Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.     

HDL-apolipoprotein A-I exchange is independently associated with cholesterol efflux capacity

HDL is the primary mediator of cholesterol mobilization from the periphery to the liver via reverse cholesterol transport (RCT). A critical first step in this process is the uptake of cholesterol from lipid-loaded macrophages by HDL, a function of HDL inversely associated with prevalent and incident cardiovascular disease. We hypothesized that the dynamic ability of HDL to undergo remodeling and exchange of apoA-I is an important and potentially rate-limiting aspect of RCT. In this study, we investigated the relationship between HDL-apoA-I exchange (HAE) and serum HDL cholesterol (HDL-C) efflux capacity. We compared HAE to the total and ABCA1-specific cholesterol efflux capacity of 77 subjects. We found that HAE was highly correlated with both total (r = 0.69, P < 0.0001) and ABCA1-specific (r = 0.47, P < 0.0001) efflux, and this relationship remained significant after adjustment for HDL-C or apoA-I. Multivariate models of sterol efflux capacity indicated that HAE accounted for approximately 25% of the model variance for both total and ABCA1-specific efflux. We conclude that the ability of HDL to exchange apoA-I and remodel, as measured by HAE, is a significant contributor to serum HDL efflux capacity, independent of HDL-C and apoA-I, indicating that HDL dynamics are an important factor in cholesterol efflux capacity and likely RCT.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

ABCA1-dependent serum cholesterol efflux capacity inversely correlates with pulse wave velocity in healthy subjects

The capacity of HDL to induce cell cholesterol efflux is considered one of its main antiatherogenic properties. Little is known about the impact of such HDL function on vascular physiology. We investigated the relationship between ABCA1-dependent serum cholesterol efflux capacity (CEC), an HDL functionality indicator, and pulse wave velocity (PWV), an indicator of arterial stiffness. Serum of 167 healthy subjects was used to conduct CEC measurement, and carotid-femoral PWV was measured with a high-fidelity tonometer. J774 macrophages, labeled with [³H]cholesterol and stimulated to express ABCA1, were exposed to sera; the difference between cholesterol efflux from stimulated and unstimulated cells provided specific ABCA1-mediated CEC. PWV is inversely correlated with ABCA1-dependent CEC (r = −0.183; P = 0.018). Moreover, controlling for age, sex, body mass index, mean arterial pressure, serum LDL, HDL-cholesterol, and fasting plasma glucose, PWV displays a significant negative regression on ABCA1-dependent CEC (β = −0.204; 95% confidence interval, −0.371 to −0.037). The finding that ABCA1-dependent CEC, but not serum HDL cholesterol level (r = −0.002; P = 0.985), is a significant predictor of PWV in healthy subjects points to the relevance of HDL function in vascular physiology and arterial stiffness prevention.     

The LXR agonist T0901317 promotes the reverse cholesterol transport from macrophages by increasing plasma efflux potential

The liver X receptors (LXRs) have been shown to affect lipoprotein plasma profile, lipid metabolism, and reverse cholesterol transport (RCT). In the present study, we investigated whether a short-term administration of the synthetic LXR agonist T0901317 (T0) to mice may affect RCT by modulating the capacity of plasma to promote cellular lipid efflux. Consistent with previous data, the pharmacological treatment of mice caused a significant increase of macrophage-derived [3H]cholesterol content in plasma, liver, and feces and resulted in improved capacity of plasma to promote cellular cholesterol release through passive diffusion and scavenger receptor class B type I (SR-BI)-mediated mechanisms. Differently, plasma from treated mice possessed similar or reduced capacity to drive lipid efflux via ABCA1. Consistent with these data, the analysis of plasma HDL fractions revealed that T0 caused the formation of larger, lipid-enriched particles. These results suggest that T0 promotes in vivo RCT from macrophages at least in part by inducing an enrichment of those HDL subclasses that increase plasma capacity to promote cholesterol efflux by passive diffusion and SR-BI-mediated mechanisms.     

Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.     

Plasma cholesterol efflux capacity from human THP-1 macrophages is reduced in HIV-infected patients: impact of HAART

The capacity of HDL to remove cholesterol from macrophages is inversely associated with the severity of angiographic coronary artery disease. The effect of human immunodeficiency virus (HIV) infection or its treatment on the ability of HDL particles to stimulate cholesterol efflux from human macrophages has never been studied. We evaluated the capacity of whole plasma and isolated HDL particles from HIV-infected subjects (n = 231) and uninfected controls (n = 200), as well as in a subset of 41 HIV subjects receiving highly active antiretroviral therapy (HAART) to mediate cholesterol efflux from human macrophages. Plasma cholesterol efflux capacity was reduced (−12%; P = 0.001) in HIV patients as compared with controls. HIV infection reduced by 27% (P < 0.05) the capacity of HDL subfractions to promote cholesterol efflux from macrophages. We observed a reduced ABCA1-dependent efflux capacity of plasma (−27%; P < 0.0001) from HIV-infected subjects as a result of a reduction in the efflux capacity of HDL3 particles. HAART administration restored the capacity of plasma from HIV patients to stimulate cholesterol efflux from human macrophages (9.4%; P = 0.04). During HIV infection, the capacity of whole plasma to remove cholesterol from macrophages is reduced, thus potentially contributing to the increased coronary heart disease in the HIV population. HAART administration restored the removal of cholesterol from macrophages by increasing HDL functionality.     

Purified human paraoxonase-1 interacts with plasma membrane lipid rafts and mediates cholesterol efflux from macrophages

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant and anti-inflammatory capacities of HDLs. However, the role of PON1 in the modulation of cholesterol efflux is poorly understood. The aim of our study was to investigate the involvement of PON1 in the regulation of cholesterol efflux, especially the mechanism by which it modulates HDL-mediated cholesterol transport. The enrichment of HDL(3) with human PON1 enhanced, in a dose-dependent manner, cholesterol efflux from THP-1 macrophage-like cells and ABCA1-enriched J774 macrophages. Moreover, an additive effect was observed when ABCA1-enriched J774 macrophages were incubated with both PON1 and apo-AI. Interestingly, PON1 alone was able to mediate cholesterol efflux from J774 macrophages and to upregulate ABCA1 expression on J774 macrophages. Immunofluorescence measurement showed an increase in PON1 levels in the cytoplasm of J774 macrophages overexpressing ABCA1. PON1 used an apo-AI-like mechanism to modulate cholesterol efflux from rapid and slow efflux pools derived from the lipid raft and nonraft domains of the plasma membrane, respectively. This was supported by the fact that ABCA1 protein was incrementally expressed by J774 macrophages within the first few hours of incubation with cholesterol-loaded J774 macrophages and that cyclodextrin significantly inhibited the capacity of PON1 to modulate cholesterol efflux from macrophages. This finding suggested that PON1 plays an important role in the antiatherogenic properties of HDLs and may exert its protective function outside the lipoprotein environment.     

Depletion of pre-beta-high density lipoprotein by human chymase impairs ATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipid efflux to high density lipoprotein

The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cellular unesterified cholesterol and phospholipid to lipid-poor apolipoprotein A-I. Chymase, a protease secreted by mast cells, selectively cleaves pre-beta-migrating particles from high density lipoprotein (HDL)(3) and reduces the efflux of cholesterol from macrophages. To evaluate whether this effect is the result of reduction of ABCA1-dependent or -independent pathways of cholesterol efflux, in this study we examined the efflux of cholesterol to preparations of chymase-treated HDL(3) in two types of cell: 1) in J774 murine macrophages endogenously expressing low levels of scavenger receptor class B, type I (SR-BI), and high levels of ABCA1 upon treatment with cAMP; and 2) in Fu5AH rat hepatoma cells endogenously expressing high levels of the SR-BI and low levels of ABCA1. Treatment of HDL(3) with the human chymase resulted in rapid depletion of pre-beta-HDL and a concomitant decrease in the efflux of cholesterol and phospholipid (2-fold and 3-fold, respectively) from the ABCA1-expressing J774 cells. In contrast, efflux of free cholesterol from Fu5AH to chymase-treated and to untreated HDL(3) was similar. Incubation of HDL(3) with phospholipid transfer protein led to an increase in pre-beta-HDL contents as well as in ABCA1-mediated cholesterol efflux. A decreased cholesterol efflux to untreated HDL(3) but not to chymase-treated HDL(3) was observed in ABCA1-expressing J774 with probucol, an inhibitor of cholesterol efflux to lipid-poor apoA-I. Similar results were obtained using brefeldin and gliburide, two inhibitors of ABCA1-mediated efflux. These results indicate that chymase treatment of HDL(3) specifically impairs the ABCA1-dependent pathway without influencing either aqueous or SR-BI-facilitated diffusion and that this effect is caused by depletion of lipid-poor pre-beta-migrating particles in HDL(3). Our results are compatible with the view that HDL(3) promotes ABCA1-mediated lipid efflux entirely through its lipid-poor fraction with pre-beta mobility.     

Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRα and PPARγ was increased by APN. In APN-KO mice, the expression of ABCA1, LXRα, PPARγ, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRα and PPARγ.     

Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1

Moderate alcohol consumption increases HDL cholesterol, which is involved in reverse cholesterol transport (RCT). The aim of this study was to investigate the effect of moderate alcohol consumption on cholesterol efflux, using J774 mouse macrophages and Fu5AH cells, and on other parameters in the RCT pathway. Twenty-three healthy men (45-65 years) participated in a randomized, partially diet-controlled, crossover trial. They consumed four glasses of whisky (40 g of alcohol) or water daily for 17 days. After 17 days of whisky consumption, serum capacity to induce ABCA1-dependent cholesterol efflux from J774 mouse macrophages was increased by 17.5% (P = 0.027) compared with water consumption. Plasma capacity to induce cholesterol efflux from Fu5AH cells increased by 4.6% (P = 0.002). Prebeta-HDL, apolipoprotein A-I (apoA-I), and lipoprotein A-I:A-II also increased by 31.6, 6.2, and 5.7% (P < 0.05), respectively, after whisky consumption compared with water consumption. Changes of cAMP-stimulated cholesterol efflux correlated (r = 0.65, P < 0.05) with changes of apoA-I but not with changes of prebeta-HDL (r = 0.30, P = 0.18). Cholesterol efflux capacities from serum of lean men were higher than those from overweight men. In conclusion, this study shows that moderate alcohol consumption increases the capacity of serum to induce cholesterol efflux from J774 mouse macrophages, which may be mediated by ABCA1.     

Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function

Objective

Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor–γ (PPARγ).

Methods and Results

Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2–3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = −0.41, p<0.001; rho = −0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = −0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression.

Conclusions

ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia- related, persistent disruption of a key component of RCT.     

Zymosan-mediated inflammation impairs in vivo reverse cholesterol transport

Inflammation has been proposed to impair HDL function and reverse cholesterol transport (RCT). We investigated the effects of inflammation mediated by zymosan, a yeast glucan, on multiple steps along the RCT pathway in vivo and ex vivo. Acute inflammation with 70 mg/kg zymosan impaired RCT to plasma, liver, and feces similarly by 17-22% (P < 0.05), with no additional block at the liver. Hepatic gene expression further demonstrated no change in ABCG5, ABCB4, and ABCB11 expression but a decline in ABCG8 mRNA (32% P < 0.05). Plasma from zymosan-treated mice had a 21% decrease in cholesterol acceptor ability (P < 0.01) and a 35% decrease in ABCA1-specific efflux capacity (P < 0.01) in vitro. Zymosan treatment also decreased HDL levels and led to HDL remodeling with increased incorporation of serum amyloid A. In addition, cholesterol efflux from cultured macrophages declined with zymosan treatment in a dose dependent manner. Taken together, our results suggest that zymosan impairs in vivo RCT primarily by decreasing macrophage-derived cholesterol entering the plasma, with minimal additional blocks downstream. Our study supports the notion that RCT impairment is one of the mechanisms for the increased atherosclerotic burden observed in inflammatory conditions.     

ATP binding cassette transporter A1 - key roles in cellular lipid transport and atherosclerosis

ATP-binding cassette transporter A1 (ABCA1) was recently recognized as the mutant molecule responsible for Tangier disease with low HDL levels, accumulation of cholesteryl esters in tissues, and increased risk of cardiovascular disease. Extensive studies for the past 2 years have recognized the critical role of ABCA1 in cholesterol and phospholipid trafficking. Since the removal of cholesterol from tissues is a key step in the prevention of atherosclerosis, significant attention has been focused on this molecule. Natural ABCA1 mutations in Tangier disease (TD) patients and WHAM chickens together with induced mutation in ABCA1 knock-out mice unequivocally established the important role of ABCA1 in maintaining circulating HDL levels and promoting cholesterol efflux from the arterial wall. Mice lacking ABCA1 showed similar phenotypes observed in Tangier disease patients with low levels of HDL. Further understanding of the roles of ABCA1 in lipid transport and atherosclerosis became clear from studies with ABCA1 transgenic mice. These mice showed enhanced cholesterol efflux from macrophages and reduced atherosclerotic lesion formation. The promoter of the ABCA1 gene has been mapped to a large extent, with the exception of cAMP response element. The present review summarizes recent developments on the role of ABCA1 in cholesterol efflux and prevention of atherosclerosis. Given the antiatherogenic properties of ABCA1, this molecule can serve as an appropriate target for developing drugs to treat individuals with low levels of HDL.     

Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis

PURPOSE OF REVIEW: The appearance of scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in macrophages and liver implicates these transporters in different stages of reverse cholesterol transport. This review focuses on the role of SR-BI and ABCA1 in reverse cholesterol transport in the context of atherosclerotic lesion development. RECENT FINDINGS: Recent studies indicate that hepatic expression of ABCA1 and SR-BI is important for the generation of nascent HDL and the delivery of HDL cholesteryl esters to the liver, respectively. Although macrophage SR-BI and ABCA1 do not contribute significantly to circulating HDL levels, the perpetual cycle of HDL lipidation and delipidation by the liver ensures the availability of acceptors for cholesterol efflux that maintain cholesterol homeostasis in arterial macrophages, thereby reducing atherogenesis. In addition to its established role in the selective uptake of HDL cholesteryl esters, there is now evidence that hepatic SR-BI facilitates postprandial lipid metabolism, and that hepatic secretion of VLDL is dependent on ABCA1-mediated nascent HDL formation. Thus, remnant and HDL metabolism are more intimately intertwined in hepatic lipid metabolism than has previously been appreciated. SUMMARY: Recent advances in the understanding of the role of ABCA1 and SR-BI in HDL metabolism and their atheroprotective properties indicate the significant potential of modulating ABCA1 and SR-BI expression in both arterial wall macrophages and the liver for the treatment of atherosclerotic coronary artery disease.     

Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes

Adipose tissue constitutes a major location for cholesterol storage and, as such, it may play a role in the regulation of circulating cholesterol levels. A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high density lipoprotein, HDL, was investigated in 3T3-L1 adipocytes. In the presence of HDL, composed of human apoA-I and phosphatidylcholine, adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion. β-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles. Activation of lipolysis promotes a rapid decrease in the cholesterol content of the plasma membrane and a concomitant increase in lipid droplet cholesterol. This change is independent of the presence of HDL. Activation of the lipolysis does not affect the levels of ABCA1 and SR-BI. Therefore, the enhancement of cholesterol efflux is not due to the level of plasma membrane cholesterol, or to the levels of the cholesterol transporters ABCA1 and scavenger receptor SR-BI. Brefeldin A did not affect the rate of cholesterol efflux under basal lipolytic conditions, but it abolished the lipolysis-dependent enhancement of cholesterol efflux to HDL. This study suggests that activation of lipolysis is accompanied by an increase in BFA-sensitive vesicular transport that in turn enhances cholesterol efflux to HDL. The study supports a metabolic link between the lipolytic activity of adipocytes and the rate of cellular cholesterol efflux to HDL.