Novel approaches to treating cardiovascular disease: lessons from Tangier disease

Atherosclerotic cardiovascular disease (CVD) remains the leading cause of morbidity and mortality in Western societies. Although cholesterol is a major CVD risk factor, therapeutic interventions to lower plasma cholesterol levels have had limited success in reducing coronary events. Thus, novel approaches are needed to reduce or eliminate CVD. A potential therapeutic target is a newly discovered ATP binding cassette transporter called ABCA1, a cell membrane protein that is the gateway for secretion of excess cholesterol from macrophages into the high density lipoprotein (HDL) metabolic pathway. Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterised by accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. Studies of Tangier disease heterozygotes revealed that the relative activity of ABCA1 determines plasma HDL levels and susceptibility to CVD. Drugs that induce ABCA1 in mice increase clearance of cholesterol from tissues and inhibit intestinal absorption of dietary cholesterol. Thus, ABCA1-stimulating drugs have the potential to both mobilise cholesterol from atherosclerotic lesions and eliminate cholesterol from the body. By reducing plaque formation and rupture independently of the atherogenic factors involved, these drugs would be powerful agents for treating CVD.     

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.     

ATP-binding cassette transporters in macrophages: promising drug targets for treatment of cardiovascular disease

Numerous ATP-binding cassette (ABC) transporters are expressed in monocyte-derived macrophages and are subject to sterol-dependent regulation. ABCA1 has been identified as a key regulator of macrophage cholesterol efflux and HDL-mediated reverse cholesterol transport. Although the precise mechanisms of ABCA1 function are not completely understood, recent data suggest that the ABCA1 pathway regulates vesicular traffic, filipodia formation and lipid microdomains, thereby controlling susceptibility to atherosclerosis. Nuclear hormone receptors including LXR/RXR and PPAR/RXR heterodimers are recognized as direct or indirect regulators of ABCA1 expression and are discussed as potential targets for pharmacological intervention in cardiovascular disease. Future studies clarifying the processes involved in the ABCA1 pathway at the cellular level are expected to identify new and possibly more specific pharmaceutical targets.     

Angiotensin‐(1–7) upregulates expression of adenosine triphosphate‐binding cassette transporter A1 and adenosine triphosphate‐binding cassette transporter G1 through the Mas receptor through the liver X receptor alpha signalling pathway in THP‐1 macrophages treated with angiotensin‐II

Adenosine triphosphate‐binding cassette transporter A1 (ABCA1) and ABCG1 play crucial roles in reverse cholesterol transport, and have anti‐atherosclerosis effects, and liver X receptor alpha (LXRα) can stimulate cholesterol efflux through these transporters. Angiotensin (Ang)‐(1–7) can protect endothelial cells, inhibit smooth muscle cell growth, ameliorate inflammation and exert anti‐atherosclerotic effects. In the present study, we attempted to clarify the effect of Ang‐(1–7) on expression of ABCA1 and ABCG1, and explored the role of LXRα in the regulation of ABCA1 and ABCG1 in THP‐1 macrophages that had been incubated with angiotensin‐II (AngII). Ang‐(1–7) increased ABCA1 and ABCG1 expression in a concentration‐dependent manner at both the mRNA and protein levels, promoted cholesterol efflux, and decreased cholesterol content in THP‐1 macrophages treated with AngII. Furthermore, Ang‐(1–7) upregulated the expression of LXRα in a concentration‐dependent manner in these cells. LXRα small interfering RNA, as well as the Mas receptor antagonist A‐779, completely abolished these effects of Ang‐(1–7). In summary, Ang‐(1–7) upregulates ABCA1 and ABCG1 expression in THP‐1 macrophages treated with AngII through the Mas receptor, via the LXRα pathway. This novel insight into the molecular mechanism underlying Ang‐(1–7) and AngII interaction could prove useful for developing new strategies for treatment of cardiovascular diseases.     

The ABCA2 transporter: intracellular roles in trafficking and metabolism of LDL-derived cholesterol and sterol-related compounds

ATP-binding cassette (ABC) transporters comprise a family of critical membrane bound proteins functioning in the translocation of molecules across cellular membranes. Substrates for transport include lipids, cholesterol and pharmacological agents. Mutations in ABC transporter genes cause a variety of human pathologies and elicit drug resistance phenotypes in cancer cells. ABCA2, the second member the A subfamily to be identified, was highly expressed in ovarian carcinoma cells resistant to the anti-cancer agent, estramustine, and more recently, in human vestibular schwannomas. Cells expressing elevated levels of ABCA2 show resistance to variety of compounds, including estradiol, mitoxantrone and a free radical initiator, 2,2'-azobis-(2-amidinopropane). ABCA2 is expressed in a variety of tissues, with greatest abundance in the central nervous system and macrophages. This transporter, along with other proteins that have a high degree of homology to ABCA2, including ABCA1 and ABCA7, are up-regulated in human macrophages during cholesterol import. Recent studies have shown ABCA2 also plays a role in the trafficking of low-density lipoprotein (LDL)-derived free cholesterol and to be coordinately expressed with sterol-responsive genes. A single nucleotide polymorphism in exon 14 of the ABCA2 gene was shown to be linked to early onset Alzheimer disease (AD) in humans, supporting an earlier study showing ABCA2 expression influences levels of APP and beta-amyloid peptide, the primary component of senile plaques. Studies thus far implicate ABCA2 as a sterol transporter, the deregulation of which may affect a cellular phenotype conducive to the pathogenesis of a variety of human diseases including AD, atherosclerosis and cancer.     

Physiological function of ABCG1

Since ATP-binding cassette transporter A1 (ABCA1) was discovered as the cause of Tangier disease and familial high-density lipoprotein (HDL) deficiency, many investigators have been interested in the relationship between ABC transporters and the mechanism underlying abnormal lipid metabolism. ABCG1 is an ABC half transporter that facilitates efflux excess cholesterol from macrophages. To elucidate the potential physiological role of ABCG1, we have initiated a series of studies overexpressing ABCG1, using an adenovirus vector (rABCG1-Adv) in C57BL mice. Overexpression of ABCG1 in the liver of mice using recombinant ABCG1 vectors results in decreased plasma HDL levels and increased biliary cholesterol excretion, and indicates that ABCG1 can modulate plasma lipoprotein levels in vivo. ABCG1 and the other ABC transporters might play an important role in cholesterol homeostasis, especially in the liver.     

Atorvastatin inhibits ABCA1 expression and cholesterol efflux in THP-1 macrophages by an LXR-dependent pathway

The effect of atorvastatin on adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and cholesterol efflux remains controversial. In an effort to clarify this issue, ABCA1 expression and apolipoprotein AI (apoAI)-mediated cholesterol efflux after atorvastatin treatment were investigated in THP-1 macrophages. Atorvastatin from 2 microM to 40 microM dose-dependently inhibited ABCA1 expression in human monocyte-derived macrophages and phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 monocytes. ApoAI-mediated cholesterol efflux was reduced in PMA-stimulated THP-1 cells treated with atorvastatin, this effect was abolished with acetylated low-density lipoprotein (LDL) pretreatment. Atorvastatin treatment also dose-dependently reduced liver X receptor alpha (LXRalpha) expression and Rho activation. Rho activation by farnysylpyophosphate (FPP) and lysophosphatidic acid (LPA) did not salvage, but further depressed, the cholesterol efflux and ABCA1 expression in the presence of atorvastatin. Without atorvastatin, Rho activation by mevalonate, FPP, and LPA diminished apoAI-mediated cholesterol efflux, and Rho activation by GTPgammaS also decreased ABCA1 messenger ribonucleic acid (mRNA) by 16%. Furthermore, Rho inhibition by C3 exoenzyme increased ABCA1 mRNA by 48% despite a 17% decrease in apoAI-mediated cholesterol efflux. LXRalpha agonists (T01901317 and 22(R)-hydroxycholesterol) prevented any reductions in cholesterol efflux or ABCA1 expression associated with atorvastatin treatment. Furthermore, Western blot analysis demonstrated the reciprocal inhibition of Rho and LXRalpha. In conclusion, atorvastatin decreases ABCA1 expression in noncholesterol-loaded macrophages in an LXRalpha- but not Rho-dependent pathway; this effect can be compromised after acetylated LDL cholesterol loading.     

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。     

Remembering your A,B, C's: Alzheimer's disease and ABCA1

The function of ATP binding cassette protein A1 (ABCA1) is central to cholesterol mobilization. Reduced ABCA1 expression or activity is implicated in Alzheimer's disease (AD) and other disorders. Therapeutic approaches to boost ABCA1 activity have yet to be translated successfully to the clinic. The risk factors for AD development and progression, including comorbid disorders such as type 2 diabetes and cardiovascular disease, highlight the intersection of cholesterol transport and inflammation. Upregulation of ABCA1 can positively impact APOE lipidation, insulin sensitivity, peripheral vascular and blood–brain barrier integrity, and anti-inflammatory signaling. Various strategies towards ABCA1-boosting compounds have been described, with a bias toward nuclear hormone receptor (NHR) agonists. These agonists display beneficial preclinical effects; however, important side effects have limited development. In particular, ligands that bind liver X receptor (LXR), the primary NHR that controls ABCA1 expression, have shown positive effects in AD mouse models; however, lipogenesis and unwanted increases in triglyceride production are often observed. The longstanding approach, focusing on LXRβ vs. LXRα selectivity, is over-simplistic and has failed. Novel approaches such as phenotypic screening may lead to small molecule NHR modulators that elevate ABCA1 function without inducing lipogenesis and are clinically translatable.     

The potential for novel anti-inflammatory therapies for coronary artery disease

Although drugs that lead to cholesterol and lipid lowering have proved to have significant effects in lowering cardiovascular morbidity and mortality, coronary artery disease remains a principal cause of death worldwide. There is a clear need to discover further therapeutic approaches to control this disease adequately. This review focuses on the mechanisms that have been implicated in the recruitment, activation and differentiation of inflammatory monocytes/macrophages in nascent vascular lesions into lipid-laden foam cells. These mechanisms might provide attractive targets for novel therapies for coronary artery disease.     

人膜转运蛋白ABCA1表达上调剂的筛选与鉴定

人ATP结合盒转运体A1(ATP-binding cassette transporter A1,ABCA1)可介导细胞内磷脂和游离胆固醇转运至贫脂或无脂的载脂蛋白A-I(apolipoprotein A-I,apoA-I),从而促进高密度脂蛋白(high density lipoprotein,HDL)生成,启动胆固醇逆转运过程,在机体清除多余脂质的过程中发挥重要作用。因此,本研究以ABCA1为靶点,前期建立了ABCA1上调剂抗动脉粥样硬化(atherosclerosis,AS)药物筛选模型,以期寻找具有新作用机制的抗AS药物。在此研究中,利用该模型对1 600个化合物进行筛选,发现2030421B上调活性大于50%,其EC50为0.50μg.mL-1。进一步研究发现,2030421B不仅能上调ABCA1的mRNA以及蛋白水平,而且能使小鼠巨噬细胞RAW264.7内胆固醇流出增加,脂滴量减少,是具有较好效果的ABCA1上调剂。     

Potential Role of Acyl-Coenzyme A:Cholesterol Transferase (ACAT) Inhibitors as Hypolipidemic and Antiatherosclerosis Drugs

Acyl-coenzyme A:cholesterol transferase (ACAT) is an integral membrane protein localized in the endoplasmic reticulum. ACAT catalyzes the formation of cholesteryl esters from cholesterol and fatty acyl coenzyme A. The cholesteryl esters are stored as cytoplasmic lipid droplets inside the cell. This process is very important to the organism as high cholesterol levels have been associated with cardiovascular disease. In mammals, two ACAT genes have been identified, ACAT1 and ACAT2. ACAT1 is ubiquitous and is responsible for cholesteryl ester formation in brain, adrenal glands, macrophages, and kidneys. ACAT2 is expressed in the liver and intestine. The inhibition of ACAT activity has been associated with decreased plasma cholesterol levels by suppressing cholesterol absorption and by diminishing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein (VLDL). ACAT inhibition also prevents the conversion of macrophages into foam cells in the arterial walls, a critical event in the development of atherosclerosis. This review paper will focus on the role of ACAT in cholesterol metabolism, in particular as a target to develop novel therapeutic agents to control hypercholesterolemia, atherosclerosis, and Alzheimer's disease.     

ATP binding cassette transporter A1 (ABCA1) associated proteins: potential drug targets in the metabolic syndrome and atherosclerotic disease?

The metabolic syndrome is defined as a cluster of disorders including visceral obesity, insulin resistance, hypertension, hypertriglyceridemia and low HDL. Patients have an increased risk to develop atherosclerotic disease characterized by excessive macrophage cholesterol deposition in the vascular wall. HDL removes excess cellular cholesterol which is subsequently transported to the liver for biliary excretion and thereby preserves cholesterol homeostasis. Circulating HDL levels are maintained by hepatic ATP-binding cassette transporter A1 (ABCA1) which also transports peripheral cell cholesterol to extracellular lipid acceptors. Lipid export activity of ABCA1 improves pancreatic -cell function and ameliorates insulin release. ABCA1 affects plasma membrane cholesterol distribution and formation of lipid rafts representing signalling platforms for diverse receptors including toll-like receptor 4 (TLR4). Pharmacological activation of ABCA1 pathways presumably progresses metabolic diseases, and current approaches demonstrate beneficial effects of small peptides mimicking ABCA1 ligands which stabilize ABCA1 and enhance lipid efflux similar to its physiological acceptor apolipoprotein A-I. Research of the last decade has resulted in the identification of several ABCA1 binding proteins influencing ABCA1 signalling, stability and activity. In the current review the proteins suggested to form a complex with ABCA1 are summarized and their up to now characterized features towards ABCA1 functions are described.     

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…     

Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice

Background and Purpose

Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo.

Experimental Approach

Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo.

Key Results

Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (−31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (−70%) and secretion (−28%) by peritoneal macrophages.

Conclusions and Implications

Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis.     

阿托伐他汀对金黄地鼠糖尿病模型巨噬细胞胆固醇外流的影响

目的：研究不同剂量阿托伐他汀对糖尿病动物模型体内巨噬细胞胆固醇含量的影响，进一步探讨他汀类药物抑制动脉粥样硬化斑块进展的机制。方法：建立糖尿病金黄地鼠动物模型，体外培养腹腔巨噬细胞，在体外与不同剂量阿托伐他汀孵育，利用酶法结合高效液相色谱法测定细胞内胆固醇及胆固醇脂含量。RT—PCR方法检测细胞ABCA1 mRNA表达量的不同。结果：在apoA1存在时，阿托伐他汀可以显著促进巨噬细胞内胆固醇外流，可能与药物增强细胞内胆固醇酯水解酶活性、促进巨噬细胞AB—CA1的表达有关。结论：阿托伐他汀促进ABCA1介导的细胞内胆固醇外流。     

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.