Effects of Oxidized Low Density Lipoprotein on the Expression and Function of ABCA1 in Macrophages

In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65 % cholesterol loaded by acetyl LDL (ac-LDL), and 9.78% cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with .100 μg/mL ac-LDL or with 100 μg/mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.     

Inhibiting NF-κB increases cholesterol efflux from THP-1 derived-foam cells treated with AngII via up-regulating the expression of ATP-binding cassette transporter A1

Objective: To study the role of nuclear factor-kappa B(NF-κB) in cholesterol efflux from THP-1 derived-foam cells treated with AngiotensinⅡ(AngⅡ). Methods:Cultured THP-1 derived-foam cells were treated with AngⅡ or preincubated with tosyl-phenylalanine chloromethyl-ketone(TPCK) NF-κB inhibitor. The levels of activated NF-κB in the cells were examined by sandwich ELISA. Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol efflux was detected by scintillation counting technique. ABCA1 mRNA and protein were quantified by RT-PCR and Western blotting. Results:Addition of TPCK to the cells before AngⅡ stimulation attenuated the response of NF-κB p65 nuclear translocation induced by AngⅡ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol efflux by 24.1%(P < 0.05) and 41.1%(P < 0.05) respectively, when compared with AngⅡgroup. In accordance, the ABCA1 mRNA and protein were increased by 30% and 19%(P < 0.05) respectively, when compared with AngⅡ group. Conclusion:AngⅡ can down-regulate ABCA1 in THP-1 derived-foam cells via NF-κB, which leads to less cholesterol efflux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.     

High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

Objective:ATP-binding cassette transporters(ABC)A1 and G1 play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation.In this study,we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells(VSMCs),the other precursor of foam cells.Methods:Incubation of human VSMCs with D-glucose(5 to 30 mM)for 1 to 7 days in the presence or absence of antioxidant and nuclear factor(NF)-κB inhibitors,the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting.Results:High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs,whereas the expression of ABCA1 was not significantly decreased.Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine(NAC)and NF-κB inhibitors,BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone(TPCK).Conclusion:High glucose suppresses the expression of ABCG1 in VSMCs,which is the possible mechanism of VSMC derived foam cell transformation.     

Inhibiting NF-κB increases cholesterol efflux from THP-1 derived-foam cells treated with AngⅡ via up-regulating the expression of ATP-binding cassette transporter A1

Objective: To study the role of nuclear factor-kappa B(NF-κB) in cholesterol efflux from THP-1 derived-foam cells treated with AngiotensinⅡ(AngⅡ). Methods:Cultured THP-1 derived-foam cells were treated with AngⅡ or preincubated with tosyl-phenylalanine chloromethyl-ketone(TPCK) NF-κB inhibitor. The levels of activated NF-κB in the cells were examined by sandwich ELISA. Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol efflux was detected by scintillation counting technique. ABCA1 mRNA and protein were quantified by RT-PCR and Western blotting. Results:Addition of TPCK to the cells before AngⅡ stimulation attenuated the response of NF-κB p65 nuclear translocation induced by AngⅡ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol efflux by 24.1%(P < 0.05) and 41.1%(P < 0.05) respectively,when compared with AngⅡgroup. In accordance,the ABCA1 mRNA and protein were increased by 30% and 19%(P < 0.05) respectively,when compared with AngⅡ group. Conclusion:AngⅡ can down-regulate ABCA1 in THP-1 derived-foam cells via NF-κB,which leads to less cholesterol efflux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.     

EFFECTS OF RAPAMYCIN ON INTRACELLULAR CHOLESTEROL HOMEOSTASIS OF GLOMERULAR MESANGIAL CELL IN THE PRESENCE OF INTERLEUKIN-1β

Objective To investigate the effects of rapamycin on cholesterol homeostasis of glomerular mesangial cells and the underlying mechanisms. Methods Intracellular cholesterol accumulation was measured by Oil Red O staining and high performance liquid chromatography. The effects of rapamycin on interleukin-1β (IL-1β)-induced mRNA and protein changes of low-density lipoprotein receptor (LDLR) and ATP-binding cassette transporter A1 (ABCA1) were assayed by quantitative real-time PCR and Western blot. Transient expressions of 3 types of mammalian target of rapamycin (mTOR), including mTOR-WT (wild type), mTOR-RR (rapamycin resistant, with kinase activity), and mTOR-RR-KD (rapamycin resistant, without kinase activity), were obtained by plasmid transfection. Results Rapamycin had no significant influence on intracellular cholesterol concentration under normal condition, but it significantly decreased the intracellular cholesterol concentration in the presence of IL-1β. Rapamycin dose-dependently suppressed the increased expression of LDLR induced by IL-1β and up-regulated the suppressed expression of ABCA1 caused by IL-1β. Transient expression of 3 types of mTOR all reduced ABCA1 mRNA expression significantly, which all could be overroded by rapamycin. Conclusions Rapamycin may contribute to the maintaining of glomerular mesangial cell intracellular cholesterol homeostasis under inflammatory state by both reducing cholesterol uptake and increasing cholesterol efflux. And the effect may be not completely mediated by mTOR.     

Thymic Stromal Lmphopoietin Pomotes Macrophage-derived Foam Cell Formation简

The effect of thymic stromal lymphopoietin（TSLP） on macrophage-derived foam cell formation and the underlying mechanism were studied. Macrophages isolated from C57BL/6 mice were co-cultured in vitro with different concentrations of TSLP or TSLPR-antibody in the presence of oxidized low density lipoprotein（ox-LDL）. The effects of TSLP on macrophage-derived foam cell formation were observed by using oil red O staining and intracellular lipid determination. The expression levels of foam cell scavenger receptors（CD36 and SRA） as well as ABCA1 and TSLPR were detected by using RT-PCR and Western blotting. As compared with the control group, TSLP treatment significantly promoted lipid accumulation in macrophages, significantly increased protein expression of CD36 and TSLPR in a dose-dependent manner, and significantly reduced the expression of ABCA1 protein in a dose-dependent manner. No significant differences were noted between the TSLPR-antibody group and the control group. TSLP may down-regulate the expression of cholesterol efflux receptor ABCA1 and up-regulate scavenger receptor expression via the TSLPR signaling pathway, thereby promoting macrophage-derived foam cell formation.     

Mitofusin2 decreases intracellular cholesterol of oxidized LDL-induced foam cells from rat vascular smooth muscle cells

Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (P<0.05), and the expression levels significantly increased when the titer of Adv-Mfn2 increased (P<0.05). At 24 or 48 h after oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P<0.05), but it was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05), and it also significantly decreased when the titer of Adv-Mfn2 increased (P<0.05). The mRNA and protein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). Though the mRNA and protein expression levels of PPARγ was not significantly increased (P>0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.     

A possible mechanism linking hyperglycemia and reduced high-density lipoprotein cholesterol levels in diabetes

This study investigated the role of glucose in the biogenesis of high-density lipoprotein cholesterol(HDL-C).Mouse primary peritoneal macrophages were harvested and maintained in Dulbecco's modified Eagle's medium(DMEM) containing glucose of various concentrations.The cells were divided into 3 groups in terms of different glucose concentrations in the cultures:Control group(5.6 mmol/L glucose),high glucose concentration groups(16.7 mmol/L and 30 mmol/L glucose).ATP-binding cassette transporter A1(ABCA1) mRN...     

Study on the relationship between serum high density lipoprotein and Pi-deficiency syndrome in patients with coronary heart disease

Objective:To explore the relationship of Pi(脾)-def iciency syndrome in TCM with the change of serum high density lipoprotein(HDL) in blood lipid metabolic disorder.Methods:Sixty-eight patients with conf irmed coronary heart disease(CHD) were selected for TCM syndrome typing into Pi-defi ciency(PD) group and non-Pi-def iciency(NPD) group.Routine blood lipids and serum lipoprotein electrophoretogram(SLPG) were determined in all patients to analyze the total content of HDL and its relative contents of sub-components HDL1-5,as well as their relation with PD syndrome.Besides,a healthy control group(62 cases) was set up.Results:The level of serum HDL-C was lowered,SLPG abnormality rate increased in the patients with CHD,with total HDL and the relative contents of sub-component HDL1 and HDL3 signifi cantly lower than those in the healthy control group(P<0.01).The total HDL,HDL1 and HDL3 in the PD group were also lower than those in the NPD group(P<0.05,P<0.01).Conclusion:Serum HDL and its sub-components showed a definite relation with TCM PD syndrome type,therefore,further exploring the granular specificity of HDL and its sub-components as well as their influence on reverse cholesterol transport(RCT) may hopefully provide clues for developing RCT regulatory Chinese new drugs and for CHD prevention and treatment.     

Dyslipidemia promotes the progression of chronic kidney disease?

Dyslipidemia,including hypercholesterolemia,hypertriglyceridemia,and low high-density lipoprotein (HDL) cholesterinemia,is a key risk factor to atherosclerosis.The detrimental effect of elevated low-density lipoprotein (LDL) cholesterol and/or decreased HDL cholesterol on cardiovascular disease risk had been well established from previous studies.1 Recently,emerging evidences suggest that dyslipidemia may also be an important contributor to morbidity and mortality of chronic kidney disease (CKD),which has received more and more attention as the prevalence of CKD increases. In our study published in this issue,2 data were collected among 4779 middle-aged and elderly Chinese population.We found that participants with hypercholesterolemia had greater prevalence of albuminuria and reduced estimated glomerular filtration rate (eGFR).     

普罗布考对动脉粥样硬化兔HDL功能的影响及其调控机制

目的观察普罗布考对动脉粥样硬化兔巨噬细胞、肝细胞HDL功能的影响,并进一步探讨普罗布考抗动脉粥样硬化的机制。方法18只新西兰大白兔随机分为:(1)正常对照组(n=6):给予普通饮食喂养;(2)动脉粥样硬化组(n=6):饲以高脂饲料;(3)普罗布考组(n=6):在饲以高脂饮食基础上给予普罗布考[400mg/(kg·d)]。12周后取血以酶法测定血脂;利用流式细胞仪检测腹腔巨噬细胞、肝细胞细胞表面ABCA1、SR-BⅠ表达量;液闪计数仪检测其[3H]胆固醇转出率。结果实验12周后,普罗布考组血清总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、非高密度脂蛋白(NHDL-C)水平明显下降。普罗布考上调动脉粥样硬化兔腹腔巨噬细胞及肝细胞ABCA1及SR-BⅠ蛋白质表达水平。普罗布考组腹腔巨噬细胞、肝细胞[3H]胆固醇转出率较动脉粥样硬化组明显上升。结论虽然普罗布考降低HDL-C,但是其通过上调腹腔巨噬细胞、肝细胞SR-BⅠ及ABCA1的表达及增加其[3H]胆固醇转出率,改善HDL功能,具有促进动脉粥样硬化兔胆固醇逆转运(RCT)作用。     

高密度脂蛋白对氧化型低密度脂蛋白刺激下脂肪细胞TNF-α表达的影响

目的观察高密度脂蛋白(high density lipoprotein,HDL)对氧化型低密度脂蛋白(oxidized low density lipoprotein,oxLDL)刺激下3T3-L1脂肪细胞肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)mRNA表达的影响,并探讨其可能的作用机制。方法 3T3-L1脂肪细胞促分化成熟后,oxLDL刺激脂肪细胞,给予不同浓度的HDL(10～100μg/ml),及H-89(10μmol/L)+HDL(100μg/ml)干预,收集细胞,测定脂肪细胞TNF-αmRNA表达水平,IκB蛋白浓度及核因子-κB(NF-κB)活性。结果 OxLDL刺激使3T3-L1脂肪细胞TNF-αmRNA表达及NF-κB活性明显增强。HDL浓度依赖性抑制TNF-αmRNA表达、NF-κB活化和IκB降解。与oxLDL刺激组比较,100μg/ml HDL使TNF-αmRNA表达降低64.5%,NF-κB活性减少49%,并明显增加IκB蛋白水平。HDL的这些抗炎效应能被蛋白激酶A(PKA)抑制剂H-89部分抑制。结论HDL能抑制oxLDL诱导的3T3-L1脂肪细胞TNF-αmRNA表达,PKA-IκB-NF-κB信号通路是其中作用途径之一,该效应不需要HDL与oxLDL的直接接触作用。     

辛伐他汀对THP-1巨噬细胞胆固醇外流及ABCA1、CD36表达影响

目的研究不同剂量辛伐他汀对氧化型低密度脂蛋白(ox-LDL)诱导的THP-1巨噬细胞胆固醇外流及ATP结合盒受体A1(ABCA1)、CD36表达的影响。方法建立ox-LDL诱导泡沫细胞模型,THP-1巨噬细胞体外培养,用辛伐他汀进行干预,用[3H]标记胆固醇,液体闪烁计数法检测胆固醇外流量,油红O染色观察细胞泡沫化程度,Western Blot、RT-PCR检测细胞内ABCA1及CD36蛋白和mRNA表达。结果辛伐他汀组较ox-LDL诱导组胆固醇外流明显增加,ABCA1受体蛋白及mRNA表达明显增加(P<0.05),同时CD36受体蛋白及mRNA表达明显下调(P<0.05)呈剂量依赖性。结论辛伐他汀可促进ox-LDL诱导的泡沫细胞胆固醇外流,抑制巨噬细胞泡沫化,该作用可能与辛伐他汀上调ABCA1及抑制CD36表达有关。     

磷脂对HDL3介导大鼠皮肤成纤维细胞内胆固醇流出能力的影响

目的　研讨磷脂对HDL3 介导大鼠皮肤成纤维细胞内胆固醇流出能力的影响。方法　在卵磷脂 (PC)或鞘磷脂 (SPM )存在条件下 ,观察HDL3 介导细胞胆固醇流出量的变化、细胞内磷脂含量变化及游离胆固醇 /胆固醇酯平衡的变化。结果　①BSA组 (对照组 )、HDL3 组、PC组、SPM组、PC HDL3 组和SPM HDL3 组分别介导 4.70 %、31.5 5 %、7.35 %、8.0 6 %、42 .95 %和 46 .98%细胞胆固醇流出 ;②BSA、HDL3 、HDL3 SPM和HDL3 PC组细胞培育后胞内卵磷脂磷含量 (PC p)和鞘磷脂磷含量 (SPM p)分别为 2 0 .0 2、5 .5 6 ,17.5 6、5 .2 8,18.6 2、7.0 0和 2 2 .5 0、5 .5 2 μg/皿 ;③PC和SPM与细胞培育后 ,胞内游离胆固醇 /总胆固醇比值分别为 49.6 5和 5 9.5 7。培育前此比值为 48.6 4。结论　①PC和SPM本身无介导细胞胆固醇流出能力 ,但它们能显著提高HDL3 介导的细胞胆固醇流出能力 ,且后者强于前者 ;②随着细胞胆固醇流出 ,部分胞内PC也流出胞外 ,而胞内SPM无明显变化 ;③SPM促进细胞内胆固醇酯向游离胆固醇转化。     

载脂蛋白A-Ⅰ模拟肽对RAW264.7巨噬细胞胆固醇流出的影响

目的：观察载脂蛋白A-Ⅰ（apoA-Ⅰ）模拟肽D-4F对RAW264.7巨噬细胞胆固醇流出的影响,并探讨其机制。方法：巨噬细胞种植于24孔板,用1.85×10^7Bq/孔3H-胆固醇和含50μg/mL氧化型低密度脂蛋白（ox-LDL）共同孵育24h后,给予不同浓度的D-4F（0～100μg/mL）干预24h,收集细胞用液体闪烁计数法检测胆固醇流出。采用酶联免疫吸附试验（ELISA）测定细胞内环磷酸腺苷（cAMP）含量,采用实时荧光定量PCR及Western印迹检测三磷酸腺苷结合盒转运体A1（ABCA1）的mRNA及蛋白表达。结果：D-4F呈浓度依赖及时间依赖性促进巨噬细胞内胆固醇流出,增加细胞内cAMP水平,上调ABCA1mRNA和蛋白表达。8-Br-cAMP显著增加D-4F介导的胆固醇流出和ABCA1表达,而蛋白激酶A（PKA）抑制剂虽然对基础胆固醇流出及ABCA1表达几乎无作用,却可以抑制8-Br-cAMP对胆固醇流出和ABCA1表达的促进作用。结论：D-4F促进巨噬细胞胆固醇流出,cAMP释放及ABCA1表达,可能与激活cAMP-PKA-ABCA1通路有关。     

ABCA1在胆固醇逆转运中作用的研究进展

ATP结合盒转运蛋白A1（ATP-binding cassette transporter A1,ABCA1）是一种以ATP为能源进行物质转运的膜蛋白,胆固醇、磷脂等脂类物质是ABCA1的主要转运底物,胆固醇负荷、cAMP、PPAR等信号在调节ABCA1表达中有重要影响。ABCA1在胆固醇逆转运（reverse cholesterol transport,RCT）过程中与载脂蛋白结合参与高密度脂蛋白（high density lipoprotein,HDL）的形成,是RCT中的第一步也是关键的一步,也是该领域中研究的热点。     

Plasma oxidized high-density lipoprotein and glycated apolipoprotein A-I concentrations in ST-segment elevation myocardial infarction patients with stress hyperglycaemia or high thrombus burden

Abstract

Background: High-density lipoprotein (HDL) particles exert many beneficial actions that may help protect against cardiovascular disease. However, recent work has demonstrated that HDL can be oxidized and glycated under certain circumstances and may become pro-atherogenic. The present study investigated the impact of oxidized high-density lipoprotein (ox-HDL) and glycated apolipoprotein A-I (gly-ApoA-I) in patients presenting with ST-elevation myocardial infarction (STEMI).

Methods: We assessed 55 consecutive patients with STEMI. Patients were divided into: (1) a stress hyperglycaemia (SH) and a no SH group; and (2) a high thrombus burden (HTB) group and a low thrombus burden (LTB) group. Meanwhile, 48 healthy volunteers were recruited as controls. Plasma ox-HDL and gly-ApoA-I concentrations were measured on admission and 7?days after admission.

Results: Higher concentrations of ox-HDL and gly-ApoA-I were found in the STEMI group than in the control group on admission and at d7. Further subgroup analysis showed that ox-HDL and gly-ApoA-I were higher in the SH group than in the no SH group at both time points; the HTB group had higher ox-HDL and ox-HDL/HDL-C levels than the LTB group on admission and at d7. However, gly-ApoA-I and the relative intensity of ApoA-I glycation showed no significant differences between the HTB and LTB groups.

Conclusions: The present data indicate that: (1) SH is associated with increased plasma concentrations of ox-HDL and gly-ApoA-I and therefore aggressive treatment is recommended; and (2) that ox-HDL and ox-HDL/HDL-C were higher in the HTB group and may be used to quantify thrombus burden.     

轻度认知功能障碍患者血脂代谢的变化

目的研究轻度认知功能障碍(MCI)患者血脂代谢的变化。方法测定并比较60例MCI患者和100例年龄相当的正常对照者(对照组)血清总胆固醇(TC)、甘油三脂(TG)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)水平。结果MCI患者TC、TG和LDL水平明显高于对照组,HDL水平明显低于对照组,差异有统计学意义(P<0.05)。结论MCI患者存在明显的脂质代谢紊乱,TC、TG和LDL水平的升高或HDL的降低可能对发病有一定的影响。