Promotion Of Reverse Cholesterol Transport Research Articles

Promoting reverse cholesterol transport contributes to the amelioration of atherosclerosis by paeoniflorin

Reverse cholesterol transport (RCT) offers a practical approach to mitigating atherosclerosis. Paeoniflorin, a monoterpenoid glycoside found in plants of the Paeoniaceae family, has shown various effects on cardiovascular and liver diseases. Nevertheless, its impact on atherosclerosis in vivo remains poorly understood. The objective of this study is to examine the effect of paeoniflorin on atherosclerosis using apolipoprotein E-deficient (ApoE−/−) mice and explore the underlying mechanisms, with a specific focus on its modulation of RCT. ApoE−/− mice were continuously administered paeoniflorin by gavage for three months. We assessed lipid parameters in serum and examined pathological changes and gene expressions related to RCT pathways in the aorta, liver, and intestine. In an in vitro study, we utilized RAW264.7 macrophages to investigate the inhibitory effect of paeoniflorin on foam cell formation and its potential to promote RCT. The results revealed that paeoniflorin reduced atherosclerosis, alleviated hyperlipidemia, and mitigated hepatic steatosis. Paeoniflorin may promote RCT by stimulating cholesterol efflux from macrophages via the liver X receptor alpha pathway, enhancing serum high-density lipoprotein cholesterol and apolipoprotein A-I levels, and regulating key genes in hepatic and intestinal RCT. Additionally, treatment ApoE−/− mice with paeoniflorin suppressed the expression of inflammation-related genes, including CD68, tumor necrosis factor alpha, and monocyte chemoattractant protein-1, and mitigated oxidative stress in both the aorta and liver. Our results indicated that paeoniflorin has the potential to be a more effective and safer treatment for atherosclerosis, thanks to its promotion of RCT and its anti-inflammatory and anti-oxidative effects.

Activation of Naturally Occurring Lecithin:Cholesterol Acyltransferase Mutants by a Novel Activator Compound.

Lecithin:cholesterol acyltransferase (LCAT) is a unique plasma enzyme able to esterify cholesterol, and it plays an important role in HDL maturation and promotion of reverse cholesterol transport. Familial LCAT deficiency (FLD; OMIM number 245900) is a rare recessive disease that results from loss-of-function mutations in the LCAT gene and has no cure. In this study, we assessed the in vitro efficacy of a novel small-molecule LCAT activator. Cholesterol esterification rate (CER) and LCAT activity were tested in plasma from six controls and five FLD homozygous carriers of various LCAT mutations at different doses of the compound (0.1, 1, and 10 µg/ml). In control plasma, the compound significantly increased both CER (P < 0.001) and LCAT activity (P = 0.007) in a dose-dependent manner. Both CER and LCAT activity increased by 4- to 5-fold, reaching maximum activation at the dose of 1 µg/ml. Interestingly, Daiichi Sankyo compound produced an increase in CER in two of the five tested LCAT mutants (Leu372--Arg and Val309--Met), while LCAT activity increased in three LCAT mutants (Arg147--Trp, Thr274--Ile and Leu372--Arg); mutant Pro254--Ser was not activated at any of the tested doses. The present findings form the basis for personalized therapeutic interventions in FLD carriers and support the potential LCAT activation in secondary LCAT defects. SIGNIFICANCE STATEMENT: We characterized the pharmacology of a novel small-molecule LCAT activator in vitro on a subset of naturally occurring LCAT mutants. Our findings form the basis for personalized therapeutic interventions for familial LCAT deficiency carriers, who can face severe complications and for whom no cure exists.

Hawthorn (Crataegus pinnatifida Bunge) leave flavonoids attenuate atherosclerosis development in apoE knock-out mice

Ethnopharmacological relevanceHawthorn (Crataegus pinnatifida Bunge) leave have been used to treat cardiovascular diseases in China and Europe. Hawthorn leave flavonoids (HLF) are the main part of extraction. Whether hawthorn leave flavonoids could attenuate the development of atherosclerosis and the possible mechanism remain unknown. Materials and methodsHigh-fat diet (HFD) mixed with HLF at concentrations of 5mg/kg and 20mg/kg were administered to apolipoprotein E (apoE) knock out mice. 16 weeks later, mouse serum was collected to determine the lipid profile while the mouse aorta dissected was prepared to measure the lesion area. Hepatic mRNA of genes involved in lipid metabolism were determined. Peritoneal macrophages were collected to study the impact of HLF on cholesterol efflux, formation of foam cell and the expression of ATP binding cassette transporter A1 (ABCA1). Besides, in vivo reverse cholesterol transport (RCT) was conducted. ResultsHLF attenuated the development of atherosclerosis that the mean atherosclerotic lesion area in en face aortas was reduced by 23.1% (P<0.05). In mice fed with 20mg/kg HLF, Total cholesterol (TC) level was decreased by 18.6% and very low density lipoprotein cholesterol plus low density lipoprotein cholesterol (VLDLc+LDLc) level were decreased by 23.1% whereas high density lipoprotein cholesterol (HDLc) and triglyceride (TG) levels were similar compared to that of the control group. Peroxisome proliferator activated receptor alpha (PPARα) mRNA was increased by 31.2% (P<0.05) and 60.9% (P<0.05) in mice fed with 5mg/kg and 20mg/kg HLF respectively. Sterol regulatory element binding protein-1c (SREBP-1c) was decreased by 59.3% in the group of 20mg/kg. Carnitine palmitoyl transferase 1 (CPT-1) mRNA level of 20mg/kg group was induced 66.7% (P<0.05). Superoxide dismutase 1 and 2 (SOD1 and SOD2) mRNA were induced 25.4% (P<0.05) and 71.4% (P<0.05) while induced by 36.3% (P<0.05) and 73.2% (P<0.05) in group of 20mg/kg. Glutathione peroxidase 3 (Gpx3) mRNA in the group of 20mg/kg was induced by 96.7% (P<0.05). Hepatic hydroxymethylglutaryl CoA reductase (HMG-CoAR) expression was as same level as the control group while LDL receptor (LDLR) mRNA and protein were induced by 84.2% (P<0.05) and 98.8% (P<0.05) in group of 20mg/kg. HLF inhibit the formation of foam cell by 27.9% (P<0.05) in the dosage of 25μg/ml, and 33.3% (P<0.05) in the dosage of 50μg/ml. HLF increased the reverse cholesterol transport (RCT) in vivo. Discussion and conclusionHawthorn leave flavonoids can slow down the development of atherosclerosis in apoE knockout mice via induced expression of genes involved in antioxidant activities, inhibition of the foam cell formation and promotion of RCT in vivo, which implies the potential use in the prevention of atherosclerosis.

Quantification of In Vitro Macrophage Cholesterol Efflux and In Vivo Macrophage-Specific Reverse Cholesterol Transport.

Promotion of reverse cholesterol transport (RCT) is thought to be a major HDL-mediated mechanism for protecting against atherosclerosis. Preclinical studies support the concept that increasing cholesterol efflux from macrophages may confer atheroprotective benefits independently of the plasma HDL-cholesterol concentration. The application of the macrophage-to-feces RCT method in genetically engineered mice has provided evidence that this major HDL property correlates closely with changes in atherosclerosis susceptibility. This chapter provides details on the methodologies currently used to measure in vitro cholesterol efflux from macrophages or in vivo macrophage-specific RCT. The general principles and techniques described herein may be applied to measure the in vitro cholesterol efflux capacity of human serum in macrophage cultures and to evaluate the effect of different experimental pathophysiological conditions or the efficacy of different therapeutic strategies on the modulation of in vivo macrophage-RCT in mice.

Influence of exercise training on HDL function

Abstract“Besides promotion of reverse cholesterol transport, HDL has been shown to exert beneficial antiatherogenic effects...”

Ezetimibe enhances macrophage reverse cholesterol transport in hamsters: Contribution of hepato–biliary pathway

Reverse cholesterol transport (RCT) is pivotal in the return of excess cholesterol from peripheral tissues to the liver for excretion in bile and eventually feces. RCT from macrophages is a critical anti-atherogenicity mechanism of HDL. As the cholesterol absorption inhibitor ezetimibe promoted RCT in mice, which lack cholesterol ester transfer protein (CETP), we investigated its effects in hamsters, which have CETP.A high-cholesterol diet (HC) increased cholesterol levels throughout lipoprotein fractions and ezetimibe markedly reduced VLDL/LDL cholesterol levels under both normal chow (NC) and HC. However, ezetimibe did not affect and reduced HDL-cholesterol levels under NC and HC, respectively. Intraperitoneal injection of 3H-cholesterol pre-labeled macrophages in an in vivo RCT assay increased tracer accumulation in the liver but reduced it in bile under HC, and these changes were completely cancelled by ezetimibe. Under both NC and HC, ezetimibe reduced tracer levels in the liver but increased them in feces, indicating promotion of RCT in vivo. We performed a RCT assay using hamsters subjected to bile duct ligation (BDL) to clarify whether a transintestinal cholesterol efflux (TICE) pathway contributes to ezetimibe's enhancement of RCT. BDL markedly inhibited macrophage-derived 3H-cholesterol excretion to feces and cancelled ezetimibe's stimulatory effect on RCT, suggesting that biliary cholesterol excretion is a major contributor in RCT promotion by ezetimibe but the contribution of the TICE pathway is minimal.In conclusions, ezetimibe exerts an additive anti-atherogenic property by enhancing RCT in hamsters. Our findings suggest that this property is independent of the TICE pathway.

Effects of Native and Myeloperoxidase-Modified Apolipoprotein A-I on Reverse Cholesterol Transport and Atherosclerosis in Mice

Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques. ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques. Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Discovery of novel indane derivatives as liver-selective thyroid hormone receptor β (TRβ) agonists for the treatment of dyslipidemia

Thyromimetics that specifically target TRβ have been shown to reduce plasma cholesterol levels and avoid atherosclerosis through the promotion of reverse cholesterol transport in an animal model. We designed novel thyromimetics with high receptor (TRβ) and organ (liver) selectivity based on the structure of eprotirome (3) and molecular modeling. We found that indane derivatives are potent and dual-selective thyromimetics expected to avoid hypothyroidism in some tissues as well as heart toxicity. KTA-439 (29), a representative indane derivative, showed the same high human TRβ selectivity in a binding assay as 3 and higher liver selectivity than 3 in a cholesterol-fed rat model.

Peroxisome Proliferator-Activated Receptor-α Activation Promotes Macrophage Reverse Cholesterol Transport Through a Liver X Receptor–Dependent Pathway

Peroxisome proliferator-activated receptor-α (PPARα) activation has been shown in vitro to increase macrophage cholesterol efflux, the initial step in reverse cholesterol transport (RCT). However, it remains unclear whether PPARα activation promotes macrophage RCT in vivo. We demonstrated that a specific potent PPARα agonist GW7647 inhibited atherosclerosis and promoted macrophage RCT in hypercholesterolemic mice expressing the human apolipoprotein A-I (apoA-I) gene. We compared the effect of GW7647 on RCT in human apoA-I transgenic (hA-ITg) mice with wild-type mice and showed that the PPARα agonist promoted RCT in hA-ITg mice to a much greater extent than in wild-type mice, indicating that human apoA-I expression is important for PPARα-induced RCT. We further investigated the dependence of the macrophage PPARα-liver X receptor (LXR) pathway on the promotion of RCT by GW7647. Primary murine macrophages lacking PPARα or LXR abolished the ability of GW7647 to promote RCT in hA-ITg mice. In concert, the PPARα agonist promoted cholesterol efflux and ATP binding cassette transporter A1/G1 expression in primary macrophages, and this was also by the PPARα-LXR pathway. Our observations demonstrate that a potent PPARα agonist promotes macrophage RCT in vivo in a manner that is enhanced by human apoA-I expression and dependent on both macrophage PPARα and LXR expression.

The protein cargo of HDL: Implications for vascular wall biology and therapeutics

High-density lipoprotein (HDL) is proposed to inhibit atherosclerosis via a number of different pathways, including promotion of reverse cholesterol transport and inhibition of inflammation. However, studies in both mice and humans suggest that quantifying HDL-cholesterol (HDL-C) levels provide limited information regarding the cardioprotective effects of HDL. This article briefly reviews current thinking regarding the functional and cardioprotective effects of HDL and the role of the HDL proteome in these processes.

High-Density Lipoprotein: Key Molecule in Cholesterol Efflux and the Prevention of Atherosclerosis

Accumulation of cholesterol by macrophages, leading to their transformation into foam cells is a key event in the initiation of atherosclerosis. As maintenance of cholesterol homeostasis in macrophages is essential to prevent foam cell formation, mechanisms by which macrophages export cellular cholesterol have been intensively investigated in recent years. Several epidemiological studies have shown that plasma levels of high-density lipoprotein (HDL) cholesterol are inversely correlated with the risk of atherosclerosis. The protective effect of HDL against macrophage foam cell formation and atherosclerosis is primarily attributed to its role in reverse cholesterol transport (RCT), a process by which excess cholesterol in peripheral tissues is transported to the liver for excretion. The present review discusses current knowledge on the biological activities of the major apolipoproteins, enzymes, lipid transfer proteins, receptors, and lipid transporters associated with HDL function and levels. In addition, current views on the molecular mechanisms underlying the atheroprotective functions of HDL beyond promotion of RCT, including the anti-oxidant, anti-thrombotic, anti-inflammatory and anti-apoptotic properties of HDL are summarized.

High-Density Lipoprotein-Mediated Anti-Atherosclerotic and Endothelial-Protective Effects: A Potential Novel Therapeutic Target in Cardiovascular Disease

Reduced levels of high-density lipoprotein cholesterol (HDL) are associated with a substantially increased risk of coronary disease and cardiovascular events. Furthermore, numerous studies have suggested that HDL may exert several potentially important antiatherosclerotic and endothelial-protective effects. In particular, the promotion of reverse cholesterol transport, i.e. cholesterol efflux from lipid-loaded macrophages in atherosclerotic lesions and the subsequent cholesterol transport back to the liver, has been proposed as an anti-atherogenic effect of HDL that may promote regression of atherosclerotic lesions. Moreover, endothelial dysfunction is thought to play a critical role in development and progression of atherosclerosis and several recent studies have suggested that HDL exerts direct endothelial-protective effects, such as stimulation of endothelial production of the anti-atherogenic molecule nitric oxide, anti-oxidant, anti-inflammatory and anti-thrombotic effects. Furthermore, it has been observed that HDL may stimulate endothelial repair processes, involving mobilisation and promotion of endothelial repair capacity of endothelial progenitor cells. The relative significance of these different potential anti-atherosclerotic effects of HDL remains still unclear at present. Importantly, at the same time it has been recognized that the vascular effects of HDL may be variable, i.e. the capacity of HDL to stimulate macrophage cholesterol efflux and endothelial-protective effects may be altered in patients with inflammatory or cardiovascular disease. The further characterisation of underlying mechanisms and the identification of the clinical relevance of this "HDL dysfunction" are currently an active field of research. HDL-targeted treatment strategies are at present intensely evaluated and may lead to increased HDL plasma levels and/or HDL-stimulated anti-atherosclerotic effects. The cardiovascular protection provided by such approaches may likely depend on HDL function or quality, i.e. the anti-atherosclerotic and endothelial-protective properties of the on-treatment HDL. Currently, several HDL-raising treatment strategies are examined in clinical trials, i.e. extended-release niacin, the CETP inhibitors dalcetrapib and anacetrapib, reconstituted forms of HDL (i.e. CSL-111) or apoA-I mimetics, and some of these are already in large clinical outcome studies on top of statin therapy to determine their efficacy and safety for cardiovascular prevention.

High-density lipoproteins: an emerging target in the prevention of cardiovascular disease

High-density lipoproteins (HDLs) have been well established to protect against the development of atherosclerotic cardiovascular disease. It has become apparent that in addition to the promotion of reverse cholesterol transport, HDLs possess a number of additional functional properties that may contribute to their beneficial influence on the arterial wall. A number of exciting therapeutic strategies have been developed that target HDL and its ability to protect against the development of atherosclerotic plaque. This paper will review how the promotion of the functional properties of HDL inhibits the formation of atherosclerotic plaque and stabilises lesions in patients with established disease.

High-density lipoproteins: a therapeutic target for atherosclerotic cardiovascular disease

Despite great progress being made during the last two decades in cardiovascular disease prevention, especially by lowering low-density lipoprotein-cholesterol with statins, cardiovascular events continue to occur. Plasma high-density lipoprotein (HDL) exerts multiple protective effects on the arterial wall, through promotion of reverse cholesterol transport, prevention of endothelial dysfunction and inhibition of lipid oxidation. Therapeutic interventions raising plasma HDL levels or directly mimicking its beneficial effects represent the next frontier in the prevention and treatment of cardiovascular disease.

Pharmacological Activation of Liver X Receptors Promotes Reverse Cholesterol Transport In Vivo

Liver X receptors (LXRs) are ligand-activated transcription factors involved in the control of lipid metabolism and inflammation. Synthetic LXR agonists have been shown to inhibit the progression of atherosclerosis in mice, but the mechanism is uncertain. LXR agonism upregulates the genes encoding ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) in macrophages, thus promoting efflux of cholesterol; it also upregulates liver and intestinal ABCG5 and ABCG8, helping to promote biliary and fecal excretion of cholesterol. Thus, LXR agonism may inhibit atherosclerosis through promotion of reverse cholesterol transport (RCT) in vivo, but this has not been proven. We previously described an in vivo method to trace the movement of cholesterol from 3H-cholesterol-labeled J774 macrophages into plasma, into liver, and ultimately into the bile and feces as free cholesterol or bile acids. In the present study we used this approach to test the hypothesis that administration of the synthetic LXR agonist GW3965 would increase the rate of macrophage RCT in vivo. Three different mouse models-wild-type C57BL/6 mice, LDLR/apobec-1 double knockout mice, and human apolipoprotein (apo)B/cholesteryl ester transfer protein (CETP) double transgenic mice-were treated with either vehicle or GW3965. Mice were injected intraperitoneally with 3H-cholesterol-labeled and cholesterol-loaded macrophages and monitored for the appearance of 3H-tracer in plasma, liver, and feces. Administration of GW3965 significantly increased the levels of 3H-tracer in plasma and feces in all 3 mouse models. These results demonstrate that administration of the LXR agonist GW3965 increases the rate of RCT from macrophages to feces in vivo.

W10-O-002 Catalyticallly active PLTP promotes HDL-facilitated cholesterol efflux from macrophage foam cells

The role of a high-affinity receptor site for high-density lipoproteins (HDL) has been investigated in parental Ob1771 adipose cells and their transformed counterparts after transfer of the complete early region of polyoma virus (Ob17PY cells). Binding of ApoAI, ApoAII and HDL3 occurs in Ob1771 cells and derived membranes, whereas no binding is observed in Ob17PY cells and derived membranes. After thymidine block, growth-arrested Ob17PY cells become able to bind ApoAI, ApoAII and HDL3; this recovery is prevented in actinomycin D- or cycloheximide-treated cells. In contrast to ApoAI, ApoAII or HDL3 binding, both growing and growth-arrested Ob17PY cells do show receptor activities for low density lipoproteins and transferrin, respectively, which are similar in affinity and maximal capacity. Following cholesterol accumulation which takes place in the presence of LDL cholesterol, subsequent exposure to HDL3 or ApoAI promotes cholesterol efflux from Ob1771 cells and growth-arrested Ob17PY cells but not from growing Ob17PY cells. These results show that the presence of a high-affinity receptor site for HDL in intact adipose cells is required for the promotion of reverse cholesterol transport.

Gene activation, apolipoprotein A-I/high density lipoprotein, atherosclerosis prevention and longevity.

Recent studies in man and human apolipoprotein A-I transgenic animals emphasize the significance of apolipoprotein A-I and high density lipoprotein in antiatherogenesis. Several drugs and other compounds, e.g. phenobarbital, gemfibrozil, fenofibrate, prednisone, estrogen and alcohol, induce apolipoprotein A-I synthesis. They commonly produce serum lipoprotein patterns typical of a low risk of coronary heart disease, and many of them have been found to prevent atherogenesis, reduce coronary heart disease mortality and increase survival. These compounds act against atherosclerosis by using one or several mechanisms that include overexpression of the apolipoprotein A-I gene with an increase in serum apolipoprotein A-I and high density lipoprotein and promotion of reverse cholesterol transport, upregulation of the low density lipoprotein receptor gene with a decrease in serum apolipoprotein B and low density lipoprotein, maintenance of endothelial cell function and protection against thrombosis. They have been found to raise high density lipoprotein cholesterol and apolipoprotein A-I together with a decrease in cholesterol ester transfer protein activity, and to induce hepatic cholesterol 7 alpha-hydroxylase and cholesterol and bile acid elimination from the body. By raising the activities of apolipoprotein A-I/high density lipoprotein-associated paraoxonase and other antioxidative enzymes, the inducers have the capacity to prevent atherogenesis in arterial walls through inhibition of the oxidative modification of low density lipoprotein. Other antiatherogenic vascular actions of high density lipoprotein include interference with low density lipoprotein aggregation and uptake by endothelial cells, and competition with low density lipoprotein for endothelial-localized low density lipoprotein receptors. Apolipoprotein A-I/high density lipoprotein beneficially enhances fibrinolysis, decreases platelet aggregation, increases prostacyclin production and stabilization and prevents atherogenic immune and inflammatory responses. This gene activation or microsomal induction can prevent atherosclerosis and is a basis for tailoring effective new agents and optimal non-invasive therapy against atherosclerotic vascular disease to promote health and enhance longevity.

Relationship in adipose cells between the presence of receptor sites for high density lipoproteins and the promotion of reverse cholesterol transport

The role of a high-affinity receptor site for high-density lipoproteins (HDL) has been investigated in parental Ob1771 adipose cells and their transformed counterparts after transfer of the complete early region of polyoma virus (Ob17PY cells). Binding of ApoA I, ApoA II and HDL 3 occurs in Ob1771 cells and derived membranes, whereas no binding is observed in Ob17PY cells and derived membranes. After thymidine block, growth-arrested Ob17PY cells become able to bind ApoA I, ApoA II and HDL 3; this recovery is prevented in actinomycin D- or cycloheximide-treated cells. In contrast to ApoA I, ApoA II or HDL 3 binding, both growing and growth-arrested Ob17PY cells do show receptor activities for low density lipoproteins and transferrin, respectively, which are similar in affinity and maximal capacity. Following cholesterol accumulation which takes place in the presence of LDL cholesterol, subsequent exposure to HDL 3 or ApoA I promotes cholesterol efflux from Ob1771 cells and growth-arrested Ob17PY cells but not from growing Ob17PY cells. These results show that the presence of a high-affinity receptor site for HDL in intact adipose cells is required for the promotion of reverse cholesterol transport.