessed, in order to explore the contributory role of CDKN2B-AS1 to the progression of atherosclerosis. RIP and ChIP assays were used to identify interactions between CDKN2B-AS1, CCCTC-binding factor (CTCF), enhancer of zeste homologue 2 (EZH2), and CDKN2B. Triplex formation was determined by RNA-DNA pull-down and capture assay as well as EMSA experiment. FindingsCDKN2B-AS1 showed high expression levels in atherosclerosis, whereas CDKN2B showed low expression levels. CDKN2B-AS1 accelerated lipid uptake and intracellular lipid accumulation whilst attenuating mRCT in THP-1 macrophage-derived foam cells, HPM-derived foam cells, and in the mouse model. EZH2 and CTCF were found to bind to the CDKN2B promoter region. An RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B promoter was found to recruit EZH2 and CTCF in the CDKN2B promoter region and consequently inhibit CDKN2B transcription by accelerating histone methylation. InterpretationThe results demonstrated that CDKN2B-AS1 promotes atherosclerotic plaque formation and inhibits mRCT in atherosclerosis by regulating CDKN2B promoter, and thereby could be a potential therapeutic target for atherosclerosis.

GPR120 facilitates cholesterol efflux in macrophages through activation of AMPK signaling pathway.

Cholesterol efflux from macrophages is the initial step of reverse cholesterol transport, an important process for high-density lipoprotein-mediated atheroprotection. G protein-coupled receptor (GPR) 120, which functions as long-chain fatty acid receptor, is well known for its anti-inflammatory and insulin-sensitizing function in macrophages. However, the role of GPR120 on macrophage foam cell formation, the hallmark of atherosclerotic plaques, has not been verified. In this study, we found for the first time that stimulation of GPR120 by its agonist GW9508 elevated the expression of ATP-binding cassette transporters (ABC) A1 and ABCG1 in THP-1 macrophage-derived foam cells and Raw264.7 macrophages, and promoted ABCA1- and ABCG1-mediated cholesterol efflux and reduced cellular cholesteryl ester (CE) content as well. In addition, GPR120 activation was accompanied with the stimulation of AMPK pathway in macrophages; however, the effect of GPR120 on macrophage cholesterol efflux was largely abolished by AMPK inhibition. Moreover, the AMPK activity and the expression of ABCA1 and ABCG1 were markedly abrogated by knockdown of GPR120, or application of phospholipase C (PLC) inhibitor, calcium chelator, or CaMKK inhibitor. Because only free cholesterol can be effluxed from macrophages, we found that activation of AMPK could lead to increase both neutral CEs hydrolysis by upregulation of neutral cholesterol ester hydrolase expression and acid CEs hydrolysis by activation of ULK1. In conclusion, these results demonstrated that GPR120 facilitated ABCA1- and ABCG1-mediated cholesterol efflux through activation of PLC/Ca2+ /CaMKK/AMPK signaling pathway, which induced CE hydrolysis and elevated the expression of ABCA1 and ABCG1 in macrophages.

LncRNA ZFAS1 confers inflammatory responses and reduces cholesterol efflux in atherosclerosis through regulating miR-654-3p-ADAM10/RAB22A axis

PurposeAtherosclerosis is the leading cause of cardiovascular diseases (CVD) with high incidence rate and mortality rate. Long non-coding RNAs (lncRNAs) are important functional molecules in atherosclerosis. Present study aimed to explore the functional role and underlying mechanism of ZFAS1 in atherosclerosis. MethodsThe in-vitro cell model of atherosclerosis was established by using oxidized low-density lipoprotein (ox-LDL) to induce THP-1 macrophage-derived foam cells. qRT-PCR measured the mRNA levels of ZFAS1, miR-654-3p, ADAM10 and RAB22A. Western blot detected the protein levels of ADAM10 and RAB22A. The levels of IL-1β, IL-6 and TNF-ɑ (inflammatory biomarkers) were tested with ELISA assay. Detection of cholesterol efflux rate was experimented. The interaction between RNAs was affirmed with luciferase reporter and RNA pull-down experiments. ResultsThe expression of ZFAS1 was significantly up-regulated in in-vitro cell model of atherosclerosis at a dose- and time-dependent manner. Knockdown of ZFAS1 impaired inflammatory responses and promoted cholesterol efflux rate. Overexpression of ZFAS1 accelerated inflammatory responses and hampered cholesterol efflux rate. Then, the cytoplasmic role of ZFAS1 was revealed. By bio-informatics analysis and mechanism assays, miR-654-3p was identified to bind with ZFAS1. Moreover, ADAM10 and RAB22A were targeted and suppressed by miR-654-3p. ZFAS1 served as a ceRNA to positively regulate ADAM10 and RAB22A expression through endogenously sponging miR-654-3p. ConclusionIn conclusion, ZFAS1 elevated ADAM10/RAB22A expression to reduce cholesterol efflux rate and facilitate inflammatory responses in atherosclerosis at a miR-654-3p-dependent way, suggesting a prospective treatment method for amelioration of atherosclerosis.

Role of Different Salmonella enterica Serotypes in THP-1 Macrophage Derived Foam Cell Autophagy and Inflammatory Response

Infected aortic aneurysm is a rare but life-threatening condition. Intimal injury, especially in arteries with atherosclerotic plaques, is vulnerable to Salmonella species. In this study, THP-1 macrophage-derived foam cells were used to mimic the atherosclerotic plaques. We investigated the role of different Salmonella serotype infections in foam cell autophagy and inflammatory response. Three different serotypes of Salmonella enterica-Typhimurium, Enteritidis, and Choleraesuis - with or without a virulence plasmid were used in this study. The foam cell model was established and observed under confocal microscopy. The expression of autophagy and inflammasome-related proteins after Salmonella infection was detected by Western blotting, and the secretion of the proinflammatory cytokines interleukin-1β and IL-18 was detected using ELISA. The plasmid-bearing S. enteritidis OU7130- and plasmid-less S. choleraesuis OU7266-infected foam cells both showed a lower expression of beclin-1 at 0.5 h post-infection (hpi) and p62 at 2 hpi as well as a higher expression of caspase-1 at 2 hpi than other infected cells. Moreover, infection of OU7266 induced the highest IL-1β and IL-18 production among the tested strains. In conclusion, the virulence plasmid of S. enteritidis OU7130 induced foam cell inflammatory responses, whereas that of S. choleraesuis played an opposite role. The different serotype of Salmonella may impact the infected aortic aneurysms through regulating inflammatory balance of the macrophage and foam cells in the atherosclerotic plaque.

Salmonella-Infected Aortic Aneurysm: Investigating Pathogenesis Using Salmonella Serotypes.

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes – Typhimurium, Enteritidis, and Choleraesuis – in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1β, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy – significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1β secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1β secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.

LncRNA SNHG16 promoted proliferation and inflammatory response of macrophages through miR-17-5p/NF-κB signaling pathway in patients with atherosclerosis.

Atherosclerosis (AS) is a leading cause of cardiovascular disease (CVD), which is also the leading reason for mortality and morbidity worldwide. Growing evidence has shown that long non-coding RNAs (lncRNAs) play some roles in the development of AS; however, their roles remain unclear. In this study, we aimed to explore the function of lncRNA SNHG16 in AS. qRT-PCR was used to detect the expressions of SNHG16 and miR-17-5p in AS serum samples and THP-1 macrophage-derived foam cells; the correlations were also analyzed. THP-1 macrophages were respectively treated with ox-LDL and several inflammatory factors to explore the affecting factors. What's more, SNHG16 overexpression lentivirus (LV-SNHG16) and downregulation lentivirus (LV-sh SNHG16) were purchased and infected into THP-1 macrophages. CCK8 assay was used to measure cell proliferation; the levels of IKKβ, p-IkBα and p-p65 were detected by western blot (WB), and the levels of TNF-α, IL-1β and IL-6 were detected by ELISA kit. Moreover, the luciferase assay was performed to explore the binding site of SNHG16 and miR-17-5p. Furthermore, we transfected miR-17-5p mimic and inhibitor into THP-1 macrophages; the proliferation, NF-κB signaling pathway factors and inflammatory factors were detected. Finally, JSH, a NF-κB signaling inhibitor, was added into LV-SNHG16 THP-1 macrophages and miR-17-5p inhibitor was transfected into LV-sh SNHG16 THP-1 macrophages to confirm that SNHG16 functions via miR-17-5p/ NF-κB signaling pathway. We found that SNHG16 was increased in AS patients and THP-1 macrophage-derived foam cells. Additionally, SNHG16 was increased in THP-1 macrophages by ox-LDL with time-dependence and dose-dependence. Furthermore, SNHG16 overexpression promoted proliferation, inflammatory response and increased levels of IKKβ, p-IkBα, p-p65 in THP-1 macrophages, while SNHG16 downregulation led to the opposite results. Most importantly, we found that miR-17-5p expressions were significantly decreased in AS patients, which were negatively correlated with SNHG16. Luciferase gene reporter assay confirmed that SNHG16 could directly bind with miR-17-5p. Moreover, the proliferation, inflammatory factors and NF-κB signaling factors were significantly repressed after transfecting miR-17-5p mimic into THP-1 macrophages, while it led to the opposite results after transfecting miR-17-5p inhibitor. Then, we added JSH, a NF-κB signaling inhibitor, into LV-SNHG16 THP-1 macrophages; as a result, the increased cell proliferation rate and inflammatory response were both decreased. Finally, we found that the repressed cell proliferation, inflammatory factors and expressions of NF-κB signaling factors in LV-sh SNHG16 group were increased after co-transfected with miR-17-5p inhibitor. According to the results, we found that SNHG16 was upregulated in AS patients. Furthermore, we firstly found that SNHG16 was increased by ox-LDL in THP-1 macrophages. Most importantly, we uncovered a previously unappreciated SNHG16/miR-17-5p/ NF-κB signaling axis in promoting proliferation and inflammatory response in AS patients and THP-1 macrophages, which might provide a potential target for treating AS.

The lncRNA DAPK-IT1 regulates cholesterol metabolism and inflammatory response in macrophages and promotes atherogenesis

Atherosclerosis is the leading cause of cardiovascular disease (CVD) and the leading reason behind mortality and morbidity in Western countries. The role of long noncoding RNAs (lncRNAs) in CVD is still unexplored with inadequate research on the involvement of lncRNAs in atherogenesis. We found the lncRNA DAPK1-IT1 and lipoprotein lipase (LPL) to be up-regulated in THP-1 macrophage-derived foam cells. We demonstrated that DAPK1-IT1 mediated its promoting effect on LPL expression via regulating an intermediary miRNA hsa-miR-590–3p. This DAPK1-IT1/hsa-miR-590–3p/LPL axis regulates cholesterol metabolism and the inflammatory response in macrophages in vitro. Overexpressing LPL using lentiviral vectors led to decreased circulation of high-density lipoprotein cholesterol (HDL-C), increased circulation of low-density lipoprotein cholesterol (LDL-C) and very-LDL-C (VLDL-C), increased circulating pro-inflammatory cytokine levels (IL-1β, IL-6, TNF-α), and enhanced atherogenesis in apolipoprotein E-deficient (apoE—/—) mice. In sum, the DAPK1-IT1/hsa-miR-590–3p/LPL axis regulates cholesterol metabolism and the inflammatory response in macrophages and may contribute to atherogenesis in vivo. These findings suggest this axis may be a promising therapeutic target in ameliorating CVD.

MiR-33-5p knockdown attenuates abdominal aortic aneurysm progression via promoting target adenosine triphosphate-binding cassette transporter A1 expression and activating the PI3K/Akt signaling pathway.

The aim of this study was to investigate the role of miR-33-5p in abdominal aortic aneurysm progression, which regulated adenosine triphosphate-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux and lipid accumulation in THP-1 macrophage-derived foam cells through the PI3K/Akt pathway. Quantitative reverse transcription polymerase chain reaction was used to evaluate the expression level of miR-33-5p and ABCA1 mRNA in abdominal aortic aneurysm patient and normal person tissues. The relationship between miR-33-5p and ABCA1 was examined by dual luciferase report assay. High-performance liquid chromatography was used to evaluate the levels of cholesterol contents. Cholesterol efflux detection was performed by liquid scintillator. The expression of inflammatory cytokines was detected by quantitative reverse transcription polymerase chain reaction. Western blot was applied to determine the expression levels of ABCA1, PI3K (p-PI3K), and Akt (p-Akt). The quantitative reverse transcription polymerase chain reaction analysis results revealed miR-33-5p overexpression in abdominal aortic aneurysm tissues, but the expression level of ABCA1 was lower in abdominal aortic aneurysm tissues than non-abdominal aortic aneurysm tissues. Subsequently, the dual luciferase report gene assay confirmed that ABCA1 was a target of miR-33-5p, and miR-33-5p-negative regulated ABCA1 expression. Moreover, the expression levels of p-PI3K, p-Akt, and ABCA1 were decreased in THP-1 cell transferred with ABCA1 siRNA, but knockdown of miR-33-5p had an opposite effect. Furthermore, knockdown of miR-33-5p decreased the expression of MMP-2, MMP-9, TNF-α, total cellular cholesterol, and promoted cholesterol efflux in THP-1-derived foam cells. Importantly, LY294002 (PI3K inhibitor) or si-ABCA1 completely inhibited the stimulatory effects of miR-33-5p inhibitor. This study has found that knockdown of miR-33-5p induced ABCA1 expression and promoted inflammatory cytokines and cholesterol efflux likely via activating the PI3K/Akt signaling pathway.

Long non-coding RNA CDKN2B-AS1 reduces inflammatory response and promotes cholesterol efflux in atherosclerosis by inhibiting ADAM10 expression.

Introduction: Long non-coding RNAs (lncRNAs) play key roles in the development of atherosclerosis through the inflammatory pathway. This study aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in atherosclerosis via its function in A disintegrin and metalloprotease 10 (ADAM10).Methods: Initially, the expression of CDKN2B-AS1 and ADAM10 in atherosclerotic plaque tissues and THP-1 macrophage-derived foam cells was determined, after which the cholesterol efflux rate of macrophages was calculated. Interaction between CDKN2B-AS1 and ADAM10 was analyzed, after which, expression of CDKN2B-AS1 and ADAM10 were altered to explore their effects on inflammatory response and cholesterol efflux. The aforementioned findings were further intended to be validated by the atherosclerosis mouse model experiments.Results: Atherosclerotic plaque tissue and THP-1 macrophage-derived foam cells exhibited downregulated CDKN2B-AS1 and upregulated ADAM10. Upon overexpressing CDKN2B-AS1 or silencing ADAM10, lipid accumulation was reduced and cholesterol efflux was increased. CDKN2B-AS1 located in the nucleus could bind to DNA methyltransferase 1 (DNMT1) to enhance methylation of ADAM10 promoter, leading to suppressed atherosclerotic inflammatory response and promoted cholesterol efflux.Conclusion: Altogether, lncRNA CDKN2B-AS1 can inhibit the transcription of ADAM10 via DNMT1-mediated ADAM10 DNA methylation, consequently preventing inflammatory response of atherosclerosis and promoting cholesterol efflux.

LncRNA DYNLRB2-2 inhibits THP-1 macrophage foam cell formation by enhancing autophagy.

The aim of this study was to investigate whether long non-coding RNA (lncRNA) DYNLRB2-2 can inhibit foam cell formation by activating autophagy. The location of DYNLRB2-2 in THP-1-derived macrophages was analyzed by fluorescence in situ hybridization (FISH). Oxidized-low-density lipoprotein (ox-LDL) was used to induce the formation of foam cells, Oil Red O (ORO) staining and high-performance liquid chromatography (HPLC) were performed to detect accumulation of lipid droplets and the level of cholesterol concentration, respectively. The mRNA and protein level of ATP-binding cassette transporter A1 (ABCA1) were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. Relative protein levels of (p-) liver kinase B1 (LKB1), (p-) AMP-activated protein kinase (AMPK), (p-) the mammalian target of rapamycin (mTOR) and autophagy markers (LC3 II, Beclin-1 and p62) in THP-1 macrophage-derived foam cells were analyzed by Western blotting. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β] in THP-1 macrophage-derived foam cells were detected by enzyme-linked immunosorbent assay (ELISA). 3-MA and compound C were used to block autophagy. Our data show that DYNLRB2-2 inhibited the formation of THP-1 macrophage-derived foam cells and promotes cholesterol efflux (CE) by activating autophagy. DYNLRB2-2 caused autophagy by activating the signaling pathway of LKB1/AMPK/mTOR in foam cells. DYNLRB2-2 activated the LKB1/AMPK/mTOR signaling pathway via the miR-298/Sirtuin 3 (SIRT3) axis. Our data indicated that DYNLRB2-2 enhanced CE by regulating the LKB1/AMPK/mTOR autophagy signaling pathway through the miR-298/SIRT3 axis, thereby blocking the formation of foam cells from THP-1 macrophages.

Long Non-Coding RNA CDKN2B-AS1 Contributes to Atherosclerotic Plaque Formation in Apolipoprotein E Knockout Mice by Forming RNA-DNA Triplex in the CDKN2B Promoter

Background: Atherosclerosis, a cardiovascular disease caused by several risk factors, is one of the major contributors to mortality in developed countries. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. In this study, we aim to explore the possible involvement of cyclin-dependent kinase inhibitor 2B antisense noncoding RNA (CDKN2B-AS1) and CDKN2B in progression of atherosclerosis. Methods: Initially, we quantified the expression ofCDKN2B-AS1 in atherosclerotic plaque tissues and THP-1 macrophage-derived foam cells by qRT-PCR and western blot, followed by a mouse model establishment of atherosclerosis using apolipoprotein E knockout (ApoE-/-) mice. Subsequently, lipid uptake, lipid accumulation and macrophage reverse cholesterol transport (mRCT) were detected to explore the contributory role of CDKN2B-AS1 in the progression of atherosclerosis. Furthermore, we performed RIP and ChIP to identify the interaction among CDKN2B-AS1, CCCTC-binding factor (CTCF), enhancer of zeste homologue 2 (EZH2) and CDKN2B. Results: Triplex formation was determined by RNA-DNA pull-down and capture assay as well as EMSA experiment. CDKN2B-AS1 showed high expression in atherosclerosis and CDKN2B showed low expression. Silencing of CDKN2B-AS1 decreased the ability of lipid uptake and intracellular lipid accumulation and promoted mRCT in THP-1 macrophage-derived foam cells and mouse models. EZH2 and CTCF were found to bind to CDKN2B promoter region. A RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B promoter was found to recruit EZH2 and CTCF in CDKN2B promoter region and subsequently inhibited CDKN2B transcription by accelerating histone methylation. Conclusion: The key findings of this study collectively propose silencing of CDKN2B-AS1 inhibits atherosclerotic plaque formation and promotes mRCT in atherosclerosis by regulating CDKN2B promoter, which can be explored to treat this vascular disease. Funding: This study was supported by the General Projects of National Natural Science Foundation of China — the Function and Regulation Mechanism of ETBR in Aortic Endothelium and Smooth Muscle Cells in Pregnancy Induced Hypertension (No. 81871187), the Regional Projects of the National Natural Science Foundation of China — Correlation Analysis Between Endothelin Receptor and Pregnancy Induced Hypertension and Abnormal Blood Pressure Regulation (No. 81460239), and the Natural Science Foundation Project of Ningxia Province — Correlation Analysis Between Anastomotic Regional Blood Flow Velocity and Intimal Hyperplasia after Revascularization (No. NZ17195). Declaration of Interest: The authors have declared that no competing interests exist. Ethical Approval: This research study was conducted with the approval of the ethics committee of Qingdao Municipal Hospital. All patients participating in the study signed informed written consent documentation, according to Helsinki Declaration. The animal experiment procedures were performed in accordance with strict protocols approved by the Institutional Animal Care and Use Committee.

ApoA-1 Mimetic Peptide ELK-2A2K2E Decreases Inflammatory Factor Levels Through the ABCA1-JAK2-STAT3-TTP Axis in THP-1-Derived Macrophages.

The aim of this study was to determine whether the apolipoprotein A-1 (apoA-1) mimetic peptide ELK-2A2K2E regulates inflammatory cytokine expression through activating the adenosine triphosphate-binding cassette transporter A1 (ABCA1)-janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3)-tristetraprolin (TTP) signaling pathway in THP-1 macrophage-derived foam cells. The cells were treated with the apoA-1 mimetic peptide ELK-2A2K2E at different concentrations (0, 20, 40, and 80 μg/mL) or incubated with ELK-2A2K2E (40 μg/mL) for different times (0, 6, 12, and 24 hours). Our results showed that the levels of the cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), were decreased at both concentration- and time-dependent manners. When the cells were exposed to lipopolysaccharides and actinomycin D, ELK-2A2K2E significantly decreased the mRNA stability of inflammatory cytokines at different time points (0, 30, 60, and 120 minutes) by increasing TTP expression as analyzed by real-time quantitative polymerase chain reaction. The effect of ELK-2A2K2E on TTP was obviously blocked by the inhibition of the JAK-STAT3 pathway. Furthermore, we found that ELK-2A2K2E activated the JAK-STAT3-TTP pathway through the upregulation of ABCA1 and then decreased inflammatory cytokine expression. ApoA-I mimetic peptide ELK-2A2K2E increases the degradation of TNF-α, IL-6, and MCP-1 mRNA and reduces the levels of inflammatory cytokines through activating the JAK2-STAT3-TTP signaling pathway that is dependent on the upregulation of ABCA1.

Validation and Application of a Novel Cholesterol Efflux Assay Using Immobilized Liposomes as a Substitute for Cultured Cells

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.

MiR-155 inhibits transformation of macrophages into foam cells via regulating CEH expression

MiR-155 can inhibit the formation of atherosclerosis by interfering with the transformation of macrophages into foam cells that plays a critical role in the pathogenesis of atherosclerosis, but the precise mechanisms of miR-155 are still unknown. Herein, we observed that mRNA and protein expression levels of CEH were significantly upregulated in a dose- and time-dependent manner by transfected with miR-155 mimics in THP-1 macrophages. Further studies showed that overexpression of miR-155 can significantly inhibit foam cells formation, reduce intracellular CE accumulation and enhance the efflux of FC and cholesterol, result in a decrease of intracellular lipid accumulation; while this effect was significantly reversed by siCEH. Meanwhile, we found that Tim-3 is associated with miR-155-mediated CEH expression in THP-1 macrophage-derived foam cells. Overexpression of Tim-3 can attenuate miR-155-mediated CEH induction. Taken together, our findings demonstrated that miR-155 can inhibit the transformation of macrophages into foam cells by enhancing CEH signaling pathway in macrophages, this effect is likely to be achieved by inhibiting the expression of Tim-3.

Lowering the n-6/n-3 PUFAs ratio inhibits the formation of THP-1 macrophage-derived foam cell

BackgroundThe balance between n-6 and n-3 PUFAs is an important determinant in the risk for cardiovascular disease. The study was to investigate the influence of the n-6 and n-3 PUFAs ratio on the formation of THP-1 monocyte-derived foam cells and explore the probable mechanism of anti-atherosclerosis.MethodsTHP-1 monocyte cells were cultured with PMA and ox-LDL to establish a foam-cell model, while treated with different ratios of n-6 to n-3 PUFAs for 48 h. The cholesterol of foam cells was measured by a cholesterol assay kit. The levels of IL-6 and TNFα in supernatant were detected with ELISA methods. The expressions of CD36, ABCA1, ACAT1, PPARγ and LXRα mRNA were detected with real-time PCR.ResultsCompared with the foam cell model group, the low and middle ratio of n-6 to n-3 PUFAs groups decreased the intracellular concentration of cholesterol (P < 0.01), but the high n-6/n-3 PUFAs ratio did not. Fatty acids decreased the level of IL-6 and TNFα in supernatant in a ratio-dependent manner. Fatty acids treatment also decreased the expressions of CD36、ACTA1、PPARγ、LXRα mRNA in a ratio-dependent manner.ConclusionsLowering the ratios of n-6 to n-3 PUFAs can decrease the secretion of inflammatory cytokines then reduce the expressions of CD36 and ACAT1 mRNA. As well, it can decrease the expressions of CD36 mRNA through the PPARγ pathway. This leads to less cholesterol ingestion into the cells and decreased synthesis of cholesteryl ester, which inhibits the formation of the foam cells, further preventing the occurrence and development of atherosclerosis.

IL-8 negatively regulates ABCA1 expression and cholesterol efflux via upregulating miR-183 in THP-1 macrophage-derived foam cells

ObjectivePrevious studies suggest that IL-8 has an important role in the regulation of cholesterol efflux, but whether miRNAs are involved in this process is still unknown. The purpose of this study is to explore whether IL-8 promotes cholesterol accumulation by enhancing miR-183 expression in macrophages and its underlying mechanism. Methods and resultsTreatment of THP-1 macrophage-derived foam cells with IL-8 decreased ABCA1 expression and cholesterol efflux. Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-183 was highly conserved during evolution and directly inhibited ABCA1 protein and mRNA expression by targeting ABCA1 3′UTR. MiR-183 directly regulated endogenous ABCA1 expression levels. Furthermore, IL-8 enhanced the expression of miR-183 and decrease ABCA1 expression. Cholesterol transport assays confirmed that IL-8 dramatically inhibited apolipoprotein AI-mediated ABCA1-dependent cholesterol efflux by increasing miR-183 expression. In contrast, treatment with anti-IL-8 antibody reversed these effects. ConclusionIL-8 enhances the expression of miR-183, which then inhibits ABCA1 expression and cholesterol efflux. Our studies suggest that the IL-8-miR-183-ABCA1 axis may play an intermediary role in the development of atherosclerosis.

Visceral adipose tissue-derived serine protease inhibitor accelerates cholesterol efflux by up-regulating ABCA1 expression via the NF-κB/miR-33a pathway in THP-1 macropahge-derived foam cells

Atherosclerosis is a dyslipidemia disease characterized by foam cell formation driven by the accumulation of lipids. Visceral adipose tissue-derived serine protease inhibitor (vaspin) is known to suppress the development of atherosclerosis via its anti-inflammatory properties, but it is not yet known whether vaspin affects cholesterol efflux in THP-1 macrophage-derived foam cells. Here, we investigated the effects of vaspin on ABCA1 expression and cholesterol efflux, and further explored the underlying mechanism. We found that vaspin decreased miR-33a levels, which in turn increased ABCA1 expression and cholesteorl efflux. We also found that inhibition of NF-κB reduced miR-33a expression and vaspin suppressed LPS-mediated NF-κB phosphorylation. Our findings suggest that vaspin is not only a regular of inflammasion but also a promoter of cholesterol efflux.

Validation and application of a novel cholesterol efflux assay using immobilized liposomes as a substitute for cultured cells.

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.