ABCA1 mRNA Expression Research Articles

Abstract 235: Atorvastatin Has No Impact on the Effects of CETP Inhibitors on HDL Structure and Cholesterol Efflux Capacity in Chow-Fed Rabbits

Introduction: In the dal-OUTCOMES trial, cholesteryl ester transfer protein (CETP) inhibition by dalcetrapib increased HDL-C but had a neutral effect on CV risk. However, these patients were also treated with standard-of-care therapies including statins, a drug class shown to reduce ABCA1 mRNA expression in human peripheral blood mononuclear cells (PBMC). Hypothesis: Given the role of ABCA1 in cholesterol efflux and HDL biogenesis, we determined the impact of atorvastatin on the potential beneficial effects provided by dalcetrapib and anacetrapib, two CETP inhibitors. Methods: Dalcetrapib (300 mg/kg) or anacetrapib (30 mg/kg) were administered to New Zealand White rabbits receiving or not atorvastatin (2.5 mg/kg) for 14 days. Lipid profiles were measured biochemically. ApoA-I distribution in HDL subclasses was evaluated by 1D-non-denaturing gradient gel electrophoresis (1D-NDGGE). BHK cells with inducible expression of human ABCA1 and HepG2 hepatocytes were used to measure ABCA1- and SR-BI-dépendent cholesterol efflux. Human THP-1 macrophages and PBMC isolated from rabbits were also used for efflux assays. Results: Dalcetrapib increased HDL-C by +81% (p<0.01) and total apoA-I (1D-NDGGE) by 1.5-fold (p<0.01). This was associated with an increase in both large and small α-migrating HDL (+52%, p<0.05; +74%, p<0.05). Cholesterol efflux showed that ABCA1- and SR-BI-dependent effluxes to apoB-depleted serum were increased in dalcetrapib-treated rabbits (+31%, p<0.01; +38%, p<0.001). Atorvastatin had no impact on all these parameters. Moreover, treatment of THP-1 with atorvastatin did not inhibit the increase of cholesterol efflux (+24%, p<0.01) induced by depleted serum from dalcetrapib-treated rabbits. Efflux capacity of PBMC isolated from rabbits treated with atorvastatin was also unchanged compared to the other groups. The absence of effect of atorvastatin on cholesterol efflux was also observed with anacetrapib. Conclusion: CETP inhibition increased HDL-C, apoA-I levels and cholesterol efflux from different cell types. Atorvastatin had no effect on these parameters, suggesting that the increased cholesterol efflux capacity induced by CETPi is not hampered by statins.

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.

Ac-hE-18A-NH2 inhibits the inflammatory response induced by ox-LDL via inhibiting NF-κB activation in RAW264.7 macrophages

To evaluate the effect of Ac-hE-18A-NH2 on TNF-α secretion and mRNA expression in ox-LDL-stimulated RAW264.7 macrophages and to elucidate the possible mechanisms. Macrophages were incubated in the medium containing various concentrations of Ac-hE18A-NH2 (1-50 μg/mL) with ox-LDL (50 μg/mL) stimulated. The TNF-α level and intracellular cholesterol content were measured by commercially available quantitation kits following the manufacturer's instructions. TNF-α and ATP-binding cassette transporter A1 (ABCA1) mRNA expression were detected by real-time PCR. ABCA1 and IκB protein -expression in the macrophages were determined by Western blot. NF-κB activity was evaluated by electrophoretic mobility shift assay (EMSA). Ox-LDL stimulation induced a significant increase in TNF-α secretion, mRNA expression, cholesterol accumulation and nuclear factor-κB (NF-κB) activity in RAW264.7 macrophages. Ac-hE-18A-NH2 reduced TNF-α secretion and mRNA expression, up-regulated the ABCA1 mRNA and protein expression, reduced the intracellular cholesterol content, and inhibited NF- κB activation in a dose-dependent manner. Under the same condition and the same concentration, Ac-hE-18A-NH2 was more efficient than D-4F (apoA-I mimetic peptide) in inhibiting the inflammatory response induced by ox-LDL in the macrophages. Ac-hE-18A-NH2 may suppress TNF-α secretion and mRNA expression in ox-LDL stimulated RAW264.7 macrophages via IκB-NF-κB signaling pathway. The anti-inflammatory effect of Ac-hE-18A-NH2 is better than that of apoA-I mimic peptide D-4F.

A novel gene regulator, pyrrole–imidazole polyamide targeting ABCA1 gene increases cholesterol efflux from macrophages and plasma HDL concentration

Pyrrole-imidazole (PI) polyamides are nuclease-resistant novel compounds that inhibit transcription factors by binding to the minor groove of DNA. A PI polyamide that targets mouse ABCA1 and increases ABCA1 gene expression was designed and evaluated as an agent to increase plasma HDL concentration. A PI polyamide was designed to bind the activator protein-2 binding site of the mouse ABCA1 promoter. The effect of this PI polyamide on ABCA1 expression was evaluated by real-time RT-PCR and Western blotting using RAW264 cells. In vivo effects of this polyamide on ABCA1 gene expression and plasma HDL level were examined in C57B6 mice. One milligram per kilogram of body weight of PI polyamide was injected via the tail veins every 2 days for 1 week, and plasma lipid profiles were evaluated. PI polyamide showed a specific binding to the target DNA in gel mobility shift assay. Treatment of RAW264 cells with 1.0 μM PI polyamide significantly increased ABCA1 mRNA expression. PI polyamide also significantly increased apolipoprotein AI-mediated HDL biogenesis in RAW264 cells. Cellular cholesterol efflux mediated by apolipoprotein AI was significantly increased by the PI polyamide treatment. PI polyamide significantly increased expression of ABCA1 mRNA in the liver of C57B6 mice. Plasma HDL concentration was increased by PI polyamide administration. All of the HDL sub-fractions showed a tendency to increase after PI polyamide administration. The designed PI polyamide that targeted ABCA1 successfully increased ABCA1 expression and HDL biogenesis. This novel gene-regulating agent is promising as a useful compound to increase plasma HDL concentration. A novel pyrrole-imidazole (PI) polyamide binds to ABCA1. PI polyamide interfered with binding of AP-2ɑ protein to the ABCA1 gene promoter. PI polyamide inhibited the AP-2ɑ-mediated reduction of ABCA1 gene and protein expression. PI polyamide increased ABCA1 protein and apolipoprotein AI mediated HDL biogenesis. PI polyamide is a new gene regulator for the prevention of atherosclerotic diseases.

In vitro steatosis hepatic cell model to compare the lipid-lowering effects of pomegranate peel polyphenols with several other plant polyphenols as well as its related cholesterol efflux mechanisms

This study was aimed to compare the relative activities of the purified pomegranate peels polyphenols (PPPs) with some other plant polyphenols including punicalagin, ellagic acid, gallic acid, phlorizin, and epigallocatechin gallate (EGCG) on the lipid metabolism regulation, and the cholesterol efflux mechanisms of PPPs and punicalagin were also investigated. In this paper, a convenient and accurate in vitro HL7702 steatosis hepatic cell model was applied to evaluate the lipid-lowering effects of the tested polyphenols. The results showed that PPPs possessed the strongest lipid-lowering effects. Prevention group (treated with polyphenols when establishing of steatosis model) was more effective than treatment group (treated with polyphenols after establishment of steatosis model). Punicalagin displayed the strongest lipid-lowering effects among all the tested components of pomegranate peel polyphenols. Moreover, PPPs and punicalagin (10, 20, 40 μg/mL) significantly increased the mRNA expression of LXRα (Liver X receptor alpha) and its target genes-ABCA1 (ATP-binding cassette transporter A1) in a dose-dependent manner in HL7702 steatosis hepatic cells. The high mRNA expression of LXRα and ABCA1, next to lovastatin, was observed in cells treated with 40 μg/mL of PPPs. These in vitro findings suggested that PPPs might have great potential in the clinic treatment of hyperlipemia.

Rosiglitazone enhances apolipoprotein M (Apom) expression in rat's liver.

Apolipoprotein M (APOM) has been suggested as a vasculoprotective constituent of high density lipoprotein (HDL), which plays a crucial role behind the mechanism of HDL-mediated anti-atherosclerosis. Previous studies demonstrated that insulin resistance could associate with decreased APOM expressions. In agreement with our previous reports, here, we further confirmed that the insulin sensitivity was also reduced in rats treated with high concentrations of glucose; such effect could be reversed by administration of rosiglitazone, a peroxisome proliferator-activated receptor-γ (PPARγ). The present study shows that Apom expression is significantly affected by either rosiglitazone or hyperglycemia alone without cross interaction with each other, which indicates that the pathway of Apom expression regulating by hyperglycemia might be differed from that by rosiglitazone. Further study indicated that hyperglycemia could significantly inhibit mRNA levels of Lxrb (P=0.0002), small heterodimer partner 1 (Shp1) (P<0.0001), liver receptor homologue-1 (Lrh1) (P=0.0012), ATP-binding cassette transporter 1 (Abca1) (P=0.0012) and Pparb/d (P=0.0043). Two-way ANOVA analysis demonstrated that the interactions between rosiglitazone and infusion of 25% glucose solution on Shp1 (P=0.0054) and Abca1 (4E, P=0.0004) mRNA expression was statistically significant. It is concluded that rosiglitazone could increase Apom expression, of which the detailed mechanism needs to be further investigated. The downregulation of Apom by hyperglycemia might be mainly through decreasing expression of Pparg and followed by inhibiting Lxrb in rats.

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2−/− mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2−/− mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.

Reduced beta2-glycoprotein I protects macrophages from ox-LDL-induced foam cell formation and cell apoptosis

BackgroundReduced beta2-glycoprotein I (beta2-GPI) is a free thiol-containing form of beta2-GPI that displays a powerful effect in protecting endothelial cells from oxidative stress-induced cell death. The present study aims to investigate the effect of beta2-GPI or reduced beta2-GPI on ox-LDL-induced foam cell formation and on cell apoptosis and to determine the possible mechanisms.MethodsThe RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining and cholesterol measurement were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to detect cell apoptosis. Real-time quantitative PCR was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as CD36, SRB1, ABCA1 and ABCG1. Western blot analysis was used to detect the protein expression of certain apoptosis-related proteins, such as caspase-9, caspase-3, p38 MAPK/p-p38 MAPK and JNK/p-JNK.ResultsBeta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.05;74.44 ± 5.27 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05). Reduced beta2-GPI decreased the ox-LDL-induced expression of CD36 mRNA and ABCA1 mRNA (p < 0.05), as well as CD36, cleaved caspase-9, cleaved caspase-3, p-p38 MAPK and p-JNK proteins (p < 0.05 or p < 0.01). Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein.ConclusionsBoth beta2-GPI and reduced beta2-GPI inhibit ox-LDL-induced foam cell formation and cell apoptosis, and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake of macrophages by downregulating CD36 as well as protein expression. Reduced beta2-GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38 MAPK and JNK, and the amount of cleaved caspase-3 and caspase-9. Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.

GW24-e1881 Effect of polydatin on peritoneal macrophage-derived foam cells of apolipoprotein E gene knockout mice

ObjectivesPolydatin, one of the major active ingredients in Rhizoma Polygonl Cuspidatl, has been recently shown to possess extensive cardiovascular pharmacological activities. However, a direct effect of Polydatin on the uptake...

GW24-e2301 17β-estradiol reduces lipid accumulation in vascular smooth muscle cells through liver X receptor α pathway

ObjectivesSeveral epidemiological and experimental evidences indicate that oestrogen is intimately involved in the pathogenesis of atherosclerosis. The anti-proliferatory, pro-apoptotic, anti-migratory, anti-oxidative, and anti-inflammatory effects of oestrogen on vascular smooth muscle...

Hyperglycemia accelerates ATP‐binding cassette transporter A1 degradation via an ERK‐dependent pathway in macrophages

An elevation in blood glucose concentration leads to increased risk of developing diabetes-associated atherosclerotic cardiovascular disease due to an excessive accumulation of cholesterol in arterial macrophages. ATP-binding cassette transporter A1 (ABCA1) is an atheroprotective protein that mediates the export of cholesterol from macrophages. The present study aims to investigate the effect of hyperglycemia on the regulation of ABCA1 expression and to explore its underlying mechanisms of regulation in macrophages. Our results show that high glucose activates the extracellular signal-regulated kinases (ERK) signaling pathway via reactive oxygen species (ROS) production, which in turn down-regulates ABCA1 mRNA and protein expression. This down-regulation is mediated by accelerating ABCA1 mRNA and protein degradation in macrophages exposed to high concentrations of glucose. Our results provide evidence for the first time that hyperglycemia inhibits ABCA1 expression by ERK-modulated ABCA1 mRNA and protein stability. Overall, these results provide a mechanism for hyperglycemia-induced reduction in ABCA1 expression, which suggests a promising strategy for the treatment of diabetes-associated atherosclerosis.

C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway

BackgroundThe aim of this study was to investigate the effect of C-reactive protein/oxidised low-density lipoprotein/β2-glycoprotein I (CRP/oxLDL/β2GPI) complex on atherosclerosis (AS) in diabetic BALB/c mice.MethodsBALB/c mice were fed high-fat and normal diet. Eight weeks later, the mice fed with high-fat diet were injected with streptozotocin (STZ) to induce diabetes. The diabetic mice were respectively injected twice monthly with 20 μg oxLDL, 20 μg β2GPI, 40 μg oxLDL/β2GPI complex, 44 μg CRP/oxLDL/β2GPI complex, and PBS. Aortas were stained with Sudan IV to investigate lipid plaque formation. The infiltration condition of smooth muscle cells (SMCs), macrophages, and T cells in the aortas were determined by immunohistochemistry (IH). The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR. The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and MKK3/6 in aorta tissues were assessed by Western blot. The expression of inflammation cytokines was evaluated by protein chip.ResultsThe lipid plaques were more extensive, the lumen area was obviously narrower, the ratio of intima and media thickness were increased, and the normal internal elastic lamia structure and endothelial cell disappeared (P < 0.05) in the oxLDL and CRP/oxLDL/β2GPI groups (P < 0.05). CRP/oxLDL/β2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05). The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/β2GPI group (P < 0.05).ConclusionsCRP/oxLDL/β2GPI complex aggravated AS in diabetic BALB/c mice by increasing lipid uptake, the mechanism of which may be mediated by the p38MAPK signal pathway.

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.

DNA topoisomerase II inhibitors induce macrophage ABCA1 expression and cholesterol efflux—An LXR-dependent mechanism

ATP-binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux and thereby inhibits lipid-laden macrophage/foam cell formation and atherosclerosis. ABCA1 expression is transcriptionally regulated by activation of liver X receptor (LXR). Both etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors and are chemotherapeutic medications used in the treatment of various cancers. Interestingly, etoposide inhibits atherosclerosis in rabbits by unclear mechanisms. Herein, we report the effects of etoposide and teniposide on macrophage ABCA1 expression and cholesterol efflux. Both etoposide and teniposide increased macrophage free cholesterol efflux. This increase was associated with increased ABCA1 mRNA and protein expression. Etoposide and teniposide also increased ABCA1 promoter activity in an LXR-dependent manner and formation of the LXRE-LXR/RXR complex indicating that transcriptional induction had occurred. Expression of ABCG1 and fatty acid synthase (FAS), another two LXR-targeted genes, was also induced by etoposide and teniposide. In vivo, administration of mice with either etoposide or teniposide induced macrophage ABCA1 expression and enhanced reverse cholesterol transport from macrophages to feces. Taken together, our study indicates that etoposide and teniposide increase macrophage ABCA1 expression and cholesterol efflux that may be attributed to the anti-atherogenic properties of etoposide. Our study also describes a new function for Topo II inhibitors in addition to their role in anti-tumorigenesis.

Curcumin Induces ABCA1 Expression and Apolipoprotein A-I-Mediated Cholesterol Transmembrane in the Chronic Cerebral Hypoperfusion Aging Rats

Cerebral hypoperfusion or aging often results in the disturbances of cholesterol and lipoprotein, which have been well depicted as a common pathological status contributing to neurodegenerative diseases such as vascular dementia (VaD) and Alzheimer's dementia (AD). The pathway of the liver X receptor-β (LXR-β)/retinoic X receptor-α (RXR-α)/ABCA1 plays a vital role in lipoprotein metabolism. Curcumin, a kind of phenolic compound, has been widely used. It has been reported that curcumin can reduce the levels of cholesterol in serum, but the underlying mechanisms are poorly understood. In this study, we evaluated the effects of curcumin on the cholesterol level in brain, vascular cognitive impairment and explored whether the mechanisms for those effects are through activating LXR-β/RXR-α and ABCA1 expression and apoA-I. With a Morris water test, we found that curcumin treatment could attenuate cognitive impairment. With HE and Nissl staining, we found that curcumin could significantly ameliorate the abnormal changes of pyramidal neurons. Meanwhile, the expression of LXR-β, RXR-α, ABCA1 and apoA-I mRNA and protein were increased in a dose-dependent manner after curcumin treatment. Interestingly, both serum HDL cholesterol and total cholesterol levels were statistically higher in the curcumin treatment group than those other groups. We conclude that curcumin has the ability to activate permissive LXR-β/RXR-α signaling and thereby modulate ABCA1 and apoA-I-mediated cholesterol transmembrane transportation, which is a new preventive and therapeutic strategy for cerevascular diseases.

Aromatase Overexpression Elicits a Paradoxical Cardiac Response to Ischaemia and Reperfusion Challenge

field, p= 0.01). Patient serum elicited increased mRNA expression of ABCA1 (7.1± 5.7 vs 2.0± 0.95 fold increase, p= 0.01) and decrease in SR-BI (0.7± 0.3 vs 1.3± 0.47 fold decrease, p= 0.001) in murine bone marrow-derived macrophages when incubated together overnight. Conclusion: The presence of chronic inflammatory disease in adolescence is associated with evidence of early impairment of the atheroprotective functional properties of HDL. The impact of these changes on the artery wall remains to be determined. http://dx.doi.org/10.1016/j.hlc.2013.05.073

Kaempferol suppresses lipid accumulation in macrophages through the downregulation of cluster of differentiation 36 and the upregulation of scavenger receptor class B type I and ATP-binding cassette transporters A1 and G1

The accumulation of foam cells in atherosclerotic lesions is a hallmark of early-stage atherosclerosis. Kaempferol has been shown to inhibit oxidized low-density lipoprotein (oxLDL) uptake by macrophages; however, the underlying molecular mechanisms are not yet fully investigated. In this study, we shown that treatment with kaempferol markedly suppresses oxLDL-induced macrophage foam cell formation, which occurs due to a decrease in lipid accumulation and an increase in cholesterol efflux from THP-1-derived macrophages. Additionally, the kaempferol treatment of macrophages led to the downregulation of cluster of differentiation 36 (CD36) protein levels, the upregulation of ATP-binding cassette (ABC) transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI) and ABCG1 protein levels, while no effects on scavenger receptor A (SR-A) expression were observed. Kaempferol had similar effects on the mRNA and protein expression of ABCA1, SR-BI, SR-A, CD36 and ABCG1. The reduced CD36 expression following kaempferol treatment involved the inhibition of c-Jun-activator protein-1 (AP-1) nuclear translocation. The inhibition of AP-1 using the inhibitor, SP600125, confirmed this involvement, as the AP-1 inhibition significantly augmented the kaempferol-induced reduction in CD36 expression. Accordingly, the kaempferol-mediated suppression of lipid accumulation in macrophages was also augmented by SP600125. The increased expression of ABCA1, SR-BI and ABCG1 following kaempferol treatment was accompanied by the enhanced protein expression of heme oxygenase-1 (HO-1). This increase was reversed following the knockdown of the HO-1 gene using small hairpin RNA (shRNA). Moreover, the kaempferol-mediated attenuation of lipid accumulation and the promotion of cholesterol efflux was also inhibited by HO-1 shRNA. In conclusion, the c-Jun-AP‑1-dependent downregulation of CD36 and the HO-1-dependent upregulation of ABCG1, SR-BI and ABCA1 may mediate the beneficial effects of kaempferol on foam cell formation.

The Expression of Cholesterol Metabolism Genes in Monocytes From HIV-Infected Subjects Suggests Intracellular Cholesterol Accumulation

Human immunodeficiency virus (HIV) infection is associated with increased cardiovascular risk and reduced high-density lipoprotein cholesterol (HDL-c). In vitro, HIV impairs monocyte-macrophage cholesterol efflux, a major determinant of circulating HDL-c, by increasing ABCA1 degradation, with compensatory upregulation of ABCA1 messenger RNA (mRNA). We examined expression of genes involved in cholesterol uptake, metabolism, and efflux in monocytes from 22 HIV-positive subjects on antiretroviral therapy (ART-Treated), 30 untreated HIV-positive subjects (ART-Naive), and 22 HIV-negative controls (HIV-Neg). HDL-c was lower and expression of ABCA1 mRNA was higher in ART-Naive subjects than in both ART-Treated and HIV-Neg subjects (both P < .01), with HDL-c inversely correlated with HIV RNA (ρ = -0.52; P < .01). Expression of genes involved in cholesterol uptake (LDLR, CD36), synthesis (HMGCR), and regulation (SREBP2, LXRA) was significantly lower in both ART-Treated and ART-Naive subjects than in HIV-Neg controls. In vivo, increased monocyte ABCA1 expression in untreated HIV-infected patients and normalization of ABCA1 expression with virological suppression by ART supports direct HIV-induced impairment of cholesterol efflux previously demonstrated in vitro. However, decreased expression of cholesterol sensing, uptake, and synthesis genes in both untreated and treated HIV infection suggests that both HIV and ART affect monocyte cholesterol metabolism in a pattern consistent with accumulation of intramonocyte cholesterol.

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.

Abstract 9110: Elevated Glucose Increases ATP-Binding Cassette Transporter A1 mRNA Expression in Human Leukocytes and Murine Bone Marrow-Derived Macrophages and Decreases Cholesterol Efflux

Background: Individuals with diabetes are at increased CVD risk. Limited evidence suggests that this is partially attributed to hyperglycemic suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux. The relationship between glucose concentrations and expression of ABC-transporters (ABCA1 and ABCG1) was assessed in human monocytes and leukocytes (study 1) and murine bone marrow-derived macrophages (BMDM) as well as cholesterol efflux in BMDM (study 2). Methods: Ten healthy subjects (4F/6M, 50-85 yrs, BMI 25-35 kg/m²) were recruited for study 1. At baseline, 1- and 2-hour post oral glucose challenge (50 g glucose), RNA was extracted from blood leukocytes (Paxgene RNA kit) and from monocytes isolated by magnetic cell sorting to assess ABC-transporter mRNA expression. For study 2, bone marrow cells were isolated from C57BL/6 mice and differentiated to BMDM with macrophage-colony stimulating factor. BMDM were stimulated with a 20mmol/L challenge of D- or L-glucose and/or 5mmol/L D-glucose (control), 16 hrs, followed by exposure to oxLDL (25 ug/mL, 24 hrs). Triglyceride (TG) and cholesteryl ester (CE) accumulation, ABC-transporter mRNA and protein expression, as well as HDL-mediated cholesterol efflux were evaluated. Results: A glucose challenge caused a 35% rise in human leukocyte ABCA1 mRNA expression after 1- (p=0.003) and 2- (p=0.020) hrs, but no significant effect on leukocyte ABCG1, or monocyte ABCA1 or ABCG1. Elevated glucose concentrations in BMDM culture media led to a 120% increase in ABCA1 mRNA (p=0.001) but no significant effects on expression of ABCG1 transcripts, ABCA1 or ABCG1 protein, or on intracellular TG or CE accumulation. Elevated glucose suppressed HDL-mediated cholesterol efflux by 10% (p=0.005). Conclusion: These results support the concept that hyperglycemia attenuates HDL-mediated cholesterol efflux from macrophages, potentially contributing to diabetes-associated CVD. Moreover, in the absence of altered ABC-transporter protein expression, our data suggest that hyperglycemia impairs HDL-mediated cholesterol efflux perhaps by attenuating transporter activity. Correspondingly, we found human monocyte ABC-transporter gene expression unresponsive to a glucose challenge.