Nifedipine Enhances Cholesterol Efflux in RAW264.7 Macrophages

Purpose

Studies have shown that nifedipine protects against atherosclerotic progression, but its underlying mechanisms remain unclear. In this study, we examined if nifedipine increases macrophage cholesterol efflux, a pathway known to inhibit atherogenesis.

Methods

We evaluated the ability of different doses of nifedipine to affect cholesterol efflux in RAW264.7 macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques.

Results

Nifedipne at 1, 10, and 100 nmol/L increased apoA-I-mediated cholesterol efflux from 2.55 % to 5.65 %, 6.20 %, and 6.10 %, as well as HDL-mediated cholesterol efflux from 31.0 % to 42.5 %, 46.0 %, and 43.5 %, respectively, in RAW264.7 macrophages (p?<?0.05), which was associated with increased mRNA expression levels of ABCA1, ABCG1, SR-BI, and LXRα (405 %, 381 %, 336 %; 890 %, 960 %, 1002 %; 285 %, 325 %, 336 %; 482 %, 445 %, 405 %, respectively, p?<?0.05), and with increased protein levels of ABCA1, ABCG1, SR-BI, and LXRα (428 %, 492 %, 361 %; 288 %, 331 %, 365 %; 283 %, 320 %, 505 %; 581 %, 678 %, 608 %, respectively, p?<?0.05). SiRNA-mediated silencing of LXRα revealed that LXRα was involved in these increases and the enhanced cholesterol efflux.

Conclusion

Nifedipine may protect against atherosclerosis partly by promoting macrophage cholesterol efflux through the stimulation of LXRα-dependent expression of ABCA1, ABCG1, and SR-BI.     

Paraoxonase 1 (PON1) enhances HDL-mediated macrophage cholesterol efflux via the ABCA1 transporter in association with increased HDL binding to the cells: a possible role for lysophosphatidylcholine

We investigated the role of HDL-associated paraoxonase 1 (PON1) in HDL-mediated macrophage cholesterol efflux by using HDL derived from wild type mice (Control-HDL), from human PON1-transgenic mice (HDL-PON1Tg) or from PON1-knockout mice (HDL-PON1(0)). Cholesterol efflux from mouse peritoneal macrophages (MPM) or from J774 A.1 macrophage cell line by HDL-PON1Tg, was significantly increased (by 60%) compared to HDL-PON1(0). We demonstrated that this PON1 effect was associated with an increased HDL binding to the cells, as the binding of HDL-PON1Tg (or HDL-PON1(0) that was enriched with PON1) was increased by 50% compared to that of HDL-PON1(0). Using either a cAMP analogue, to increase ABCA1 receptor expression, or rabbit anti-mouse SR-BI specific antibody to block the SR-BI receptor, PON1 stimulation of HDL binding and of HDL-mediated macrophage cholesterol efflux, were both found to involve the ABCA1 transporter. Studies with PON1 specific inhibitors revealed that PON1 activity was required for its stimulation of HDL-mediated macrophage cholesterol efflux. Upon incubation of macrophages with Control-HDL or with HDL-PON1Tg, macrophage lysophosphatidylcholine (LPC) content was increased by 3.7- and 7.5-fold, respectively. Such an LPC enrichment of macrophages resulted in up to 60% increased HDL binding to the cells, and a 41% increased HDL-mediated cholesterol efflux. Similarly, macrophage loading with LPC (by either adding LPC, or PON1 or phospholipase A(2)) significantly increased apolipoprotein A-I (apoA-I) mediated cholesterol efflux by 104, 65 and 56%, respectively, in ABCA1 overexpressing macrophages. We conclude that HDL-associated PON1 may contribute to the attenuation of atherosclerosis development by its ability to act on macrophage phospholipids, to form LPC, in turn, stimulates HDL binding and HDL-mediated macrophage cholesterol efflux via the ABCA1 transporter.     

A novel liver X receptor agonist establishes species differences in the regulation of cholesterol 7alpha-hydroxylase (CYP7a)

The liver X receptors, LXRalpha and LXRbeta, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRalpha knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F(3)MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F(3)MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F(3)MethylAA induced cyp7a mRNA, confirming conclusions from the knockout mouse studies. Furthermore, in rat in vivo studies, F(3)MethylAA induced liver cyp7a mRNA and enzyme activity. A critical species difference is also reported in that neither F(3)MethylAA nor 22(R)-hydroxycholesterol induced cyp7a in human primary hepatocytes. However, other LXR target genes, ABCA1, ABCG1, and SREBP1, were regulated.     

Protein kinase C alpha modulates liver X receptor alpha transactivation

Liver X receptor alpha (LXRalpha), an oxysterol-activated nuclear hormone receptor, regulates the expression of genes involved in lipid and cholesterol homeostasis and inflammation. We show here that transactivation by LXRalpha in monkey kidney COS-1 (Cos-1) cells is decreased by activation of the protein kinase C (PKC) signaling pathway. In transient co-transfection assays, phorbol myristate acetate (PMA) suppressed LXR-dependent transactivation of LXR-responsive reporter genes or the natural promoter of the human ATP-binding cassette (ABC), ABCA1 gene. The decrease in LXR transactivation after PMA treatment was also observed in human embryonic kidney (HEK) 293 and human hepatocellular carcinoma (HepG2) cells. Moreover, endogenous LXR target genes, ABCA1 and sterol response element-binding protein-1c, were also decreased by PMA treatment in HEK293 cells as assessed by real-time PCR. The PMA-mediated decrease of LXR activity was blocked by the PKC inhibitor bisindolylmaleimide and mimicked by constitutively active PKCalpha. Nuclear extracts treated with PMA show no decrease in LXRalpha DNA binding as assessed by mobility shift and chromatin immunoprecipitation assays. Additionally, in vitro kinase assays demonstrate that PKCalpha can phosphorylate LXRalpha. Our findings reveal a mode of regulation of LXRalpha that may be relevant to disease conditions where aberrant PKC signaling is observed, such as diabetes.     

Pharmacological regulation of cholesterol efflux in human monocyte-derived macrophages in the absence of exogenous cholesterol acceptors

Cholesterol efflux from human monocyte-derived macrophages in the absence of exogenous acceptors has been described, but is unclear in mechanism. We investigated this process in relation to the expression of relevant genes, intracellular cholesterol storage and apoE secretion using drugs affecting different aspects of cholesterol metabolism. Both natural (22R-hydroxycholesterol/9-cis-retinoic acid) and synthetic (T0901317 and RO264456) LXR/RXR ligands increased ABCA1 and ABCG1 mRNAs in native macrophages and in cells loaded with acetylated LDL (acLDL). The ACAT inhibitor avasimibe increased only ABCG1 mRNA, whereas no treatment affected apoE mRNA. Avasimibe, progesterone, and natural but not synthetic LXR/RXR ligands prevented cholesterol esterification after acLDL-loading. Cholesterol efflux into acceptor-free medium was increased only by synthetic LXR/RXR ligands and avasimibe in acLDL-loaded cells. ApoE secretion was reduced by drugs affecting cholesterol trafficking but enhanced by LXR/RXR ligands. Incubation with an anti-apoE antibody virtually removed immunodetectable apoE from the medium, significantly increasing cholesterol storage and decreasing efflux. These findings indicate that in human macrophages spontaneous cholesterol efflux: (i) is not necessarily promoted by increasing intracellular free cholesterol, (ii) is increased by compounds that activate ABCA1 and, to a greater extent, ABCG1 and (iii) is only partially correlated with secretion of endogenous apoE, which acted as a cholesterol acceptor.     

Adiponectin reduces lipid accumulation in macrophage foam cells

Adiponectin is one of several, important metabolically active cytokines secreted from adipocytes. Low circulating levels of this adipokine have been associated epidemiologically with obesity, insulin resistance, type II diabetes, and cardiovascular disease. To determine if adiponectin can modulate lipid metabolism in macrophages, we expressed the adiponectin gene in human THP-1 macrophage foam cells using a lentiviral vector expression system and demonstrated that macrophages transduced with the adiponectin gene had decreased lipid accumulation compared with control macrophages transduced with the LacZ gene. Macrophages transduced with the adiponectin gene also exhibited decreased oxidized low-density lipoprotein (oxLDL) uptake and increased HDL-mediated cholesterol efflux. Additional studies suggest two potential mechanisms for the reduced lipid accumulation in these adiponectin-transduced macrophage foam cells. The first mechanism involves the PPARgamma and LXR signaling pathways which up-regulate the expression of ABCA1 and promote lipid efflux from these cells. The second mechanism involves decreased lipid uptake and increased lipid hydrolysis which may result from decreased SR-AI and increased SR-BI and HSL gene activities in the transformed macrophage foam cells. We also demonstrated that the expression of two proatherogenic cytokines, MCP-1 and TNFalpha, were decreased in the adiponectin-transduced macrophage foam cells. These results suggest that adiponectin may modulate multiple pathways of lipid metabolism in macrophages. Our studies provide new insights into potential mechanisms of adiponectin-mediated alterations in lipid metabolism and macrophage foam cell formation which may impact the development of atherosclerosis.     

Enhanced synthesis of the oxysterol 24(S),25-epoxycholesterol in macrophages by inhibitors of 2,3-oxidosqualene:lanosterol cyclase: a novel mechanism for the attenuation of foam cell formation

Oxysterols are key regulators of lipid metabolism and regulate gene expression by activating the liver X receptor (LXR). LXR plays a vital role in macrophage foam cell formation, a central event in atherosclerosis. It is known that addition of exogenous oxysterols to cultured macrophages activates LXR, leading to increased expression of ABCA1 and cholesterol efflux. In this study, we tested the novel hypothesis that stimulation of endogenous oxysterol synthesis would block foam cell formation induced by atherogenic lipoproteins. Macrophage synthesis of 24(S),25-epoxycholesterol, a potent LXR ligand, increased 60-fold by partial inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), a microsomal enzyme in both the cholesterol biosynthetic pathway and the alternative oxysterol synthetic pathway. When macrophages were challenged with human hypertriglyceridemic VLDL (HTG-VLDL), cellular cholesteryl ester accumulation increased 12-fold. This was reduced dramatically, by 65%, after preincubation with an OSC inhibitor (OSCi). The HTG-VLDL-induced accumulation of macrophage TG (70-fold) was unaffected by the OSCi or exogenous 24(S),25-epoxycholesterol, an effect associated with suppression of SREBP-1 processing. By contrast, TO901317, a synthetic LXR agonist, increased cellular TG significantly and markedly increased SREBP-1 processing. OSC inhibition decreased HTG-VLDL uptake through downregulation of LDL-receptor expression, despite substantial inhibition of cholesterol synthesis. Furthermore, OSC inhibition significantly upregulated ABCA1 and ABCG1 expression, which led to enhanced macrophage cholesterol efflux, an effect mediated through LXR activation. Therefore, increased macrophage synthesis of endogenous oxysterols represents a new mechanism for the dual regulation of LXR- and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis.     

Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages

Liver X receptors (LXRs) are nuclear receptors that regulate macrophage cholesterol efflux by inducing ATP-binding cassette transporter A1 (ABCA1) and ABCG1/ABCG4 gene expression. The Niemann-Pick C (NPC) proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant governing its availability for efflux to extracellular acceptors. Here we investigated the influence of LXR activation on intracellular cholesterol trafficking in primary human macrophages. Synthetic LXR activators increase the amount of free cholesterol in the plasma membrane by inducing NPC1 and NPC2 gene expression. Moreover, ABCA1-dependent cholesterol efflux induced by LXR activators was drastically decreased in the presence of progesterone, which blocks postlysosomal cholesterol trafficking, and reduced when NPC1 and NPC2 mRNA expression was depleted using small interfering RNA. The stimulation of cholesterol mobilization to the plasma membrane by LXRs led to a decrease in cholesteryl ester formation and Acyl-coenzyme A cholesterol acyltransferase-1 activity. These data indicate that LXR activation enhances cholesterol trafficking to the plasma membrane, where it becomes available for efflux, at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.     

Determinants of leukocyte adenosine triphosphate-binding cassette transporter G1 gene expression in type 2 diabetes mellitus

Cellular cholesterol efflux is regulated by cholesterol transporters including adenosine triphosphate–binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B type I (SR-BI). We have investigated whether the expression of these transporters is affected by type 2 diabetes mellitus and the association with glycemic indexes and oxidized low-density lipoprotein (oxLDL). Messenger RNA of ABCA1, ABCG1, and SR-BI in peripheral monocytes was measured in 30 diabetic patients and 30 matched controls. Plasma oxLDL and advanced glycation end products (AGEs) were assayed by enzyme-linked immunosorbent assay. Cellular cholesterol efflux from monocytes to serum was determined in a subgroup chosen randomly. The expression of ABCG1 was decreased in diabetic patients (P < .05), whereas the levels of ABCA1 and SR-BI were comparable between the 2 groups. Fasting glucose, hemoglobin A_1c, AGEs, and oxLDL were all significantly increased in diabetic patients. There was an inverse correlation between serum AGEs and ABCG1 (r = −0.44, P < .05) that remained significant after adjusting for potential confounding factors. No associations between fasting glucose, hemoglobin A_1c, plasma lipids, or oxLDL and the expression of ABCG1, ABCA1, or SR-BI were found. Cholesterol efflux from monocytes to standard serum or autologous serum was significantly impaired in diabetic patients, and the reduction in efflux to autologous serum correlated with the expression of ABCG1 (r = 0.60, P < .05). The expression of ABCG1 in monocytes is reduced in type 2 diabetes mellitus and is partly related to serum AGEs concentration. The reduction in ABCG1 is associated with impairment in cholesterol efflux and may contribute to accelerated foam cell formation in diabetic patients.     

肝脏X受体α对肝细胞HepG2胆固醇代谢基因表达的调控

目的:研究肝细胞X受体α(liver X receptorα,LXRα)基因对HepG2肝细胞胆固醇代谢相关基因表达的调控作用。方法:将特异性LXRα小片段干扰RNA(siRNA)经脂质体Lipofectamine 2000介导转染人HepG2肝细胞,同时行HepG2肝细胞LXRα的激动(T0901317)实验,采用实时定量PCR方法分别测定干扰和激动前后的LXRα以及胆固醇代谢相关基因ATP结合盒(ABC)G5、ABCG8、ABCA1 mRNA的表达。结果:LXRαsiRNA干扰24h后,HepG2实验组LXRα mRNA相对表达量显著低于阴性对照组(P0.05)。HepG2加入激动剂(T0901317)48h后,实验组ABCG8和ABCA1基因表达均显著升高(P0.05)。结论:RNA干扰使HepG2肝细胞LXRα表达受抑制,继而有使靶基因ABCG5、ABCG8、ABCA1表达降低趋势;人工合成配体T0901317与LXRα结合,使其活性增加,上调了ABCG8、ABCA1和ABCG5 mRNA的表达。提示LXRα可调控HepG2肝细胞的胆固醇代谢基因。     

ABCA1 and atherosclerosis

ATP binding cassette transporter A1 (ABCA1) mediates the cellular efflux of phospholipids and cholesterol to lipid-poor apolipoprotein A1 (apoA1) and plays a significant role in high density lipoprotein (HDL) metabolism. ABCA1's role in the causation of Tangier disease, characterized by absent HDL and premature atherosclerosis, has implicated this transporter and its regulators liver-X-receptoralpha (LXRalpha) and peroxisome proliferator activated receptorgamma (PPARgamma) as new candidates potentially influencing the progression of atherosclerosis. In addition to lipid regulation, these genes are involved in apoptosis and inflammation, processes thought to be central to atherosclerotic plaque progression. A Medline-based review of the literature was carried out. Tangier disease and human heterozygotes with ABCA1 mutations provide good evidence that ABCA1 is a major candidate influencing atherosclerosis. Animal and in vitro experiments suggest that ABCA1 not only mediates cholesterol and phospholipid efflux, but is also involved in the regulation of apoptosis and inflammation. The complex and beneficial interactions between apoA1 and ABCA1 seem to be pivotal for cholesterol efflux. The expression of the ABCA1 is tightly regulated. Furthermore the plaque microenvironment could potentially promote ABCA1 protein degradation thus compromising cholesterol efflux. PPAR-LXR-ABCA1 interactions are integral to cholesterol homeostasis and these nuclear receptors have proven anti-inflammatory and anti-matrix metalloproteinase activity. Therapeutic manipulation of the ABCA1 transporter is feasible using PPAR and LXR agonists. PPAR agonists like glitazones and ABCA1 protein stabilization could potentially modify the clinical progression of atherosclerotic lesions.     

Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice

BACKGROUND/AIMS: Cystathionine beta synthase (CBS) deficiency leads to severe hyperhomocysteinemia, which confers diverse clinical manifestations, notably fatty liver. Recently, abnormal lipid metabolism has been demonstrated in CBS-deficient mice, a murine model of severe hyperhomocysteinemia. To gain further insights into effects of CBS deficiency on hepatic cholesterol metabolism, the expression of hepatic genes involved in biosynthesis, uptake and efflux was determined in CBS-deficient mice. METHODS: Gene expression analysis was performed on liver of CBS-deficient mice using quantitative real-time PCR. RESULTS: We found that CBS-deficiency in liver mice significantly increases expression of genes induced by endoplasmic reticulum stress and genes that regulate the expression of enzymes required for cholesterol and fatty acid biosynthesis and uptake, notably the scavenger receptor class B type I (SR-BI), concomitant with overexpression of SR-BI at the protein level. Moreover, we also found increased mRNA levels of ABCG5, ABCG8, ABCG1 and ABCA1, which play important roles in reverse cholesterol transport, associated with an upregulation of liver X receptors and a downregulation of the peroxisome proliferators-activated receptor alpha. CONCLUSIONS: We found that several ATP-binding cassette transporters and nuclear hormone receptors involved in liver lipid homeostasis are differentially expressed in liver of CBS-deficient mice.     

Reduction of atherosclerosis in apolipoprotein E knockout mice by activation of the retinoid X receptor

A common feature of many metabolic pathways is their control by retinoid X receptor (RXR) heterodimers. Dysregulation of such metabolic pathways can lead to the development of atherosclerosis, a disease influenced by both systemic and local factors. Here we analyzed the effects of activation of RXR and some of its heterodimers in apolipoprotein E -/- mice, a well established animal model of atherosclerosis. An RXR agonist drastically reduced the development of atherosclerosis. In addition, a ligand for the peroxisome proliferator-activated receptor (PPAR)gamma and a dual agonist of both PPARalpha and PPARgamma had moderate inhibitory effects. Both RXR and liver X receptor (LXR) agonists induced ATP-binding cassette protein 1 (ABC-1) expression and stimulated ABC-1-mediated cholesterol efflux from macrophages from wild-type, but not from LXRalpha and beta double -/-, mice. Hence, activation of ABC-1-mediated cholesterol efflux by the RXR/LXR heterodimer might contribute to the beneficial effects of rexinoids on atherosclerosis and warrant further evaluation of RXR/LXR agonists in prevention and treatment of atherosclerosis.