Expression Of ATP-binding Cassette Transporter G1 Research Articles

Cav3.1 T-type calcium channel blocker NNC 55-0396 reduces atherosclerosis by increasing cholesterol efflux

Calcium channel blockers (CCBs) are commonly used as antihypertensive agents. While certain L-type CCBs exhibit antiatherogenic effects, the impact of Cav3.1 T-type CCBs on antiatherogenesis and lipid metabolism remains unexplored. NNC 55–0396 (NNC) is a highly selective blocker of T-type calcium channels (Cav3.1 channels). We investigated the effects of NNC on relevant molecules and molecular mechanisms in human THP-1 macrophages. Cholesterol efflux, an indicator of reverse cholesterol transport (RCT) efficiency, was assessed using [3H]-labeled cholesterol. In vivo, high cholesterol diet (HCD)-fed LDL receptor knockout (Ldlr-/-) mice, an atherosclerosis-prone model, underwent histochemical staining to analyze plaque burden. Treatment of THP-1 macrophages with NNC facilitated cholesterol efflux and reduced intracellular cholesterol accumulation. Pharmacological and genetic interventions demonstrated that NNC treatment or Cav3.1 knockdown significantly enhanced the protein expression of scavenger receptor B1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and liver X receptor alpha (LXRα) transcription factor. Mechanistic analysis revealed that NNC activates p38 and c-Jun N-terminal kinase (JNK) phosphorylation, leading to increased expression of ABCA1, ABCG1, and LXRα-without involving the microRNA pathway. LXRα isrequired for NNC–induced ABCA1 and ABCG1 expression. Administering NNC diminished atherosclerotic lesion area and lipid deposition in HCD-fed Ldlr-/- mice. NNC's anti-atherosclerotic effects, achieved through enhanced cholesterol efflux and inhibition of lipid accumulation, suggest a promising therapeutic approach for hypertensive patients with atherosclerosis. This research highlights the potential of Cav3.1 T-type CCBs in addressing cardiovascular complications associated with hypertension.

Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is a prevalent hematologic malignancy in children, and methotrexate (MTX) is a widely employed curative treatment. Despite its common use, clinical resistance to MTX is frequently encountered. In this study, an MTX-resistant cell line (Reh-MTXR) was established through a stepwise selection process from the ALL cell line Reh. Comparative analysis revealed that Reh-MTXR cells exhibited resistance to MTX in contrast to the parental Reh cells. RNA-seq analysis identified an upregulation of ATP-binding cassette transporter G1 (ABCG1) in Reh-MTXR cells. Knockdown of ABCG1 in Reh-MTXR cells reversed the MTX-resistant phenotype, while overexpression of ABCG1 in Reh cells conferred resistance to MTX. Mechanistically, the heightened expression of ABCG1 accelerated MTX efflux, leading to a reduced accumulation of MTX polyglutamated metabolites. Notably, the ABCG1 inhibitor benzamil effectively sensitized Reh-MTXR cells to MTX treatment. Moreover, the observed upregulation of ABCG1 in Reh-MTXR cells was not induced by alterations in DNA methylation or histone acetylation. This study provides insight into the mechanistic basis of MTX resistance in ALL and also suggests a potential therapeutic approach for MTX-resistant ALL in the future.

Artesunate attenuates foam cell formation by enhancing cholesterol efflux.

BackgroundAtherosclerosis is the main cause of many cardiovascular diseases and the second leading cause of death in elderly people. The formation of intimal macrophage-derived foam cells is a major feature of early atherosclerotic lesions. Little is known about the effects of artesunate (ART) on macrophage-derived foam cell formation.MethodsOil red O staining was employed to detect foam cell formation; colorimetric analysis was employed for cholesterol measurement; quantitative real time polymerase chain reaction (qRT-PCR) and western blot analysis were employed to assess messenger RNA (mRNA) and protein expression, respectively; enzyme-linked immunosorbent assay (ELISA) analyses were used to observe interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) release; and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to examine cell viability.ResultsIt was revealed that ART attenuated oxidized low-density lipoprotein (ox-LDL)-induced foam cell formation from THP-1-derived macrophages by decreasing cholesterol accumulation, and the effect might have occurred via enhanced cholesterol efflux. Additionally, ART decreased toll-like receptor 4 (TLR4) expression, increased adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) expression, and reduced the secretion of IL-6 and TNF-α.ConclusionsThis study showed that ART attenuated the ox-LDL-induced formation of foam cells from THP-1-derived macrophages by increasing ABCA1 and ABCG1 expression via inhibiting TLR4 expression and reducing TNF-α and IL-6 secretion from macrophages induced by ox-LDL, which ultimately decreased the accumulation of cholesterol. It is worthwhile further investigate ART as a potential drug for the treatment of atherosclerosis.

Atrial Natriuretic Peptide Inhibited ABCA1/G1-dependent Cholesterol Efflux Related to Low HDL-C in Hypertensive Pregnant Patients.

Objective: It has been reported that atrial natriuretic peptide (ANP) regulates lipid metabolism by stimulating adipocyte browning, lipolysis, and lipid oxidation, and by impacting the secretion of adipokines. In our previous study, we found that the plasma ANP concentration of hypertensive disorders of pregnancy (HDP) was significantly increased in comparison to that of normotensive pregnancy patients. Thus, this study’s objective was to investigate the lipid profile in patients with HDP and determine the effects of ANP on the cholesterol efflux in THP-1 macrophages.Methods: A total of 265 HDP patients and 178 normotensive women as the control group were recruited. Clinical demographic characteristics and laboratory profile data were collected. Plasma total triglycerides (TGs), total cholesterol (TC), low-density cholesterol (LDL-C), and high-density cholesterol (HDL-C) were compared between the two groups. THP-1 monocytes were incubated with different concentrations of ANP. ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) mRNA and protein were evaluated. ABCA1- and ABCG1-mediated cholesterol efflux to apolipoprotein A-Ⅰ (apoA-Ⅰ) and HDL, respectively, were measured by green fluorescent labeled NBD cholesterol. Natriuretic peptide receptor A (NPR-A) siRNA and specific agonists of the peroxisome proliferator–activated receptor-γ (PPAR-γ) and liver X receptor α (LXRα) were studied to investigate the mechanism involved.Results: Plasma TG, TC, LDL-C, and LDL-C/HDL-C were significantly increased, and HDL-C was significantly decreased in the HDP group in comparison to the control (all p < 0.001). ANP inhibited the expression of ABCA1 and ABCG1 at both the mRNA and protein levels in a dose-dependent manner. The functions of ABCA1- and ABCG1-mediated cholesterol efflux to apoA-I and HDL were significantly decreased. NPR-A siRNA further confirmed that ANP binding to its receptor inhibited ABCA1/G1 expression through the PPAR-γ/LXRα pathway.Conclusions: ABCA1/G1 was inhibited by the stimulation of ANP when combined with NPR-A through the PPAR-γ/LXRα pathway in THP-1 macrophages. The ABCA1/G1-mediated cholesterol efflux was also impaired by the stimulation of ANP. This may provide a new explanation for the decreased level of HDL-C in HDP patients.

Lipopolysaccharide Augments Foam Cell Formation through the Downregulation of ABCG1 in Murine Macrophages: Role of ERK1/2 Signaling

Bacterial lipopolysaccharide (LPS) has been reported to suppress the expression of ATP-binding cassette transporter G1 (ABCG1), a molecule facilitating cholesterol efflux, in macrophages and augmented the formation of foam cells, a critical step in the pathogenesis of atherosclerosis. The essential role of MAP kinases in ABCG1 expression in macrophage and foam cell formation is also reported. However, the role of MAP kinases in LPS-induced downregulation of ABCG1 is still unknown. Therefore, this study aimed to examine whether the overexpression of ABCG1 could attenuate the foam cell formation augmented by LPS and to explore the role of MAP kinase pathways in LPS-induced downregulation of ABCG1. In RAW 264.7 macrophages, LPS stimulation time- and dose-dependently reduced the expression of ABCG1 without affecting ABCA1. Moreover, the overexpression of ABCG1 significantly reduced the foam cell formation augmented by LPS. Although LPS induced the activation of MAP kinases (p38, JNK1/2, ERK1/2), AP1 (c-FOS/JUN), and NF-kB signaling molecules, a specific ERK1/2 inhibitor (U0128) effectively inhibited the downregulation of ABCG1 mediated by LPS. In addition, transient knockdown of ERK1/2 by small interference RNA similarly prevented the downregulation of ABCG1 mediated by LPS. Furthermore, we confirmed that T0901317, a specific agonist of the Liver X receptor, reduced LPS-induced downregulation of ABCG1 and partly inhibited the activation of ERK1/2 mediated by LPS. Overall, our study suggests that ERK1/2 signaling plays a crucial role in LPS-induced downregulation of ABCG1, which may be further implicated in atherogenic foam cell formation. Additional in vivo and in vitro studies are certainly warranted to gain a deeper understanding of these effects.

Synergetic Effect of rHDL and LXR Agonist on Reduction of Atherosclerosis in Mice.

High-density lipoproteins (HDLs) are unique in that they play an important role in the reverse cholesterol transport process. However, reconstituted HDL (rHDL) infusions have demonstrated limited beneficial effect in clinical practice. This is perhaps a consequence of the limited cholesterol efflux abilities of atheroma macrophages due to decreased expression of cholesterol transporters in advanced atheromas and following rHDL infusion treatment. Thus, we propose that a combination therapy of rHDL and a liver X receptor (LXR) agonist could maximize the therapeutic benefit of rHDL by upregulating ATP-binding cassette transporters A-1 (ABCA1) and ATP-binding cassette transporter G-1 (ABCG1), and enhancing cholesterol efflux to rHDL. In macrophages, rHDL downregulated the expression of ABCA1/G1 in a dose- and rHDL composition-dependent manner. Although LXR agonist, T0901317 (T1317), upregulated the expression of ABCA1 and ABCG1, the drug itself did not have any effect on cholesterol efflux (6.6 ± 0.5%) while the combination of rHDL and T1317 exhibited enhanced cholesterol efflux from [3H]-cholesterol loaded J774A.1 macrophages (23.3 ± 1.3%). Treatment with rHDL + T1317 significantly reduced the area of aortic plaque in ApoE−/− mice compared to PBS treated control animals (24.16 ± 1.42% vs. 31.59 ± 1.93%, p < 0.001), while neither rHDL nor T1317 treatment alone had a significant effect. Together, we show that rHDL paired with an LXR agonist can induce a synergetic effect in reducing atheroma burden. This synergy could lead to lower overall effective dose for both drugs, potentially overcoming the existing barriers in clinical development and renewing pharmaceutical interest in these two drug classes.

Zafirlukast prevented ox-LDL-induced formation of foam cells

Atherosclerosis (AS), a common arterial disease, is one of the main pathological roots of cardiovascular disease. The formation and accumulation of foam cells is an important event in early AS. An imbalance between cholesterol uptake and efflux is the primary cause of foam cell formation. Although research has focused on preventing the formation of foam cells, a safe and effective therapy has to be found. Zafirlukast is a widely useful type 1 cysteinyl leukotriene receptor (CysLT1R) antagonist with a good safety profile. Zafirlukast is the most used for the treatment of asthma and allergic rhinitis. However, the effect of zafirlukast on preventing the formation of foam cells has not been determined. The aim of this study was to investigate whether zafirlukast prevented macrophages from transforming into foam cells. Our data show that zafirlukast reduced the expression of CD36 and lipoprotein receptor-1 (LOX-1), which are responsible for lipid uptake. In addition, zafirlukast enhanced the activity of ATP-Binding Cassette A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1), leading to the acceleration of cholesterol efflux. Furthermore, zafirlukast influenced the activity of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway, which mediates the expression of ABCA1 and ABCG1. In summary, our data indicate that zafirlukast might be a potential treatment strategy for AS by mediating lipid metabolism and preventing the formation of foam cells.

Luteolin improves hypercholesterolemia and glucose intolerance through LXRα-dependent pathway in diet-induced obese mice.

Luteolin, a naturally derived flavonoid, exerts beneficial effects such as antitumor, antioxidant, and anti-inflammatory effects. However, the molecular mechanism underlying the effect of luteolin in hypercholesterolemia remains unclear. In this study, we demonstrated that luteolin upregulated the expression of liver X receptor (LXR) α, ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor class B member 1 (SRB1), which play a major role in cholesterol efflux, in HepG2 hepatocytes. Luteolin-stimulated expression of ABCG1 and SRB1 was reversed by inhibitory compound of LXRα. Luteolin administration also upregulated the expression of ABCG1, and SRB1 as well as cholesterol 7 α-hydroxylase (Cyp7α1) in the liver of diet-induced obese mice. Luteolin decreased the level of blood cholesterol and non-high-density lipoprotein cholesterol in obese mice. In addition, luteolin ameliorated glucose intolerance and reduced expression of gluconeogenesis-associated enzymes in an LXRα-dependent manner. PRACTICAL APPLICATIONS: Luteolin is known to possess various pharmacological activities. This research revealed that luteolin ameliorates hypercholesterolemia and glucose intolerance in diet-induced obesity. The results indicate that the potential properties of luteolin in cholesterol metabolism could be explained, at least in part, as being due to upregulated expression of ABCG1, and SRB1 through activation of liver X receptor, LXRα signaling pathway in HepG2 cells.

Oxaliplatin resistance is enhanced by saracatinib via upregulation Wnt-ABCG1 signaling in hepatocellular carcinoma

BackgroundChemo-resistance in hepatocellular carcinoma (HCC) is a major problem, and acquired drug resistance prevents cancer therapies from achieving complete responses. Molecular targeting therapy presents an opportunity to impede tumor through combination or sequential therapy, while the accurate effect is vague.MethodsThe efficacy of combinations between oxaliplatin and anti-cancer molecular targeting drugs was screened. Strangely, the combined chemotherapy with oxaliplatin and saracatinib induced significantly antagonistic effects. Then the antitumor effects of combined treatment with saracatinib and oxaliplatin were confirmed in wide type HCC as well as in saracatinib- and oxaliplatin-resistant HCC. RNA sequencing was used to explore the resistance mechanism, and the roles of ATP-binding cassette transporter G1 (ABCG1) and Wnt signaling in oxaliplatin resistance were confirmed.ResultsChemotherapy with oxaliplatin and saracatinib individually induced strong anti-HCC effects, while combined or sequential treatment of HCC cells with these two drugs exhibited reduced efficacy compared to treatment with the single drugs. And it was saracatinib treatment caused oxaliplatin resistance. RNA sequencing revealed 458 genes that were altered by treatment with saracatinib and oxaliplatin. Of these, the gene encoding ABCG1 and Wnt-associated genes were significantly upregulated. Upregulation of ABCG1 and oxaliplatin resistance were associated with activation of Wnt signaling. Interference with ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin resistance in HCC.ConclusionsThese studies demonstrated that combined or sequential chemotherapy with oxaliplatin and saracatinib reduced antitumor efficacy, and this antagonism was attributed to the activation of Wnt signaling and upregulation of ABCG1 by saracatinib.

The effect of genistein on lipid levels and LDLR, LXR\u03b1 and ABCG1 expression in postmenopausal women with hyperlipidemia

BackgroundThis study investigates the effect of genistein (Gen) on the lipid profiles and expression of low-density lipoprotein receptor (LDLR), liver X receptor α (LXRα) and ATP-binding cassette transporter G1 (ABCG1) in the plasma macrophages of postmenopausal women with hyperlipidemia in China.MethodsThis study considered 187 cases, where 160 postmenopausal women had hyperlipidemia. The subjects were divided into placebo group (PG) and experimental group (EG). EG received 60 mg/day of Gen, PG received placebo for 6 months. Body weight, height, waist circumference, body mass index and glucose levels were determined according to standard operating procedures. The triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), apolipoprotein-A1 (Apo-A1) and apolipoprotein-B (Apo-B) levels were detected in the plasma macrophages using ELISA. The protein and mRNA expression levels of LDLR, LXRα and ABCG1 were detected by western blot and real-time PCR techniques, respectively.ResultsCompared to the baseline, Gen effectively lowered TG, TC and LDL-C levels, whereas HDL-C levels as well as the protein and mRNA expression levels of LDLR, LXRα and ABCG1 (p < 0.05) were increased. There was a significant difference in the expression of LDLR protein between the two groups (p < 0.05). The mRNA expression levels of LDLR, LXRα and ABCG1 were significantly increased in the EG compared to the PG.ConclusionGen effectively modulated the plasma lipid indices. The cholesterol-lowering effects of Gen may be attributed to its regulation on some of the key genes involved in cholesterol homeostasis.

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells.

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10–35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

Placental mechanism of prenatal nicotine exposure-reduced blood cholesterol levels in female fetal rats

Clinical studies showed that intrauterine growth retardation (IUGR) neonatus had lower cholesterol concentrations in cord blood, which might be associated with increased risk of metabolic syndrome and cardiovascular diseases in adulthood. We previously observed lower blood cholesterol levels in prenatal nicotine exposure (PNE)-induced IUGR fetal rats, and this study aimed to elucidate the placental mechanism. Pregnant Wistar rats were subcutaneously injected with nicotine (2.0 mg/kg⋅d) on gestational day 9–20. In vivo, PNE increased levels of total cholesterol (TCH), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) in maternal serum, while decreased levels of TCH and LDL-C in female fetal serum. Meanwhile, the expression of scavenger receptor class B type 1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) were decreased, and the expression of liver X receptor (LXR) α and β were also decreased in female placentas. In vitro, nicotine (0.1–10 μM) reduced the expression of LXRα, LXRβ, SR-B1, ABCA1 and ABCG1 in a concentration dependent manner, which could be annulled by nAChR antagonist and LXR agonist. Taken together, nicotine could inhibit the expression of SR-B1, ABCA1 and ABCG1 via nAChR and LXR α/β in female placentas, finally leading to reduced blood cholesterol levels in fetal rats.

Role of Hyaluronic Acid on Apolipoprotein A1 and HDL-Mediated Cholesterol Efflux in Macrophages

Diabetic macroangiopathy are characterized by increased arterial stiffness and hyaluronic acid (HA) deposition in the large elastic arteries. It is noteworthy that systemic inhibition of HA synthesis by 4-methylumbelliferone interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, inflammation, and progression of atherosclerosis. Apolipoprotein A1 (ApoA1) and High-density lipoprotein (HDL) mediates cholesterol efflux, which is the initial and rate-limiting step of reverse cholesterol transport preventing from atherosclerosis. The aim of the present study was to investigate the role of HA on ApoA1 and HDL-mediated cholesterol efflux in macrophages. Murine J774.1 macrophages and human THP-1 macrophages were labeled with 3H-cholesterol and incubated with ApoA1 (10 μg/ml) for 24 hours or HDL (50 μg/ml) for 4 hours. The percentage cholesterol efflux was calculated by dividing the media-derived radioactivity by the sum of the radioactivity in the media and the cells. The effect of HA on the cholesterol efflux was studied, adding that into the medium before the analysis of the cholesterol efflux. Under the existence of medium molecular weight HA (75-350 kDa), the HDL-mediated cholesterol efflux had significantly increased by 10.9% (n=4, p&amp;lt;0.01) in J774.1 macrophages. We also researched the relationship between HA and ATP-binding cassette transporter G1 (ABCG1), because expression of ABCG1 enhances cholesterol efflux to HDL. Predictably, HA significantly increased ABCG1 mRNA expression in macrophage (n=4, p&amp;lt;0.01), determined by Real-time Quantitative RT-PCR. These data suggest that HA increases HDL-mediated cholesterol efflux via ABCG1 in macrophages and the difference of molecular weight on HA may influence ApoA1 and HDL-mediated cholesterol efflux. The relationship between HA and cholesterol efflux might be one of the important mechanisms to prevent progression of atherosclerosis and diabetic macroangiopathy. Disclosure K. Matsuki: None. M. Ogura: None. M. Harada-Shiba: Advisory Panel; Self; Aegerion Pharmaceuticals. Speaker's Bureau; Self; Amgen Inc., Astellas Pharma US, Inc., Sanofi, Boehringer Ingelheim Pharmaceuticals, Inc., AstraZeneca, Daiichi Sankyo Company, Limited, MSD K.K.. Research Support; Self; Sanofi, Aegerion Pharmaceuticals, Kowa Pharmaceuticals America, Inc..

LAL (Lysosomal Acid Lipase) Promotes Reverse Cholesterol Transport In Vitro and In Vivo.

To explore the role of LAL (lysosomal acid lipase) in macrophage cholesterol efflux and whole-body reverse cholesterol transport. Immortalized peritoneal macrophages from lal-/- mice showed reduced expression of ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1), reduced production of the regulatory oxysterol 27-hydroxycholesterol, and impaired suppression of cholesterol synthesis on exposure to acetylated low-density lipoprotein when compared with lal+/+ macrophages. LAL-deficient mice also showed reduced hepatic ABCG5 (ATP-binding cassette transporter G5) and ABCG8 (ATP-binding cassette transporter G8) expression compared with lal+/+ mice. LAL-deficient macrophages loaded with [3H]-cholesteryl oleate-labeled acetylated low-density lipoprotein showed impaired efflux of released [3H]-cholesterol to apoA-I (apolipoprotein A-I), with normalization of [3H]-cholesteryl ester levels and partial correction of ABCA1 expression and cholesterol efflux to apoA-I when treated with exogenous rhLAL (recombinant human LAL protein). LAL-deficient mice injected intraperitoneally with lal-/- macrophages cholesterol loaded and labeled in the same way exhibited only 1.55±0.35% total injected [3H]-cholesterol counts appearing in the feces for 48 h (n=30), compared with 5.38±0.92% in lal+/+ mice injected with labeled lal+/+ macrophages (n=27), P<0.001. To mimic the therapeutic condition of delivery of supplemental LAL in vivo, injection of labeled lal-/- macrophages into lal+/+ mice resulted in a significant increase in reverse cholesterol transport (2.60±0.46% of 3H-cholesterol counts in feces at 48 hours [n=19]; P<0.001 when compared with injection into lal-/- mice). These results indicate a critical role for LAL in promoting both macrophage and whole-body reverse cholesterol transport and the ability of supplemental LAL to be taken up and correct reverse cholesterol transport in vivo.

ABCG1 as a potential oncogene in lung cancer.

ATP-binding cassette transporter G1 (ABCG1) is a member of the ABC transporter family and regulates cellular cholesterol homeostasis. It has important roles in cholesterol homeostasis and tumor immunity, which has, however, not been reported in lung cancer. The present study showed that ABCG1 protein is upregulated in lung cancer compared with adjacent normal tissues and furthermore, aberrant ABCG1 expression was detected in lung cancer cell lines. ABCG1 was shown to promote the proliferation of HKULC4 lung cancer cells. Moreover, ABCG1 was found to regulate proliferation-, apoptosis- and cancer stem cell-associated markers in HKULC4 cells, implying that ABCG1 has important roles not only in the regulation of proliferation but also of apoptosis and cancer stem cells. A microRNA microarray analysis showed that ABCG1 significantly downregulated miR-29a, -b and -c expression in HKULC4 cells. Finally, it was demonstrated that ABCG1 promoted the migration and invasion in HKULC4 cells. Thus, ABCG1 may be a novel therapeutic target to improve the treatment of non-small cell lung cancer.

Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier

Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [3H]-cholesterol efflux (by 50%) but did not reduce [3H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.

Abstract 13229: Varying ABCG1 Expression is Associated With Macrophage Apoptosis&amp;Phagocytosis

Introduction: ATP-binding cassette transporter G1(ABCG1)is a transmembrane protein mediated efflux of cellular cholesterol to HDL, which plays a vital part in macrophage lipid homeostasis, but the role of ABCG1 in atherosclerosis still remains controversial. Our previous study demonstrated that human ABCG1 -367 G&gt;A polymorphism in promoter region, which leads to lower ABCG1 protein expression, was associated with a reduced risk of CAD. However, the mechanism behind is not fully understood. Hypothesis: Oxysterols, the oxidized derivatives of cholesterol, whose efflux are mediated by ABCG1, exert cytotoxic effect on macrophage and accelerate cell apoptosis. Low ABCG1 expression induces excess oxysterols accumulation in macrophage, which augments the inflammatory response and accelerates apoptosis. Since defective apoptotic macrophage clearance in late lesions promotes atherosclerosis progression, we hypothesize that macrophage with lower ABCG1 expression may remove apoptotic cells more efficiently, thereby generating an antiatherogenic effect. Methods: To investigate the effect of ABCG1 expression on macrophage, we established ABCG1 knockdown cell line by means of lentivirus mediated shRNA. Apoptosis was determined by flow cytometry (FCM). Fluorescence labeled apoptotic model was generated and macrophage phagocytosis was evaluated. The inflammatory factors level and phagocytosis associated gene expression were measured by ELISA and realtime PCR. Results: Compared with control group, macrophage with lower ABCG1 expression had a higher secretion level of Inflammatory factors, such as TNF-α and IL-1β, showing an increased expression of classic inflammatory M1 phenotype markers,such as CD86 and TGF-β, and a down-regulation of CD163,Arg and IL-6 expression, which are markers of anti-inflammatory M2 phenotype macrophage. The apoptosis was significantly accelerated and the phagocytic clearance was enhanced after ABCG1 expression decreased, which means apoptotic cells could be removed more quickly. Both CD36 and Mertk showed a higher mRNA level in ABCG1 knockdown group. Conclusions: Decreased expression of macrophage ABCG1 may exert antiatherogenic function through accelerating apoptosis and enhancing phagocytosis.

The critical role of ABCG1 and PPARγ/LXRα signaling in TLR4 mediates inflammatory responses and lipid accumulation in vascular smooth muscle cells.

Toll-like receptor 4 (TLR4) plays critical roles in vascular inflammation, lipid accumulation and atherosclerosis development. However, the mechanisms underlying these processes are still not well established, especially in vascular smooth muscle cells (VSMCs). ATP-binding cassette transporter G1 (ABCG1) is one of the key genes mediating inflammation and cellular lipid accumulation. The function of TLR4 in regulating the expression of ABCG1 and the underlying molecular mechanisms remain to be elucidated. In this study, we cultured VSMCs from the thoracic aortas of mice and treated the cells with 50μg/ml oxidized low-density lipoprotein (oxLDL) to activate TLR4 signaling. We observed that activating TLR4 with oxLDL induced inflammatory responses and lipid accumulation in VSMCs. The expression of peroxisome proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα) and ABCG1 was inhibited by TLR4 activation. However, these effects could be reversed by knocking out TLR4. PPARγ activation by rosiglitazone rescued LXRα and ABCG1 expression and reduced TLR4-induced inflammation and lipid accumulation. Silencing PPARγ expression with a specific small interfering RNA (siRNA) inhibited LXRα and ABCG1 expression and, importantly, enhanced TLR4-induced inflammation and lipid accumulation. In conclusion, ABCG1 expression was down-regulated by TLR4, which induces inflammation and lipid accumulation in VSMCs via PPARγ/LXRα signaling. These findings indicate a novel molecular mechanism underlying TLR4-induced inflammation and lipid accumulation.

Loss of ABCG1 influences regulatory T cell differentiation and atherosclerosis.

ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol accumulation and alters T cell homeostasis, which may contribute to progression of atherosclerosis. Here, we investigated how the selective loss of ABCG1 in T cells impacts atherosclerosis in LDL receptor-deficient (LDLR-deficient) mice, a model of the disease. In LDLR-deficient mice fed a high-cholesterol diet, T cell-specific ABCG1 deficiency protected against atherosclerotic lesions. Furthermore, T cell-specific ABCG1 deficiency led to a 30% increase in Treg percentages in aorta and aorta-draining lymph nodes (LNs) of these mice compared with animals with only LDLR deficiency. When Abcg1 was selectively deleted in Tregs of LDLR-deficient mice, we observed a 30% increase in Treg percentages in aorta and aorta-draining LNs and reduced atherosclerosis. In the absence of ABCG1, intracellular cholesterol accumulation led to downregulation of the mTOR pathway, which increased the differentiation of naive CD4 T cells into Tregs. The increase in Tregs resulted in reduced T cell activation and increased IL-10 production by T cells. Last, we found that higher ABCG1 expression in Tregs was associated with a higher frequency of these cells in human blood samples. Our study indicates that ABCG1 regulates T cell differentiation into Tregs, highlighting a pathway by which cholesterol accumulation can influence T cell homeostasis in atherosclerosis.