MicroRNA-27a/b regulates cellular cholesterol efflux,influx and esterification/hydrolysis in THP-1 macrophages

Rationale

Macrophage cholesterol homeostasis maintenance is the result of a balance between influx, endogenous synthesis, esterification/hydrolysis and efflux. Excessive accumulation of cholesterol leads to foam cell formation, which is the major pathology of atherosclerosis. Previous studies have shown that miR-27 (miR-27a and miR-27b) may play a key role in the progression of atherosclerosis.

Objective

We set out to investigate the molecular mechanisms of miR-27a/b in intracellular cholesterol homeostasis.

Methods and results

In the present study, our results have shown that the miR-27 family is highly conserved during evolution, present in mammals and directly targets the 3′ UTR of ABCA1, LPL, and ACAT1. apoA1, ABCG1 and SR-B1 lacking miR-27 bind sites should not be influenced by miR-27 directly. miR-27a and miR-27b directly regulated the expression of endogenous ABCA1 in different cells. Treatment with miR-27a and miR-27b mimics reduced apoA1-mediated cholesterol efflux by 33.08% and 44.61% in THP-1 cells, respectively. miR-27a/b also regulated HDL-mediated cholesterol efflux in THP-1 macrophages and affected the expression of apoA1 in HepG2 cells. However, miR-27a/b had no effect on total cellular cholesterol accumulation, but regulated the levels of cellular free cholesterol and cholesterol ester. We further found that miR-27a/b regulated the expression of LPL and CD36, and then affected the ability of THP-1 macrophages to uptake Dil-oxLDL. Finally, we identified that miR-27a/b regulated cholesterol ester formation by targeting ACAT1 in THP-1 macrophages.

Conclusion

These findings indicate that miR-27a/b affects the efflux, influx, esterification and hydrolysis of cellular cholesterol by regulating the expression of ABCA1, apoA1, LPL, CD36 and ACAT1.     

Probucol enhances cholesterol efflux from cultured human skin fibroblasts

To explore whether the reported xanthoma-reducing effects of probucol are related to tissue-specific mechanisms of the drug, the influence of probucol on cholesterol efflux from cultured human skin fibroblasts was investigated. Incubation of cells with probucol led to a 2-fold enhancement of high-density lipoprotein-mediated cholesterol efflux. This result raises the possibility that probucol may reduce cholesterol accumulation in tissues through a cholesterol-mobilizing action.     

Impaired mobilisation of cholesterol from stored cholesteryl esters in human (THP-1) macrophages

The formation of macrophage-derived foam cells is central to the development of fatty streaks within the arterial wall, and to the progression of atherosclerosis. The unregulated deposition of cholesteryl esters, as lipid droplets within the cytoplasm of these cells, is responsible for the formation of foam cells; this process is thought to be regulated by the balance between cholesterol esterification, by acyl CoA:cholesterol acyltransferase (ACAT), and hydrolysis, by neutral cholesteryl ester hydrolase (nCEH). This study examines the importance of the balance between these two enzymes in determining the efflux of cholesterol from human (THP-1) macrophages. The presence of modified lipoprotein, or of 25-hydroxycholesterol, markedly increased cholesterol esterification in these cells and these effects were potently inhibited by the presence of the ACAT inhibitor, 447C88. In the absence of HDL, an acceptor particle, there was little or no hydrolysis of the cholesteryl ester pool and no efflux of cholesterol to the extracellular milieu; addition of HDL led to a partial (36%) reduction in cholesteryl esters, an effect which was not enhanced by the inhibition of ACAT. This suggested that the stored cholesteryl esters in human (THP-1) macrophages, unlike those in mouse peritoneal macrophages, were relatively resistant to removal by efflux to HDL. Efflux of newly synthesised free cholesterol from these macrophages was increased by HDL in a saturable manner, suggesting that the lack of reduction of stored cholesteryl esters was due to impaired mobilisation of cholesteryl esters to free cholesterol via nCEH. Indeed, nCEH activity in these macrophages was much lower than in mouse peritoneal macrophages, and appeared to be down-regulated in the presence of 25-hydroxycholesterol or modified lipoproteins; this loss of nCEH activity was prevented by the ACAT inhibitor 447C88. The efflux of stored cholesteryl esters from THP-1 macrophages therefore appears to be limited by the activity of nCEH.     

牛磺酸下调单核-巨噬细胞ACAT-1表达

目的:观察牛磺酸对THP-1及THP-1源性巨噬细胞酰基辅酶A:胆固醇酰基转移酶1(ACAT-1)表达及活性的影响。方法:将THP-1细胞与PMA孵育48h使之分化为巨噬细胞,在有或无干扰素-γ(IFN-γ)的条件下,用不同浓度的牛磺酸与THP-1持续作用不同的时间,RTPCR检测ACAT-1mRNA水平,WesternBlot检测其蛋白表达,并测定其酶活性。结果:牛磺酸显著下调THP1及THP1源性巨噬细胞的ACAT1mRNA和蛋白表达水平以及ACAT-1活性(P<0.05～0.01,n=3);IFNγ促进ACAT1mRNA和蛋白表达、增强ACAT-1活性(P<0.05,n=3)的作用被牛磺酸部分抑制(P<0.05,n=3)。结论:牛磺酸不但能降低单核巨噬细胞ACAT-1的基础表达和活性,而且能抑制由IFN-γ诱导的ACAT-1表达和活性,这可能是其抗动脉粥样硬化的机制之一。     

HDL2 of heavy alcohol drinkers enhances cholesterol efflux from raw macrophages via phospholipid-rich HDL 2b particles

Background: Alcohol consumption is associated with increased serum high density lipoprotein (HDL) cholesterol levels and a decreased risk for the development of atherosclerosis. However, the effects of heavy alcohol intake on reverse cholesterol transport, one of the key anti‐atherogenic processes related to HDL, are poorly known. Methods: The ability of total HDL as well as HDL₂ and HDL₃ subclasses to promote cholesterol efflux from ³H‐cholesterol‐labeled RAW 264.7 macrophages was studied among 6 heavy alcohol drinkers and 6 controls. Distribution of HDL subclasses was analyzed by 4 to 30% native gradient gels. Serum phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) activities were analyzed among several other biochemical measures. Results: Cholesterol efflux to HDL₂ of heavy drinkers was 22% (p = 0.025) higher relative to controls. The increase in HDL₂ phospholipids, with a concomitant 2‐fold (p = 0.055) increase in large HDL_2b particles, was associated with enhanced cholesterol efflux to HDL₂. Interestingly, the cholesterol efflux to HDL₃ did not differ between the 2 study groups. These findings may be partially explained by a decreased CETP activity (?26%, p = 0.037) and an increased PLTP activity (39%, p = 0.045) in heavy drinkers. Conclusions: The increased cholesterol efflux potential of HDL₂ is most likely an anti‐atherogenic feature linked to heavy alcohol consumption. The cholesterol efflux and HDL phospholipids also associated strongly within the whole study group (r_s = 0.910, p ≤ 0.01) suggesting a common pathway of enhanced cholesterol efflux via enlarged phospholipid‐rich HDL particles.     

Enhanced efflux of cholesterol from ABCA1-expressing macrophages to serum from type IV hypertriglyceridemic subjects

Since elevated plasma triglycerides (TGs) are an independent cardiovascular risk factor, we have compared the cholesterol efflux potential of sera from asymptomatic hypertriglyceridemic (HTG) type IIb, type IV or normolipidemic (NLP) individuals using two different cell systems. In both type IIb and IV HTG, the efflux of cholesterol from SR-BI-rich Fu5AH cells was similar to that obtained with NLP. The maintenance of efflux efficiency in spite of reduced HDL-cholesterol levels can be mainly attributed to the relative enrichment of HDL with phospholipid. In the J774 macrophage cell system, pretreatment with cAMP, which upregulates ABCA1, induced a markedly higher increase in efflux to type IV sera compared with type IIb or NLP. In addition, type IV sera exhibited two-fold higher pre-beta HDL relative concentration (percentage of total apo AI) compared with NLP. Moreover, positive correlations were established between ABCA1-mediated efflux and the serum pre-beta HDL levels or TG concentrations. Thus, the hyperTGemia is associated with a higher fraction of apo AI recovered as pre-beta HDL which appear to be partly responsible for enhanced efflux obtained upon the cAMP stimulation of J774 cells. In conclusion, we demonstrated for the first time that the ABCA1-expressing J774 cell system is responsive to the percent of apo AI present in human serum as pre-beta HDL. Our results suggest that high-plasma TG, accompanied by low HDL may not result in an impaired cholesterol efflux capacity.     

Endothelial lipase enhances low density lipoprotein binding and cell association in THP-1 macrophages

OBJECTIVE: Endothelial lipase (EL) is expressed in macrophages in human atherosclerotic lesions. However, its specific metabolic role in human macrophages has not been fully explored. METHODS: The present study used lentivirus containing either shRNA or cDNA for EL to decrease or increase EL expression, respectively in THP-1 macrophages to investigate the consequence on LDL binding and cell association. RESULTS: EL suppression significantly decreased the binding and cell association of native LDL (52% and 33%) and mildly oxLDL (43% and 36%) as well as extensively oxLDL binding (36%) in THP-1 macrophages. EL overexpression markedly increased the binding and cell association of native LDL (3.1- and 2.2-fold), mildly oxLDL (1.9- and 1.4-fold), and extensively oxLDL (1.5- and 1.5-fold). An inactive mutant EL compromised EL-mediated cell association of native and mildly oxLDL but not extensively oxLDL. Heparinase treatment almost completely eliminated EL-mediated native and oxLDL binding and cell association in THP-1 macrophages. LDL receptor blocking by antibodies decreased EL-mediated native LDL binding and cell association by 24% and 54%, respectively. Neither receptor associated protein or CD36 antibody treatment led to changes in EL-mediated lipoprotein binding and cell association. Furthermore, wild-type and the catalytically inactive mutant EL increased lipid accumulation in THP-1 macrophages. CONCLUSIONS: EL expression promotes the binding and uptake of native and oxidized LDL in THP-1 macrophages in a heparan sulfate proteoglycan-dependent manner, and the LDL receptor was partly responsible for the EL-enhanced uptake of native LDL.     

Improved plasma cholesterol efflux capacity from human macrophages in patients with hyperalphalipoproteinemia

Objectives

CETP or HL deficiencies lead to a marked increase in HDL-C levels however the atheroprotective effect of this phenotype, in particular the ability of HDL particles to remove cholesterol from human macrophages, remains to be determined.

Methods

We measured cholesterol efflux from human THP-1 macrophages to total plasma or to isolated HDL subfractions in patients with HALP carrying molecular defect in either the CETP or LIPC gene.

Results

We demonstrate that HALP is associated with an increased plasma cholesterol efflux capacity from human macrophages. This observation is primarily related to a stimulation of both SR-BI and ABCA1 dependent efflux pathways as a result of quantitative elevation in HDL2 and enhanced intrinsic capacity of HDL3 subspecies, respectively.

Conclusion

HDL particles from HALP patients with molecular defect within either CETP or LIPC gene are not dysfunctional and are efficient to stimulate cholesterol efflux from human macrophages.     

HDL does not promote cholesterol efflux from macrophages of hypercholesterolemic rabbit: efflux differences between species.

The purpose of this study was to investigate the difference between species (mouse and rabbit) and the effect of hypercholesterolemia on the ability of their peritoneal macrophages to release unesterified cholesterol to an exogenous acceptor. The macrophages from mouse, normocholesterolemic rabbits and hypercholesterolemic rabbits (Mm, Mnr, Mhr) were loaded with cholesterol esters by incubation with oxidatively modified low density lipoprotein or plasma lipoprotein (beta-migrating very low density lipoproteins). When human HDL was used as cholesterol acceptor for 24 h of incubation, the Mm Cells, the Mnr cells and Mhr cells retained about 40%, 70%, and more than 90% of their cholesterol esters. The difference between species of lipoproteins were not effective for the ability to release cholesterol esters. ACAT (acylcoenzyme A: cholesterol acyltransferase) inhibitor 58-035 increased these capacities. These data suggest that the limited capacity of macrophages from hypercholesterolemic rabbits to release cholesterol may be related to the progression and resistance to regression of atherosclerosis, and that ACAT activity might influence this capacity.     

G蛋白偶联受体120激动剂促进巨噬细胞胆固醇流出的研究

目的:探讨G蛋白偶联受体120(GPR120)在巨噬细胞胆固醇流出中的作用及机制。方法:培养Raw264.7巨噬细胞,以50-200μmol/L的GPR120激动剂GW9508处理Raw264.7细胞18h。NBD-胆固醇负载法检测细胞胆固醇的流出率;MTS方法检测细胞的存活率;定量PCR和Western blot方法检测胆固醇流出相关转运体ABCA1、ABCG1和SR-B1的mRNA及蛋白表达水平;定量PCR检测核受体LXRα、LXRβ和PPARα、PPARγ的mRNA水平。结果:与对照组相比,100、150和200μmol/L的GW9508均提高Raw264.7细胞NBD-胆固醇流出率,分别提高了8%、10%和18%;ABCA1的蛋白水平分别上调了1.4倍、1.8倍和4.8倍, ABCG1的蛋白水平分别上调了1.3倍、3.2倍和5.1倍,但是不影响SR-B1的蛋白水平;而且,100和200μmol/L的GW9508分别上调ABCA1的mRNA水平3.6倍和6.8倍,上调ABCG1的mRNA水平1.6倍和3.3倍;同时,LXRα的mRNA水平也分别上调了0.6倍和1.25倍,且差异有统计学意义;但是不影响SR-B1及LXRβ、PPARα和PPARγ的mRNA水平。另外,50-200μmol/L的GW9508均不影响Raw264.7细胞的存活率。结论:GPR120通过LXRα上调ABCA1和ABCG1的表达而促进Raw264.7巨噬细胞的胆固醇流出。     

葡萄糖对THP-1源性巨噬细胞酰基辅酶A:胆固醇酰基转移酶1表达的影响

研究葡萄糖对人THP-1单核分化巨噬细胞酰基辅酶A：胆固醇酰基转移酶1（ACAT-1）表达的影响。发现高糖作用下，巨噬细胞ACAT-1的mRNA及蛋白表达增加，这可能是糖尿病血管病变机制之一。