ATP-Binding Cassette Sterol Transporters Are Differentially Expressed in Normal and Diseased Human Gallbladder

Background and Aims

Gallbladder epithelial cells (GBEC) are exposed to high cholesterol concentrations in bile, and export cholesterol via an ATP-binding cassette (ABC) transporter-mediated pathway in vitro. These findings suggest that aberrant expression and/or function of ABC sterol transporters may be associated with cholesterol-related gallbladder diseases (CAGD). In this study, we investigated the relative levels of the sterol transporters ABCA1, ABCG5, and ABCG8 in human gallbladders in CAGD, and the relationship between ABCA1 and inflammation.

Methods

Expression of ABCA1, ABCG5, and ABCG8 was evaluated in 31 gallbladders with CAGD and 6 normal gallbladders by western blotting and immunohistochemistry. RT-PCR was used to measure ABCA1 mRNA expression. To investigate the relationship between ABCA1 and inflammation, wWestern blots were performed on cultured dog GBEC treated with lipopolysaccharide (LPS) using an anti-ABCA1 antibody.

Results

Immunohistochemistry showed ABCA1 to be localized predominantly to the basolateral membrane, while ABCG8 formed a diffuse intracellular pattern at the apical pole of human GBEC. ABCA1 and ABCG8 expression was more prominent in GBEC that were surrounded by cholesterol-laden macrophages. ABCA1 and ABCG8 expression was increased in gallbladders with CAGD. Western blots showed increased ABCA1, ABCG5, and ABCG8 expression in CAGD. ABCA1 mRNA levels were increased in all gallbladders with CAGD. LPS treatment of cultured dog GBEC enhanced ABCA1 expression.

Conclusions

The sterol transporters ABCA1, ABCG5, and ABCG8 may play a role in the pathogenesis of human CAGD. Inflammation appears to be a key factor that increases ABCA1 expression and activity in the human gallbladder.     

高糖对THP-1巨噬细胞ABC转运体的表达和功能的影响

目的:探讨高糖对体外培养的THP-1巨噬细胞中三磷酸腺苷结合盒（ABC）转运体的表达及功能的影响。方法: 以不同浓度的D-葡萄糖干预培养的THP-1单核巨噬细胞5 d,用实时定量PCR和Western blot检测巨噬细胞中ABCG1、ABCA1 mRNA和其蛋白的表达。用酶荧光化学法检测培养基中及细胞内胆固醇的含量。结果: 高糖可抑制巨噬细胞中ABCG1的表达,但是对ABCA1的表达影响不明显。随着D-葡萄糖浓度的增加,从巨噬细胞中流出的胆固醇量减少,同时细胞内胆固醇的含量增加。结论: 高糖可抑制巨噬细胞中ABCG1的表达及功能,有助于促进巨噬细胞内脂质堆积。     

Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion

Cholesterol and other sterols exit the body primarily by secretion into bile. In patients with sitosterolemia, mutations in either of two ATP-binding cassette (ABC) half-transporters, ABCG5 or ABCG8, lead to reduced secretion of sterols into bile, implicating these transporters in this process. To elucidate the roles of ABCG5 and ABCG8 in the trafficking of sterols, we disrupted Abcg5 and Abcg8 in mice (G5G8(-/-)). The G5G8(-/-) mice had a 2- to 3-fold increase in the fractional absorption of dietary plant sterols, which was associated with an approximately 30-fold increase in plasma sitosterol. Biliary cholesterol concentrations were extremely low in the G5G8(-/-) mice when compared with wild-type animals (mean = 0.4 vs. 5.5 micromol ml) and increased only modestly with cholesterol feeding. Plasma and liver cholesterol levels were reduced by 50% in the chow-fed G5G8(-/-) mice and increased 2.4- and 18-fold, respectively, after cholesterol feeding. These data indicate that ABCG5 and ABCG8 are required for efficient secretion of cholesterol into bile and that disruption of these genes increases dramatically the responsiveness of plasma and hepatic cholesterol levels to changes in dietary cholesterol content.     

New insights into the molecular actions of plant sterols and stanols in cholesterol metabolism

Plant sterols and stanols (phytosterols/phytostanols) are known to reduce serum low-density lipoprotein (LDL)-cholesterol level, and food products containing these plant compounds are widely used as a therapeutic dietary option to reduce plasma cholesterol and atherosclerotic risk. The cholesterol-lowering action of phytosterols/phytostanols is thought to occur, at least in part, through competition with dietary and biliary cholesterol for intestinal absorption in mixed micelles. However, recent evidence suggests that phytosterols/phytostanols may regulate proteins implicated in cholesterol metabolism both in enterocytes and hepatocytes. Important advances in the understanding of intestinal sterol absorption have provided potential molecular targets of phytosterols. An increased activity of ATP-binding cassette transporter A1 (ABCA1) and ABCG5/G8 heterodimer has been proposed as a mechanism underlying the hypocholesterolaemic effect of phytosterols. Conclusive studies using ABCA1 and ABCG5/G8-deficient mice have demonstrated that the phytosterol-mediated inhibition of intestinal cholesterol absorption is independent of these ATP-binding cassette (ABC) transporters. Other reports have proposed a phytosterol/phytostanol action on cholesterol esterification and lipoprotein assembly, cholesterol synthesis and apolipoprotein (apo) B100-containing lipoprotein removal. The accumulation of phytosterols in ABCG5/G8-deficient mice, which develop features of human sitosterolaemia, disrupts cholesterol homeostasis by affecting sterol regulatory element-binding protein (SREBP)-2 processing and liver X receptor (LXR) regulatory pathways. This article reviews the progress to date in studying these effects of phytosterols/phytostanols and the molecular mechanisms involved.     

RIP140 contributes to foam cell formation and atherosclerosis by regulating cholesterol homeostasis in macrophages

Atherosclerosis, a syndrome with abnormal arterial walls, is one of the major causes that lead to the development of various cardiovascular diseases. The key initiator of atherosclerosis is cholesterol accumulation. The uncontrolled cholesterol deposition, mainly involving low-density lipoprotein (LDL), causes atheroma plaque formation, which initiates chronic inflammation due to the recruitment of inflammatory cells such as macrophages. Macrophages scavenge excess peripheral cholesterol and transport intracellular cholesterol to high-density lipoprotein (HDL) for excretion or storage. Cholesterol-laden macrophage-derived foam cell formation is the main cause of atherogenesis. It is critical to understand the regulatory mechanism of cholesterol homeostasis in the macrophage in order to prevent foam cells formation and further develop novel therapeutic strategies against atherosclerosis. Here we identified a protein, RIP140 (receptor interacting protein 140), which enhances macrophage-derived foam cell formation by reducing expression of reverse cholesterol transport genes, A TP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1). In animal models, we found that reducing RIP140 levels by crossing macrophage-specific RIP140 knockdown (MϕRIP140KD) mice with ApoE null mice effectively ameliorates high-cholesterol diet-induced atherosclerosis. Our data suggest that reducing RIP140 levels in macrophages significantly inhibits atherosclerosis, along with markers of inflammation and the number of macrophages in a western diet fed ApoE null mouse. This study provides a proof-of-concept for RIP140 as a risk biomarker of, and a therapeutic target for, atherosclerosis.     

Coexistence of foam cells and hypocholesterolemia in mice lacking the ABC transporters A1 and G1

The concept that macrophages can become foam cells as a result of a disturbed balance between the uptake of cholesterol from lipoproteins and cholesterol efflux is generally accepted. ABCA1 and ABCG1 are two cholesterol transporters that may act sequentially to remove cellular cholesterol, but currently their combined role in vivo is unknown. We report here that targeted disruption of both ABCA1 and ABCG1 in mice, despite severe plasma hypocholesterolemia, leads to massive lipid accumulation and foam cell formation of tissue macrophages. A complete ablation of cellular cholesterol efflux in vitro is observed, whereas in vivo macrophage-specific reverse cholesterol transport to the feces is markedly decreased. Despite the massive foam cell formation of tissue macrophages, no lipid accumulation was observed in the vascular wall, even in mice of 1 year old, indicating that the double knockout mice, possibly because of their hypocholesterolemia, lack the trigger to attract macrophages to the vessel wall. In conclusion, even under hypocholesterolemic conditions macrophages can be converted into foam cells, and ABCA1 and ABCG1 play an essential role in the prevention of foam cell formation.     

ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport

Excessive uptake of atherogenic lipoproteins such as modified low-density lipoprotein complexes by vascular macrophages leads to foam cell formation, a critical step in atherogenesis. Cholesterol efflux mediated by high-density lipoproteins (HDL) constitutes a protective mechanism against macrophage lipid overloading. The molecular mechanisms underlying this reverse cholesterol transport process are currently not fully understood. To identify effector proteins that are involved in macrophage lipid uptake and release, we searched for genes that are regulated during lipid influx and efflux in human macrophages using a differential display approach. We report here that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is induced in monocyte-derived macrophages during cholesterol influx mediated by acetylated low-density lipoprotein. Conversely, lipid efflux in cholesterol-laden macrophages, mediated by the cholesterol acceptor HDL(3), suppresses the expression of ABCG1. Immunocytochemical and flow cytometric analyses revealed that ABCG1 is expressed on the cell surface and in intracellular compartments of cholesterol-laden macrophages. Inhibition of ABCG1 protein expression using an antisense strategy resulted in reduced HDL(3)-dependent efflux of cholesterol and choline-phospholipids. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified an additional set of ABC genes whose expression is regulated by cholesterol uptake or HDL(3)-mediated lipid release, suggesting a potential function for these transporters in macrophage lipid homeostasis. Our results demonstrating a regulator function for ABCG1 in cholesterol and phospholipid transport define a biologic activity for ABC transporters in macrophages.     

ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins

The mechanisms responsible for the inverse relationship between plasma high-density lipoprotein (HDL) levels and atherosclerotic cardiovascular disease are poorly understood. The ATP-binding cassette transporter A1 (ABCA1) mediates efflux of cellular cholesterol to lipid-poor apolipoproteins but not to HDL particles that constitute the bulk of plasma HDL. We show that two ABC transporters of unknown function, ABCG1 and ABCG4, mediate isotopic and net mass efflux of cellular cholesterol to HDL. In transfected 293 cells, ABCG1 and ABCG4 stimulate cholesterol efflux to both smaller (HDL-3) and larger (HDL-2) subclasses but not to lipid-poor apoA-I. Treatment of macrophages with an liver X receptor activator results in up-regulation of ABCG1 and increases cholesterol efflux to HDL. RNA interference reduced the expression of ABCG1 in liver X receptor-activated macrophages and caused a parallel decrease in cholesterol efflux to HDL. These studies indicate that ABCG1 and ABCG4 promote cholesterol efflux from cells to HDL. ABCG1 is highly expressed in macrophages and probably mediates cholesterol efflux from macrophage foam cells to the major HDL fractions, providing a mechanism to explain the relationship between HDL levels and atherosclerosis risk.     

Macrophage ABCA2 deletion modulates intracellular cholesterol deposition, affects macrophage apoptosis, and decreases early atherosclerosis in LDL receptor knockout mice

ObjectiveThe ABCA2 transporter shares high structural homology to ABCA1, which is crucial for the removal of excess cholesterol from macrophages and, by extension, in atherosclerosis. It has been suggested that ABCA2 sequesters cholesterol inside the lysosomes, however, little is known of the macrophage-specific role of ABCA2 in regulating lipid homeostasis in vivo and in modulating susceptibility to atherosclerosis.MethodsChimeras with dysfunctional macrophage ABCA2 were generated by transplantation of bone marrow from ABCA2 knockout (KO) mice into irradiated LDL receptor (LDLr) KO mice.ResultsInterestingly, lack of ABCA2 in macrophages resulted in a diminished lesion size in the aortic root (?24.5%) and descending thoracic aorta (?36.6%) associated with a 3-fold increase in apoptotic cells, as measured by both caspase 3 and TUNEL. Upon oxidized LDL exposure, macrophages from wildtype (WT) transplanted animals developed filipin-positive droplets in lysosomal-like compartments, corresponding to free cholesterol (FC) accumulation. In contrast, ABCA2-deficient macrophages displayed an abnormal diffuse distribution of FC over peripheral regions. The accumulation of neutral sterols in lipid droplets was increased in ABCA2-deficient macrophages, but primarily in cytoplasmic clusters and not in lysosomes. Importantly, apoptosis of oxLDL loaded macrophages lacking ABCA2 was increased 2.7-fold, probably as a consequence of the broad cellular distribution of FC.ConclusionsLack of functional ABCA2 generates abnormalities in intracellular lipid distribution/trafficking in macrophages consistent with its lysosomal sequestering role, leading to an increased susceptibility to apoptosis in response to oxidized lipids and reduced atherosclerotic lesion development.     

High-density lipoprotein and atherosclerosis: Roles of lipid transporters

Various previous studies have found a negative cor-relation between the risk of cardiovascular events and serum high-density lipoprotein(HDL) cholesterol levels. The reverse cholesterol transport, a pathway of choles-terol from peripheral tissue to liver which has several potent antiatherogenic properties. For instance, the particles of HDL mediate to transport cholesterol from cells in arterial tissues, particularly from atherosclerotic plaques, to the liver. Both ATP-binding cassette trans-porters(ABC) A1 and ABCG1 are membrane cholesterol transporters and have been implicated in mediating cholesterol effluxes from cells in the presence of HDL and apolipoprotein A-I, a major protein constituent of HDL. Previous studies demonstrated that ABCA1 and ABCG1 or the interaction between ABCA1 and ABCG1 exerted antiatherosclerotic effects. As a therapeutic approach for increasing HDL cholesterol levels, much focus has been placed on increasing HDL cholesterol levels as well as enhancing HDL biochemical functions. HDL therapies that use injections of reconstituted HDL, apoA-I mimetics, or full-length apoA-I have shown dramatic effectiveness. In particular, a novel apoA-I mi-metic peptide, Fukuoka University ApoA-I Mimetic Pep-tide, effectively removes cholesterol via specific ABCA1 and other transporters, such as ABCG1, and has an an-tiatherosclerotic effect by enhancing the biological func-tions of HDL without changing circulating HDL choles-terol levels. Thus, HDL-targeting therapy has significant atheroprotective potential, as it uses lipid transporter-targeting agents, and may prove to be a therapeutic tool for atherosclerotic cardiovascular diseases.     

Relevance of hereditary defects in lipid transport proteins for the pathogenesis of cholesterol gallstone disease

In the formation of cholesterol gallstones, cholesterol hypersecretion into bile causing cholesterol supersaturation and crystallization appears to be the primary factor, with disturbed gallbladder and intestinal motility as secondary factors. Although intestinal uptake mechanisms have not yet been fully elucidated, the HDL receptor scavenger receptor B1 (SRB1) may be involved. Since HDL-cholesterol, both from the intestine and peripheral sources, is the preferred type of cholesterol for biliary secretion, increased HDL transport to the liver can also cause cholesterol hypersecretion in bile. In the hepatocyte, bile formation is regulated by several transmembrane proteins, all belonging to the ABC family. A change in the activity in one of these proteins can have a profound impact on biliary lipid secretion. The bile salt export pump (BSEP or ABCB11) regulates the excretion of bile salts into bile and mutations cause severe cholestasis. The second ABC transporter, ABCB4 (MDR3) regulates the secretion in bile of phosphatidylcholine (PC), while ABCG5/G8 is active in the excretion of cholesterol and sterols into bile. These transporters also facilitate transport of sterols back into the intestinal lumen. Mutations in either of these genes cause sitosterolaemia with increased absorption of plant sterols and cholesterol. Until now, evidence for a genetic background of human gallstone disease is mostly indirect and based on ethnic differences. Only two single gene defects are associated with gallstones. One is an ABCB4 mutation which causes a deficiency in biliary PC secretion and the other is a CYP7A1 mutation, the rate-limiting enzyme in the synthesis of bile salts from cholesterol in the liver. Recently, several common DNA polymorphisms in the ABCG8 gene were discovered that are associated with variations in plasma sterols, which could also influence biliary cholesterol secretion, but there is still a paucity of human studies.     

The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg x apoE-knockout (KO) mice. Overexpression of hABCA1 in C57BL/6 mice resulted in a unique anti-atherogenic profile characterized by decreased plasma cholesterol (63%), cholesteryl ester (63%), free cholesterol (67%), non-high density lipoprotein (HDL)-cholesterol (53%), and apolipoprotein (apo) B (64%) but markedly increased HDL-cholesterol (2.8-fold), apoA-I (2.2-fold), and apoE (2.8-fold) levels. These beneficial changes in the lipid profile led to significantly lower (65%) aortic atherosclerosis in hABCA1-Tg mice. In marked contrast, ABCA1 overexpression had a minimal effect on the plasma lipid profile of apoE-KO mice and resulted in a 2- to 2.6-fold increase in aortic lesion area. These combined results indicate that overexpression of ABCA1 in C57BL/6 mice on a high cholesterol diet results in an atheroprotective lipoprotein profile and decreased atherosclerosis, and thus provide previously undocumented in vivo evidence of an anti-atherogenic role for the ABCA1 transporter. In contrast, overexpression of ABCA1 in an apoE-KO background led to increased atherosclerosis, further substantiating the important role of apoE in macrophage cholesterol metabolism and atherogenesis. In summary, these results establish that, in the presence of apoE, overexpression of ABCA1 modulates HDL as well as apoB-containing lipoprotein metabolism and reduces atherosclerosis in vivo, and indicate that pharmacological agents that will increase ABCA1 expression may reduce atherogenic risk in humans.     

High-density lipoprotein protects macrophages from oxidized low-density lipoprotein-induced apoptosis by promoting efflux of 7-ketocholesterol via ABCG1

Oxidized sterols consumed in the diet or formed on low-density lipoprotein (LDL) are toxic to endothelial cells and macrophages and are thought to have a central role in promoting atherogenesis. The ATP-binding cassette transporter ABCG1 was recently shown to promote efflux of cholesterol from macrophages to high-denisty lipoprotein (HDL). We show that HDL protects macrophages from apoptosis induced by loading with free cholesterol or oxidized LDL. The protective effect of HDL was reduced in Abcg1(-/-) macrophages, especially after loading with oxidized LDL. Similarly, HDL exerted a protective effect against apoptosis induced by 7-ketocholesterol, the major oxysterol present in oxidized LDL and atherosclerotic lesions, in Abcg1(+/+), but not in Abcg1(-/-) macrophages. In transfected 293 cells, efflux of 7-ketocholesterol and related oxysterols was completely dependent on expression of ABCG1 and the presence of HDL in media. In contrast, ABCA1 and apoA-1 did not stimulate the efflux of 7-ketocholesterol into media. HDL stimulated the efflux of 7-ketocholesterol from Abcg1(+/+), but not from Abcg1(-/-) macrophages. In Abcg1(-/-) mice fed a high-cholesterol diet, plasma levels of 7-ketocholesterol were reduced, whereas their macrophages accumulated 7-ketocholesterol. These findings indicate a specific role for ABCG1 in promoting efflux of 7-ketocholesterol and related oxysterols from macrophages onto HDL and in protecting these cells from oxysterol-induced cytotoxicity.     

MicroRNA-19b promotes macrophage cholesterol accumulation and aortic atherosclerosis by targeting ATP-binding cassette transporter A1

Rationale

Macrophage accumulation of cholesterol leads to foam cell formation which is a major pathological event of atherosclerosis. Recent studies have shown that microRNA (miR)-19b might play an important role in cholesterol metabolism and atherosclerotic diseases. Here, we have identified miR-19b binding to the 3′UTR of ATP-binding cassette transporter A1 (ABCA1) transporters, and further determined the potential roles of this novel interaction in atherogenesis.

Objective

To investigate the molecular mechanisms involved in a miR-19b promotion of macrophage cholesterol accumulation and the development of aortic atherosclerosis.

Methods and results

We performed bioinformatics analysis using online websites, and found that miR-19b was highly conserved during evolution and directly bound to ABCA1 mRNA with very low binding free energy. Luciferase reporter assay confirmed that miR-19b bound to 3110-3116 sites within ABCA1 3′UTR. MiR-19b directly regulated the expression levels of endogenous ABCA1 in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by qRT-PCR and western blot. Cholesterol transport assays revealed that miR-19b dramatically suppressed apolipoprotein AI-mediated ABCA1-dependent cholesterol efflux, resulting in the increased levels of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) as revealed by HPLC. The excretion of ³H-cholesterol originating from cholesterol-laden MPMs into feces was decreased in mice overexpressing miR-19b. Finally, we evaluated the proatherosclerotic role of miR-19b in apolipoprotein E deficient (apoE^−/−) mice. Treatment with miR-19b precursor reduced plasma high-density lipoprotein (HDL) levels, but increased plasma low-density lipoprotein (LDL) levels. Consistently, miR-19b precursor treatment increased aortic plaque size and lipid content, but reduced collagen content and ABCA1 expression. In contrast, treatment with the inhibitory miR-19b antisense oligonucleotides (ASO) prevented or reversed these effects.

Conclusion

MiR-19b promotes macrophage cholesterol accumulation, foam cell formation and aortic atherosclerotic development by targeting ABCA1.     

ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism

BACKGROUND AND AIMS: Members of the family of ABC transporters are involved in different processes of sterol metabolism, and ABCA1 was recently identified as a key regulator of high-density lipoprotein (HDL) metabolism. Our aim was to further analyze the role of ABCA1 in cholesterol metabolism. METHODS: ABCA1-deficient mice (ABCA1-/-) and wild-type mice were compared for different aspects of sterol metabolism. Intestinal cholesterol absorption was determined by a dual stable isotope technique, and analysis of fecal, plasma, and tissue sterols was performed by gas chromatography/mass spectrometry. Key regulators of sterol metabolism were investigated by Northern and Western blot analyses or enzyme activity assays. RESULTS: ABCA1-disrupted sv129/C57BL/6 hybrid mice showed a significant reduction in intestinal cholesterol absorption. The decrease in cholesterol absorption was followed by an enhanced fecal loss of neutral sterols, whereas fecal bile acid excretion was not affected. Total body cholesterol synthesis was significantly increased, with enhanced 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase observed in adrenals and spleen. In addition, ABCA1-/- mice showed markedly increased concentrations of cholesterol precursors in the plasma, lung, intestine, and feces. Reduced HMG-CoA reductase messenger RNA and enzyme activity in the liver suggest that enhanced cholesterol synthesis in ABCA1-/- mice occurs in peripheral tissues rather than the liver. CONCLUSIONS: The metabolism of cholesterol and cholesterol precursors is markedly affected by a lack of ABCA1 function.