Malaria in pregnancy regulates P-glycoprotein (P-gp/Abcb1a) and ABCA1 efflux transporters in the Mouse Visceral Yolk Sac.

Malaria in pregnancy (MiP) induces intrauterine growth restriction (IUGR) and preterm labour (PTL). However, its effects on yolk sac morphology and function are largely unexplored. We hypothesized that MiP modifies yolk sac morphology and efflux transport potential by modulating ABC efflux transporters. C57BL/6 mice injected with Plasmodium berghei ANKA (5 × 105 infected erythrocytes) at gestational day (GD) 13.5 were subjected to yolk sac membrane harvesting at GD 18.5 for histology, qPCR and immunohistochemistry. MiP did not alter the volumetric proportion of the yolk sac's histological components. However, it increased levels of Abcb1a mRNA (encoding P-glycoprotein) and macrophage migration inhibitory factor (Mif chemokine), while decreasing Abcg1 (P < 0.05); without altering Abca1, Abcb1b, Abcg2, Snat1, Snat2, interleukin (Il)-1ß and C-C Motif chemokine ligand 2 (Ccl2). Transcripts of Il-6, chemokine (C-X-C motif) ligand 1 (Cxcl1), Glut1 and Snat4 were not detectible. ABCA1, ABCG1, breast cancer resistance protein (BCRP) and P-gp were primarily immunolocalized to the cell membranes and cytoplasm of endodermic epithelium but also in the mesothelium and in the endothelium of mesodermic blood vessels. Intensity of P-gp labelling was stronger in both endodermic epithelium and mesothelium, whereas ABCA1 labelling increased in the endothelium of the mesodermic blood vessels. The presence of ABC transporters in the yolk sac wall suggests that this fetal membrane acts as an important protective gestational barrier. Changes in ABCA1 and P-gp in MiP may alter the biodistribution of toxic substances, xenobiotics, nutrients and immunological factors within the fetal compartment and participate in the pathogenesis of malaria-induced IUGR and PTL.

Regulation of ATP binding cassette transporter A1 (ABCA1) expression: cholesterol-dependent and - independent signaling pathways with relevance to inflammatory lung disease.

The role of the ATP binding cassette transporter A1 (ABCA1) in maintaining cellular lipid homeostasis in cardiovascular disease is well established. More recently, the important beneficial role played by ABCA1 in modulating pathogenic disease mechanisms, such as inflammation, in a broad range of chronic conditions has been realised. These studies position ABCA1 as a potential therapeutic target in a diverse range of diseases where inflammation is an underlying cause. Chronic respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD) are driven by inflammation, and as such, there is now a growing recognition that we need a greater understanding of the signaling pathways responsible for regulation of ABCA1 expression in this clinical context. While the signaling pathways responsible for cholesterol-mediated ABCA1 expression have been clearly delineated through decades of studies in the atherosclerosis field, and thus far appear to be translatable to the respiratory field, less is known about the cholesterol-independent signaling pathways that can modulate ABCA1 expression in inflammatory lung disease. This review will identify the various signaling pathways and ligands that are associated with the regulation of ABCA1 expression and may be exploited in future as therapeutic targets in the setting of chronic inflammatory lung diseases.

Interleukin-5 promotes ATP-binding cassette transporter A1 expression through miR-211/JAK2/STAT3 pathways in THP-1-dervied macrophages.

Interleukin-5 (IL-5) is manifested as its involvement in the process of atherosclerosis, but the mechanism is still unknown. In this study, we explored the effect of IL-5 on lipid metabolism and its underlying mechanisms in THP-1-derived macrophages. The quantitative polymerase chain reaction (qPCR) and western blot analysis results showed that IL-5 significantly up-regulated ATP-binding cassette transporter A1 (ABCA1) expression in a dose-dependent and time-dependent manner. [3H]-labeled cholesterol was used to assess the levels of cholesterol efflux, and the results showed that IL-5 increased ABCA1-mediated cholesterol efflux. A high-performance liquid chromatography assay indicated that cellular cholesterol content was decreased by IL-5 treatment in THP-1-derived macrophages. The selective inhibitor and small interfering RNA were used to block the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway. The results of the qPCR and western blot analysis showed that IL-5 activated JAK2/STAT3 pathway to up-regulate ABCA1 expression. Meanwhile, IL-5 reduced the expression level of miR-211. Furthermore, we found that JAK2 is a target gene of miR-211 and miR-211 mimic inhibited the expression of JAK2 and reduced the levels of p-STAT3 and ABCA1 as revealed by luciferase reporter assay, qPCR and western blot analysis. In summary, these findings indicated that IL-5 promotes ABCA1 expression and cholesterol efflux through the miR-211/JAK2/STAT3 signaling pathway in THP-1-derived macrophages.

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

OBJECTIVE: Previous 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 RESULTS: Treatment 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. CONCLUSION: IL-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.

Apigenin Retards Atherogenesis by Promoting ABCA1-Mediated Cholesterol Efflux and Suppressing Inflammation.

BACKGROUND/AIMS: The development of atherosclerosis is accompanied by escalating inflammation and lipid accumulation within blood vessel walls. ABCA1 plays a crucial role in mediating cholesterol efflux from macrophages, which protects against atherogenesis. This research was designed to explore the effects and underlying mechanisms of apigenin (4', 5, 7-trihydroxyflavone) on ABCA1-mediated cellular cholesterol efflux and LPS-stimulated inflammation in RAW264.7 macrophages and apoE-/- mice. METHODS: Expression of genes or proteins was examined by RT-PCR or western blot analysis. Liquid scintillation counting was used to detect percent cholesterol efflux. Cellular cholesterol content was measured using HPLC assay. The secretion levels of pro-inflammatory cytokines were quantified by ELISA assay. Atherosclerotic lesion sizes were determined with Oil Red O staining. The contents of macrophages and smooth muscle cells in atherosclerotic lesion were evaluated using immunohistochemistry. Plasma TC, TG, HDL-C and LDL-C levels in apoE-/- mice were evaluated using commercial test kits. RESULTS: Apigenin potently increased ABCA1 expression through miR-33 repression in a dose- and time-dependent manner. Treatment with apigenin significantly increased ABCA1-mediated cholesterol efflux, and reduced TC, FC and CE levels in macrophage-derived foam cells. In LPS-treated macrophages, the expression levels of TLR-4, MyD88 and p-IκB-α as well as nuclear NF-κB p65 were decreased by the addition of apigenin. Moreover, apigenin markedly decreased secretion levels of several pro-inflammatory cytokines. Lastly, in LPS-challenged apoE-/- mice, apigenin administration augmented ABCA1 expression, decreased the contents of macrophages and smooth muscle cells in atherosclerotic lesion, reduced miR-33, TLR-4, and NF-κB p65 levels, improved plasma lipid profile and relieved inflammation, which results in less atherosclerotic lesion size. CONCLUSIONS: Taken together, these results suggest that apigenin may attenuate atherogenesis through up-regulating ABCA1-mediated cholesterol efflux and inhibiting inflammation.

E3317 promotes cholesterol efflux in macrophage cells via enhancing ABCA1 expression.

Reverse cholesterol transport (RCT) plays an important role in cholesterol and lipid metabolism. Regulating the activities of key transporters and receptors in RCT, such as ATP-binding cassette transporter A1 (ABCA1), helps to prevent atherosclerotic cardiovascular disease. In this study, we used an ABCA1 promoter luciferase reporter assay to screen 20,000 compounds for ABCA1 upregulators. Compound E3317 (N-(6-butylbenzo[d]thiazol-2(3H)-ylidene)-3-(N-(2-cyanoethyl)sulfamoyl)benzamide)) was identified as a positive hit with an EC50 value of 0.2 µM in ABCA1p-LUC HepG2 cells. Thus, we hypothesized that E3317 might have cholesterol- and lipid metabolism-regulating effects through ABCA1 upregulation. E3317 significantly increased ABCA1 mRNA and protein expression in hepatic L02 cells and RAW264.7 macrophages. E3317 promoted cholesterol efflux to apolipoprotein A-I in RAW264.7 macrophages and significantly decreased lipid accumulation in oxidized low-density lipoprotein-induced murine RAW264.7 macrophages. Further studies using ABCA1 siRNA showed that the promotion of cholesterol efflux and decrease of lipid accumulation by E3317 depended on ABCA1 expression. Mechanistic studies indicated that E3317 regulated ABCA1 expression via activating nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), which plays an important role in the regulation of glucose homeostasis and lipid metabolism. The structure of E3317 was docked in the ligand-binding domain of PPARγ (PBD code: 4EMA) to find the key binding amino acids. Site mutation assays confirmed that Y327 and F363 were the key PPARγ binding epitopes of E3317. Our results revealed that E3317 upregulates ABCA1 expression and thereby promotes cholesterol efflux. E3317 may regulate ABCA1 expression through PPARγ. Our findings provide a new compound, E3317, which may have beneficial cardiovascular effects.

Kuwanon G attenuates atherosclerosis by upregulation of LXRα-ABCA1/ABCG1 and inhibition of NFκB activity in macrophages.

BACKGROUND: Atherosclerosis is characterized by chronic inflammation in vascular wall. Previous studies suggest that Kuwanon G (KWG) exerts anti-inflammatory activities. However, the effect of KWG on atherosclerosis remains unexplored. AIMS: To explore whether KWG affects macrophage foam cell formation in vitro and atherogenesis in vivo. METHODS: RAW 264.7 macrophages were stimulated with ox-LDL for 24h to induce foam cell formation and treated with KWG. Foam cell formation was determined by ORO staining and enzymatic analysis. Pro-inflammatory cytokines mRNA levels were tested by Real-time PCR method. Further molecular mechanism was investigated using Western blot. In vivo, ApoE-/- mice were fed with high-fat diet and intraperitoneally injected with KWG. Atherosclerotic lesion was accessed by H&E and ORO staining. Plaque composition was evaluated by immunohistochemistry and Sirius Red staining. Serum lipid profile and inflammatory cytokines were evaluated by enzymatic method and ELISA. RESULTS: KWG significantly decreased intracellular lipid accumulation and inflammatory cytokines mRNA levels in macrophages through enhancing LXRα-ABCA1/ABCG1 pathway and inhibiting NFκB activation. Administrated with KWG remarkably reduced the atherosclerotic lesion areas and macrophage content in the plaque of high-fat diet fed ApoE-/- mice. KWG also reduced hyperlipidemia and serum inflammatory cytokines in vivo. CONCLUSION: Taken together, these data highlight that KWG can attenuate atherosclerosis through inhibiting foam cell formation and inflammatory response.

Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression.

Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.

TRAK2, a novel regulator of ABCA1 expression, cholesterol efflux and HDL biogenesis.

Aims: The recent failures of HDL-raising therapies have underscored our incomplete understanding of HDL biology. Therefore there is an urgent need to comprehensively investigate HDL metabolism to enable the development of effective HDL-centric therapies. To identify novel regulators of HDL metabolism, we performed a joint analysis of human genetic, transcriptomic, and plasma HDL-cholesterol (HDL-C) concentration data and identified a novel association between trafficking protein, kinesin binding 2 (TRAK2) and HDL-C concentration. Here we characterize the molecular basis of the novel association between TRAK2 and HDL-cholesterol concentration. Methods and results: Analysis of lymphocyte transcriptomic data together with plasma HDL from the San Antonio Family Heart Study (n = 1240) revealed a significant negative correlation between TRAK2 mRNA levels and HDL-C concentration, HDL particle diameter and HDL subspecies heterogeneity. TRAK2 siRNA-mediated knockdown significantly increased cholesterol efflux to apolipoprotein A-I and isolated HDL from human macrophage (THP-1) and liver (HepG2) cells by increasing the mRNA and protein expression of the cholesterol transporter ATP-binding cassette, sub-family A member 1 (ABCA1). The effect of TRAK2 knockdown on cholesterol efflux was abolished in the absence of ABCA1, indicating that TRAK2 functions in an ABCA1-dependent efflux pathway. TRAK2 knockdown significantly increased liver X receptor (LXR) binding at the ABCA1 promoter, establishing TRAK2 as a regulator of LXR-mediated transcription of ABCA1. Conclusion: We show, for the first time, that TRAK2 is a novel regulator of LXR-mediated ABCA1 expression, cholesterol efflux, and HDL biogenesis. TRAK2 may therefore be an important target in the development of anti-atherosclerotic therapies.

ABCA1/ApoE/HDL Pathway Mediates GW3965-Induced Neurorestoration After Stroke.

BACKGROUND AND PURPOSE: ATP-binding cassette transporter A1 (ABCA1) is a major reverse cholesterol transporter and plays critical role in the formation of brain high-density lipoprotein (HDL) cholesterol. Apolipoprotein E (ApoE) is the most abundant apolipoprotein and transports cholesterol into cells in brain. ABCA1 and ApoE are upregulated by liver-X receptors. Activation of liver-X receptors has neurorestorative benefit for stroke. The current study investigates whether ABCA1/ApoE/HDL pathway mediates GW3965, a synthetic dual liver-X receptor agonist, induced neurorestoration after stroke. METHODS: Middle-aged male specific brain ABCA1-deficient (ABCA1-B/-B) and floxed-control (ABCA1fl/fl) mice were subjected to distal middle-cerebral artery occlusion (dMCAo) and gavaged with saline or GW3965 (10 mg/kg) or intracerebral infusion of artificial cerebrospinal fluid or human plasma HDL3 in ABCA1-B/-B stroke mice, starting 24 hours after dMCAo and daily until euthanization 14 days after dMCAo. RESULTS: No differences in the blood level of total cholesterol and triglyceride and lesion volume were found among the groups. Compared with ABCA1fl/fl ischemic mice, ABCA1-B/-B ischemic mice exhibited impairment functional outcome and decreased ABCA1/ApoE expression and decreased gray/white matter densities in the ischemic boundary zone 14 days after dMCAo. GW3965 treatment of ABCA1fl/fl ischemic mice led to increased brain ABCA1/ApoE expression, concomitantly to increased blood HDL, gray/white matter densities and oligodendrocyte progenitor cell numbers in the ischemic boundary zone, as well as improved functional outcome 14 days after dMCAo. GW3965 treatment had negligible beneficial effects in ABCA1-B/-B ischemic mice. However, intracerebral infusion of human plasma HDL3 significantly attenuated ABCA1-B/-B-induced deficits. In vitro, GW3965 treatment (5 µM) increased ABCA1/synaptophysin level and neurite/axonal outgrowth in primary cortical neurons derived from ABCA1fl/fl embryos, but not in neurons derived from ABCA1-B/-B embryos. HDL treatment (80 µg/mL) attenuated the reduction of neurite/axonal outgrowth in neurons derived from ABCA1-B/-B embryos. CONCLUSIONS: ABCA1/ApoE/HDL pathway, at least partially, contributes to GW3965-induced neurorestoration after stroke.

Eicosapentaenoic acid membrane incorporation impairs cholesterol efflux from cholesterol-loaded human macrophages by reducing the cholesteryl ester mobilization from lipid droplets.

A diet containing a high n-3/n-6 polyunsaturated fatty acids (PUFA) ratio has cardioprotective properties. PUFAs incorporation into membranes influences the function of membrane proteins. We investigated the impact of the membrane incorporation of PUFAs, especially eicosapentaenoic acid (EPA) (C20:5 n-3), on the anti-atherogenic cholesterol efflux pathways. We used cholesteryl esters (CE)-loaded human monocyte-derived macrophages (HMDM) to mimic foam cells exposed to the FAs for a long period of time to ensure their incorporation into cellular membranes. Phospholipid fraction of EPA cells exhibited high levels of EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which was associated with a decreased level of arachidonic acid (AA) (C20:4 n-6). EPA 70µM reduced ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 30% without any alteration in ABCA1 expression. The other tested PUFAs, DPA, docosahexaenoic acid (DHA) (C22:6 n-3), and AA, were also able to reduce ABCA1 functionality while the monounsaturated oleic FA slightly decreased efflux and the saturated palmitic FA had no impact. Moreover, EPA also reduced cholesterol efflux to HDL mediated by the Cla-1 and ABCG1 pathways. EPA incorporation did not hinder efflux in free cholesterol-loaded HMDM and did not promote esterification of cholesterol. Conversely, EPA reduced the neutral hydrolysis of cytoplasmic CE by 24%. The reduced CE hydrolysis was likely attributed to the increase in cellular TG contents and/or the decrease in apo E secretion after EPA treatment. In conclusion, EPA membrane incorporation reduces cholesterol efflux in human foam cells by reducing the cholesteryl ester mobilization from lipid droplets.

Inhibition of ABCA1 Protein Expression and Cholesterol Efflux by TNF α in MLO-Y4 Osteocytes.

Hip fracture and myocardial infarction cause significant morbidity and mortality. In vivo studies raising serum cholesterol levels as well as pro-inflammatory cytokines such as TNF α manifest bone loss and atherosclerotic vascular disease, suggesting that abnormalities of cholesterol transport may contribute to osteoporosis. We used the mouse osteocyte cell line (MLO-Y4) to investigate the effects of TNF α on the expression of cholesterol acceptor proteins such as apolipoprotein A-I (apo A-I) and apolipoprotein E (apo E), as well as on the cholesterol transporters ATP-binding cassette-1 (ABCA1), scavenger receptor class B type 1 (SRB1), and cluster of differentiation 36 (CD36). MLO-Y4 cells do not express apo A-I or apo E; however, they do express all three cholesterol transporters (ABCA1, SRB1, and CD36). Treatment of MLO-Y4 cells with TNF α had no effect on SRB1, CD36, and osteocalcin levels; however, TNF α reduced ABCA1 protein levels in a dose-dependent manner and cholesterol efflux to apo A-I. Interestingly, TNF α treatment increased ABCA1 promoter activity and ABCA1 mRNA levels, and increased liver X receptor α protein expression, but had no effect on retinoid X receptor α and retinoic acid receptor α levels. Pharmacological inhibition of p38 mitogen-activated protein (MAP) kinase, but not c-jun-N-terminal kinase 1 or mitogen-activated protein kinase (MEK), restored ABCA1 protein levels in TNF α-treated cells. These results suggest that pro-inflammatory cytokines regulate cholesterol metabolism in osteocytes in part by suppressing ABCA1 levels post-translationally in a p38 MAP kinase-dependent manner.

Apolipoprotein A-I Mimetic Peptide D-4F Reduces Cardiac Hypertrophy and Improves Apolipoprotein A-I-Mediated Reverse Cholesterol Transport From Cardiac Tissue in LDL Receptor-null Mice Fed a Western Diet.

Epidemiological studies have suggested that hypercholesterolemia is an independent determinant of increased left ventricular (LV) mass. Because high-density lipoprotein and its major protein apolipoprotein A-I (apoA-I) mediate reverse cholesterol transport (RCT) and have cardiac protective effects, we hypothesized that the apoA-I mimetic peptide D-4F could promote RCT in cardiac tissue and decrease cardiac hypertrophy induced by hypercholesterolemia. Low-density lipoprotein receptor-null mice were fed by a Western diet for 18 weeks and then randomized to receive water, or D-4F 0.3 mg/mL, or D-4F 0.5 mg/mL added to drinking water for 6 weeks. After D-4F administration, an increase in high-density lipoprotein cholesterol and a decrease in low-density lipoprotein cholesterol, total cholesterol, and triglyceride in a trend toward dose-responsivity were found in cardiac tissue. Ultrasound biomicroscopy revealed a reduction in LV posterior wall end-diastolic dimension, and an increase in mitral valve E/A ratio and LV ejection fraction. Hematoxylin-eosin staining showed reduced LV wall thickness and myocardial cell diameter. The protein levels of ABCA1 and LXRα were elevated in cardiac tissue of D-4F treated mice compared with the controls (P < 0.05). These results demonstrated that D-4F treatment reduced cardiac hypertrophy, and improved cardiac performance in low-density lipoprotein receptor-null mice fed a Western diet, presumably through the LXRα-ABCA1 pathway associated with enhanced myocardial RCT.

Saikosaponin-a Attenuates Oxidized LDL Uptake and Prompts Cholesterol Efflux in THP-1 Cells.

Saikosaponins-a (Ssa) is a major bioactive extract of Radix Bupleuri which is a traditional Chinese medicine. The roles of inflammatory response and lipid transportation in the process of atherosclerosis have drawn increasing attention. We explored the regulation of lipid transportation and immune-inflammatory role of Ssa in early atherosclerosis. The antiatherogenic actions and possible molecular mechanisms of Ssa were texted in THP-1 cells. We examined the effect of Ssa on oxidized low-density lipoprotein (ox-LDL)-induced lipid uptake, cholesterol efflux, immune-inflammatory response. THP-1 macrophages were treated with Ssa followed by ox-LDL for 24 hours. Results from western blot showed that Ssa obviously reduced lipoprotein uptake to block foam cell formation and the expression of Density Lipoprotein Receptor-1 and CD36. Ssa also significantly boosted cholesterol efflux and the expression of ATP binding cassettetransporter A1 and peroxisome proliferator-activated receptor γ. The results also indicated that Ssa inhibited ox-LDL-induced activation of AKT and nuclear factor-κB, assembly of NLRP3 inflammasome and production of proinflammatory cytokines. It is suggested that the ability against immune inflammatory response of Ssa is due to modulation of the PI3K/AKT/NF-κB/NLRP3 pathway. In conclusion, this study provides new insight into Ssa's molecular mechanism and its therapeutic potential in the treatment of atherosclerosis.

Chronic vitamin A-enriched diet feeding regulates hypercholesterolaemia through transcriptional regulation of reverse cholesterol transport pathway genes in obese rat model of WNIN/GR-Ob strain.

BACKGROUND & OBJECTIVES: Hepatic scavenger receptor class B1 (SR-B1), a high-density lipoprotein (HDL) receptor, is involved in the selective uptake of HDL-associated esterified cholesterol (EC), thereby regulates cholesterol homoeostasis and improves reverse cholesterol transport. Previously, we reported in euglycaemic obese rats (WNIN/Ob strain) that feeding of vitamin A-enriched diet normalized hypercholesterolaemia, possibly through hepatic SR-B1-mediated pathway. This study was aimed to test whether it would be possible to normalize hypercholesterolaemia in glucose-intolerant obese rat model (WNIN/GR/Ob) through similar mechanism by feeding identical vitamin A-enriched diet. METHODS: In this study, 30 wk old male lean and obese rats of WNIN/GR-Ob strain were divided into two groups and received either stock diet or vitamin A-enriched diet (2.6 mg or 129 mg vitamin A/kg diet) for 14 wk. Blood and other tissues were collected for various biochemical analyses. RESULTS: Chronic vitamin A-enriched diet feeding decreased hypercholesterolaemia and normalized abnormally elevated plasma HDL-cholesterol (HDL-C) levels in obese rats as compared to stock diet-fed obese groups. Further, decreased free cholesterol (FC) and increased esterified cholesterol (EC) contents of plasma cholesterol were observed, which were reflected in higher EC to FC ratio of vitamin A-enriched diet-fed obese rats. However, neither lecithin-cholesterol acyltransferase (LCAT) activity of plasma nor its expression (both gene and protein) in the liver were altered. On the contrary, hepatic cholesterol levels significantly increased in vitamin A-enriched diet fed obese rats. Hepatic SR-B1 expression (both mRNA and protein) remained unaltered among groups. Vitamin A-enriched diet fed obese rats showed a significant increase in hepatic low-density lipoprotein receptor mRNA levels, while the expression of genes involved in HDL synthesis, namely, ATP-binding cassette protein 1 (ABCA1) and apolipoprotein A-I, were downregulated. No such response was seen in vitamin A-supplemented lean rats as compared with their stock diet-fed lean counterparts. INTERPRETATION & CONCLUSIONS: Chronic vitamin A-enriched diet feeding decreased hypercholesterolaemia and normalized HDL-C levels, possibly by regulating pathways involved in HDL synthesis and degradation, independent of hepatic SR-B1 in this glucose-intolerant obese rat model.

The implication of cigarette smoking and cessation on macrophage cholesterol efflux in coronary artery disease patients.

We investigated ATP-binding cassette transporters A1/G1 expression and function in mediating cholesterol efflux by examining the macrophages of cigarette-smoking patients with coronary artery disease (CAD) before and after smoking abstinence. Peripheral blood monocyte cells were collected from nonsmokers (n = 17), non-CAD (NCAD) smokers (n = 35), and CAD smokers (n = 32) before and after 3 months of smoking cessation. We found that the ABCA1 expression level was lower in macrophages from NCAD and CAD smokers than from nonsmokers at baseline. The ABCA1 function of mediating cholesterol efflux was reduced in NCAD and CAD smokers as compared with nonsmokers. After 3 months of smoking cessation, ABCA1 expression and function were improved in CAD smokers. However, ABCG1 expression and function did not change after smoking cessation. Furthermore, ABCA1 expression was inhibited by tar in human acute monocytic leukemia cell line THP-1-derived macrophages through the inhibition of liver X receptors. Nicotine and carbon monoxide did not inhibit ABCA1 expression. Our results indicate that chronic cigarette smoking impaired ABCA1-mediated cholesterol efflux in macrophages and that tobacco abstinence reversed the function and expression of ABCA1, especially in CAD patients. It was tobacco tar, rather than nicotine or carbon monoxide, that played a major role in the tobacco-induced disturbance of cellular cholesterol homeostasis.

MicroRNA-33a/b in lipid metabolism ­ novel "thrifty" models.

MicroRNAs (miRNAs; miRs) are small non-protein-coding RNAs that negatively regulate gene expression. They bind to the 3' UTR of specific mRNAs and either inhibit translation or promote mRNA degradation. There is emerging evidence linking miR-33a/b to lipid homoeostasis, targeting ABCA1,SREBF1, etc and it would appear that they have acted as "thrifty genes" during evolution to maintain cholesterol levels both at the cellular and whole body level. As we are now living in a period of "satiation", miR-33a/b no longer seem to be useful and could be potential therapeutic targets for lipid disorders and/or atherosclerosis. In this review, we describe the current understanding of the function of miR-33a/b in lipid homeostasis, focusing on the "thrifty" aspect.

Rutaecarpine suppresses atherosclerosis in ApoE-/- mice through upregulating ABCA1 and SR-BI within RCT.

ABCA1 and scavenger receptor class B type I (SR-BI)/CD36 and lysosomal integral membrane protein II analogous 1 (CLA-1) are the key transporter and receptor in reverse cholesterol transport (RCT). Increasing the expression level of ABCA1 and SR-BI/CLA-1 is antiatherogenic. The aim of the study was to find novel antiatherosclerotic agents upregulating expression of ABCA1 and SR-BI/CLA-1 from natural compounds. Using the ABCA1p-LUC and CLA-1p-LUC HepG2 cell lines, we found that rutaecarpine (RUT) triggered promoters of ABCA1 and CLA-1 genes. RUT increased ABCA1 and SR-BI/CLA-1 expression in vitro related to liver X receptor alpha and liver X receptor beta. RUT induced cholesterol efflux in RAW264.7 cells. ApoE-deficient (ApoE(-/-)) mice treated with RUT for 8 weeks showed ∼68.43, 70.23, and 85.56% less en face lesions for RUT (L), RUT (M), and RUT (H) groups, respectively, compared with the model group. Mouse macrophage-specific antibody and filipin staining indicated that RUT attenuated macrophages and cholesterol accumulations in atherosclerotic lesions, respectively. Additionally, ABCA1 and SR-BI expression was highly induced by RUT in livers of ApoE(-/-) mice. Meanwhile, RUT treatment significantly increased the fecal (3)H-cholesterol excretion, which demonstrated that RUT could promote RCT in vivo. RUT was identified to be a candidate that protected ApoE(-/-) mice from developing atherosclerosis through preferentially promoting activities of ABCA1 and SR-BI within RCT.

RNA binding protein HuR regulates the expression of ABCA1.

ABCA1 is a major regulator of cellular cholesterol efflux and plasma HDL biogenesis. Even though the transcriptional activation of ABCA1 is well established, the posttranscriptional regulation of ABCA1 expression is poorly understood. Here, we investigate the potential contribution of the RNA binding protein (RBP) human antigen R (HuR) on the posttranscriptional regulation of ABCA1 expression. RNA immunoprecipitation assays demonstrate a direct interaction between HuR and ABCA1 mRNA. We found that HuR binds to the 3' untranslated region of ABCA1 and increases ABCA1 translation, while HuR silencing reduces ABCA1 expression and cholesterol efflux to ApoA1 in human hepatic (Huh-7) and monocytic (THP-1) cells. Interestingly, cellular cholesterol levels regulate the expression, intracellular localization, and interaction between HuR and ABCA1 mRNA. Finally, we found that HuR expression was significantly increased in macrophages from human atherosclerotic plaques, suggesting an important role for this RBP in controlling macrophage cholesterol metabolism in vivo. In summary, we have identified HuR as a novel posttranscriptional regulator of ABCA1 expression and cellular cholesterol homeostasis, thereby opening new avenues for increasing cholesterol efflux from atherosclerotic foam macrophages and raising circulat-ing HDL cholesterol levels.

Excess nitric oxide impairs LXR(α)-ABCA1-dependent cholesterol efflux in macrophage foam cells.

Excess nitric oxide (NO) promotes the progression of atherosclerosis by increasing the oxidation of low-density lipoprotein (LDL) and inflammatory responses. However, little is known about the impact of NO and its underlying molecular mechanism on lipid metabolism of macrophage foam cells. In this study, Oil-red O staining, cholesterol and triglyceride assay, Dil-oxidized LDL (oxLDL) binding assay, cholesterol efflux assay, real-time RT-PCR and Western blot analysis were used for in vitro experiments. Apolipoprotein E-deficient (apoE(-/-) ) and apoE and inducible nitric oxide synthase-deficient (apoE(-/-) iNOS(-/-) ) mice were as our in vivo models. Treatment with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), an NO donor, exacerbated oxLDL-induced cholesterol accumulation in macrophages, because of reduced efficacy of cholesterol efflux. In addition, SNAP decreased the protein level of ATP-binding cassette transporter A1 (ABCA1) without affecting scavenger receptor type A (SR-A), CD36, ABCG1, or SR-B1 levels. This SNAP-mediated downregulation of ABCA1 was mainly through the effect of NO but not peroxynitrite. Furthermore, the SNAP-downregulated ABCA1 was due to the decrease in the liver X receptor α (LXRα)-dependent transcriptional regulation. Moreover, genetic deletion of iNOS increased the serum capacity of reverse cholesterol efflux and protein expression of LXRα, ABCA1, and SR-BI in aortas and retarded atherosclerosis in apoE(-/-) mice. Our findings provide new insights in the pro-atherogenic effect of excess NO on cholesterol metabolism in macrophages.