Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1 macrophages

INTRODUCTIONOxidized lipid signaling in macrophages is centralto the pathogenesis of atherosclerosis[1]. Exposure ofmacrophages and other vascular cells to oxidized low-density lipoprotein (ox-LDL) leads to complex changesin gene expression that are collectively thought to influ-ence the development of the atherosclerotic lesion[2].Using two-dimensional gel electrophoresis, the overallprotein map in U937 control cells and U937 foam cellswas obtained. Compared with U937 cells, 37 spots…     

Angiotensin‐(1–7) treatment ameliorates angiotensin II‐induced apoptosis of human umbilical vein endothelial cells

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Rolipram对THP-1巨噬细胞源性泡沫细胞胆固醇流出和ABCA1表达的影响

目的　以THP 1巨噬细胞源性泡沫细胞为研究对象 ,探讨磷酸二酯酶抑制剂rolipram对THP 1巨噬细胞源性泡沫细胞ABCA1表达及细胞内胆固醇流出的影响。方法　用液体闪烁计数器检测细胞内胆固醇流出 ,运用逆转录 -多聚酶链反应和Westernblot印迹分别检测ABCA1mRNA与ABCA1蛋白的表达 ,采用低pH值EIA法测定细胞内cAMP水平。结果　实验显示PDE4抑制剂rolipram能引起THP 1巨噬细胞源性泡沫细胞cAMP水平、ABCA1表达和apoA 1介导的胆固醇流出成平衡增加 ,而细胞内总胆固醇、游离胆固醇与胆固醇酯明显减少。结论　PDE4抑制剂rolipram增加THP 1巨噬细胞源性泡沫细胞ABCA1表达及细胞内胆固醇流出 ,这为防治动脉粥样硬化发生发展提供一个新的策略     

Protective arms of the renin–angiotensin‐system in neurological disease

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载脂蛋白嵌合模拟肽对巨噬细胞胆固醇流出的影响

目的观察载脂蛋白嵌合模拟肽Ac-hE-18A-NH2对RAW264.7巨噬细胞胆固醇流出的影响,并探讨其机制。方法 RAW264.7巨噬细胞种植于24孔板,用0.5μCi/孔3H-胆固醇和含50mg·mL-1氧化低密度脂蛋白共同孵育24h之后,给予不同浓度的Ac-hE-18A-NH2(0～100mg·mL-1)干预24h,收集细胞用液体闪烁计数法检测胆固醇流出。采用ELISA测定细胞内cAMP含量,采用实时荧光定量PCR及Westernblot检测ABCA1、LXRα和PPARγ的mRNA及蛋白表达。结果 Ac-hE-18A-NH2以浓度依赖方式介导胆固醇流出;50mg·mL-1Ac-hE-18A-NH2干预不同时间,其介导的胆固醇流出率分别为(10.86±1.46)%(6h),(13.43±1.55)%(12h),(20.58±1.34)%(18h),和(26.93±4.37)%(24h)。同时,Ac-hE-18A-NH2还以浓度依赖方式增加细胞内cAMP水平,上调ABCA1、LXRα和PPARγmR-NA和蛋白表达。加用cAMP刺激剂8-Br-cAMP,Ac-hE-18A-NH2介导的胆固醇流出率由26.93±4.37增加至35.81±2.73,ABCA1mRNA表达增加了66.67%。而加用PPARγ特异性抑制剂预处理细胞后,PPARγ的表达几乎完全抑制,ABCA1和LXRα的表达也受到一定程度抑制,Ac-hE-18A-NH2介导的胆固醇流出率明显减少。结论模拟肽Ac-hE-18A-NH2可以明显促进巨噬细胞胆固醇流出,其机制可能与cAMP-ABCA1和PPARγ-LXRα-ABCA1两种途径有关。     

Angiotensin‐(1‐7)‐mediated Mas1 receptor/NF‐κB‐p65 signaling is involved in a cigarette smoke‐induced chronic obstructive pulmonary disease mouse model

Angiotensin‐(1‐7) [Ang‐(1‐7)] has been shown to play a significant role in the pathogenesis of lung inflammation via Mas receptor; however, its effect in chronic obstructive pulmonary disease (COPD) remains unknown. To explore the effect of Ang‐(1‐7) on a cigarette smoke (CS) exposure‐induced COPD model, 40 C57BL/6J mice were divided into four groups (n = 10) and exposed to air or CS for 8 weeks. After that, they were treated with saline or Ang‐(1‐7) at 0.3 mg/kg for 2 weeks by subcutaneous infusion using osmotic pump. The day following drug/vehicle challenge, lung function was examined and bronchoalveolar lavage (BAL) was performed. Chemokine (C–X–C motif) ligand 1, interleukin‐6, and tumor necrosis factor‐α protein levels in BAL fluid were determined using ELISA; the corresponding mRNA levels in lung tissues were measured using RT‐PCR. Mas1 receptor, pIκBα, IκBα, nuclear NF‐κB‐p65 protein, pERK1/2, ERK2, pp38, and p38 proteins expression in lung tissues were examined by immunohistochemical staining and western blotting. Ang‐(1‐7) challenge had no effect on the decreased lung function and emphysema induced by CS exposure. However, Ang‐(1‐7) treatment blocked CS exposure‐induced lung inflammatory responses and lung fibrosis, as determined by Masson's Trichrome staining. Exposure to CS for 8 weeks caused irreversible loss of lung function and emphysema, which could not be reversed by Ang‐(1‐7) treatment. Thus, the beneficial effect of Ang‐(1‐7) may be confined to pulmonary inflammation and fibrosis.     

ATP-binding cassette transporters A1 and G1, HDL metabolism, cholesterol efflux, and inflammation: important targets for the treatment of atherosclerosis

Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries. Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. One central antiatherogenic role of HDL is believed to be its ability to remove excessive peripheral cholesterol back to the liver for subsequent catabolism and excretion, a physiologic process termed reverse cholesterol transport (RCT). Cholesterol efflux from macrophage foam cells, the initial step of RCT is the most relevant step with respect to atherosclerosis. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play crucial roles in the efflux of cellular cholesterol to HDL and its apolipoproteins. Moreover, ABCA1 and ABCG1 affect cellular inflammatory cytokine secretion by modulating cholesterol content in the plasma membrane and within intracellular compartments. In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excessive cholesterol in macrophages, and physiological manipulation of ABCA1 expression affects atherogenesis. Here we review recent advances in the role of ABCA1 and ABCG1 in HDL metabolism, macrophage cholesterol efflux, inflammation, and atherogenesis. Next, we summarize the structure, expression, and regulation of ABCA1 and ABCG1. Finally, we give an update on the progress and pitfalls of therapeutic approaches that target ABCA1 and ABCG1 to stimulate the flux of lipids through the RCT pathway.     

Tributyltin chloride induces ABCA1 expression and apolipoprotein A-I-mediated cellular cholesterol efflux by activating LXRalpha/RXR

Organotins, including tri-butyltin chloride (TBTC), are widely used in agricultural and chemical industries and cause persistent and widespread pollution. TBTC has been shown to activate nuclear receptor retinoid X receptor (RXR)/PPARγ signaling by interacting with RXR to modulate adipogenesis. However, whether TBTC affects liver X receptor (LXR)/RXR activity and subsequently the expression of cholesterol mobilizing genes is not known. In this study, we evaluated the ability of TBTC to activate LXR/RXR and ABC transporter A1 (ABCA1) expression. ABCA1 plays a critical role in HDL generation, maintaining cholesterol homeostasis, and cholesterol accumulation-induced diseases, such as atherosclerosis and pancreatic islet dysfunction. In a reporter gene assay, TBTC activated LXRα/RXR but not LXRβ/RXR. In mouse macrophage RAW264 cells, TBTC activated the ABCA1 promoter in an LXR-responsive element dependent manner and increased ABCA1 mRNA expression. TBTC augmented ABCA1 protein levels and apolipoprotein A-I-dependent cellular cholesterol efflux (HDL generation). The LXR-target fatty acid synthase and Spα mRNA levels were also increased by TBTC exposure. We conclude that TBTC has the ability to activate permissive LXRα/RXR signaling and thereby modulate cellular cholesterol efflux.     

ABC家族转运体与糖尿病

糖尿病是一种常见的全身性慢性代谢疾病,其发病过程伴随着血糖、血脂和激素水平的失调,并由此引发高血脂、动脉粥样硬化、心肌病等并发症.ABCA,ABCB,ABCC,ABCG等ABC家族转运体有可能参与糖尿病及其并发症发病机制.如ABCA1和ABCG1促使细胞内的胆固醇泵出,进入高密度脂蛋白通路,减少巨噬细胞中的胆固醇沉积,防止巨噬细胞转化为泡沫细胞;ABCG5和ABCG8抑制肠道膳食中脂类吸收,并促进胆固醇通过胆汁排泄;表达在胰岛β细胞上的ABCC8作为钾通道的调节亚基控制胰岛素的释放,维持血糖平衡;ABCB1,ABCC1-ABCC5以及ABCC2是药物转运相关的外排蛋白,影响着其底物药物的体内过程,也参与体内内源性物质的转运.上述ABC家族转运体缺失或变异有可能诱发糖尿病及其并发症;而糖尿病状态下体内环境的变化也会改变这些ABC家族转运体表达与功能,进一步加剧了病情的发展.本文讨论了糖尿病及与之相关的四类ABC家族转运体,以期深入理解两者之间的相互作用,为糖尿病研究和治疗提供新思路.     

The effect of T0901317 on ATP-binding cassette transporter A1 and Niemann-Pick type C1 in apoE-/- mice

Although a range of studies indicated Liver X receptor (LXR) activation inhibited the development of atherosclerosis in animal models, the mechanism of this effect for LXR agonists has not been fully understood. A recent study has suggested LXR activators increased the amount of free cholesterol in the plasma membrane of human macrophages by inducing Niemann-Pick type C1 (NPC1) gene expression. Therefore, we hypothesize that LXRs may also promote NPC1 expression in vivo. Here we investigated the effect of a synthetic LXR agonist T0901317 on ATP-binding cassette transporter A1 (ABCA1) and NPC1 in apolipoprotein E knockout (apoE-/-) mice. Male apoE-/- mice were randomized into four groups: baseline group (n = 10), vehicle group (n = 14), prevention group (n = 14), and treatment group (n = 14). En face analysis and Oil red O staining were used to examine the aortic atherosclerotic lesions. Macrophage content of aortic root atherosclerotic lesions and cholesterol efflux form peritoneal macrophages were measured. Gene and protein expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting, respectively. T0901317 treatment reduced aortic atherosclerotic lesion area by 64.2% in prevention group (P < 0.001) and 58.3% in treatment group (P < 0.001) and resulted in a reduction in macrophage content. Plasma triglyceride, total cholesterol, high-density lipoprotein cholesterol, and apoA-I concentrations were markedly increased in T0901317-treated groups. T0901317 also promoted ABCA1 and NPC1 gene and protein levels in the aorta, liver, and small intestine of apoE-/- mice and significantly increased cholesterol efflux from peritoneal macrophages. T0901317 upregulates ABCA1 and NPC1. This study gives us a new insight into the mechanism for antiatherogenic effect of LXR synthetic agonists.     

Rate‐limiting factors of cholesterol efflux in reverse cholesterol transport: Acceptors and donors

1. Plasma levels of high‐density lipoprotein (HDL) are believed to be inversely related to coronary artery disease. High‐density lipoprotein plays a key role in the process of reverse cholesterol transport, by which HDL is able to extract excess cholesterol from peripheral tissues and transfer it to the liver for biliary excretion. 2. Efflux of lipids (cholesterol and phospholipids) is the first step in reverse cholesterol transport. Several cellular membrane transporters, including ABCA1 and ABCG1, as well as scavenger receptor (SR)‐BI receptor, are believed to facilitate the active efflux of cholesterol to lipid‐poor apolipoprotein A‐I and mature HDL, respectively. Furthermore, overexpression or deletion of one or more specific genes supports the view that HDL is involved in cholesterol efflux. 3. In conclusion, current evidence supports a critical role for HDL in atheroprotection via an active efflux pathway through reverse cholesterol transport, with the substantial support of appropriate functions of cell donors.     

Tributyltin chloride induces ABCA1 expression and apolipoprotein A-I-mediated cellular cholesterol efflux by activating LXRα/RXR

Organotins, including tri-butyltin chloride (TBTC), are widely used in agricultural and chemical industries and cause persistent and widespread pollution. TBTC has been shown to activate nuclear receptor retinoid X receptor (RXR)/PPARγ signaling by interacting with RXR to modulate adipogenesis. However, whether TBTC affects liver X receptor (LXR)/RXR activity and subsequently the expression of cholesterol mobilizing genes is not known. In this study, we evaluated the ability of TBTC to activate LXR/RXR and ABC transporter A1 (ABCA1) expression. ABCA1 plays a critical role in HDL generation, maintaining cholesterol homeostasis, and cholesterol accumulation-induced diseases, such as atherosclerosis and pancreatic islet dysfunction. In a reporter gene assay, TBTC activated LXRα/RXR but not LXRβ/RXR. In mouse macrophage RAW264 cells, TBTC activated the ABCA1 promoter in an LXR-responsive element dependent manner and increased ABCA1 mRNA expression. TBTC augmented ABCA1 protein levels and apolipoprotein A-I-dependent cellular cholesterol efflux (HDL generation). The LXR-target fatty acid synthase and Spα mRNA levels were also increased by TBTC exposure. We conclude that TBTC has the ability to activate permissive LXRα/RXR signaling and thereby modulate cellular cholesterol efflux.     

Ibrolipim increases ABCA1/G1 expression by the LXRα signaling pathway in THP-1 macrophage-derived foam cells

Aim:

To determine the effects and potential mechanisms of ibrolipim on ATP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1) expression from human macrophage foam cells, which may play a critical role in atherogenesis.

Methods:

Human THP-1 cells pre-incubated with ox-LDL served as foam cell models. Specific mRNA was quantified using real-time RT-PCR and protein expression using Western blotting. Cellular cholesterol handling was studied using cholesterol efflux experiments and high performance liquid chromatography assays.

Results:

Ibrolipim 5 and 50 μmol/L significantly increased cholesterol efflux from THP-1 macrophage-derived foam cells to apoA-I or HDL. Moreover, it upregulated the expression of ABCA1 and ABCG1. In addition, LXRα was also upregulated by the ibrolipim treatment. In addition, LXRα small interfering RNA completely abolished the promotion effect that was induced by ibrolipim.

Conclusion:

Ibrolipim increased ABCA1 and ABCG1 expression and promoted cholesterol efflux, which was mediated by the LXRα signaling pathway.     

Combination of virtual screening and high throughput gene profiling for identification of novel liver X receptor modulators

We conducted virtual docking studies using GLIDE with modified LXRbeta ligand-binding domain (LBD) on internal compound collection followed by the gene profiling with ArrayPlate mRNA assay. A total of 69 compounds were found to upregulate LXRalpha and certain LXR regulated genes from 1308 compounds selected by virtual screen (hit rate: 5.3%). Compound 4 was shown to significantly induce the expression of LXR target genes such as ABCA1, ABCG1, APOE, SCD-1, and SREBP-1c in THP-1 differentiated macrophages. In vitro binding assay confirmed that 4 binds to both LXRalpha and LXRbeta directly and recruits coactivator peptide SRC-1. It functions as a full LXR agonist in stimulating cholesterol efflux in THP-1 differentiated macrophages and induces lipogenesis in HepG2 cells. This study demonstrates that the combination of virtual screen and high throughput gene profiling is an efficient approach for rapid identification of novel LXR modulators.     

Functional expressions of adenosine triphosphate‐binding cassette transporters during the development of zebrafish embryos and their effects on the detoxification of cadmium chloride and β‐naphthoflavone

Adenosine triphosphate‐binding cassette (ABC) transporters, including ABCB, ABCC and ABCG families represent general biological defenses against environmental toxicants in varieties of marine and freshwater organisms, but their physiological functions at differential developmental stages of zebrafish embryos remain undefined. In this work, functional expressions of typical ABC transporters including P‐glycoprotein (Pgp), multiresistance associated protein 1 (Mrp1) and Mrp2 were studied in zebrafish embryos at 4, 24, 48 and 72 h post‐fertilization (hpf). As a result, both the gene expressions and activities of Pgp and Mrps increased with the development of embryos. Correspondingly, 4–72 hpf embryos exhibited an increased tolerance to the toxicity caused by cadmium chloride (CdCl₂) and β‐naphthoflavone (BNF) with time. Such a correlation was assumed caused by the involvement of ABC transporters in the detoxification of chemicals. In addition, the assumption was supported by the fact that model efflux inhibitors of Pgp and Mrps such as reversine 205 and MK571 significantly inhibited the efflux of toxicants and increased the toxicity of Cd and BNF in zebrafish embryos. Moreover, exposure to CdCl₂ and BNF induced the gene expressions of Pgp and Mrp1 in 72 hpf embryos. Thus, functional expressions of Pgp and Mrps increased with the development of zebrafish embryos, which could cause an increasing tolerance of zebrafish embryos to CdCl₂ and BNF. Copyright © 2015 John Wiley & Sons, Ltd.