Ilaria Zanotti

Overexpression of Apolipoprotein A-I Promotes Reverse Transport of Cholesterol From Macrophages to Feces In Vivo

Background—Abundant data indicate that overexpression of apolipoprotein A-I (apoA-I) in mice inhibits atherosclerosis. One mechanism is believed to be promotion of reverse cholesterol transport, but no direct proof of this concept exists. We developed a novel approach to trace reverse transport of labeled cholesterol specifically from macrophages to the liver and feces in vivo and have applied this approach to investigate the ability of apoA-I overexpression to promote macrophage-specific reverse cholesterol transport. Method and Results—J774 macrophages were loaded with cholesterol by incubation with acetylated LDL, labeled with 3H-cholesterol, and then injected intraperitoneally into mice. Plasma and feces were collected at 24 hours and 48 hours, when mice were exsanguinated, tissues were harvested, and all were analyzed for tracer counts. 3H-cholesterol was found in the plasma, liver, and feces. For apoA-I overexpression, mice were injected intravenously with apoA-I adenovirus (1011 particles per animal) 3 days before labeled macrophages were injected. ApoA-I overexpression led to significantly higher 3H-cholesterol in plasma, liver, and feces. The amount of 3H-tracer in the liver was 35% higher (P <0.05) and the 3H-tracer excreted into feces over 48 hours was 63% higher (P <0.05) in apoA-I–expressing mice than in control mice. Conclusion—Injection of 3H-cholesterol–labeled macrophage foam cells is a method of measuring reverse cholesterol transport specifically from macrophages to feces in vivo, and apoA-I overexpression promotes macrophage-specific reverse cholesterol transport.

Probucol Inhibits ABCA1-Mediated Cellular Lipid Efflux

Objective—ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of lipids from cells to lipid-poor apolipoproteins. In this article, we characterize the effect of probucol on cellular ABCA1-mediated lipid efflux. Methods and Results—Probucol inhibited cholesterol efflux up to 80% in J774 macrophages expressing ABCA1. In Fu5AH hepatoma cells that contain scavenger receptor class B, type I, but not functional ABCA1, we observed no effect of probucol on cholesterol efflux. Probucol inhibited cholesterol efflux from normal human skin fibroblasts but not from fibroblasts from a Tangier patient. Fluorescent confocal microscopy and biotinylation assay demonstrated that in J774 cells probucol impaired the translocation of ABCA1 from intracellular compartments to the plasma membrane. Probucol also inhibited the formation of an ABCA1-linked cholesterol oxidase sensitive plasma membrane domain. Consistent with the inhibitory effect on ABCA1 translocation to the plasma membrane, probucol reduced cell surface–specific [125I]-labeled apolipoprotein-AI binding. Conclusions—We conclude that probucol is an effective inhibitor of ABCA1-mediated cholesterol efflux without influencing scavenger receptor class B type I–mediated efflux. The inhibition of ABCA1 translocation to the plasma membrane may in part explain the reported in vivo high-density lipoprotein–lowering action of probucol.

Relative Contributions of ABCA1 and SR-BI to Cholesterol Efflux to Serum From Fibroblasts and Macrophages

Objectives—Cholesterol efflux is achieved by several mechanisms. This study examines contributions of these pathways to efflux to human serum. Methods and Results—Human fibroblasts were stably transfected with SR-BI while ABCA1 was upregulated. Quantitation of cholesterol efflux to human serum demonstrated that there was efflux from cells without either protein. Expression of ABCA1 produced a small increase in efflux, whereas SR-BI expression had a dramatic impact. To quantitate ABCA1 and SR-BI contribution, fibroblasts were pretreated with Probucol and BLT-1 to, respectively, inhibit these efflux proteins. Exposing SR-BI–expressing fibroblasts to BLT-1 inhibited efflux by 67%. Probucol pretreatment of ABCA1-expressing fibroblasts reduced efflux to serum by 26%. A large fraction of total efflux was uninhibited. For both J774 and mouse peritoneal macrophages, contributions of either ABCA1 or SR-BI to efflux to serum were low, with background/uninhibited efflux contributing from 70% to 90% of total efflux. Conclusions—We have shown that ABCA1-mediated efflux to serum responds to the pool of lipid-free/poor apolipoproteins, whereas phospholipid-containing particles mediate SR-BI efflux. Although SR-BI and ABCA1 contribute to efflux from fibroblasts and cholesterol-enriched macrophages, a large proportion of the total efflux to human serum is mediated by a mechanism that is neither SR-BI nor ABCA1.

A unique protease-sensitive high density lipoprotein particle containing the apolipoprotein A-I(Milano) dimer effectively promotes ATP-binding Cassette A1-mediated cell cholesterol efflux

Carriers of the apolipoprotein A-IMilano (A-IM) variant present with severe reductions of plasma HDL levels, not associated with premature coronary heart disease (CHD). Sera from 14 A-IM carriers and matched controls were compared for their ability to promote ABCA1-driven cholesterol efflux from J774 macrophages and human fibroblasts. When both cell types are stimulated to express ABCA1, the efflux of cholesterol through this pathway is greater with A-IM than control sera (3.4 ± 1.0% versus 2.3 ± 1.0% in macrophages; 5.2 ± 2.4% versus 1.9 ± 0.1% in fibroblasts). A-IM and control sera are instead equally effective in removing cholesterol from unstimulated cells and from fibroblasts not expressing ABCA1. The A-IM sera contain normal amounts of apoA-I-containing preβ-HDL and varying concentrations of a unique small HDL particle containing a single molecule of the A-IM dimer; chymase treatment of serum degrades both particles and abolishes ABCA1-mediated cholesterol efflux. The serum content of chymase-sensitive HDL correlates strongly and significantly with ABCA1-mediated cholesterol efflux (r = 0.542, p = 0.004). The enhanced capacity of A-IM serum for ABCA1 cholesterol efflux is thus explained by the combined occurrence in serum of normal amounts of apoA-I-containing preβ-HDL, together with a unique protease-sensitive, small HDL particle containing the A-IM dimer, both effective in removing cell cholesterol via ABCA1.

Macrophage, But Not Systemic, Apolipoprotein E Is Necessary for Macrophage Reverse Cholesterol Transport In Vivo

Objective—To assess the role of apolipoprotein (apo) E in macrophage reverse cholesterol transport (RCT) in vivo. Methods and Results—ApoE exerts an antiatherosclerotic activity by regulating lipoprotein metabolism and promoting cell cholesterol efflux. We discriminated between macrophage and systemic apoE contribution using an assay of macrophage RCT in mice. The complete absence of apoE lead to an overall impairment of the process and, similarly, the absence of apoE exclusively in macrophages resulted in the reduction of cholesterol mobilization from macrophages to plasma, liver, and feces. Conversely, expression of apoE in macrophages is sufficient to promote normal RCT even in apoE-deficient mice. The mechanisms accounting for these results were investigated by evaluating the first step of RCT (ie, cholesterol efflux from cells). Macrophages isolated from apoE-deficient mice showed a reduced ability to release cholesterol into the culture medium, whereas the apoB-depleted plasma from apoE-deficient and healthy mice possessed a similar capacity to promote cellular lipid release from cultured macrophages. Conclusion—Our data demonstrate, for the first time to our knowledge, that apoE significantly contributes to macrophage RCT in vivo and that this role is fully attributable to apoE expressed in macrophages.

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.

Cholesterol Efflux and Reverse Cholesterol Transport

Both alterations of lipid/lipoprotein metabolism and inflammatory events contribute to the formation of the atherosclerotic plaque, characterized by the accumulation of abnormal amounts of cholesterol and macrophages in the artery wall. Reverse cholesterol transport (RCT) may counteract the pathogenic events leading to the formation and development of atheroma, by promoting the high-density lipoprotein (HDL)-mediated removal of cholesterol from the artery wall. Recent in vivo studies established the inverse relationship between RCT efficiency and atherosclerotic cardiovascular diseases (CVD), thus suggesting that the promotion of this process may represent a novel strategy to reduce atherosclerotic plaque burden and subsequent cardiovascular events. HDL plays a primary role in all stages of RCT: (1) cholesterol efflux, where these lipoproteins remove excess cholesterol from cells; (2) lipoprotein remodeling, where HDL undergo structural modifications with possible impact on their function; and (3) hepatic lipid uptake, where HDL releases cholesterol to the liver, for the final excretion into bile and feces. Although the inverse association between HDL plasma levels and CVD risk has been postulated for years, recently this concept has been challenged by studies reporting that HDL antiatherogenic functions may be independent of their plasma levels. Therefore, assessment of HDL function, evaluated as the capacity to promote cell cholesterol efflux may offer a better prediction of CVD than HDL levels alone. Consistent with this idea, it has been recently demonstrated that the evaluation of serum cholesterol efflux capacity (CEC) is a predictor of atherosclerosis extent in humans.

Cellular Cholesterol Efflux Pathways: Impact on Intracellular Lipid Trafficking and Methodological Considerations

Intracellular lipid homeostasis is regulated by multiple mechanisms devoted to the tight control of cholesterol levels. Cholesterol efflux to extracellular acceptors represents a cellular response to excess accumulation of lipids that occurs by both passive and active processes and was shown to exert a beneficial, antiatherosclerotic activity. Up to now 3 lipid transporters responsible for cholesterol removal from cells have been characterized: ATP Binding Cassette A1 (ABCA1), ATP Binding Cassette G1 (ABCG1) and Scavenger Receptor Class B Type I (SR-BI). These proteins widely differ in the pathway of efflux they mediate, as well as in the nature of extracellular acceptors they interact with. The experimental investigation of cholesterol efflux pathways can be efficiently performed in vitro, following precise criteria in the selection of cell types and extracellular acceptors to specifically investigate the mechanism involved. The aim of this review is to describe how lipid transporter-mediated cholesterol efflux influences the intracellular trafficking of cholesterol and to provide methodological considerations for experimental evaluation of this process.

Therapeutic perspectives of Eph–ephrin system modulation

Eph receptors are the largest class of kinase receptors and, together with their ligands ephrins, they have a primary role in embryogenesis. Their expression has been found deregulated in several cancer tissues and, in many cases, abnormal levels of these proteins have been correlated to a poor prognosis. Recently, the Eph-ephrin system was found to be deregulated in other pathological processes, involving the nervous and cardiovascular systems. The increasing body of evidence supports the Eph-ephrin system as a target not only for the treatment of solid tumors, but also to face other critical diseases such as amyotrophic lateral sclerosis and diabetes driving current efforts toward the development of pharmacological tools potentially able to treat these pathologies.

Severe HDL deficiency due to novel defects in the ABCA1 transporter.

Objectives.  The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency.