Lipid Accumulation In THP-1 Macrophages Research Articles

IGFBPL1 inhibits macrophage lipid accumulation by enhancing the activation of IGR1R/LXRα/ABCG1 pathway.

Lipid accumulation in macrophages plays an important role in atherosclerosis and is the major cause of atherosclerotic cardiovascular disease. Reducing lipid accumulation in macrophages is an effective therapeutic target for atherosclerosis. Insulin-like growth factor 1 (IGF-1) exerts the anti-atherosclerotic effects by inhibiting lipid accumulation in macrophages. Furthermore, almost all circulating IGF-1 combines with IGF binding proteins (IGFBPs) to activate or inhibit the IGF signaling. However, the mechanism of IGFBPs in macrophage lipid accumulation is still unknown. GEO database analysis showed that among IGFBPS family members, IGFBPL1 has the largest expression change in unstable plaque. We found that IGFBPL1 was decreased in lipid-laden THP-1 macrophages. Through oil red O staining, NBD-cholesterol efflux, liver X receptor α (LXRα) transcription factor and IGR-1 receptor blocking experiments, our results showed that IGFBPL1 inhibits lipid accumulation in THP-1 macrophages through promoting ABCG1-meditated cholesterol efflux, and IGFBPL1 regulates ABCG1 expression and macrophage lipid metabolism through IGF-1R/LXRα pathway. Our results provide a theoretical basis of IGFBPL1 in the alternative or adjunct treatment options for atherosclerosis by reducing lipid accumulation in macrophages.

2-(2-Phenylethyl)chromone-enriched extract of Chinese agarwood (Aquilaria sinensis) inhibits atherosclerosis progression through endoplasmic reticulum stress-mediated CD36 expression in macrophages

Ethnopharmacological relevanceChinese agarwood, derived from the Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae), has a long history of use in Traditional Chinese Medicine for the management of cardiovascular disease. However, the specific active ingredients responsible for its impact on atherosclerosis are yet to be fully understood.Aim of the study: The aim of this study is to investigate the anti-atherosclerotic effectiveness of the 2-(2-phenylethyl)chromone-enriched extract derived from Chinese agarwood (CPE) through the ER stress-mediated CD36 pathway. Materials and methodsTo assess the effectiveness of CPE, an atherosclerotic mouse model was established using ApoE−/− mice with a high-fat diet. Then we assessed the impact of CPE on lipid accumulation in THP-1 macrophages that were exposed to oxLDL. Subsequently, the effect of CPE on the expression of CD36 and markers related to ER stress was characterized. ResultsOur in vivo research confirmed that CPE effectively reduces the formation of aortic plaques in atherosclerotic ApoE−/− mice. Additionally, our in vitro study observed that CPE inhibits the uptake of oxLDL and hinders the generation of foam cells. This effect is achieved by downregulating the level of CD36 in macrophages. Furthermore, our study revealed that the increase in CD36 expression, resulting from oxLDL exposure, is governed by the activation of JNK1/2/3 signaling pathways and the initiation of ER stress. ConclusionCPE demonstrated significant efficacy to inhibit the atherosclerosis. The ER stress/P-JNK/PPARγ/CD36 signaling pathway plays critical involvement in modulating the foam cell formation in vitro and in vivo. These findings underscore the efficacy of CPE as a viable therapeutic intervention for the treatment of atherosclerosis.

Atheroprotective Effect of Fucoidan in THP-1 Macrophages by Potential Upregulation of ABCA1.

Targeting foam cells reduces the risk and pathophysiology of atherosclerosis, of which they are one of its early hallmarks. The precise mechanism of action of fucoidan, a potential anti-atherogenic drug, is still unknown. Our objective was to assess the ability of fucoidan to regulate expression of ATP-binding cassette transporter A1 (ABCA1) in ox-LDL-induced THP-1 macrophages. Molecular docking was used to predict how fucoidan interacts with anti-foam cell markers, and further in vitro experiments were performed to evaluate the protective effect of fucoidan on modulating uptake and efflux of lipids. THP-1 macrophages were protected by 50 µg/mL of fucoidan and were then induced to form foam cells with 25 µg/mL of ox-LDL. Expression levels were assessed using RT-qPCR, and an Oil Red O stain was used to observe lipid accumulation in THP-1 macrophages. In addition, ABCA1 protein was examined by Western blot, and cellular cholesterol efflux was determined using fluorescently labeled cholesterol. Under a light microscope, decreased lipid accumulation in ox-LDL-induced-THP-1 macrophages pre-treated with fucoidan showed a significant effect, although it did not affect the expression of scavenger receptors (SR-AI and CD36). It is interesting to note that fucoidan dramatically increased the gene and protein expression of ABCA1, perhaps via the liver X receptor-α (LXR-α). Moreover, fucoidan's ability to increase and control the efflux of cholesterol from ox-LDL-induced THP-1 macrophages revealed how it may alter ABCA1's conformation and have a major effect on how it interacts with apolipoprotein A (ApoA1). In vitro results support a rationale for predicting fucoidan and its interaction with its receptor targets' predicted data, hence validating its anti-atherogenic properties and suggesting that fucoidan could be promising as an atheroprotective.

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.

TCDD induced lipid accumulation by impairment of autophagic flux in THP-1 macrophages.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental and food contaminant generated as a byproduct of various industrial activities. It is found in a lot of foods, especially in dairy products, eggs, fish, and meat. Autophagy is a highly conserved cellular degradation and cycling process, which plays an important role in lipid metabolism. This study aimed to explore the effects of TCDD on autophagic flux and lipid metabolism in THP-1 macrophages. The data showed that TCDD promoted the accumulation of autophagosomes in THP-1 macrophages, and subsequent findings revealed that this autophagosome accumulation was caused by the inhibition of autophagic flux by testing the expression of LC3II, p62 levels, and mRFP-GFP-LC3. Further, we found that TCDD treatment significantly increased the amount of triglyceride (TG) and total cholesterol (TC) in THP-1 macrophages. Meanwhile, pretreatment with autophagy activator (rapamycin, Rapa) efficiently relieved TCDD-induced lipid accumulation. On the contrary, pretreatment with autophagy inhibitor (Chloroquine, CQ) promoted TCDD-induced lipid accumulation. In the experiment of co-localization of LC3 and lipid droplets, the co-localization of LC3 and lipid droplets increased after TCDD induction. These results indicated that TCDD promoted lipid accumulation in THP-1 macrophages by inhibiting autophagic flux. Our findings revealed new insights into the toxicity mechanisms of TCDD.

Effect of oxytocin on lipid accumulation under inflammatory conditions in human macrophages

Introduction and aimsOxytocin (OT) is a neuropeptide hormone secreted by the posterior pituitary gland. Deficits in OT action have been observed in patients with behavioral and mood disorders, some of which correlate with an increased risk of cardiovascular disease (CVD). Recent research has revealed a wider systemic role that OT plays in inflammatory modulation and development of atherosclerotic plaques. This study investigated the role that OT plays in cholesterol transport and foam cell formation in LPS-stimulated THP-1 human macrophages. MethodsTHP-1 differentiated macrophages were treated with media, LPS (100 ng/ml), LPS + OT (10 pM), or LPS + OT (100 pM). Changes in gene expression and protein levels of cholesterol transporters were analyzed by real time quantitative PCR (RT-qPCR) and Western blot, while oxLDL uptake and cholesterol efflux capacity were evaluated with fluorometric assays. ResultsRT-qPCR analysis revealed a significant increase in ABCG1 gene expression upon OT + LPS treatment, compared to LPS alone (p = 0.0081), with Western blotting supporting the increase in expression of the ABCG1 protein. Analysis of oxLDL uptake showed a significantly lower fluorescent value in LPS + OT (100pM) -treated cells when compared to LPS alone (p < 0.0001). While not statistically significant (p = 0.06), cholesterol efflux capacity increased with LPS + OT treatment. ConclusionWe demonstrate here that OT can attenuate LPS-mediated lipid accumulation in THP-1 macrophages. These findings support the hypothesis that OT could be used to reduce pro-inflammatory and potentially atherogenic changes observed in patients with heightened CVD risk. This study suggests further exploration of OT effects on monocyte and macrophage cholesterol handling in vivo.

LncRNA kcnq1ot1 promotes lipid accumulation and accelerates atherosclerosis via functioning as a ceRNA through the miR-452-3p/HDAC3/ABCA1 axis

Kcnq1 overlapping transcript 1 (kcnq1ot1), an imprinted antisense lncRNA in the kcnq1 locus, acts as a potential contributor to cardiovascular disease, but its role in atherosclerosis remains unknown. The aim of this study was to explore the effects of kcnq1ot1 on atherogenesis and the underlying mechanism. Our results showed that kcnq1ot1 expression was significantly increased in mouse aorta with atherosclerosis and lipid-loaded macrophages. Lentivirus-mediated kcnq1ot1 overexpression markedly increased atherosclerotic plaque area and decreased plasma HDL-C levels and RCT efficiency in apoE−/− mice fed a Western diet. Upregulation of kcnq1ot1 also reduced the expression of miR-452-3p and ABCA1 but increased HDAC3 levels in mouse aorta and THP-1 macrophages. Accordingly, kcnq1ot1 overexpression inhibited cholesterol efflux and promoted lipid accumulation in THP-1 macrophages. In contrast, kcnq1ot1 knockdown protected against atherosclerosis in apoE−/− mice and suppressed lipid accumulation in THP-1 macrophages. Mechanistically, kcnq1ot1 enhanced HDAC3 expression by competitively binding to miR-452-3p, thereby inhibiting ABCA1 expression and subsequent cholesterol efflux. Taken together, these findings suggest that kcnq1ot1 promotes macrophage lipid accumulation and accelerates the development of atherosclerosis through the miR-452-3p/HDAC3/ABCA1 pathway.

Circulating Levels of CILP2 Are Elevated in Coronary Heart Disease and Associated with Atherosclerosis.

Methods and Results Circulating CILP2 levels (measured by ELISA) were compared to various insulin resistance- and atherosclerosis-related parameters in normal subjects and newly diagnosed CHD patients. THP-1 cells were cultured and treated with indicated stimulators. Western blots and RT-PCR were performed to examine protein and mRNA expressions. The results showed that there were significantly higher circulating CILP2 levels in CHD patients relative to healthy controls. Circulating CILP2 correlated positively with waist-hip ratio (WHR), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), HbA1c, homeostasis model assessment of insulin resistance (HOMA-IR), and Gensini scores. In an in vitro study, we found that CILP2 increased oxidatively modified LDL-stimulated lipid accumulation in THP-1 macrophages via the upregulation of CD36 expression. Inhibition of PPARγ signaling eliminated the CILP2 regulation of CD36 expression in THP-1 macrophages. CILP2 positively regulated CD36 transcription through PPARγ-mediated action on two peroxisome-proliferator-responsive elements (PPREs) binding sites of CD36 promoter, PPRE-G, and PPRE-J. Conclusions Our findings have uncovered a novel role for CILP2 in lipid uptake and foam cell formation. This role is mediated by CD36 through the activation of PPARγ pathway.

Di-n-butyl phthalate promotes lipid accumulation via the miR200c-5p-ABCA1 pathway in THP-1 macrophages

Di-n-butyl phthalate (DBP) is ubiquitously in the environment and has been detected in almost all of human bodies. Few data could be found about the effects of DBP on cardiovascular system, though its reproductive toxicities have been studied extensively. This study aimed to explore the effects of DBP on lipid metabolism, a key step during the formation of atherosclerosis, since DBP was recently reported to be associated with atherosclerosis. THP-1 macrophages were employed and exposed to various levels of DBP (10−8, 10−7, 10−6, 10−5 and 10−4 mol/L) or DMSO as control. Lipid accumulation was determined by detection of cellular total cholesterol, free cholesterol, cholesterol ester and content of lipid drops. Expressions of mRNA/miRNAs and proteins were measured by qRT-PCR and western blotting, respectively. Bioinformatic analysis and dual luciferase reporter assay were used to analyze the combination between miR200c-5p and ATP-binding cassette transporter A1 (ABCA1). Cholesterol efflux assay was executed to study the inhibitory effects of DBP on cholesterol efflux capability. Results revealed that DBP at 10−7 mol/L prompted THP-1 macrophages lipid accumulation by inhibiting cholesterol efflux via suppressing ABCA1 expression. In addition, a non-linear inverted U-shaped relationship between DBP and lipid accumulation could be observed. Moreover, miR200c-5p could directly targets to ABCA1 3′UTR and modulate ABCA1 expression. Besides, downregulation of ABCA1 expression and reduction of lipid efflux induced by DBP were due to the miR200c-5p upregulation. Collectively, these data suggested that DBP at levels relative to human exposure could increase lipid accumulation in THP-1 macrophages by decreasing cholesterol efflux through miR200c-5p-ABCA1, then potentiate the formation of atherosclerosis.

Role of caffeic and chlorogenic acid in the modulation of cellular fatty acid uptake

AbstractPolyphenols are bioactive molecules widely distributed in numerous foods such as fruits, vegetables, tea, coffee, cocoa and beverages. Their main classification include flavonoids (i.e. flavonols, flavones, flavanones, flavanols, anthocyanins, and isoflavones), non-flavonoids (i.e. lignans and stilbens) and phenolic acids (i.e. hydroxycinnamic and hydroxybenzoic acids)(1). Caffeic acid (CA) and chlorogenic acid (CGA; an ester of CA and quinic acid) are the major representatives of hydroxycinnamic acids. Accumulating evidence has demonstrated that CA and CGA may exert different biological activities, including antioxidant, anti-inflammatory, antidiabetic, and antihypertensive(2). Despite these promising and diverse anti-atherosclerotic actions, investigations addressing the effect of CA and CGA on atherogenesis are scarce.The present study evaluated the capacity of CA and CGA to reduce lipid accumulation in macrophages derived from monocytic THP-1 cells. THP-1-derived macrophages were incubated with fatty acids (500 μM oleic/palmitic acid, 2:1 ratio) and different concentrations (from 0.03 to 3 μM) of CA and CGA, alone or in combination. Lipid accumulation was quantified spectrophotometrically (excitation: 544 nm, emission: 590 nm) with the fluorescent dye, Nile red. The fold increase compared to the control (without fatty acids) was calculated. In addition, the expression of several transcription factors including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein (CEBP), as potential mechanisms involved in the regulation of lipid accumulation, was evaluated by real time PCR.Analysis of variance (ANOVA) was used to assess the effect of the different concentrations of CA and CGA on lipid accumulation in THP-1 macrophages following stimulation with FA.The preliminary results obtained have shown a significant increase in lipid accumulation following fatty acid exposure (p &lt; 0.0001). Incubation with CA and CGA did not reduce lipid accumulation in THP-1 derived macrophages, while the combination of CA + CGA at 0.03, 0.3 and 3 μM (p &lt; 0.01) decreased cellular fatty acid uptake at all concentrations tested by -28%, -32%, -23%, respectively. An apparent modulation of the transcriptional activity of PPARγ, but not CEBP, was observed following the combination of phenolic acids.In conclusion, the incubation of CA + CGA at physiologically relevant concentrations, but not the single compounds, seem to reduce the uptake of fatty acids in THP-1-derived macrophages. Further experiments are ongoing in order to confirm the findings obtained and to better identify the mechanisms of action involved in the reduction of lipid accumulation as a key phenomenon of atherogenesis.

Atorvastatin Blocks Advanced Glycation End Products Induced Reduction in Macrophage Cholesterol Efflux Mediated With ATP-Binding Cassette Transporters G 1.

There is accumulating evidence that the AGEs-RAGE interaction plays an important role in accelerated atherosclerosis in diabetes. Our previous study showed that the AGEs-RAGE axis can reduce the cholesterol efflux of THP-1 macrophages through suppression of the expression of ABCG1 and that statins can inhibit the expression of RAGE. However, the role of statins in recovering the cholesterol efflux of macrophages reduced by AGEs has not been assessed.Methods and Results:ApoE-/-mice and THP-1 macrophages were both treated by AGEs or AGEs combined with anti-RAGE antibody (only in THP-1 cells), ALT711 and atorvastatin separately. Cholesterol efflux of THP-1 macrophages and murine peritoneal macrophages was tested by fluorescence microplate technique. RT-PCR and western blot analysis were used to measure the expression of RAGE and molecules included in cholesterol efflux. After co-incubating with atorvastatin and AGEs, reduction in lipid accumulation in THP-1 macrophages and improvement of lesions complexity occurred compared with treating by AGEs only. Atorvastatin increased cholesterol efflux and ABCG1 expression of macrophages, which were reduced by AGEs, and decreased the expression of RAGE at the same time. This study demonstrated that atorvastatin can recover the deleterious ABCG1-mediated cholesterol efflux induced by AGEs in THP-1 macrophages and murine peritoneal macrophages by downregulating RAGE expression. It may contribute to the protective action of atorvastatin in diabetic subjects with atherosclerosis.

Multi-walled carbon nanotubes (MWCNTs) promoted lipid accumulation in THP-1 macrophages through modulation of endoplasmic reticulum (ER) stress

Recent studies from us and others have shown that nanoparticle (NP) exposure could modulate endoplasmic reticulum (ER) stress. Since ER stress is closely related to lipid droplet biogenesis, in this study, we investigated if multi-walled carbon nanotubes (MWCNTs) might influence lipid accumulation in THP-1 macrophages through the modulation of ER stress. Exposure to up to 64 μg/mL pristine MWCNTs (p-MWCNTs), hydroxylated MWCNTs (h-MWCNTs), and carboxylated MWCNTs (c-MWCNTs) led to NP internalization, which was associated with a modest increase of cytotoxicity. Oxidative stress was also induced, showing as increased intracellular reactive oxygen species (ROS) and decreased glutathione. Only h-MWCNTs and c-MWCNTs significantly promoted the release of interleukin-8 (IL-8), which was significantly higher compared with that after p-MWCNT exposure. All types of MWCNTs significantly induced lipid accumulation in THP-1 macrophages and more modestly in the co-culture model consisting of 16HBE human bronchial epithelial cells and THP-1 macrophages. MWCNT-induced lipid accumulation in THP-1 macrophages was decreased modestly by antioxidant N-acetyl-l-cysteine and more effectively by ER stress inhibitor 4-phenylbutyric acid. Moreover, MWCNT exposure promoted the expression of ER stress gene DDIT3 as well as ER stress protein p-chop, whereas the transcription factor XBP-1s was decreased. In addition, the expression of scavenger receptors, namely CD36 and MSR1, was also elevated after MWCNT exposure. In conclusion, this study suggested that MWCNT exposure could promote lipid accumulation in THP-1 macrophages, which could be related to the modulation of ER stress leading to upregulation of scavenger receptors.

MicroRNA–21 attenuates BDE-209-induced lipid accumulation in THP-1 macrophages by downregulating Toll-like receptor 4 expression

Growing evidence demonstrates a possible response of specific microRNA (miRNA) to environmental pollutant stimuli in multiple biological processes. We previously reported that a persistent organic pollutant, decabromodiphenyl ether (BDE-209), can enhance Toll-like receptor 4 (TLR4)-dependent lipid uptake in THP-1 macrophages; whether miRNAs are involved in this process remains unclear. In the present study, we investigated the levels of several miRNAs related to TLR4 signaling, including miRs–9, –21, −27b, −125b, −132, −146a, −147, −155, and –let-7e, in THP-1 macrophages after stimulation by BDE-209 and oxidized low-density lipoprotein. The results showed that the levels of miR–21 were significantly suppressed by BDE-209 at concentrations of 6.25, 12.5 and 25 μM, in a dose-dependent manner; whereas there was no significant changes for the other miRNAs investigated. Moreover, the suppression of miR–21 was accompanied by an upregulated TLR4 expression, at both mRNA and protein levels. Further analysis showed that the up-regulated TLR4 induced by BDE-209 was inhibited in macrophages transfected with miR–21 mimic; meanwhile opposite results were exhibited when an anti-miR–21 inhibitor was transfected to the macrophages. Additionally, transfection with miR–21 mimic effectively attenuated BDE-209-induced lipid accumulation in macrophages. Together, these data illustrate that miR–21 inhibits BDE-209-triggered lipid accumulation in macrophages through down-regulating TLR4 expression.

Inflammation-induced mTORC2-Akt-mTORC1 signaling promotes macrophage foam cell formation

The transformation of macrophages into lipid-loaded foam cells is a critical and early event in the pathogenesis of atherosclerosis. Several recent reports highlighted that induction of TLR4 signaling promotes macrophage foam cell formation; however, the underlying molecular mechanisms have not been clearly elucidated. Here, we found that the TLR4 mediated inflammatory signaling communicated with mTORC2-Akt-mTORC1 metabolic cascade in macrophage and thereby promoting lipid uptake and foam cell formation. Mechanistically, LPS treatment markedly upregulates TLR4 mediated inflammatory pathway which by activating mTORC2 induces Akt phosphorylation at serine 473 and that aggravate mTORC1 dependent scavenger receptors expression and consequent lipid accumulation in THP-1 macrophages. Inhibition of mTORC2 either by silencing Rictor expression or inhibiting its association with mTOR notably prevents LPS induced Akt activation, scavenger receptors expression, and macrophage lipid accumulation. Although suppression of mTORC1 expression by genetic knockdown of Raptor did not produce any significant change in Akt S473 phosphorylation, however, incubation with Akt activator in Rictor silenced cells failed to promote scavenger receptors expression and macrophage foam cell formation. Thus, present research explored the signaling pathway involved in inflammation-induced macrophage foam cells formation and therefore, targeting this pathway might be useful for preventing macrophage foam cell formation.

Blocking Wnt5a signaling decreases CD36 expression and foam cell formation in atherosclerosis.

Wnt5a is a highly studied member of the Wnt family and recently has been implicated in the pathogenesis of atherosclerosis, but its precise role is unknown. Foam cell development is a critical process to atherosclerotic plaque formation. In the present study, we investigated the role of noncanonical Wnt5a signaling in the development of foam cells. Human carotid atherosclerotic tissue and THP-1-derived macrophages were used to investigate the contribution of Wnt5a signaling in the formation of foam cells. Immunohistochemistry was used to evaluate protein expression of scavenger receptors and noncanonical Wnt5a receptors [frizzled 5 (Fz5) and receptor tyrosine kinase-like orphan receptor 2 (Ror2)] in human atherosclerotic macrophages/foam cells. Changes in protein expression in response to Wnt5a stimulation/inhibition were determined by Western blot, and lipid accumulation was evaluated by fluorescent lipid droplet staining. Wnt5a (P<.05), Fz5 (P<.01), and Ror2 (P<.01) were significantly expressed in advanced atherosclerotic lesions compared to less advanced lesions (N=10). Wnt5a, Fz5, and Ror2 were expressed in macrophages/foam cells within the plaque. In vitro studies revealed that Wnt5a significantly increased the expression of the lipid uptake receptor CD36 (P<.05) but not the lipid efflux receptor ATP-binding cassette transporter (P>.05). rWnt5a also significantly increased lipid accumulation in THP-1 macrophages (P<.05). Furthermore, inhibition of Wnt5a signaling with Box5 prevented lipid accumulation (P<.01) and prevented CD36 up-regulation (P<.01). These results suggest a direct role for Wnt5a signaling in the pathogenesis of atherosclerosis, specifically the accumulation of lipid in macrophages and the formation of foam cells.

MiR-497 accelerates oxidized low-density lipoprotein-induced lipid accumulation in macrophages by repressing the expression of apelin.

microRNAs (miRNAs) play important roles in the pathogenesis of atherosclerosis. A previous study has reported that miR-497 is elevated in advanced atherosclerotic lesions in an apoE-deficient (apoE-/-) mouse model. The purpose of this study is to test whether miR-497 can modulate macrophage foam cell formation, an initiating event in atherosclerosis. We found that miR-497 was upregulated in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). Enforced expression of miR-497 promoted lipid accumulation and decreased cholesterol efflux in oxLDL-exposed THP-1 macrophages. In contrast, downregulation of miR-497 suppressed oxLDL-induced lipid accumulation in THP-1 macrophages. Apelin was identified to be a downstream target of miR-497. Overexpression of miR-497 significantly reduced the expression of apelin in THP-1 macrophages. Interestingly, delivery of a miR-497-resistant variant of apelin significantly inhibited lipid accumulation and enhanced cholesterol efflux in miR-497-overexpressing THP-1 macrophages in response to oxLDL. In addition, miR-497 expression was increased and negatively correlated with apelin protein expression in human atherosclerotic lesions. In conclusion, miR-497 contributes to oxLDL-induced lipid deposition in macrophages largely via targeting of apelin and thus represents a potential therapeutic target for atherosclerosis.

Puerarin promotes ABCA1-mediated cholesterol efflux and decreases cellular lipid accumulation in THP-1 macrophages

It was reported that puerarin decreases the total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and increases high-density lipoprotein cholesterol (HDL-C) level, but the underlying mechanism is unclear. This study was designed to determine whether puerarin decreased lipid accumulation via up-regulation of ABCA1-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. Our results showed that puerarin significantly promoted the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via the AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor-alpha (LXR-α) pathway and decreased cellular lipid accumulation in human THP-1 macrophage-derived foam cells. The miR-7 directly targeted 3′ untranslated region of STK11 (Serine/Threonine Kinase 11), which activated the AMPK pathway. Transfection with miR-7 mimic significantly reduced STK11 expression in puerarin-treated macrophages, decreased the phosphorylation of AMPK, down-regulated the expression of the PPARγ-LXR-α-ABCA1 expression. Additionally, treatment with miR-7 decreased cholesterol efflux and increased cholesterol levels in THP-1 macrophage-derived foam cells. Our study demonstrates that puerarin promotes ABCA1-mediated cholesterol efflux and decreases intracellular cholesterol levels through the pathway involving miR-7, STK11, and the AMPK-PPARγ-LXR-α-ABCA1 cascade.

MicroRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2

Transformation of macrophages into foam cells plays a critical role in the pathogenesis of atherosclerosis. The aim of this study was to determine the expression and biological roles of microRNA (miR)-150 in the formation of macrophage foam cells and to identify its functional target(s). Exposure to 50 μg/ml oxidized low-density lipoprotein (oxLDL) led to a significant upregulation of miR-150 in THP-1 macrophages. Overexpression of miR-150 inhibited oxLDL-induced lipid accumulation in THP-1 macrophages, while knockdown of miR-150 enhanced lipid accumulation. apoA-I- and HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in miR-150-overexpressing macrophages relative to control cells. In contrast, downregulation of miR-150 significantly reduced cholesterol efflux from oxLDL-laden macrophages. Bioinformatic analysis and luciferase reporter assay revealed adiponectin receptor 2 (AdipoR2) as a direct target of miR-150. Small interfering RNA-mediated downregulation of AdipoR2 phenocopied the effects of miR-150 overexpression, reducing lipid accumulation and facilitating cholesterol efflux in oxLDL-treated THP-1 macrophages. Knockdown of AdipoR2 induced the expression of proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα), ABCA1, and ABCG1. Moreover, pharmacological inhibition of PPARγ or LXRα impaired AdipoR2 silencing-induced upregulation of ABCA1 and ABCG1. Taken together, our results indicate that miR-150 can attenuate oxLDL-induced lipid accumulation in macrophages via promotion of cholesterol efflux. The suppressive effects of miR-150 on macrophage foam cell formation are mediated through targeting of AdipoR2. Delivery of miR-150 may represent a potential approach to prevent macrophage foam cell formation in atherosclerosis.

MicroRNA-186 promotes macrophage lipid accumulation and secretion of pro-inflammatory cytokines by targeting cystathionine γ-lyase in THP-1 macrophages

Background and aimsSeveral studies suggest that cardiomyocyte-enriched miR-186 is involved in cardiac injury and myocardial infarction, and also plays an important role in atherosclerotic diseases, but the underlying mechanism is unknown. Cystathionine-γ-lyase (CSE) is the predominant enzyme to produce H2S in the cardiovascular system. Here, miR-186 was identified to bind to the 3′UTR of CSE. In this study, we aimed at exploring whether miR-186 affects lipid accumulation and secretion of pro-inflammatory cytokines by targeting CSE and its underlying mechanism in human THP-1 macrophages and peripheral blood monocyte-derived macrophages (PBMDM). PBMDM just as a control group for the comparison with the THP-1 macrophages. MethodsMiR-186 target genes, CSE 3′UTR sequence and free energy were predicted and analyzed by bioinformatics analyses and dual-luciferase reporter assays. The expression of CSE mRNA and protein were measured by real-time quantitative PCR and western blot analyses. The lipid accumulation in THP-1 macrophages was detected by high performance liquid chromatography (HPLC). The effects of miR-186 on secretion of IL-6, IL-1β and TNF-α were examined by ELISA. Endogenous H2S was detected by spectrophotometry. Using small interfering RNA (siRNA) approach to decrease the expression of CSE protein and mRNA. ResultsWe found that miR-186 directly inhibited CSE protein and mRNA expression through targeting CSE 3′UTR by bioinformatics analyses and dual-luciferase reporter assays. HPLC assays showed that miR-186 increased the lipid accumulation in human THP-1 macrophages. We also showed that miR-186 enhanced secretion of pro-inflammatory cytokines in human THP-1 macrophages. Using siRNA approach, we found that CSE siRNA could inhibit the miR-186 inhibitor-induced decrease in the expression of LPL protein and mRNA in human THP-1 macrophages, which was accompanied a decrease in the level of H2S. ConclusionsMicroRNA-186 promotes macrophage lipid accumulation and pro-inflammatory cytokine secretion by targeting cystathionine γ-lyase in THP-1 macrophages.

UFM1 Protects Macrophages from oxLDL-Induced Foam Cell Formation Through a Liver X Receptor α Dependent Pathway.

Macrophage foam cell formation is the most prominent characteristic of the early stages of atherosclerosis. Ubiquitin Fold Modifier 1 (UFM1) is a new member of the ubiquitin-like protein family, and its underlying mechanism of action in macrophage foam cell formation is poorly understood. Our current study focuses on UFM1 and investigates its role in macrophage foam cell formation. Using real-time quantitative PCR (qRT-PCR) and western blot analysis, we first analyzed the UFM1 expression in mouse peritoneal macrophages (MPMs) from ApoE－/－ mice in vivo and in human macrophages treated with oxLDL in vitro. Subsequently, the effects of UFM1 on macrophages foam cell formation were determined by Nile Red staining and direct lipid analysis. We then examined whether UFM1 affects the process of lipid metabolism in macrophages. Lastly, with the method of small interfering RNA (siRNA), we delineated the mechanism of UFM1 to attenuate lipid accumulation in THP-1 macrophages. UFM1 is dramatically upregulated under atherosclerosis conditions both in vivo and in vitro. Moreover, UFM1 markedly decreased macrophage foam cell formation. Mechanistic studies revealed that UFM1 increased the macrophage cholesterol efflux, which was due to the increased expression of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Furthermore, the upregulation of ABCA1 and ABCG1 by UFM1 resulted from liver X receptor α (LXRα) activation, which was confirmed by the observation that LXRα siRNA prevented the expression of ABCA1 and ABCG1. Consistent with this, the UFM1-mediated attenuation of lipid accumulation was abolished by such inhibition. Taken together, our results showed that UFM1 could suppress foam cell formation via the LXRα-dependent pathway.