ABCA1 mRNA Levels Research Articles

Differential regulation of ABCA1 and ABCG1 gene expressions in the remodeling mouse hippocampus after entorhinal cortex lesion and liver-X receptor agonist treatment

Entorhinal cortex lesioning (ECL) causes an extensive deafferentation of the hippocampus that is classically followed by a compensatory reinnervation, where apolipoprotein E, the main extracellular lipid-carrier in the CNS, has been shown to play a crucial role by shuttling cholesterol to reconstructing neurons terminals. Hence, we investigated whether the ATP-binding cassette (ABC) transporters -A1 and -G1, known to regulate cellular cholesterol efflux and lipidation of the apolipoprotein E-containing lipoprotein complex are actively involved in this context of brain׳s plastic response to neurodegeneration and deafferentation. We assessed ABCA1 and ABCG1 mRNA and protein levels throughout the degenerative phase and the reinnervation process and evaluated the associated cholinergic sprouting following ECL in the adult mouse brain. We subsequently tested the effect of the pharmacological activation of the nuclear receptor LXR, prior to versus after ECL, on hippocampal ABCA1 and G1 expression and on reinnervation. ECL induced a time-dependent up-regulation of ABCA1, but not G1, that coincided with a significant increase in acetylcholine esterase (AChE) activity in the ipsilateral hippocampus. Pre-ECL, but not post-ECL i.p. treatment with the LXR agonist TO901317 also led to a significant increase solely in hippocampal ABCA1 expression, paralleled by increases in both AchE and synaptophysin protein levels in the deafferented hippocampus. Thus, ABCA1 and -G1 are differentially regulated in the lesioned brain and upon treatment with an LXR agonist. Further, TO901317-induced up-regulation of ABCA1 appears to be more beneficial in a prevention (pre-lesion) than rescue (post-lesion) treatment; both findings support a central role for ABC transporters in brain plasticity.

Linoleic Acid Suppresses Cholesterol Efflux and ATP‐Binding Cassette Transporters in Murine Bone Marrow‐Derived Macrophages

Individuals with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease (CVD), possibly associated with elevated plasma free fatty acid concentrations. Paradoxically, evidence suggests that unsaturated, compared to saturated fatty acids, suppress macrophage cholesterol efflux, favoring cholesterol accumulation in the artery wall. Murine bone marrow-derived macrophages (BMDM) were used to further explore the relationship between saturated and unsaturated fatty acids, and cholesterol efflux mediated by ATP-binding cassette transporters (ABCA1 and ABCG1) through transcription factors liver-x-receptor-alpha (LXR-α) and sterol receptor element binding protein (SREBP)-1. BMDM isolated from C57BL/6 mice were exposed to 100 μM linoleic acid (18:2) or palmitic acid (16:0) for 16h, and 25 μg/mL oxidized low density lipoprotein for an additional 24h. ABCA1 and ABCG1 mRNA expression was suppressed to a greater extent by 18:2 (60% and 54%, respectively) than 16:0 (30% and 29%, respectively) relative to the control (all p<0.01). 18:2 decreased ABCA1 protein levels by 94% and high density lipoprotein (HDL) mediated cholesterol efflux by 53% (both p<0.05), and had no significant effect on ABCG1, LXR-α or SREBP-1 protein levels. 16:0 had no effect on ABCA1, ABCG1, LXR-α or SREBP-1 protein expression or HDL-mediated cholesterol efflux. These results suggest that 18:2, relative to 16:0, attenuated macrophage HDL-mediated cholesterol efflux through down regulation of ABCA1 mRNA and protein levels but not through changes in LXR-α or SREBP-1 expression. The effect of 18:2 relative to 16:0 on macrophages cholesterol homeostasis may exacerbate the predisposition of individuals with T2DM to increased CVD risk.

Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters.

Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30–49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL.

Adiponectin Receptor 2 Deficiency Results in Reduced Atherosclerosis in the Brachiocephalic Artery in Apolipoprotein E Deficient Mice

Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2. The original aim of this study was to investigate the effect of combined AdipoR1 and AdipoR2 deficiency (AdipoR1-/-AdipoR2-/-) on atherosclerosis. However, we made the interesting observation that AdipoR1 -/- AdipoR2 -/- leads to embryonic lethality demonstrating the critical importance of the adiponectin signalling system during development. We then investigated the effect of AdipoR2-ablation on the progression of atherosclerosis in apolipoprotein E deficient (ApoE -/-) mice. AdipoR2-/-ApoE-/- mice fed an atherogenic diet had decreased plaque area in the brachiocephalic artery compared with AdipoR2 +/+ApoE-/- littermate controls as visualized in vivo using an ultrasound biomicroscope and confirmed by histological analyses. The decreased plaque area in the brachiocephalic artery could not be explained by plasma cholesterol levels or inflammatory status. However, accumulation of neutral lipids was decreased in peritoneal macrophages from AdipoR2-/-ApoE-/- mice after incubation with oxidized LDL. This effect was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages from AdipoR2-/-ApoE-/- mice compared with AdipoR2+/+ApoE-/- controls after incubation with oxidized LDL. In summary, we show that adiponectin receptors are crucial during embryonic development and that AdipoR2-deficiency slows down the progression of atherosclerosis in the brachiocephalic artery of ApoE-deficient mice.

Subjects with Low Plasma HDL Cholesterol Levels Are Characterized by an Inflammatory and Oxidative Phenotype

BackgroundEpidemiological studies have shown that low plasma levels of high-density lipoprotein (HDL) cholesterol are associated with increased risk of cardiovascular disease, but the mechanisms for the possible atheroprotective effects of HDL cholesterol have still not been fully clarified, in particular in relation to clinical studies.ObjectiveTo examine the inflammatory, anti-oxidative and metabolic phenotype of subjects with low plasma HDL cholesterol levels.Methods and ResultsFifteen subjects with low HDL cholesterol levels (eleven males and four females) and 19 subjects with high HDL (three males and 16 females) were recruited. Low HDL cholesterol was defined as ≤10th age/sex specific percentile and high HDL-C was defined as ≥90 age/sex specific percentile. Inflammatory markers in circulation and PBMC gene expression of cholesterol efflux mediators were measured. Our main findings were: (i) subjects with low plasma HDL cholesterol levels were characterized by increased plasma levels of CRP, MMP-9, neopterin, CXCL16 and ICAM-1 as well as low plasma levels of adiponectin, suggesting an inflammatory phenotype; (ii) these individuals also had reduced paraoxonase (PON)1 activity in plasma and PON2 gene expression in peripheral blood mononuclear cells (PBMC) accompanied by increased plasma levels of oxidized LDL suggesting decreased anti-oxidative capacity; and (iii) PBMC from low HDL subjects also had decreased mRNA levels of ABCA1 and ABCG1, suggesting impaired reverse cholesterol transport.ConclusionSubjects with low plasma HDL cholesterol levels are characterized by an inflammatory and oxidative phenotype that could contribute to the increased risk of atherosclerotic disorders in these subjects with low HDL levels.

ABCA1 mRNA and protein levels in M-CSF-activated macrophages from patients with arterial stenosis

ABCA1 transporter is one of the key factors defining the level of antiatherogenic HDL in plasma. It is actively involved in the removal of cholesterol from peripheral tissues by reverse cholesterol transport. However, the influence of the level of ABCA1 mRNA and the level of ABCA1 protein 1 in macrophages in atherosclerosis remains unexplored. Using real time PCR we determined ABCA1 mRNA level in macrophages cultured for 5 days with macrophage colony-stimulating factor (M-CSF). ABCA1 mRNA levels in macrophages from patients with arterial stenosis were increased when compared with the control group, P = 0.04. According to a Western blot analysis ABCA1 protein level in macrophages from patients was significantly lower than in the control group, P = 0.01. Our results suggest that the level of ABCA1 mRNA and level of ABCA1 protein in macrophages may be important factors in the development of atherosclerosis.

TNFa alter cholesterol metabolism in human macrophages via PKC-θ-dependent pathway

BackgroundStudies have shown that inflammation promoted atherosclerotic progression; however, it remains unclear whether inflammation promoted atherosclerotic progression properties by altering cholesterol metabolism in human macrophages. In the present study, we evaluated a potential mechanism of inflammation on atherogenic effects. We evaluated the ability of TNFa to affect Reverse cholesterol transport (RCT) and cholesterol uptake and its mechanism(s) of action in human macrophages.ResultsWe initially determined the potential effects of TNFa on cholesterol efflux in the human macrophages. We also determined alterations in mRNA and protein levels of ABCA1, ABCG1, LXRa, CD-36, SR-A in human macrophages using quantitative real-time polymerase chain reaction (PCR) and Western immunoblot analyses. The cholesterol efflux rate and protein expression of ABCA1, ABCG1, LXRa, CD-36, SR-A were quantified in human macrophages under PKC-θ inhibition using PKC-θ siRNA. Our results showed that TNFa inhibited the rate of cholesterol efflux and down-regulation the expression levels of ABCA1, ABCG1 and LXRa and up-regulation the expression levels of CD-36, SR-A in human macrophages; PKC-θ inhibition by PKC-θ siRNA attenuated the effect of TNFa on ABCA1, ABCG1, LXRa, SR-A, CD-36 expression.ConclusionsOur results suggest TNFa alter cholesterol metabolism in human macrophages through the inhibition of Reverse cholesterol transport and enhancing cholesterol uptake via PKC-θ-dependent pathway, implicating a potential mechanism of inflammation on atherogenic effects.

Abstract 276: Shockwave Reduces Macrophage Foam Cell Formation Via Increased Expression of ATP Binding Cassette Transporters.

Background: Excessive lipid accumulation by macrophages plays a crucial role in atherosclerosis. Foam cells are generated by uncontrolled uptake of modified LDL, especially oxidized LDL (oxLDL), and/or impaired cholesterol efflux mediated by ATP-binding cassette (ABC) family transporters, ABCA-1 and ABCG-1. Shockwave, elicited by transient pressure disturbance, have been used for extracorporeal lithotripsy or for treating musculoskeletal disorders. Our current study suggests an evidence that shockwave may have anti-atherogenic effect by inhibiting foam cell formation. Methods/Results: Murine peritoneal macrophages were exposed to shockwaves at 0.04 mJ/mm 2 with 1000 impulses, lysed after 6, 18 and 24 hours, and tested for expression of ABCA-1 and ABCG-1. The western blot showed that shockwave induced 2.0-2.8 fold increase of ABCA-1 and ABCG-1 within 18-24 hours. mRNA levels of ABCA-1 and ABCG-1 were also increased by shockwave with 2.0 fold of peak increase in 18 hours. The increased expression of ABCA-1 and ABCG-1 was mediated by phosphorylation of ERK 1/2 (Tyr204). Western blot analysis revealed that shockwave induced phosphorylation of ERK 1/2 (Tyr204) in murine macrophages. Shockwave-induced increase of ABCA-1 and ABCG-1 was blocked by U0126 (40µM), a specific inhibitor for ERK. Oil-red O staining showed that macrophages exposed to shockwave had 25% less intracellular lipid droplets. Intracellular cholesterol measured by cholesterol oxidase and esterase revealed that macrophages exposed to shockwave had 23% less intracellular cholesterol when incubated with oxLDL (50µg/ml) for 16 hours. In vitro migration assays including modified Boyden chamber migration assay and scratch wound healing migration assay showed that macrophages exposed to shockwave had 1.2 fold more migration and had diminished migration-inhibitory effect of oxLDL. Conclusions: Shockwave reduces macrophage foam cell formation via ERK-mediated increase of ABCA-1 and ABCG-1 mediating lipid efflux and promotes macrophage migration which may induce macrophage egress from atherosclerotic lesion. Our study suggests anti-atherogenic effects of shockwave as a potential treatment modality for atherosclerosis.

Pro-apoptotic miRNA-128-2 modulates ABCA1, ABCG1 and RXRα expression and cholesterol homeostasis

Aberrant regulation of cholesterol homeostasis is associated with obesity as well as multiple types of cancer. However, the mechanism behind these is largely missing. Here, we show that microRNA (miRNA)-128-2 is not only a pro-apoptotic microRNA but it also alters the expression of genes involved in cellular cholesterol homeostasis. Cholesterol efflux via ATP-binding cassette transporters (ABCA1 and ABCG1) is a mechanism for cells to eliminate excess cholesterol and prevent cellular cholesterol accumulation. The regulation of these pathways is complex with transcriptional regulation by sterol-regulatory element-binding protein (SREBP) and liver X receptor/retinoid X receptor (RXR) transcription factors but poorly understood at the post-transcriptional levels. MiR-128-2 increases the expression of SREBP2 and decreases the expression of SREBP1 in HepG2, MCF7 and HEK293T cells independent of sirtuin 1 (SIRT1) status. MiR-128-2 inhibits the expression of ABCA1, ABCG1 and RXRα directly through a miR-128-2-binding site within their respective 3′untranslated regions. The administration of miR-128-2 leads to decline in the protein and mRNA levels of ABCA1, ABCG1 and RXRα. Conversely, anti-miRNA treatment leads to increased ABCA1, ABCG1 and RXRα expression. The inverse correlation between miR-128-2 and its targets viz. ABCA1 and ABCG1 was also established during high-fat diet in different mice tissues. Our data show that cholesterol efflux is attenuated by miR-128-2 overexpression and, conversely, stimulated by miR-128-2 silencing. Further, we also observed the induction of ER stress response by miR-128-2. In this study, we provide the first evidence of miR-128-2 to be a new regulator of cholesterol homeostasis. Our study shows dual role of miR-128-2, as a pro-apoptotic molecule as well as a regulator of cholesterol homeostasis.

The inhibitory effect of black soybean on hepatic cholesterol accumulation in high cholesterol and high fat diet-induced non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is defined as excess of fat in the liver. We investigated the effects of black soybean on the cholesterol metabolism and insulin resistance of mice fed high cholesterol/fat diets. Mice were randomly allocated into four groups that were fed different diets: the normal cholesterol/fat diet; high cholesterol/fat diets (HCD); and HCD with 1%, and 4% black soybean powder (1B-HCD, and 4B-HCD). Liver total cholesterol and triglyceride concentrations were significantly lower in the black soybean-supplemented groups than that in the HCD group. PCR revealed significantly lower hepatic SREBP2 and HMG-CoA reductase mRNA levels of black soybean-supplemented mice. Real-time PCR revealed significantly higher hepatic ABCA1 mRNA level of black soybean-supplemented mice, which may increase cholesterol efflux. Liver bile acids concentration was significantly high in the 4B-HCD group. Black soybean stimulated secretion of adiponectin, activation of pAMPK, and eliminated free fatty acids in the liver. Black soybean supplementation decreased MDA and nitrate level. The activities of SOD, catalase, and GPx were restored by black soybean supplementation. Our data strongly indicate that black soybean influences the balance between oxidative and antioxidative stress. We suggest that black soybean improves cholesterol metabolism, insulin resistance, and alleviates oxidative damage in NAFLD.

Porphyromonas gingivalis lipopolysaccharide increases lipid accumulation by affecting CD36 and ATP-binding cassette transporter A1 in macrophages

Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) promotes macrophage-derived foam cell formation, however, the mechanisms are not well established. In macrophages, lipid uptake is mediated by scavenger receptors including SR-A and CD36, while the cholesterol efflux is mediated by ATP-binding cassette transporter G1 (ABCG1), ABCA1 and SR-BI. We further investigated the mechanisms underlying the dysregulation by P. gingivalis LPS of these regulators resulting in the promotion of lipid accumulation in THP-1-derived macrophages. Our results showed that P. gingivalis LPS exacerbated lipid accumulation in oxidized low-density lipoprotein (oxLDL)-treated macrophages. However, cholesterol efflux was inhibited by P. gingivalis LPS in THP-1-derived macrophages. In oxLDL-untreated macrophages, P. gingivalis LPS treatment caused an increase in CD36 mRNA and protein levels, and a decrease in ABCA1 mRNA and protein levels, while having no effect on SR-A, SR-BI or ABCG1 expression. Upregulation of CD36 by P. gingivalis LPS resulted from activation of c-Jun/AP-1, and this was confirmed by the inhibition of increased CD36 expression after AP-1 inhibition using SP600125. However, the decreased protein stability of ABCA1 by P. gingivalis LPS was a result of increased calpain activity. Moreover, small hairpin RNA (shRNA) targeting heme oxygenase-1 (HO-1) augmented the P. gingivalis LPS-induced atherogenic effects on the expression of c-Jun/AP-1, CD36, ABCA1 and calpain activity. Accordingly, P. gingivalis LPS-regulated promotion of lipid accumulation in foam cells was also exacerbated by HO-1 shRNA. These results indicate that P. gingivalis LPS confers a exacerbation effect on the formation of foam cells by a novel HO-1-dependent mediation of cholesterol efflux and lipid accumulation in macrophages.

A novel LXRα- or RX-independent mechanism for increasing brain APOE levels

Apolipoprotein E (apoE) is a key lipid transport protein in brain. The e 4 allele is strongly linked to late onset and sporadic forms of Alzheimer's disease (AD). Since apoE plays an important role in neuronal repair, synaptogenesis, and clearance of toxic Aß fragments from brain, understanding how brain apoE levels are regulated is critical. In addition to known LXRα and RXR mediated mechanisms, we identified a novel mechanism mediated through pan-class I HDAC inhibition (HDAC1i) for increasing apoE levels in astrocytes. Using MS275, a pan-class I HDAC inhibitor as a tool compound, we compared the effect of HDAC1i on lipogenic gene expression in cells lines of hepatic or astrocytic origin. We also investigated the expression of these same lipogenic genes and apoE following exposure to a pan LXR agonist, T0901317 and a RXR agonist, bexarotene. Human hepatoblastoma (HepG2) cells and astrocytoma (CCF-STTG1) cells were treated with varying concentrations (0–10 μM) of MS275, TO0901317, or Bexarotene for 24 hours and mRNA expression was assessed using RT-qPCR. The amount of apoE secreted by CCF-STTG1 cells was also measured using alphaLISA after 48 hours of compound treatment. Following treatment of HepG2 cells with T0901317 we observed a dose-dependent increase in mRNA e xpression for lipogenic genes: LXRα, ABCA1, ABCG5 and ABCG8, SREPB1c and FASN. In CCF-STTG1 cells, following treatment with T0901317, we observed a dose-dependent increase in LXRα, apoE and apoE lipidating genes: ABCA1 and ABCG1. In contrast, a bell-shaped dose-response was observed in HepG2 as well as CCF-STTG1 cells for all end-points measured following treatment with bexarotene, Interestingly, treatment of HepG2 cells with MS275 did not elevate the expression of LXRα and its target genes (ABCA1, ABCG5 and ABCG8), but resulted in a significant dose-dependent increase in apoE, SREBP1c and FASN mRNA levels. Following treatment of CCF-STTG1 cells with MS275, we observed a significant increase in both apoE mRNA expression and protein secretion, as well as an increase in ABCA1 and ABCG1 mRNA levels without any significant effects on LXRα mRNA. Using MS275, T0901317 and Bexarotene as tool compounds, we demonstrate differential regulation of apoE and lipogenic gene expression that is cell type-dependent.

Resveratrol Suppresses T0901317-Induced Hepatic Fat Accumulation in Mice

Liver X receptor (LXR) has been identified as a potential target for treatment of atherosclerosis and diabetes. Activation of LXR, however, is associated with increased lipogenesis and fat accumulation in the liver. The objective of the current study was to examine the effect of resveratrol on LXR activator-induced fat accumulation in liver using mice as an animal model. Three groups of C57BL/6 mice were studied. Animals in group 1 were treated with T0901317, a potent activator of LXR in mice. Animals in group 2 served as the control and were treated with carrier solution and those in group 3 were treated with T0901317/resveratrol combination. Using histochemical and biochemical methods, we demonstrate that resveratrol treatment significantly suppressed fat accumulation in the liver induced by T0901317. In addition, resveratrol completely blocked elevation of blood levels of triglyceride and cholesterol and reduced blood glucose level. Quantitative PCR analysis revealed that resveratrol treatment did not change the mRNA levels of abca1, abcg1, cyp7a1, srebp-1c, chrebp, and acc genes compared to that of animals treated with T0901317 alone but reduced pepck and g6p gene expressions. Immunohistochemistry and Western blot analyses show resveratrol treatment activated AMP-activated protein kinase (AMPK) and increased phosphorylation of acetyl-CoA carboxylase. Treatment with T0901317 on hepatocytes increased intracellular fat accumulation and this increase was suppressed by resveratrol; the suppressive effect of resveratrol was greatly repressed by Compound C which is an inhibitor of AMPK. Collectively, these data suggest that resveratrol blocks T0901317-induced lipid accumulation in the liver and can be considered for inclusion into the treatment of diseases involving activation of liver X receptor.

Fermentation improved gene modulatory effects of soy milk to lower serum triglyceride and elevate HDL‐cholesterol in SD rats on high cholesterol diets.

Soy is known to improve blood lipid profiles and fermentation was implicated to increase bioavailability of isoflavones. Here we aimed to investigate the effects of soy milk (SOY) or fermented soy milk (FSOY) on lipid profiles and expression of genes in cholesterol and triglyceride (TG) metabolism in rats fed high cholesterol diets : Control AIN76A diet, high cholesterol (CHOL) diet containing 1% cholesterol, SOY diet (a CHOL diet with SOY substituting 20% of casein), and FSOY diet (a CHOL diet with FSOY substitution). We examined blood lipid profiles, lipid contents in liver and feces, and gene expression levels in liver and adipose tissues. SOY and FSOY lowered liver total cholesterol (TOTAL‐C) compared to CHOL but failed to affect serum TOTAL‐C and LDL‐C. This effect was associated with clear reduction of HMG‐CoAR mRNA levels in FSOY. Serum HDL‐C elevation detected in only FSOY was related to increased ABCA1 mRNA levels in adipose tissue in comparison to CHOL. CHOL‐elevated serum TG levels were markedly reduced by SOY or FSOY, which didn¡ t affect SREBP1c mRNA in the liver. Instead a slight recovery of CHOL suppression of VLDLR, Lrp1, or UCP2 transcript levels in adipose tissues was observed by SOY, which effect was much greater in FSOY. Our data suggested that FSOY may be more effective in improving CHOL‐induced aberrant lipid metabolism than SOY via greater changes in gene expression in liver and adipose tissues.

Ligation of RAGE with ligand S100B attenuates ABCA1 expression in monocytes

HypothesisATP Binding Cassette Transporter (ABC) A1 is one of the key regulators of HDL synthesis and reverse cholesterol transport. Activation of Receptors for Advanced Glycation End products (RAGE) is involved in the pathogenesis of diabetes, and its complications. The aim of the present study is to examine the effect of RAGE ligand S100B on ABCA1 expression. MethodsS100B mediated regulation of LXR target genes like ABCA1, ABCG1, ABCG8, LXR-α and LXR-β in THP-1 cells was analyzed by real-time PCR, RT-PCR and western blots. ABCA1 mRNA expression in monocytes from diabetic patients was studied. Effect of LXR ligand on S100B induced changes in LXR target genes was also studied. Luciferase reporter assay was used for S100B induced ABCA1 promoter regulation. ResultsS100B treatment resulted in a significant 2–3 fold reduction (p<0.01) in ABCA1 and ABCG1 mRNA in dose and time dependent manner in THP1 cells. ABCA1 protein level was also significantly (p<0.01) reduced. S100B-induced reduction on ABCA1 mRNA expression was blocked by treating THP-1 cell with anti-RAGE antibody. Reduced ABCA1 mRNA levels seen in peripheral blood monocytes from diabetes patients showed the in-vivo relevance of our in-vitro results. Effect of S100B on ABCA1 and ABCG1 expression was reversed by LXR ligand treatment. S100B treatment showed significant 2 fold (p<0.01) decrease in T1317 induced ABCA1 promoter activation. ConclusionsThese results show for the first time that ligation of RAGE with S100B can attenuate the expression of ABCA1 and ABCG1 through the LXRs. This could reduce ApoA-I-mediated cholesterol efflux from monocytes.

Effects of compound X, a novel potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, on the adrenal gland of rats

To investigate the adrenal toxicity of a novel inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, compound X (CX), histopathological examinations, fat staining, adrenal cholesterol measurement, blood biochemistry, plasma corticosterone and ACTH measurement, ACTH-stimulation assay, and adrenal gene-expression analyses were done in rats in repeated-dose studies (experiment 1: 0, 3, 10, 30 and 150mg/kg for 4, 8, 15 and 28 days; experiment 2: 0, 3, 10,30 and 150mg/kg for 28 days; experiment 3: 0, 10, 30, 100 and 300mg/kg for 28 days). CX induced morphologic changes such as vacuolation and hypertrophy in the zona fasciculata (ZF) at ≥10mg/kg, and eosinophilic changes in the ZF at 150mg/kg. Vacuolation decreased in a dose-dependent manner and was replaced by eosinophilic changes. Inflammatory and fibrous changes were observed at ≥30mg/kg. These changes were expressed at early stages of dosing and were not exacerbated by extension of the administration period. Oil-red-O/Filipin staining showed depletion of cholesterol ester in dose-dependent manner and enabled adrenal cholesterol measurement. Filipin staining also revealed vacuoles to be composed of cholesterol esters. No significant changes were observed during the dosing period of CX for plasma corticosterone and ACTH levels. Gene-expression analyses showed up-regulation of Star and Abca1 mRNA levels at 300mg/kg. In conclusion, CX induced adrenal toxicity, but CX did not influence adrenocortical functions, and exacerbation of adrenal toxicities by extension of the administration period was not observed. Up-regulation of genes related to the transport of FC, such as Star and Abca1, were observed in CX groups, and these genes may be involved in the maintenance of adrenal structure and function in rats given CX.

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

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. 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. 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. 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.

Keratinocyte ATP binding cassette transporter expression is regulated by ultraviolet light

Many ATP binding cassette (ABC) transporters are important regulators of lipid homeostasis and have been implicated in keratinocyte lipid transport. Ultraviolet (UV) light exposure is a known epidermal stressor, which amongst other effects causes lipid alterations and defective lamellar body biogenesis. To elucidate the background of these lipid changes we studied the effect of UVB light on ABC transporter expression. The effect of UVB treatment on the levels of 47 known human ABC transporter mRNAs was analyzed in normal human epidermal keratinocytes. Immunoblots and promoter assays were carried out for ABCA1 and ABCG1. The mRNA levels of cholesterol transport regulators ABCA1 and ABCG1 were markedly downregulated by UVB, parallel to the lamellar ichthyosis related glucosylceramide transporter ABCA12 and the suspected sphingosine-1-phosphate and cholesterol sulfate transporter ABCC1. The long but not the short alternative splice variant of the ABCF2 was found to be markedly upregulated rapidly after UVB irradiation. Immunoblot confirmed ABCA1 and ABCG1 protein downregulation, and luciferase assays showed suppression of their promoters by UVB. These proteins mostly transport lipids, which account for the integrity of the epidermal barrier; therefore our findings on the UVB regulation of ABC transporters may explain the appearance of barrier dysfunction after UVB exposure.

The Transcription Levels of ABCA1, ABCG1 and SR-BI are Negatively Associated with Plasma CRP in Chinese Populations with Various Risk Factors for Atherosclerosis

ATP binding cassette transporters (ABCA1, ABCG1) and scavenger receptor class B type I (SR-BI) are the three most important cellular cholesterol transporters that may prevent atherogenesis. The aim of this study was to investigate whether they were altered in Chinese populations with various risk factors for atherosclerosis and their potential associations with C-reactive protein (CRP). Healthy female controls (n = 30) and populations with various risk factors for atherosclerosis, such as type 2 diabetes (n = 17), hypertension (n = 12), overweight/obesity (n = 10), incipient nephropathy (n = 10), postmenopausal women (n = 9), male (n = 19), ageing male (n = 22), or smoking (n = 16), were recruited. ABCA1, ABCG1 and SR-BI mRNA levels in peripheral monocytes was determined. ABCG1 was decreased in all the risk populations except ageing. ABCA1 was decreased in all the risk populations except diabetes and male. SR-BI was decreased in those with overweight/obesity and incipient nephropathy. Circulating CRP was increased almost in all the risk populations except in males. The levels of ABCA1, ABCG1 and SR-BI were reduced in those with subclinically high CRP, and negatively associated with CRP level. These data indicates that ABCA1, ABCG1, and SR-BI are reduced in various populations under subclinically inflammatory conditions, which may potentially lead to impairing reverse cholesterol transport and developing atherosclerosis.

Abstract 281: Resveratrol Enhances Cholesterol Efflux Through PPAR-γ--Dependent Signaling Pathways in Human Macrophages

Introduction: We previously reported that resveratrol, a plant derived polyphenol with numerous cardioprotective properties, improves cholesterol homeostasis by upregulating the expression of reverse cholesterol transport (RCT) proteins critical for preventing lipid overload and macrophage foam cell formation. However, the mechanism(s) by which resveratrol exerts this effect are unclear. The present study explores a possible mechanism for the atheroprotective actions of resveratrol on cholesterol metabolism. Hypothesis: We hypothesize that the anti-atherogenic effects of resveratrol are mediated through PPAR-γ dependent signaling pathways leading to upregulation of RCT proteins and enhanced cholesterol efflux. Methods: THP-1 macrophages, a pertinent model for atherosclerosis, were incubated for 20h with resveratrol (10μM) with/without the specific PPAR-γ antagonist GW6992 (1μM). Alternatively, cells were transfected with 100 nM of human PPAR-γ small interfering RNA (siRNA) for gene silencing prior to resveratrol treatment. Total RNA was isolated, converted to cDNA, and evaluated by QRT-PCR. Each reaction was done in triplicate. Western blot analysis was performed to confirm results of gene expression. Results: Resveratrol significantly increased expression of PPAR-γ, ABCA1, and 27-hydroxylase mRNA (mean±SEM, 136.2±8.5%, 168.3±9.2%, and 171.7±11.0% of control respectively, P&lt;0.001). PPAR-γ gene silencing and PPAR-γ antagonist GW6992 effectively reduced PPAR-γ message by 90.4% and 54.7%, respectively (P&lt;0.001). Both pharmacological blockade and gene knockdown of PPAR-γ nullified resveratrol effects on cholesterol efflux proteins. In cells treated with GW6992, mRNA levels of ABCA1 and 27-hydroxylase were decreased to 66.1±3.3% and 55.0±2.9% of control, respectively (P&lt;0.001). Similarly, PPAR-γ silencing resulted in downregulation of ABCA1 and 27-hydroxylase expression to the level of control, which is significantly lower than in resveratrol treated cells (P&lt;0.001). Data from gene expression studies were subsequently confirmed by Western blot analysis. Conclusions: We propose here a mechanistic model for the atheroprotective effects of resveratrol. Our data strongly suggests that resveratrol regulates cholesterol efflux and intracellular cholesterol processing via the PPAR-γ/LXR-α pathway.