ABCA1 Expression In Macrophages Research Articles

Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment

ABCA1 has been found to be critical for cholesterol efflux in macrophages. Understanding the mechanism regulating ABCA1 expression is important for the prevention and treatment of atherosclerosis. In the present study, a G-quadruplex (G4) structure was identified in the ABCA1 promoter region. This G4 was shown to be essential for ABCA1 transcription. Stabilizing the G4 by ligands surprisingly upregulated ABCA1 expression in macrophages. Knocking out the G4 remarkably reduced ABCA1 expression, and abolished the increase of ABCA1 expression induced by the G4 ligand. By pull-down assays, the protein NONO was identified as an ABCA1 G4 binder. Overexpression or repression of NONO significantly induced upregulation and downregulation of ABCA1 expression, respectively. ChIP and EMSA experiments showed that the G4 ligand promoted the binding between the ABCA1 G4 and NONO, which led to more recruitment of NONO to the promoter region and enhanced ABCA1 transcription. Finally, the G4 ligand was shown to significantly reduce the accumulation of cholesterol in macrophages. This study showed a new insight into the regulation of gene expression by G4, and provided a new molecular mechanism regulating ABCA1 expression in macrophages. Furthermore, the study showed a possible novel application of the G4 ligand: preventing and treating atherosclerosis.

Administration of an LXR agonist promotes atherosclerotic lesion remodelling in murine inflammatory arthritis.

The leading cause of mortality in patients with rheumatoid arthritis is atherosclerotic cardiovascular disease (CVD). We have shown that murine arthritis impairs atherosclerotic lesion regression, because of cellular cholesterol efflux defects in haematopoietic stem and progenitor cells (HSPCs), causing monocytosis and impaired atherosclerotic regression. Therefore, we hypothesised that improving cholesterol efflux using a Liver X Receptor (LXR) agonist would improve cholesterol efflux and improve atherosclerotic lesion regression in arthritis. Ldlr -/- mice were fed a western-type diet for 14 weeks to initiate atherogenesis, then switched to a chow diet to induce lesion regression and divided into three groups; (1) control, (2) K/BxN serum transfer inflammatory arthritis (K/BxN) or (3) K/BxN arthritis and LXR agonist T0901317 daily for 2 weeks. LXR activation during murine inflammatory arthritis completely restored atherosclerotic lesion regression in arthritic mice, evidenced by reduced lesion size, macrophage abundance and lipid content. Mechanistically, serum from arthritic mice promoted foam cell formation, demonstrated by increased cellular lipid accumulation in macrophages and paralleled by a reduction in mRNA of the cholesterol efflux transporters Abca1, Abcg1 and Apoe. T0901317 reduced lipid loading and increased Abca1 and Abcg1 expression in macrophages exposed to arthritic serum and increased ABCA1 levels in atherosclerotic lesions of arthritic mice. Moreover, arthritic clinical score was also attenuated with T0901317. Taken together, we show that the LXR agonist T0901317 rescues impaired atherosclerotic lesion regression in murine arthritis because of enhanced cholesterol efflux transporter expression and reduced foam cell development in atherosclerotic lesions.

Role of miR-199a-5p in the post-transcriptional regulation of ABCA1 in response to hypoxia in peritoneal macrophages.

Hypoxia is a crucial factor contributing to maintenance of atherosclerotic lesions. The ability of ABCA1 to stimulate the efflux of cholesterol from cells in the periphery, particularly foam cells in atherosclerotic plaques, is an important anti-atherosclerotic mechanism. The posttranscriptional regulation by miRNAs represents a key regulatory mechanism of a number of signaling pathways involved in atherosclerosis. Previously, miR-199a-5p has been shown to be implicated in the endocytic and retrograde intracellular transport. Although the regulation of miR-199a-5p and ABCA1 by hypoxia has been already reported independently, the role of miR-199a-5p in macrophages and its possible role in atherogenic processes such us regulation of lipid homeostasis through ABCA1 has not been yet investigated. Here, we demonstrate that both ABCA1 and miR-199a-5p show an inverse regulation by hypoxia and Ac-LDL in primary macrophages. Moreover, we demonstrated that miR-199a-5p regulates ABCA1 mRNA and protein levels by directly binding to its 3'UTR. As a result, manipulation of cellular miR-199a-5p levels alters ABCA1 expression and cholesterol efflux in primary mouse macrophages. Taken together, these results indicate that the correlation between ABCA1-miR-199a-5p could be exploited to control macrophage cholesterol efflux during the onset of atherosclerosis, where cholesterol alterations and hypoxia play a pathogenic role.

Retinoic Acid Receptor Alpha (RARα) in Macrophages Protects from Diet-Induced Atherosclerosis in Mice.

Retinoic acid signaling plays an important role in regulating lipid metabolism and inflammation. However, the role of retinoic acid receptor alpha (RARα) in atherosclerosis remains to be determined. In the current study, we investigated the role of macrophage RARα in the development of atherosclerosis. Macrophages isolated from myeloid-specific Rarα-/- (RarαMac-/-) mice showed increased lipid accumulation and inflammation and reduced cholesterol efflux compared to Rarαfl/fl (control) mice. All-trans retinoic acid (AtRA) induced ATP-binding cassette subfamily A member 1 (Abca1) and Abcg1 expression and cholesterol efflux in both RarαMac-/- mice and Rarαfl/fl mice. In Ldlr-/- mice, myeloid ablation of RARα significantly reduced macrophage Abca1 and Abcg1 expression and cholesterol efflux, induced inflammatory genes, and aggravated Western diet-induced atherosclerosis. Our data demonstrate that macrophage RARα protects against atherosclerosis, likely via inducing cholesterol efflux and inhibiting inflammation.

Deletion of Macrophage-Specific Glycogen Synthase Kinase (GSK)-3α Promotes Atherosclerotic Regression in Ldlr-/- Mice.

Recent evidence from our laboratory suggests that impeding ER stress–GSK3α/β signaling attenuates the progression and development of atherosclerosis in mouse model systems. The objective of this study was to determine if the tissue-specific genetic ablation of GSK3α/β could promote the regression of established atherosclerotic plaques. Five-week-old low-density lipoprotein receptor knockout (Ldlr−/−) mice were fed a high-fat diet for 16 weeks to promote atherosclerotic lesion formation. Mice were then injected with tamoxifen to induce macrophage-specific GSK3α/β deletion, and switched to standard diet for 12 weeks. All mice were sacrificed at 33 weeks of age and atherosclerosis was quantified and characterized. Female mice with induced macrophage-specific GSK3α deficiency, but not GSK3β deficiency, had reduced plaque volume (~25%) and necrosis (~40%) in the aortic sinus, compared to baseline mice. Atherosclerosis was also significantly reduced (~60%) in the descending aorta. Macrophage-specific GSK3α-deficient mice showed indications of increased plaque stability and reduced inflammation in plaques, as well as increased CCR7 and ABCA1 expression in lesional macrophages, consistent with regressive plaques. These results suggest that GSK3α ablation promotes atherosclerotic plaque regression and identify GSK3α as a potential target for the development of new therapies to treat existing atherosclerotic lesions in patients with cardiovascular disease.

Yin-xing-tong-mai decoction attenuates atherosclerosis via activating PPARγ-LXRα-ABCA1/ABCG1 pathway

Atherosclerosis is now the major cause of mortality and morbidity worldwide. Formation of macrophage-derived foam cells is a hallmark of atherosclerosis, which is regulated by cholesterol uptake, intracellular metabolism, and efflux. PPARγ-LXRα-ABCA1/ABCG1 pathway plays an important part in regulating cholesterol efflux and this pathway could be a promising target for treating atherosclerosis. However, due to undesirable systemic effects, PPARγ agonist therapy for atherosclerosis remains challenging. Many traditional Chinese medicine has been well accepted and applied in atherosclerosis treatment. Yin-xing-tong-mai decoction (YXTMD) has been applied for treating atherosclerosis for decades. However, the mechanism remains to be explored. Here, we showed that YXTMD effectively attenuated atherosclerosis in ApoE-/- mice. YXTMD increased cholesterol efflux of foam cell by upregulation of ABCA1 and ABCG1 in vivo and in vitro. Through bioinformatic analysis and experimental validation, we found that PPARγ was an important downstream effector of YXTMD in macrophages. Reduction of PPARγ significantly decreased LXRα, ABCA1, and ABCG1 expression in macrophages, with reduced cholesterol efflux. In conclusion, these findings confirmed that YXTMD attenuated atherosclerosis by activating the PPARγ-LXRα- ABCA1/ABCG1 pathway to enhance cholesterol efflux.

Salmonella Typhimurium manipulates macrophage cholesterol homeostasis through the SseJ-mediated suppression of the host cholesterol transport protein ABCA1.

Upon infection of host cells, Salmonella enterica serovar Typhimurium resides in a modified-endosomal compartment referred to as the Salmonella-containing vacuole (SCV). SCV biogenesis is driven by multiple effector proteins translocated through two type III secretion systems (T3SS-1 and T3SS-2). While many host proteins targeted by these effector proteins have been characterised, the role of host lipids in SCV dynamics remains poorly understood. Previous studies have shown that S. Typhimurium infection in macrophages leads to accumulation of intracellular cholesterol, some of which concentrates in and around SCVs; however, the underlying mechanisms remain unknown. Here, we show that S. Typhimurium utilises the T3SS-2 effector SseJ to downregulate expression of the host cholesterol transporter ABCA1 in macrophages, leading to a ~45% increase in cellular cholesterol. Mechanistically, SseJ activates a signalling cascade involving the host kinases FAK and Akt to suppress Abca1 expression. Mutational inactivation of SseJ acyltransferase activity, silencing FAK, or inhibiting Akt prevents Abca1 downregulation and the corresponding accumulation of cholesterol during infection. Importantly, RNAi-mediated silencing of ABCA1 rescued bacterial survival in FAK-deficient macrophages, suggesting that Abca1 downregulation and cholesterol accumulation are important for intracellular survival.

Long non-coding RNA PCA3 inhibits lipid accumulation and atherosclerosis through the miR-140-5p/RFX7/ABCA1 axis

ObjectiveThe purpose of this study was to explore the role of long noncoding RNA (lncRNA) prostate cancer antigen 3 (PCA3) in atherosclerosis and the underlying mechanism. MethodsThe Gene Expression Omnibus (GEO) datasets were used to divide differentially expressed lncRNAs, microRNAs (miRNAs), and mRNAs. The expression of PCA3, miR-140-5p, RFX7 and ABCA1 were determined by qPCR or Western blot in ox-LDL-treated macrophages. Macrophage lipid accumulation s was evaluated using the Oil Red O staining and high-performance liquid chromatography. Target relationships among PCA3, miR-140-5p, RFX7, and ABCA1 promoter area were validated via dual-luciferase reporter gene assay or chromatin immunoprecipitation assay. The apoE−/− mouse model in vivo was designed to evaluate the effect of PCA3 on the reverse cholesterol transport (RCT) and atherosclerosis. ResultsPCA3 was down-regulated in foam cells, whereas miR-140-5p was highly expressed. Overexpression of PCA3 promoted ABCA1-mediated cholesterol efflux and reduced lipid accumulation in macrophages. Besides, RFX7 bound to the ABCA1 promoter and increased ABCA1 expression. Targeted relationships and interactions on the expression between miR-140-5p and PCA3 or RFX7 were elucidated. PCA3 up-regulated ABCA1 expression by binding to miR-140-5p to up-regulate RFX7 and ABCA1 expression in macrophages. PCA3 promoted RCT and impeded the progression of atherosclerosis by sponging miR-140-5p in apoE−/− mice. Meanwhile, miR-140-5p also inhibit ABCA1 expression via downregulation of RFX7 to impede RCT and aggravate atherosclerosis. ConclusionslncRNA PCA3 promotes ABCA1-mediated cholesterol efflux to inhibit atherosclerosis through sponging miR-140-5p and up-regulating RFX7.

Astragalin Retards Atherosclerosis by Promoting Cholesterol Efflux and Inhibiting the Inflammatory Response via Upregulating ABCA1 and ABCG1 Expression in Macrophages.

Lipid metabolism disorder and inflammatory response are considered to be the major causes of atherosclerogenesis. Astragalin, the most important functional component of flavonoid obtained from persimmon leaves, has the hypolipidemic effects. However, it is unknown, how astragalin protects against atherosclerosis. The aim of this study was to observe the effects of astragalin on cholesterol efflux and inflammatory response and to explore the underlying mechanisms. Our results showed that astragalin upregulated the expression of ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1), promoted cholesterol efflux, and suppressed foam cell formation. Inhibition of the PPARγ/LXRα pathway abrogated the promotive effects of astragalin on both transporter expression and cholesterol efflux. In addition, treatment of astragalin markedly decreased the secretion of inflammatory factors, including interleukin 6, monocyte chemotactic protein 1, tumor necrosis factor α, and interleukin 1β. Mechanistically, astragalin upregulated ABCA1 and ABCG1 expression, which in turn reduced TLR4 surface levels and inhibited NF-κB nuclear translocation. Consistently, astragalin reduced atherosclerotic plaque area in apoE-/- mice. Taken together, these findings suggest that astragalin protects against atherosclerosis by promoting ABCA1- and ABCG1-mediated cholesterol efflux and inhibiting proinflammatory mediator release.

Endoplasmic Reticulum Stress Affects Cholesterol Homeostasis by Inhibiting LXRα Expression in Hepatocytes and Macrophages.

Atherosclerosis (AS) is the most common cardiovascular disease, and reverse cholesterol transport (RCT) plays an important role in maintaining cholesterol homeostasis. Both endoplasmic reticulum (ER) stress and LXRα can affect the metabolism of cholesterol. However, whether ER stress can modulate cholesterol metabolism by LXRα in hepatocytes and macrophages remains unclear. Therefore, in this study, we aimed to explore the relationship between ER stress induced by tunicamycin and LXRα in hepatocytes and macrophages and clarify their possible mechanisms and roles in AS. C57BL/6 mice and Huh-7 and THP-1 cells were treated with tunicamycin and LXR-623 (an agonist of LXRα) alone or in combination. Tunicamycin-induced ER stress caused liver injury; promoted the accumulation of cholesterol and triglycerides; inhibited the expression of LXRα, ABCA1 and ABCG1 in the livers of mice, thus reducing serum high-density lipoprotein (HDL)-C, low-density lipoprotein (LDL)-C, total cholesterol and triglyceride levels; however, LXR-623 could attenuate ER stress and reverse these changes. We also obtained the same results in Huh-7 and THP-1 cells. ER stress induced by tunicamycin could clearly be reversed by activating LXRα because it promoted cholesterol efflux by enhancing the expression of ABCA1 and ABCG1 in hepatocytes and macrophages, contributing to attenuation of the development of AS.

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.

Macrophage lipid accumulation in the presence of immunosuppressive drugs mycophenolate mofetil and cyclosporin A.

Mycophenolate (MPA) and cyclosporin A (CsA) are two immunosuppressive agents currently used for the treatment of autoimmune diseases. However, reports regarding their effects on inflammation and lipid handling are controversial. Here, we compare the effect of these two drugs on the expression of proteins involved in cholesterol handling and lipid accumulation in a macrophage cell system utilizing M0, M1 and M2 human macrophages and in murine bone marrow-derived macrophages (BMDM). Differentiated M0, M1 and M2 subsets of THP-1 human macrophages were subjected to various concentrations of either MPA or CsA. Expression of proteins involved in reverse cholesterol transport (ABCA1 and 27-hydroxylase) and scavenger receptors, responsible for uptake of modified lipids (CD36, ScR-A1, CXCL16 and LOX-1), were evaluated by real-time PCR and confirmed with Western blot. DiI-oxidized LDL internalization assay was used to assess foam cell formation. The influence of MPA was also evaluated in BMDM obtained from atherosclerosis-prone transgenic mice, ApoE-/- and ApoE-/-Fas-/-. In M0 macrophages, MPA increased expression of ABCA1 and CXCL16 in a concentration-dependent manner. In M1 THP-1 macrophages, MPA caused a significant increase of 27-hydroxylase mRNA and CD36 and SR-A1 receptor mRNAs. Exposure of M2 macrophages to MPA also stimulated expression of 27-hydroxylase, while downregulating all evaluated scavenger receptors. In contrast, CsA had no impact on cholesterol efflux in M0 and M1 macrophages, but significantly augmented expression of ABCA1 and 27-hydroxylase in M2 macrophages. CsA significantly increased expression of the LOX1 receptor in naïve macrophages, downregulated expression of CD36 and SR-A1 in the M1 subpopulation and upregulated expression of all evaluated scavenger receptors. However, CsA enhanced foam cell transformation in M0 and M2 macrophages, while MPA had no effect on foam cell formation unless used at a high concentration in the M2 subtype. Our results clearly underline the importance of further evaluation of the effects of these drugs when used in atherosclerosis-prone patients with autoimmune or renal disease.

G protein pathway suppressor 2 (GPS2) links inflammation and cholesterol efflux by controlling lipopolysaccharide-induced ATP-binding cassette transporter A1 expression in macrophages.

Macrophages play important roles in linking alterations of cholesterol metabolism and inflammation to the development of atherosclerosis. Previous studies have identified several positive and negative crosstalk mechanisms that connect cholesterol efflux and inflammation at the transcriptional level. Of particular relevance is that the expression of ATP-binding cassette transporter A1 ( Abca1), a main regulator of cholesterol efflux, can be induced by oxysterol receptor LXR agonists but also by bacterial endotoxins, such as LPS, that activate TLR4 signaling. However, the extent to which these pathways influence each other has remained incompletely understood. We investigated the possible role of the transcriptional coregulator G protein pathway suppressor 2 (GPS2) in LPS-induced Abca1 expression and cholesterol efflux in mouse and human macrophages. To activate Abca1, GPS2 cooperates with the LPS-inducible NF-κB subunit p65, but not with LXRs nor with corepressor complex subunits that otherwise cooperate with GPS2 to repress proinflammatory gene expression. Overall, our work identifies a regulatory chromatin component of crosstalk mechanisms between cholesterol efflux and inflammation that specifically affects ABCA1. Because GPS2 expression is down-regulated in some humans with obese and type 2 diabetes, the macrophage GPS-2/ABC-A1 pathway could be altered and contribute to atherogenesis.-Huang, Z., Liang, N., Damdimopoulos, A., Fan, R., Treuter, E. G protein pathway suppressor 2 (GPS2) links inflammation and cholesterol efflux by controlling lipopolysaccharide-induced ATP-binding cassette transporter A1 expression in macrophages.

Apoptotic cell induction of miR-10b in macrophages contributes to advanced atherosclerosis progression in ApoE-/- mice.

We previously found that miR-10b inhibits cholesterol efflux from thioglycollate-elicited mouse peritoneal macrophages through repressing ATP binding cassette transporter (ABCA1). Herein, we deciphered the mechanism underlying macrophage miR-10b expression and the role of miR-10b in atherosclerosis. MiR-10b expression was increased in the arteries with advanced but not early atherosclerotic plaques of ApoE-/- mice. Free cholesterol-induced macrophage apoptotic cells (FC-AM) promoted miR-10b expression in mouse resident peritoneal macrophages (RPM) by up-regulation of Twist1/2 in a Mer receptor tyrosine kinase dependent manner. Surprisingly, antagomiR-10b de-repressed ABCA1 in RPM engulfing FC-AM but not in RPM alone or RPM-derived foam cells; systemic delivery of antagomiR-10b enhanced reverse cholesterol transport from RPM engulfing FC-AM but not from RPM or the foam cells in ApoE-/- mice. Mechanistically, RPM engulfing FC-AM possessed sufficient miR-10b functionally repressing ABCA1 expression, whereas RPM and foam cells had little miR-10b incompetently repressing ABCA1 expression. Notably, antagomiR-10b administration reduced advanced plaque size and also enhanced plaque stability in ApoE-/-mice, which were associated with increased plaque macrophage ABCA1 expression and reduced plaque apoptosis and inflammation. However, antagomiR-10b administration did not affect early atherosclerotic plaque formation in ApoE-/- mice. These data suggest that apoptotic cell induction of miR-10b in macrophages is important in advanced atherosclerosis progression.

MicroRNA-212 promotes lipid accumulation and attenuates cholesterol efflux in THP-1 human macrophages by targeting SIRT1

Macrophage foam cell formation is a key initiating event in the pathogenesis of atherosclerosis. This work was conducted to determine the role of microRNA (miR)-212 in the transformation of foam cells from macrophages. We examined the expression of miR-212 in atherosclerotic lesions in an apoE-deficient (apoE−/−) mouse model. The effects of miR-212 overexpression and knockdown on lipid accumulation and cholesterol homeostasis in THP-1 macrophages after exposure to oxidized low-density lipoprotein (oxLDL). The mechanism underlying the activity of miR-212 was explored. It was found that miR-212 was downregulated in atherosclerotic lesions and macrophages from apoE−/− mice fed high-fat diet, compared to the equivalents from apoE−/− mice fed standard diet. Overexpression of miR-212 promoted lipid accumulation in oxLDL-treated THP-1 macrophages, whereas miR-212 depletion exerted an opposite effect. Macrophage cholesterol efflux to apolipoprotein A-I was significantly reduced by miR-212, which was accompanied by reduced ABCA1 expression. Mechanistically, miR-212 targeted sirtuin 1 (SIRT1) to repress the expression of ABCA1 in THP-1 macrophages. Rescue experiments confirmed that co-expression of SIRT1 attenuated lipid accumulation and restored cholesterol efflux in miR-212-overexpressing THP-1 macrophages. Collectively, miR-212 facilitates macrophage foam cell formation and suppresses ABCA1-dependent cholesterol efflux through downregulation of SIRT1. Targeting miR-212 may provide a potential therapeutic strategy for atherosclerosis.

ABCA1 affects placental function via trophoblast and macrophage

AimsTo study the potential impact of ABCA1 on the function of the placenta. Main methodsTrophoblasts and macrophages were isolated from the placenta with enzymatic digestion; Immunofluorescence assay was used to detect the location of ABCA1 in cells; RT-PCR and Western-blot were used to detect the expression of ABCA1; The cholesterol efflux assays of primary trophoblasts was detected by Amplex Red cholesterol assay kit (Invitrogen);Inflammatory factor secretion from primary macrophages was detected by Elisa. Key findingsABCA1 was mainly located on trophoblast membranes. Decreased ABCA1 expression in trophoblasts reduced the cholesterol efflux of trophoblasts (P<0.01). while increased ABCA1 expression in trophoblasts reduced the cholesterol efflux of trophoblasts (P<0.05). ABCA1 was uniformly expressed on the cell membrane, cytoplasm, and nucleus of macrophages. Decreased ABCA1 expression in macrophages, increased inflammatory factors but reduced IL-10 (P<0.01). While increased ABCA1 expression in macrophages, reduced inflammatory factors but increased IL-10 (P<0.01). SignificanceABCA1 may be a potential target for the prevention of gestation diseases.

AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE−/− mice

HDL plays crucial roles at multiple stages of the pathogenesis of atherosclerosis. AMP-activated protein kinase (AMPK) is a therapeutic candidate for the treatment of cardiovascular disease. However, the effect of AMPK activation on HDL functionality has not been established in vivo. We assessed the effects of pharmacological AMPK activation using A-769662, AICAR, metformin, and IMM-H007 on the atheroprotective functions of HDL in apoE-deficient (apoE−/−) mice fed with a high-fat diet. After administration, there were no changes in serum lipid levels among the groups. However, mice treated with AMPK activators showed significantly enhanced reverse cholesterol transport in vivo and in vitro. AMPK activation also increased the expression of ABCA1 and ABCG1 in macrophages and scavenger receptor class B type I and LCAT in the liver. HDL from AMPK activation mice exhibited lower HDL inflammatory index and myeloperoxidase activity and higher paraoxonase 1 activity than HDL from untreated mice, implying superior antioxidant and anti-inflammatory capacities. Pharmacological AMPK activation also induced polarization of macrophages to the M2 state and reduced plasma lipid peroxidation, inflammatory cytokine production, and atherosclerotic plaque formation in apoE−/− mice. These observations suggest that pharmacological AMPK activation enhances the anti-atherogenic properties of HDL in vivo. This likely represents a key mechanism by which AMPK activation attenuates atherosclerosis.

Activation of PPARγ does not contribute to macrophage ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI

Activation of macrophage ABCA1/G1 expression and cholesterol efflux is believed one of the mechanisms by which PPARγ inhibits atherosclerosis. PPARγ can also activate CD36 expression, a receptor for oxLDL, which may supply LXR ligands to activate LXR-ABCA1/G1 pathways. However, the controversial effects of PPARγ on ABCA1 expression have been reported. In this study, we used peritoneal macrophages isolated from wild type and CD36 deficient (CD36−/−) mice to clarify if PPARγ ligands can influence ABCA1 expression by CD36 function. We found that CD36 deficiency had no effect on cholesterol efflux and ABCA1/ABCG1 expression at basal levels. In both cell types, PPARγ ligands (15d-PGJ2, troglitazone and pioglitazone) reduced ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI, with most by troglitazone. LXR ligand-induced ABCA1 expression and cholesterol efflux was attenuated by PPARγ ligands. Associated with decreased ABCA1 protein levels, ABCA1 mRNA and promoter activity were reduced by PPARγ ligands. Furthermore, high expressing PPARγ reduced ABCA1 expression and LXR-activated ABCA1 promoter in a CD36-independent manner. In contrast, ABCG1 expression was induced by PPARγ ligands while inhibited by PPARγ inactivation. Taken together, our study suggests that enhancement of macrophage cholesterol metabolism by PPARγ is not contributed by activating ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI, which is not involved by CD36 expression either.

Betulin attenuates atherosclerosis in apoE−/− mice by up-regulating ABCA1 and ABCG1

Betulin is a pentacyclic triterpenoid isolated from the bark of yellow and white birch trees with anti-cancer and anti-malaria activities. In this study we examined the effects of betulin on atherosclerosis in apoE-/- mice and the underlying mechanisms. Murine macrophage RAW264.7 cells and human monocyte-derived THP-1 cells were tested. Foam cell formation was detected with Oil Red O staining. Cholesterol efflux was assessed using [3H]-cholesterol efflux assay. The expression of ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) was examined using RT-PCR and Western-blotting. The ABCA1 promoter activity was evaluated using luciferase activity assay. Male apoE-/- mice fed on a high-fat-diet (HFD), and received betulin (20 and 40 mg·kg-1·d-1, ig) for 12 weeks. The macrophage content and ABCA1 expression in the aortic sinuses were evaluated with immunofluorescence staining. The hepatic, intestinal and fecal cholesterol were also analyzed in the mice. In RAW264.7 cells, betulin (0.1-2.5 μg/mL) dose-dependently ameliorated oxLDL-induced cholesterol accumulation and enhanced cholesterol efflux. In both RAW264.7 and THP-1 cells, betulin increased the expression of ABCA1 and ABCG1 via suppressing the transcriptional repressors sterol-responsive element-binding proteins (SREBPs) that bound to E-box motifs in ABCA1 promoter, whereas E-box binding site mutation markedly attenuated betulin-induced ABCA1 promoter activity. In HFD-fed apoE-/- mice, betulin administration significantly reduced lesions in en face aortas and aortic sinuses. Furthermore, betulin administration significantly increased ABCA1 expression and suppressed macrophage positive areas in the aortic sinuses. Moreover, betulin administration improved plasma lipid profiles and enhanced fecal cholesterol excretion in the mice. Betulin attenuates atherosclerosis in apoE-/- mice by promoting cholesterol efflux in macrophages.

Metformin promotes cholesterol efflux in macrophages by up-regulating FGF21 expression: a novel anti-atherosclerotic mechanism.

Metformin is widely used as a glucose-lowering agent in patients with type 2 diabetes (T2D). Recently, several studies have confirmed that metformin possessed multiple cardiovascular protective effects such as decreasing cardiovascular mortality, attenuating adverse cardiac and vascular remodeling and ameliorating atherosclerosis [1–3]. Hong et al. [4] in a multicenter, randomized, double-blind, placebo-controlled clinical trial found treatment with metformin for 3 years substantially reduced major cardiovascular events in a median follow-up of 5.0 years compared with glipizide in type 2 diabetes patients who had a history of coronary artery disease. Forouzandeh et al. [5] demonstrated metformin treatment significantly attenuated high-fat diet-induced atherosclerosis in apolipoprotein E-knockout (ApoE−/−) mice. These studies suggest metformin could ameliorate atherosclerosis, but the anti-atherosclerotic mechanism of metformin remains to be fully clarified. Previous studies reported that metformin could decrease plasma cholesterol levels in diabetic and non-diabetic patients beyond its glucose-lowering effect [6–9], and the lipid-lowering effect of metformin was also confirmed in a meta-analysis of randomized controlled trials (RCTs) [10]. Besides, it was found that metformin treatment could lower inflammation markers levels in patients at high risk of cardiovascular disease and restore impaired HDL-mediated cholesterol efflux from macrophages due to glycation [11, 12]. These effects may contribute to the anti-atherosclerotic properties of metformin. The deposition of excessive cholesterol in macrophages plays a key role in atherosclerotic plaque formation, thus removal of excess cholesterol from macrophages may attenuate and even regress the atherosclerosis [13]. It has been identified that removal of cholesterol from macrophages was mediated by several transmembrane transporters, including adenosine triphosphate binding cassette (ABC) transporters A1 and G1 [14]. Yvan-Charvet et al. [15] evidenced that combined deficiency of Abca1 and Abcg1 in macrophages severely damaged cholesterol efflux and accelerated atherosclerosis in low-density lipoprotein receptor-knockout (Ldlr-/-) mice. Bochem et al. [16] demonstrated that carriers of ABCA1 loss-of-function mutations presented with lower cholesterol efflux capacity and higher atherosclerotic burden compared with age and sex-matched controls. Conversely, increased expression of ABCA1 and ABCG1 was involved in redistribution of cholesterol from inner to outer leaflet of the plasma membrane, facilitating cholesterol efflux from cholesterol-loaded macrophages [17]. Overexpression of ABCA1 in macrophages increased cholesterol efflux by about 60 % (P = 0.0006) and reduced atherosclerotic plaques by about 68 % (P = 0.0008) in Ldlr-/- mice fed a Western-type diet for 12 weeks [18]. Thus, ABCA1- and ABCG1-driven cholesterol efflux in macrophages was a key regulator in anti-atherosclerosis. Fibroblast growth factor (FGF) 21, a member of FGF subfamily and mainly secreted by liver and adipose tissue, is a novel metabolic regulator with multiple cardioprotective efficacy [19]. It was found that FGF21 deficiency caused a marked exacerbation of atherosclerotic plaque formation in ApoE−/− mice [20]. Moreover, previous studies also demonstrated that FGF21 could activate ABCA1 and ABCG1 expression of macrophages and in turn promoted cholesterol efflux in macrophages [21, 22], suggesting up-regulation of FGF21 exerted function of reducing atherosclerotic plaques. Intriguingly, Nygaard et al. [23] found metformin significantly increased FGF21 expression in both rat and human hepatocytes. Another study showed metformin could increase serum FGF21 levels both in mice and human [24]. Thus, we speculate metformin stimulates ABCA1 and ABCG1 expression via up-regulation of FGF21. Taken together, we hypothesize metformin increases FGF21 expression, and subsequently promotes expression of ABCA1 and ABCG1 in macrophages, which promotes cholesterol efflux from macrophages and attenuated atherosclerotic plaques. Further exploring cardioprotective mechanism of metformin will be useful for insight into novel therapeutic targets.