ABCA1 Levels Research Articles

Ursolic acid enhances macrophage autophagy and attenuates atherogenesis

Macrophage autophagy has been shown to be protective against atherosclerosis. We previously discovered that ursolic acid (UA) promoted cancer cell autophagy. In the present study, we aimed to examine whether UA enhances macrophage autophagy in the context of atherogenesis. Cell culture study showed that UA enhanced autophagy of macrophages by increasing the expression of Atg5 and Atg16l1, which led to altered macrophage function. UA reduced pro-interleukin (IL)-1β protein levels and mature IL-1β secretion in macrophages in response to lipopolysaccharide (LPS), without reducing IL-1β mRNA expression. Confocal microscopy showed that in LPS-treated macrophages, UA increased LC3 protein levels and LC3 appeared to colocalize with IL-1β. In cholesterol-loaded macrophages, UA increased cholesterol efflux to apoAI, although it did not alter mRNA or protein levels of ABCA1 and ABCG1. Electron microscopy showed that UA induced lipophagy in acetylated LDL-loaded macrophages, which may result in increased cholesterol ester hydrolysis in autophagolysosomes and presentation of free cholesterol to the cell membrane. In LDLR(-/-) mice fed a Western diet to induce atherogenesis, UA treatment significantly reduced atherosclerotic lesion size, accompanied by increased macrophage autophagy. In conclusion, the data suggest that UA promotes macrophage autophagy and, thereby, suppresses IL-1β secretion, promotes cholesterol efflux, and attenuates atherosclerosis in mice.

β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells.

Atherosclerosis is an inflammatory disease that leads to an aberrant accumulation of cholesterol in vessel walls forming atherosclerotic plaques. During this process, the mechanism regulating complex cellular cholesterol pools defined as the reverse cholesterol transport (RCT) is altered as well as expression and functionality of transporters involved in this process, namely ABCA1, ABCG1, and SR-BI. Macrophages, arterial endothelial and smooth muscle cells (SMCs) have been involved in the atherosclerotic plaque formation. As macrophages are widely described as the major cell type forming the foam cells by accumulating intracellular cholesterol, RCT alterations have been poorly studied at the arterial endothelial cell and SMC levels. Amongst the therapeutics tested to actively counteract cellular cholesterol accumulation, the methylated β-cyclodextrin, KLEPTOSE® CRYSMEβ, has recently shown promising effects on decreasing the atherosclerotic plaque size in atherosclerotic mouse models. Therefore we investigated in vitro the RCT process occurring in SMCs and in arterial endothelial cells (ABAE) as well as the ability of some modified β-CDs with different methylation degree to modify RCT in these cells. To this aim, cells were incubated in the presence of different methylated β-CDs, including KLEPTOSE® CRYSMEβ. Both cell types were shown to express basal levels of ABCA1 and SR-BI whereas ABCG1 was solely found in ABAE. Upon CD treatments, the percentage of membrane-extracted cholesterol correlated to the methylation degree of the CDs independently of the lipid composition of the cell membranes. Decreasing the cellular cholesterol content with CDs led to reduce the expression levels of ABCA1 and ABCG1. In addition, the cholesterol efflux to ApoA-I and HDL particles was significantly decreased suggesting that cells forming the blood vessel wall are able to counteract the CD-induced loss of cholesterol. Taken together, our observations suggest that methylated β-CDs can significantly reduce the cellular cholesterol content of cells forming atherosclerotic lesions and can subsequently modulate the expression of ABC transporters involved in RCT. The use of methylated β-CDs would represent a valuable and efficient tool to interfere with atherosclerosis pathogenesis in patients, nonetheless their mode of action still needs further investigations to be fully understood and finely controlled at the cellular level.

Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages.

Leoligin is a natural lignan found in Edelweiss (Leontopodium nivale ssp. alpinum). The aim of this study was to examine its influence on cholesterol efflux and to address the underlying mechanism of action. Leoligin increases apo A1- as well as 1% human plasma-mediated cholesterol efflux in THP-1 macrophages without affecting cell viability as determined by resazurin conversion. Western blot analysis revealed that the protein levels of the cholesterol efflux transporters ABCA1 and ABCG1 were upregulated, whereas the SR-B1 protein level remained unchanged upon treatment with leoligin (10 μM, 24 h). Quantitative reverse transcription PCR further uncovered that leoligin also increased ABCA1 and ABCG1 mRNA levels without affecting the half-life of the two mRNAs in the presence of actinomycin D, a transcription inhibitor. Proteome analysis revealed the modulation of protein expression fingerprint in the presence of leoligin. Taken together, these results suggest that leoligin induces cholesterol efflux in THP-1-derived macrophages by upregulating ABCA1 and ABCG1 expression. This novel activity suggests leoligin as a promising candidate for further studies addressing a possible preventive or therapeutic application in the context of atherosclerosis.

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

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

Abstract 634: Silencing miR144 Enhances Regression and Attenutates Progression of Atherosclerosis

Cholesterol imbalance plays a central role in multiple diseases, particularly cardiovascular disease, the leading cause of death in the United States. Lipid-loaded macrophage foam cells are a critical determining factor in the pathogenesis of atherosclerosis. MicroRNAs are small non-coding RNA molecules that regulate both cholesterol and lipoprotein metabolism, and may significantly contribute to disease progression. We previously identified miR-144 as a novel regulator of lipid metabolism that is also regulated by the nuclear receptor farnesoid X receptor (FXR). We identified the cholesterol transporter ABCA1 as a miR-144 target gene, and showed that increased hepatic expression of miR-144 decreased ABCA1 protein and plasma cholesterol levels. Consequently, we hypothesized that silencing miR-144 may have therapeutic potential because it may increase the levels of atheroprotective HDL particles and enhance reverse cholesterol transport (RCT) due to increased ABCA1 levels in macrophages. Here we present en face analysis of atherosclerosis showing that silencing miR-144 both enhanced regression and attenuated progression of atherosclerosis. In addition, we demonstrate that silencing miR-144 enhances RCT, the process by which macrophage cholesterol is returned to the liver for excretion. Together, these data suggest that this pathway may represent a bona-fide strategy to treat atherosclerosis.

Effects of TanshinoneIIA on the modulation of miR‑33a and the SREBP‑2/Pcsk9 signaling pathway in hyperlipidemic rats.

Tanshinone IIA is the active compound isolated from Salvia miltiorrhiza bunge, which is a traditional Chinese medicine known as Danshen. The aim of the present study was to assess the effect of Tanshinone IIA on the regulation of lipid metabolism in the livers of hyperlipidemic rats and the underlying molecular events. An in vivo model of hyperlipidemia was established in rats, with the animals receiving a daily dose of Tanshinone IIA. The serum lipid profiles were analyzed using an automatic biochemical analyzer, and the histopathological alterations and lipid deposition in liver tissue were assessed using hematoxylin and eosin staining, and oil red O staining, respectively. The mRNA expression levels of microRNA (miR)-33a, ATP-binding cassette transporter (ABC)A1, ABCG1, sterol regulatory element-binding protein 2 (SREBP-2), proprotein convertase subtilisin/kexin type 9 (Pcsk9) and low-density lipoprotein receptor (LDL-R) in liver tissues were measured using reverse transcription-quantitative polymerase chain reaction, and the protein expression levels of ABCA1, ABCG1, SREBP-2, Pcsk9, and LDL-R were analyzed using western blotting. Tanshinone IIA reduced lipid deposition and improved histopathology in the rat liver tissue, however, did not alter the lipid profile in rat serum. In addition, Tanshinone IIA treatment suppressed the expression of miR-33a, whereas the protein expression levels of ABCA1, SREBP-2, Pcsk9 in addition to LDL-R mRNA and protein were upregulated. In conclusion, the present study indicated that Tanshinone IIA attenuated lipid deposition in the livers of hyperlipidemic rats and modulated the expression of miR-33a and SREBP-2/Pcsk9 signaling pathway proteins.

Persimmon tannin regulates the expression of genes critical for cholesterol absorption and cholesterol efflux by LXRα independent pathway

Persimmon tannin (proanthocyanidins) has been proven to exert significant hypocholesterolaemic effects in a variety of models. However, the regulatory mechanisms of persimmon tannin on cholesterol metabolism have yet to be fully understood. The present study investigated the regulation of persimmon tannin on the expression of genes critical for cholesterol absorption and cholesterol efflux. Our results indicated that persimmon tannin reduced the uptake of 22-NBD-cholesterol and inhibited the accumulation of cholesterol significantly in HepG2 and Caco-2 cell lines. The mRNA and proteins were quantified using real time PCR and western blot, respectively. The expression of SREBP-2 gene, critical for cholesterol synthesis, and NPC1L1 involved in cholesterol absorption were significantly inhibited by persimmon tannin. Moreover, persimmon tannin showed potential in promoting cholesterol efflux through up-regulating mRNA and/or protein levels of ABCA1, ABCG1, SR-BI, CYP7A1 and ABCG5/G8. However, the regulation effects of persimmon tannin on cholesterol homeostasis in both cell lines were LXRα independent.

Difference in expression patterns of placental cholesterol transporters, ABCA1 and SR-BI, in Meishan and Yorkshire pigs with different placental efficiency.

Cholesterol is a key cell membrane component and precursor of steroid hormones. The maternal cholesterol is an important exogenous cholesterol source for the developing embryos and its transportation is mediated by ABCA1 and SR-BI. Here we reported that during the peri-implantation period in pigs, ABCA1 was expressed by uterine luminal epithelium (LE) and interestingly, its expression was more abundantly in LE on mesometrial side of uterus. However, SR-BI was expressed primarily by LE, glandular epithelial cells (GE) and trophoblast cells (Tr). During the placentation period, the expression levels of ABCA1 and SR-BI proteins at epithelial bilayer and placental areolae were significantly higher in Chinese Meishan pigs compared to Yorkshire pigs. Consisitently, mRNA levels of HMGCR, the rate-limiting enzyme for cholesterol synthesis, were significantly higher in Meishan placentas than in Yorkshire placentas. Our findings revealed the routes of transplacental cholesterol transport mediated by ABCA1 and SR-BI in pigs and indicated that ABCA1 related pathway may participate in anchoring the conceptus to the mesometrial side of uterus. Additionally, an ABCA1 dependent compensatory mechanism related to the placental efficiency in response to the smaller placenta size in Meishan pigs was suggested.

High Uric Acid Activates the ROS-AMPK Pathway, Impairs CD68 Expression and Inhibits OxLDL-Induced Foam-Cell Formation in a Human Monocytic Cell Line, THP-1

Background/Aims: Hyperuricemia is part of the metabolic-syndrome cluster of abdominal obesity, impaired glucose tolerance, insulin resistance, dyslipidemia, and hypertension. Monocytes/macrophages are critical in the development of metabolic syndrome, including gout, obesity and atherosclerosis. However, how high uric acid (HUA) exposure affects monocyte/macrophage function remains unclear. In this study, we investigated the molecular mechanism of HUA exposure in monocytes/macrophages and its impact on oxidized low-density lipoprotein (oxLDL)-induced foam-cell formation in a human monocytic cell line, THP-1. Methods: We primed THP-1 cells with phorbol-12-myristate-13-acetate (PMA) for differentiation, then exposed cells to HUA and detected the production of reactive oxygen species (ROS) and analyzed the level of phospho-AMPKα. THP-1 cells were pre-incubated with Compound C, an AMPK inhibitor, or N-acetyl-L-cysteine (NAC), a ROS scavenger, or HUA before PMA, to assess CD68 expression and phospho-AMPKα level. PMA-primed THP-1 cells were pre-treated with oxLDL before Compound C and HUA treatment. Western blot analysis was used to examine the levels of phospho-AMPKα, CD68, ABCG1, ABCA1, cyclooxygenase-2 (COX-2) and NF-κB (p65). Flow cytometry was used to assess ROS production and CD68 expression in live cells. Oil-red O staining was used to observe oxLDL uptake in cells. Results: HUA treatment increased ROS production in PMA-primed THP-1 cells; NAC blocked HUA-induced oxidative stress. HUA treatment time-dependently increased phospho-AMPKα level in PMA-primed THP-1 cells. The HUA-induced oxidative stress increased phospho-AMPKα levels, which was blocked by NAC. HUA treatment impaired CD68 expression during cell differentiation by activating the AMPK pathway, which was reversed by Compound C treatment. Finally, HUA treatment inhibited oxLDL uptake in the formation of foam cells in THP-1 cells, which was blocked by Compound C treatment. HUA treatment significantly increased the expression of ABCG1 and reversed the oxLDL-reduced ABCG1 expression but did not affect the expression of ABCA1, NF-κB (p65) or COX-2. Conclusions: HUA exposure activated the ROS-AMPK pathway, impaired CD68 expression, and inhibited oxLDL-induced foam-cell formation in a human monocytic cell line, THP-1.

Enhanced cytotoxicity of nitidine against camptothecin-resistant A549 cells

Aim Drug resistance has been a major issue in cancer chemotherapy. It is therefore important to screen more potent natural anti-cancer agents for use against the drug-resistant cancer phenotype. The present study investigated the cytotoxicity of the traditional and natural medicinal agent nitidine (NTD) against the drug-resistant cancer phenotype of A549 human lung adenocarcinoma cells. Methods The camptothecin (CPT) tolerant phenotype was selected after A549 cells were exposed to CPT for 1 week. The cytotoxicity of NTD in these CPT-resistant cells (CRC) was studied in vitro in relation to its accumulation and expression of the ATP binding cassette (ABC) transporter gene. Results CRC expressing higher levels of ABCC1, ABCC2, ABCC3, and ABCA1 were more resistant to CPT compared with normal A549 cells. CRC were, however, more susceptible to NTD compared with normal A549 cells with preferential accumulation of NTD. A statistically significant difference in protein level between normal A549 and CRC was noted only for ABCC1 and ABCC2. Furthermore, gene expression of ABCC2 and ABCC3 had a positive correlation with NTD accumulation, and negative correlation with the effective dose that inhibited 50% of cell growth (D50) of this agent in nine cancer cell panels. Conclusion CRC were found to be more susceptible to NTD compared with the normal phenotype. The cytotoxicity of NTD was associated with its accumulation and the expression of ABCC2, suggesting the involvement of this transporter in the selective incorporation of NTD into cancer cells, resulting in enhanced cytotoxicity.

Abstract 19546: Silencing miR-144 Enhances Regression and Reduces Progression of Atheroscleoris

Enhancing cholesterol removal from macrophages in atherosclerotic plaques remains a major unmet pharmacological need. MicroRNA therapeutics are one approach that is currently being evaluated. MicroRNAs fine tune gene expression, and those that target ABCA1, the major cholesterol efflux transporter, are candidates for therapeutic approaches. Silencing microRNAs that target ABCA1 result in increased ABCA1 levels and activity and therefore enhance lipid removal from lesion macrophages. We previously showed that miR-144 targets ABCA1 by binding to at least 2 binding sites in the 3’ UTR region. We have now tested whether silencing miR-144, which increases ABCA1, affects atherosclerosis in two models. First, LDLR-/- mice were fed a western-type diet for 16 weeks, before being switched to regular rodent chow to promote lesion regression. One group of mice were sacrificed after 16 weeks of western diet (baseline), and the remaining animals were randomly assigned to one of 3 groups; vehicle (PBS), control anti-miR, or anti-miR-144 for 4 weeks. Silencing miR-144 significantly decreased atherosclerosis by en face analysis by 20% compared to either control. Further, silencing miR-144 modestly increased plasma HDL levels by 14%, in line with previous studies. To determine whether silencing miR-144 enhanced macrophage cholesterol efflux in mice, we used the same experimental approach. However, 2 days prior to the sacrifice, mice were injected with macrophages that had been loaded with radiolabelled cholesterol. Silencing miR-144 in vivo enhanced macrophage cholesterol efflux to plasma, liver and feces, suggesting the mechanism for the reduced atherosclerosis is due to increased cholesterol removal from plaques. Finally, we also determined whether silencing miR-144 decreased progression of atherosclerosis. LDLR-/- mice were fed a western-style diet for 16 weeks, and, at the same time, mice were treated mice with either anti miR-144, control anti miR or vehicle (PBS). Silencing miR-144 decreased atherosclerosis progression by 33% relative to controls, while HDL levels were also modestly increased (19%). Our results indicate that silencing miR-144 is a potential therapeutic strategy to reduce atherosclerosis, in both regression and progression models.

RAGE Suppresses ABCG1-Mediated Macrophage Cholesterol Efflux in Diabetes.

Diabetes exacerbates cardiovascular disease, at least in part through suppression of macrophage cholesterol efflux and levels of the cholesterol transporters ATP binding cassette transporter A1 (ABCA1) and ABCG1. The receptor for advanced glycation end products (RAGE) is highly expressed in human and murine diabetic atherosclerotic plaques, particularly in macrophages. We tested the hypothesis that RAGE suppresses macrophage cholesterol efflux and probed the mechanisms by which RAGE downregulates ABCA1 and ABCG1. Macrophage cholesterol efflux to apolipoprotein A1 and HDL and reverse cholesterol transport to plasma, liver, and feces were reduced in diabetic macrophages through RAGE. In vitro, RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through peroxisome proliferator–activated receptor-γ (PPARG)–responsive promoter elements but not through liver X receptor elements. Plasma levels of HDL were reduced in diabetic mice in a RAGE-dependent manner. Laser capture microdissected CD68+ macrophages from atherosclerotic plaques of Ldlr−/− mice devoid of Ager (RAGE) displayed higher levels of Abca1, Abcg1, and Pparg mRNA transcripts versus Ager-expressing Ldlr−/− mice independently of glycemia or plasma levels of total cholesterol and triglycerides. Antagonism of RAGE may fill an important therapeutic gap in the treatment of diabetic macrovascular complications.

Abstract 532: The Receptor for Advanced Glycation End Products (RAGE) Suppresses Macrophage Cholesterol Transport in Diabetes

Diabetes is a leading cause of cardiovascular morbidity and mortality. In human subjects, diabetes suppresses macrophage cholesterol efflux capacity, at least in part via reduction in levels of the major cholesterol transporters, ABCA1 and ABCG1. We tested the hypothesis that the receptor for advanced glycation end products (RAGE) contributes to these processes. We report that ligand-RAGE interaction suppresses cholesterol efflux to ApoA1 and HDL in primary murine bone marrow-derived macrophages (BMDMs) and in human THP-1 cells, at least in part via downregulation of ABCA1 and ABCG1. In vivo, compared to cholesterol-loaded wild-type (WT) diabetic murine BMDMs, diabetic BMDMs devoid of Ager (gene encoding RAGE) displayed significantly higher reverse cholesterol transport (RCT) to plasma, liver and feces when injected into WT non-diabetic mice. In atherosclerotic plaques from mice devoid of both Ldlr and Ager and fed a high cholesterol diet, higher Abca1 and Abcg1 mRNA transcript levels were also observed compared to Ager-expressing LDL receptor null mice. RAGE ligand AGEs suppress ABCG1 promoter luciferase activity and transcription of ABCG1 through reduced binding of PPARgamma (PPARG) to PPARG-responsive elements in the ABCG1 promoter. These data reveal that RAGE contributes to dysregulation of macrophage cholesterol metabolism, particularly in diabetes. We infer that antagonism of RAGE may mitigate accelerated atherosclerosis in the diabetic state, at least in part due to modulation of impaired cholesterol transport.

Abstract 449: ApoA-I Suppresses Neointimal Hyperplasia Following Stent Deployment and Modulates Neointimal Cellular Phenotype

Background: Increases in smooth muscle cell (SMC) cholesterol content reduce the expression of SMC phenotypic markers and increase macrophage related markers. The balance of neointimal SMCs to macrophage-like cells may influence neointimal hyperplasia following stent deployment. Apolipoprotein (apo) A-I, the main protein component of HDL, mediates the efflux of cholesterol from cells via the cholesterol transporter ABCA1. Through cholesterol loading of SMCs in vitro and in murine models of balloon angioplasty and stenting, we aimed to investigate the effect of apoA-I on SMC cellular phenotype and neointimal hyperplasia. Methods and Results: SMCs loaded with cholesterol (Cholesterol:MβCD complex) showed a reduction in SMC α-actin mRNA. Incubation with apoA-I restored SMC α-actin mRNA to control levels. Thoracic aortic segments from apoE-/- mice underwent balloon angioplasty or stenting then were carotid-interposition grafted into recipient mice. Mice received three PBS or apoA-I (40mg/kg) injections prior to surgery and then on alternate days until sacrifice (28 days). There were no changes in total cholesterol, LDL and HDL between groups. Thrombosis rates were 16% lower in apoA-I injected mice. Histological analysis revealed that stented aortic sections from apoA-I injected mice had smaller neointimal areas (18%±6), compared to control mice p&lt;0.05. SMC α-actin staining was strikingly higher in the neointimas of apoA-I stented arteries (76%±34, p&lt;0.05), while RT-PCR analysis of balloon angioplasty injured aortas found mRNA levels of macrophage marker CD68 were lower in apoA-I infused mice (30%±8, p&lt;0.01). Furthermore, mRNA levels of ABCA1 and the cholesterol efflux regulator PPAR-γ were significantly higher in apoA-I aortic segments (30%±13 and 48%±20, respectively), p&lt;0.05. Conclusion: ApoA-I infusions reduce neointimal area following stent deployment. Neointimas of apoA-I infused mice had higher SMC α-actin and lower macrophage CD68 expression. Mechanistically, this may be via increased SMC cholesterol efflux as apoA-I increased the expression of ABCA1 and PPAR-γ in balloon-injured arteries. These findings have significant implications for therapeutic modulation of neointimal hyperplasia following stent deployment.

MiR-146a-5p Antagonized AGEs- and P.g-LPS-Induced ABCA1 and ABCG1 Dysregulation in Macrophages via IRAK-1 Downregulation.

The miR-146-mediated IL-1 receptor-associated kinase 1 (IRAK-1) feedback circuit has been shown to inhibit inflammatory response in macrophages against lipopolysaccharide (LPS). The aim of this study is to compare the antagonized effects of miR-146a-5p on Porphyromonas gingivalis (P.g) lipolysaccharide (LPS)- and advanced glycation end products (AGEs)-triggered ABCA1 and ABCG1 dysregulation and explore the underlying mechanism. THP-1-derived macrophages transfected with miRNA mimics or not were treated with P.g LPS or AGE-BSA, respectively. The mechanism of endotoxin tolerance was mimicked. miR-146a-5p levels and protein levels of IRAK-1, LXRα/β, ABCA1, and ABCG1 were detected by stem-loopreversetranscriptionfollowed by TaqMan PCR analysis and Western blotting. Our results showed that miR-146a-5p levels were significantly increased in macrophages after 24h of stimulation with high dose of P.g LPS or AGE-BSA. Macrophages transfected with miR-146a-5p mimics attenuated the dysregulation of ABCA1/G1 induced by P.g LPS and AGEs through IRAK-1 downregulation. In low-dose LPS-tolerized cells, elevated miR-146a-5p antagonized the increase of ABCA1, ABCG1, and IRAK-1. However, low-dose AGE-BSA did not increase miR-146a-5p levels. In conclusion, the model of endotoxin tolerance is suitable for the antagonistic effects on the dysregulation of ABCA1/G1 induced by high dose of P.g LPS. Conversely, low-dose AGEs did not induce the model of P.g LPS-mediated tolerance.

Methyl protodioscin increases ABCA1 expression and cholesterol efflux while inhibiting gene expressions for synthesis of cholesterol and triglycerides by suppressing SREBP transcription and microRNA 33a/b levels

Sterol regulatory element-binding proteins (SREBPs) regulate homeostasis of LDL, HDL and triglycerides. This study was aimed to determine if inhibition of SREBPs by methyl protodioscin (MPD) regulates downstream gene and protein expressions of lipid metabolisms. In THP-1 macrophages, MPD increases levels of ABCA1 mRNA and protein in dose- and time-dependent manners, and apoA-1-mediated cholesterol efflux. The underlying mechanisms for the effects is that MPD inhibits the transcription of SREBP1c and SREBP2, and decreases levels of microRNA 33a/b hosted in the introns of SREBPs, which leads to reciprocally increase ABCA1 levels. In HepG2 cells, MPD shows the same effects as these observed in THP-1 macrophages. MPD also decreases the gene expressions of HMGCR, FAS and ACC for cholesterol and fatty acid synthesis. MPD further promotes LDL receptor through reducing the PCSK9 level. Collectively, the study demonstrates that MPD potentially increase HDL cholesterol while reducing LDL cholesterol and triglycerides.

Activation of liver X receptor protects inner retinal damage induced by N-methyl-D-aspartate.

To investigate whether activation of liver X receptors (LXRs) protects N-methyl-D-aspartic (NMDA)-induced retinal neurotoxicity in mice and to explore the underlying mechanism. Inner retinal damage was induced by intravitreal injection of NMDA. A synthetic LXR ligand TO901317 (TO90, 50 mg/kg/d) or vehicle was intragastrically administrated from 3 days before to 1 day or 7 days after NMDA injection. The severity of retinal damage was evaluated with histological analysis and TUNEL staining, and retinal functions were evaluated by ERG. The expressions of caspase-3, bax, bcl-2, TNF-α, and BACE1, the rate-limiting enzyme in the formation of amyloid β (Aβ), in the retina were examined by real-time PCR and ELISA. The levels of LXRs, NF-κB subunit p65, p-p38 mitogen-activated protein kinase (MAPK), and an LXR target gene ABCA1 were detected with real-time PCR and Western blotting. The localization and protein expression of Aβ in the retina was assessed by immunohistochemistry and Western blotting. The NMDA enhanced the expression of LXRβ but not LXRα and ABCA1 in mouse retina. Nevertheless, administration of TO90 after NMDA injection not only enhanced the expression of LXRβ but also upregulated the level of ABCA1, suggesting retinal LXRs were activated in a ligand-dependent manner. The LXRα expression was unchanged in the vehicle and the TO90-treated groups. Activation of LXRβ with TO90 inhibited cell death in the ganglion cell layer (GCL) and inner nuclear layer (INL), preserved ERG b- and a-wave amplitudes, and the b/a ratio in the NMDA-treated mice. Meanwhile, TO90 suppressed the elevation of apoptosis factors caspase-3 and bax induced by NMDA and upregulated the level of an antiapoptotic factor bcl-2. The TO90 also inhibited the increase of p-p38 MAPK and proinflammatory cytokine TNF-α after NMDA injection. Furthermore, activation of LXR attenuated the activation of NF-κB, and reduced gene expression of BACE1 and accumulation of Aβ induced by NMDA. Activation of LXRβ with a synthetic LXR ligand TO90 protects the inner retinal damage induced by NMDA in mice. We speculate the protective effect is associated with inhibition of the NF-κB signaling pathway and reduction of Aβ formation in retina. The LXR agonists may become a new class of neuroprotective agent for retinal diseases associated with glutamate-induced excitotoxicity.

Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients.

BackgroundThe dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.ResultsWe first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).ConclusionsABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0036-2) contains supplementary material, which is available to authorized users.

Bone marrow angiotensin AT2 receptor deficiency aggravates atherosclerosis development by eliminating macrophage liver X receptor-mediated anti-atherogenic actions.

Bone marrow (BM) Angiotensin II (Ang II) type 1 (AT1) receptor plays a crucial role in atherosclerosis development; however, the effect of BM Ang II type 2 (AT2) receptor on atherogenesis remains undefined. We generated BM chimera apoE-deficient (apoE(-/-)) mice whose BM cells were repopulated with AT2-deficient (Agtr2(-/-)) or wild-type (Agtr2(+/+)) cells. After 2 months of a high-cholesterol diet, the atherosclerotic lesion area was significantly increased in the apoE(-/-)/BM-Agtr2(-/-) mice compared with the apoE(-/-)/BM-Agtr2(+/+) mice (51%, P < 0.05), accompanied by an augmented accumulation of lesion macrophages. Although phenotypic polarization in BM-derived macrophages and lipopolysaccharide-induced expression of proinflammatory cytokines in thioglycollate-induced peritoneal macrophages (TGPMs) were not affected by AT2-deficiency, mRNA and protein expression levels of macrophage liver X receptor β (LXRβ) were significantly decreased in Agtr2(-/-) TGPMs compared with Agtr2(+/+) TGPMs. Anti-inflammatory effects of LXR agonist (GW3965) were markedly inhibited in Agtr2(-/-) TGPMs. Furthermore, the expression levels of ATP-binding cassette transporter ABCA1 and CCR7 were much lower in Agtr2(-/-) TGPMs than Agtr2(+/+) TGPMs, accompanied by a significantly reduced cholesterol efflux. Our findings demonstrate that BM-AT2 deficiency aggravates atherosclerosis, at least in part, by eliminating the anti-atherogenic properties of macrophages elicited by LXRβ activation.

Abstract 18363: ApoA-I Suppresses Neointimal Hyperplasia Following Stent Deployment and Modulates Neointimal Cellular Phenotype

Background: Increases in smooth muscle cell (SMC) cholesterol content reduce the expression of SMC phenotypic markers and increase macrophage related markers. The balance of neointimal SMCs to macrophage-like cells may influence neointimal hyperplasia following stent deployment. Apolipoprotein (apo) A-I, the main protein component of high-density lipoproteins (HDL), mediates the efflux of cholesterol from cells via the cholesterol transporter ABCA1. In a murine model of balloon angioplasty and stenting, we aimed to investigate the effect of apoA-I on neointimal hyperplasia and neointimal cellular phenotype. Methods and Results: Thoracic aortic segments from apoE knockout mice underwent balloon angioplasty alone or balloon angioplasty with stenting then were carotid-interposition grafted into recipient mice. Mice received PBS or apoA-I (40mg/kg) injections on alternate days in the week prior to surgery and then until sacrifice (28 days). There were no changes in total cholesterol, LDL and HDL between groups at sacrifice and thrombosis rates were 16.7% lower in apoA-I injected mice. Histological analysis revealed that stented aortic sections from mice injected with apoA-I had significantly smaller neointimal areas (18%±6.9), compared to PBS control mice p&lt;0.05. The percentage of SMC alpha-actin positive staining was strikingly higher in the neointimas of apoA-I stented arteries (76%±34.3, p&lt;0.05), while RT-PCR analysis of balloon angioplasty injured aortas found mRNA levels of macrophage marker CD68 were lower in apoA-I infused mice (30%±8.9, p&lt;0.01). Furthermore, mRNA levels of ABCA1 and the cholesterol efflux regulator PPAR-gamma were significantly higher in apoA-I balloon-injured aortic segments (30%±13.8 and 48%±20.7, respectively), p&lt;0.05. Conclusion: ApoA-I infusions reduce neointimal area following stent deployment. In apoA-I infused mice, the cells of the neointima had significantly higher SMC alpha-actin expression and lower macrophage CD68 expression. Mechanistically, this may be due to increased SMC cholesterol efflux as apoA-I increased the expression of ABCA1 and PPAR-gamma. These findings have significant implications for therapeutic modulation of neointimal hyperplasia following stent deployment.