Nascent High-density Lipoprotein Research Articles

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Abstract 1025: Structural Basis Of Apolipoprotein A1 Activation Of Lecithin:Cholesterol Acyl Transferase In High Density Lipoproteins

Lecithin:cholesterol acyl transferase (LCAT) is responsible for the formation of extracellular cholesteryl ester, which is critical for high density lipoprotein (HDL) maturation. It also modulates numerous HDL physiological functions by impacting particle subspeciation patterns. LCAT is activated by apolipoprotein A1 (APOA1) which forms antiparallel dimers around the edge of nascent HDL. Previous work has suggested that the interface of APOA1 helices 4 and 6, when properly aligned, creates two symmetrical sites for productive LCAT binding. Size exclusion chromatography was used to isolate reconstituted HDL discs containing two molecules of APOA1 chemically crosslinked to 2 LCAT molecules. Mass spectrometry using multiple crosslinkers, including a novel application of formaldehyde, indicated that LCAT interacts with both APOA1 helices 4 and 6. Cryo-electron microscopy (cryoEM) was used to determine a 10 Å structure of the complex. Using the HADDOCK suite of molecular docking tools, we developed a structural model of LCAT bound to APOA1 discs that is consistent with the preponderance of data accumulated so far. The model shows that LCAT may bind at the two symmetrical sites in different orientations, possibly reflecting different steps of the LCAT reaction. Work to improve the resolution of the cryoEM datasets is ongoing. Further work, using strategically designed cysteine mutations in APOA1 to lock helices 4 and 6 together, suggests that the helices do not move apart during the LCAT reaction process. Determination of the structural interactions between LCAT and its activator, APOA1, is crucial to understanding HDL maturation, which has significant clinical implications. This work provides the highest resolution structural model available for this important interaction.

Abstract 114: Inhibition Of Lecithin Cholesterol Acyl Transferase By A Novel Anti-apolipoprotein A1 Antibody Fragment

Apolipoprotein A1 (APOA1), the most abundant protein in high-density lipoproteins (HDL), is the primary activator of lecithin cholesterol acyl transferase (LCAT). LCAT’s cholesteryl esterification activity is key for maturing nascent HDL discs into the spherical forms commonly found in plasma. However, the molecular mechanism for APOA1 activation of LCAT is poorly understood. Using phage display selection against nanodiscs (lipid complexes stabilized by an APOA1 variant commonly used in transmembrane protein structural biology), we have identified, isolated, and expressed a novel Fab fragment (Fab16). Size exclusion chromatography elution shift experiments showed that Fab16 readily bound reconstituted HDL (rHDL) particles containing human purified plasma APOA1 with nearly quantitative binding at a 1:1 molar ratio of Fab16 to APOA1. Negative stain electron microscopy (NSEM), confirmed that 1-2 Fab16 bound to each rHDL particle in a specific pattern around the disc periphery. Additionally, Fab16 effectively bound both large and small spherical HDL particles isolated from human plasma. Chemical cross-linking / mass spectrometry experiments indicated that Fab16 binds an APOA1 epitope between residues 94 and 118 (i.e., within amphipathic helix 4). As our previous work has shown that APOA1 helix 4 is important for LCAT activation, we hypothesized that Fab16 would inhibit LCAT activity. Using a novel gas chromatography cholesterol esterification assay, we found that Fab16 applied at a 1.5:1 molar ratio to APOA1 inhibited LCAT cholesterol esterification activity by 56±7%. The symmetrical nature of both discoidal and spherical HDL particles has hampered the application of cryogenic electron microscopy (cryo EM) for understanding HDL structure. Fab16 may provide a valuable ‘landmark’ to assist in deriving a high-resolution structure of the complex. Work to optimize conditions for collecting high quality cryo EM datasets of discoidal and spherical HDL in the presence and absence of LCAT is ongoing. Deriving a high-resolution understanding of HDL structure and its interaction with remodeling factors such LCAT has been a long-standing problem but is crucial for deriving new strategies to manipulate HDL function for therapeutic benefit.

Integrated meta-omics reveals the regulatory landscape involved in lipid metabolism between pig breeds

BackgroundDomesticated pigs serve as an ideal animal model for biomedical research and also provide the majority of meat for human consumption in China. Porcine intramuscular fat content associates with human health and diseases and is essential in pork quality. The molecular mechanisms controlling lipid metabolism and intramuscular fat accretion across tissues in pigs, and how these changes in response to pig breeds, remain largely unknown.ResultsWe surveyed the tissue-resident cell types of the porcine jejunum, colon, liver, and longissimus dorsi muscle between Lantang and Landrace breeds by single-cell RNA sequencing. Combining lipidomics and metagenomics approaches, we also characterized gene signatures and determined key discriminating markers of lipid digestibility, absorption, conversion, and deposition across tissues in two pig breeds. In Landrace, lean-meat swine mainly exhibited breed-specific advantages in lipid absorption and oxidation for energy supply in small and large intestinal epitheliums, nascent high-density lipoprotein synthesis for reverse cholesterol transport in enterocytes and hepatocytes, bile acid formation, and secretion for fat emulsification in hepatocytes, as well as intestinal-microbiota gene expression involved in lipid accumulation product. In Lantang, obese-meat swine showed a higher synthesis capacity of chylomicrons responsible for high serum triacylglycerol levels in small intestinal epitheliums, the predominant characteristics of lipid absorption in muscle tissue, and greater intramuscular adipcytogenesis potentials from muscular fibro-adipogenic progenitor subpopulation.ConclusionsThe findings enhanced our understanding of the cellular biology of lipid metabolism and opened new avenues to improve animal production and human diseases.D7xkEMpXE5aNHjWuhXhcDpVideo

The ATPase activity of ABCA1 is increased by cholesterol in the presence of anionic lipids.

High-density lipoprotein (HDL) transports excess cholesterol from peripheral tissues back to the liver, and plasma HDL levels are inversely related to cardiovascular disease incidence. ATP-binding cassette A1 (ABCA1) is a member of the ABC protein superfamily, and generates nascent HDL, which consists of several hundreds of phospholipids and cholesterol wrapped by apolipoprotein A-I (apoA-I). However, it remains unclear whether cholesterol is a transport substrate of ABCA1. Since ATP hydrolysis of ABC proteins is typically increased by their transport substrates, we characterized the effects of cholesterol on the ATPase activity of purified ABCA1 using liposomes of various lipid compositions. ABCA1 showed substantial ATPase activity (20-30 nmol∙min-1∙mg-1) only in liposomes containing anionic lipids, including phosphatidylserine. Cholesterol increased the ATPase activity by 1.6- to 3-fold in the presence of anionic lipids. Moreover, phosphatidylserine addition to BHK/ABCA1 cells increased phosphatidylcholine and cholesterol efflux to apoA-I. Next, we investigated the sterol specificity of ABCA1. The ATPase activity of ABCA1 was strongly enhanced by desmosterol and zymosterol, similar to cholesterol. In contrast, 7-dehydrocholesterol and lathosterol weakly increased the ATPase activity, and no increase was observed with stigmasterol or brassicasterol. These findings suggest that ABCA1 transports cholesterol, and prefers cholesterol over plant sterols as a transport substrate.

Applications of Synthetic Polymer Discoidal Lipid Nanoparticles to Biomedical Research.

Liposomes are artificially prepared vesicular lipid nanoparticles with a bilayer structure, resembling cell membrane. Their ability to encapsulate various molecules along with excellent biocompatibility makes them ideal delivery vehicles for pharmaceuticals. They can also serve as platforms for membrane proteins to elucidate the structure and function in lipid membranes. Nascent high-density lipoproteins are discoidal lipid nanoparticles with a bilayer structure, which can be reconstituted with their constituents. Such reconstituted nanoparticles, nanodisks, were originally generated in terms of elucidation for mechanisms of lipoprotein metabolisms. At the same time, like liposomes, nanodisks have been developed as delivery vehicles and platforms for membrane proteins in structural biology. From a developmental background, apolipoproteins, their analogs, or fragment peptides were initially used as scaffolding molecules to wrap around the edge of the disk-shaped lipid bilayer. Since the discovery that styrene-maleic acid copolymers produce nanodisks instead of apolipoproteins, variously modified or novel polymers have been synthesized to broaden the applications of polymer nanodisks. This review provides an overview of the types of synthetic polymers used to produce nanodisks, and the biomedical applications of nanodisks to the developments of delivery vehicles and to the structural studies of membrane proteins.

Different Pathways of Cellular Cholesterol Efflux.

Cholesterol efflux is the first and rate-limiting step of reverse cholesterol transport (RCT) from peripheric cells to the liver. The involvement of high-density lipoprotein (HDL) in RCT determines the atheroprotective properties of HDL. Cholesterol efflux from different membrane pools includes both passive and energy-dependent processes. The first type of route consists of cholesterol desorption from the cell membrane into the unstirred layer adjacent to the cell surface and diffusion in the water phase. Moreover, the selective uptake and facilitated diffusion of cholesterol and cholesteryl ester molecules through the hydrophobic tunnel in the scavenger receptor BI molecule does not require energy consumption. The second type of route includes active cholesterol export by the ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Several cholesterol acceptors specifically bind cholesterol and phospholipid molecules, and cholesterol binding to the albumin molecule, which acts as a shuttle, significantly increases cholesterol movement between acceptors and red blood cells, thus functioning as a sink for cholesterol. Cholesterol and phospholipid molecules effluxed from macrophages by ABCA1 are accepted exclusively by the lipid-free apolipoprotein apoA-I, which is the major protein moiety of HDL, whereas those effluxed by ABCG1 are accepted by HDL. ABCA1- and ABCG1-mediated cholesterol transport, together with cholesterol diffusion, largely determine cholesterol turnover at the physiological level of intracellular cholesterol. However, at cholesterol overload, ABCA1-mediated efflux prevails over other routes. The exchange of apoA-I between lipid-free and lipid-associated states and the synergism of nascent and mature HDL contribute to cholesterol efflux efficiency. Moreover, extracellular cholesterol deposits and microvesicles may be involved in RCT.

Role of ABCA1 in Cardiovascular Disease.

Cholesterol homeostasis plays a significant role in cardiovascular disease. Previous studies have indicated that ATP-binding cassette transporter A1 (ABCA1) is one of the most important proteins that maintains cholesterol homeostasis. ABCA1 mediates nascent high-density lipoprotein biogenesis. Upon binding with apolipoprotein A-I, ABCA1 facilitates the efflux of excess intracellular cholesterol and phospholipids and controls the rate-limiting step of reverse cholesterol transport. In addition, ABCA1 interacts with the apolipoprotein receptor and suppresses inflammation through a series of signaling pathways. Thus, ABCA1 may prevent cardiovascular disease by inhibiting inflammation and maintaining lipid homeostasis. Several studies have indicated that post-transcriptional modifications play a critical role in the regulation of ABCA1 transportation and plasma membrane localization, which affects its biological function. Meanwhile, carriers of the loss-of-function ABCA1 gene are often accompanied by decreased expression of ABCA1 and an increased risk of cardiovascular diseases. We summarized the ABCA1 transcription regulation mechanism, mutations, post-translational modifications, and their roles in the development of dyslipidemia, atherosclerosis, ischemia/reperfusion, myocardial infarction, and coronary heart disease.

A Review: Molecular Mechanism of Regulation of ABCA1 Expression.

ATP-binding cassette subfamily A member 1 (ABCA1) protein plays an essential role in a variety of events, such as cholesterol and phospholipid efflux, nascent high-density lipoprotein (HDL) biosynthesis, phospholipid translocation. Thus, there has been much research activity aimed at understanding the molecular mechanisms of regulating ABCA1 expression. In this review, we first discuss ABCA1 structure, tissue distribution, cellular localization, and trafficking, as well as its function. Furthermore, current understanding of the molecular mechanisms involved in the regulation of ABCA1 expression is summarized. ABCA1 transcriptional regulation is mediated by a very complicated system, including nuclear receptor systems, factors binding to other sites in the ABCA1 promoter, cytokines, hormones, growth factors, lipid metabolites, enzymes, and other messengers/factors/pathways. In addition, ABCA1 posttranscriptional regulation is mediated by microRNA, long noncoding RNA, RNA-binding proteins, proteases, fatty acids, PDZ proteins, signaling proteins, and other factors. Compared to the transcriptional regulation of ABCA1, which is well established, the post-transcriptional regulation of ABCA1 expression is poorly understood.

ABCA1, ABCG1, and Cholesterol Homeostasis.

Cholesterol is a major component of mammalian cell membranes and plays important structural and functional roles. However, excessive cholesterol accumulation is toxic to cells and constitutes the molecular basis for many diseases, especially atherosclerotic cardiovascular disease. Thus, cellular cholesterol is tightly regulated to maintain a homeostasis. Reverse cholesterol transport (RCT) is thought to be one primary pathway to eliminate excessive cholesterol from the body. The first and rate-limiting step of RCT is ATP-binding cassette (ABC) transports A1 (ABCA1)- and ABCG1-dependent cholesterol efflux. In the process, ABCA1 mediates initial transport of cellular cholesterol to apolipoprotein A-I (apoA-I) for forming nascent high-density lipoprotein (HDL) particles, and ABCG1 facilitates subsequent continued cholesterol efflux to HDL for further maturation. In this chapter, we summarize the roles of ABCA1 and ABCG1 in maintaining cellular cholesterol homoeostasis and discuss the underlying mechanisms by which they mediate cholesterol export.

Structure and transport mechanism of the human cholesterol transporter ABCG1.

The reverse cholesterol transport pathway is responsible for the maintenance of human cholesterol homeostasis, an imbalance of which usually leads to atherosclerosis. As a key component of this pathway, the ATP-binding cassette transporter ABCG1 forwards cellular cholesterol to the extracellular acceptor nascent high-density lipoprotein (HDL). Here, we report a 3.26-Å cryo-electron microscopy structure of cholesterol-bound ABCG1 in an inward-facing conformation, which represents a turnover condition upon ATP binding. Structural analyses combined with functional assays reveals that a cluster of conserved hydrophobic residues, in addition to two sphingomyelins, constitute a well-defined cholesterol-binding cavity. The exit of this cavity is closed by three pairs of conserved Phe residues, which constitute a hydrophobic path for the release of cholesterol in an acceptor concentration-dependent manner. Overall, we propose an ABCG1-driven cholesterol transport cycle initiated by sphingomyelin-assisted cholesterol recruitment and accomplished by the release of cholesterol to HDL.

Current models of apolipoprotein A-I lipidation by adenosine triphosphate binding cassette transporter A1.

The primary cardioprotective function of high-density lipoprotein (HDL) is to remove excess cellular free cholesterol (FC) from peripheral tissues and deliver it to the liver. Here, we summarize recent research that examines apolipoprotein A-I (apoA-I) lipidation models by adenosine triphosphate binding cassette transporter A1 (ABCA1) and discuss its relevance in atherosclerotic cardiovascular disease (ASCVD). The first step in HDL formation involves the interaction between apoA-I and ABCA1, where ABCA1 mediates the removal of FC and phospholipids from lipid-laden macrophages to form discoidal nascent HDL (nHDL). However, there are currently no clear-cut systematic models that characterize HDL formation. A number of recent studies have investigated the importance of apoA-I C- and N-terminal domains required for optimal cholesterol efflux and nHDL production. Furthermore, functional ABCA1 is required for direct or indirect binding to apoA-I where ABCA1 dimer-monomer interconversion facilitates apoA-I lipidation from plasma membrane microdomains. Microparticles are also another lipid source for apoA-I solubilization into nHDL. ApoA-I and ABCA1 are key factors in macrophage-mediated cholesterol efflux and nHDL production. Understanding of the key steps in HDL formation may unlock the therapeutic potential of HDL and improve clinical management of ASCVD.

Novel Cocrystallization of Apolipoprotein A‐I with Butyric Acid

Diseases of lipid metabolism, in particular cardiovascular atherosclerosis, remain the number one cause of morbidity and mortality in Western society. The well-documented anti-atherogenic role of high-density lipoprotein (HDL) is related to its involvement in reverse cholesterol transport. When the transporter ABCA1 initiates cholesterol efflux from peripheral and hepatic cells, apolipoprotein A-I (apoA-I) binds the free cholesterol together with phospholipid to form discoidal nascent HDL (“good cholesterol”). The enzyme lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of the cholesterol carried by HDL. ApoA-I activates LCAT for reverse cholesterol transport, wherein LCAT esterifies the cholesterol within the nascent HDL2 and facilitates maturation of HDL to a mature spherical form. After esterification, cholesterol can no longer diffuse back to the peripheral and hepatic cells. The esterified cholesterol, or cholesteryl ester, molecules form the hydrophobic core of the mature HDL particle and leave the circulation upon uptake by a hepatic scavenger receptor for excretion by the liver. Research in the Atkinson laboratory has implicated the arginine 123 residue of apoA-I in the function of LCAT. Based on the X-ray crystal structure, the Arg123 residues of each apoA-I monomer enclose a hydrophobic tunnel into which the lipid substrates of LCAT may be sequestered. ApoA-I has a closed conformation without lipid bound that transforms to a “double belt” encircling the nascent HDL disc. When apoA-I activates LCAT, the tunnel should widen in order to present the substrates to LCAT for esterification. However, the mechanism by which apoA-I interfaces with LCAT substrates remains unknown. Cocrystallization of apoA-I with the lipid component, butyric acid, will enable us to assess how the “double belt” of apoA-I opens in three-dimensional space.

Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

ObjectiveProbucol is a cholesterol-lowering agent whose ability to prevent atherosclerosis is currently under study. Herein, we investigate the putative mechanism of probucol by observation of changes in cellular cholesterol efflux and lipid droplet morphology in macrophages. ResultsThe inhibitory activity of probucol was assessed in non-foam or foam cell macrophages expressing ABCA1 generated by treatment with fetal calf serum (FCS) alone or in combination with acetylated LDL, respectively. Probucol inhibited cholesterol efflux to apolipoprotein A-I (apoA-I) by 31.5±0.1% in THP-1 non-foam cells and by 18.5±0.2% in foam cells. In probucol-treated non-foam THP-1 cells, nascent high density lipoprotein (nHDL) particles with a diameter < 7 nm were generated, while in probucol-treated THP-1 foam cells nHDL particles of > 7 nm in diameter containing cholesterol were produced. Foam cells also displayed a significant accumulation of free cholesterol at the plasma membrane, as measured by percent cholestenone formed. Intracellularly, there was a significant decrease in lipid droplet number and an increase in size in probucol-treated THP-1 foam cells when compared to non-treated cells. ConclusionsWe report for the first time that probucol is unable to completely inhibit cholesterol efflux in foam cells to the same extent as in non-foam cells. Indeed, functional nHDL is released from foam cells in the presence of probucol. This difference in inhibitory effect could potentially be explained by changes in the plasma membrane pool as well as intracellular cholesterol storage independently of ABCA1.

Adiponectin's mechanisms in high-density lipoprotein biogenesis and cholesterol efflux

AimAmong adiponectin's beneficial properties is its ability to promote cellular cholesterol efflux, thereby generating high-density lipoprotein (HDL) particles. However, adiponectin's role in the regulation of macrophage lipid metabolism, a crucial process in atherogenesis, remains poorly investigated. The aim of this study was to characterize the adiponectin's role in HDL biogenesis. Methods and resultsWe perform kinetics studies in baby hamster kidney (BHK) and Tamm-Horsfall protein 1 (THP-1) cell lines to elucidate adiponectin's role in HDL biogenesis. In cholesterol-enriched cells, specific molar doses of adiponectin stimulated cholesterol efflux with high efficiency to apoA-I. In the presence of adiponectin, BHK cells expressing ATP binding cassette transporter A1 (ABCA1) or ABCG1 generated lipidated particles having α electrophoretic mobility (α-HDL) and a molecular size of 7.5–20 nm. Interestingly, in THP-1 macrophages, cholesterol efflux was associated with more lipidated preβ1-HDL particles. Direct molecular interaction of adiponectin with apoA-I enhanced the affinity of apoA-I to free cholesterol and resulted in an increase in preβ1-HDL particles from plasma ex vivo. Adiponectin increased ABCA1 and ABCG1 protein expression and activated the formation of ABCA1-linked cholesterol oxidase sensitive plasma membrane domains. ConclusionAdiponectin upregulated ABCA1 and ABCG1 protein expression, reduced lipid accumulation, and efficiently promoted nascent HDL formation. These results highlight that these cellular processes are interconnected through adiponectin and ABCA1- and ABCG1-dependent. In this pathway, adiponectin increased the affinity of apoA-I to cholesterol and effectively accelerated cholesterol removal from the plasma membrane to HDL particles. Thus, by accelerating HDL biogenesis, adiponectin may have therapeutic potential for atherosclerotic cardiovascular disease prevention and management.

Cholesterol Efflux Efficiency of Reconstituted HDL Is Affected by Nanoparticle Lipid Composition.

Cardiovascular disease (CVD), the leading cause of mortality worldwide is primarily caused by atherosclerosis, which is promoted by the accumulation of low-density lipoproteins into the intima of large arteries. Multiple nanoparticles mimicking natural HDL (rHDL) have been designed to remove cholesterol excess in CVD therapy. The goal of this investigation was to assess the cholesterol efflux efficiency of rHDLs with different lipid compositions, mimicking different maturation stages of high-density lipoproteins (HDLs) occurring in vivo. Methods: the cholesterol efflux activity of soybean PC (Soy-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC:Chol:1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC) and DPPC:18:2 cholesteryl ester (CE):LysoPC rHDLs was determined in several cell models to investigate the contribution of lipid composition to the effectiveness of cholesterol removal. Results: DPPC rHDLs are the most efficient particles, inducing cholesterol efflux in all cellular models and in all conditions the effect was potentiated when the ABCA1 transporter was upregulated. Conclusions: DPPC rHDLs, which resemble nascent HDL, are the most effective particles in inducing cholesterol efflux due to the higher physical binding affinity of cholesterol to the saturated long-chain-length phospholipids and the favored cholesterol transfer from a highly positively curved bilayer, to an accepting planar bilayer such as DPPC rHDLs. The physicochemical characteristics of rHDLs should be taken into consideration to design more efficient nanoparticles to promote cholesterol efflux.

Protective Effect of ApoA1 (Apolipoprotein A1)-Milano in a Rat Model of Large Vessel Occlusion Stroke.

Background and Purpose- Previous experimental studies found that the infusion of human purified nascent HDL (high-density lipoprotein) significantly reduced infarct volume and hemorrhagic transformation rate by decreasing neutrophil recruitment. ApoA1-M (apolipoprotein A1-Milano) is a natural variant of human ApoA1 that confers protection against atherosclerosis. Recombinant ApoA1-M has been formulated as a complex with phospholipids to mimic the properties of nascent HDL. The aim of this study was to assess the impact of intravenous ApoA1-M in a transient middle cerebral artery occlusion stroke model in rats. Methods- In a first experiment, rats were subjected to 120-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused immediately or 4 hours after occlusion. In a second experiment, rats were subjected to 240-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused with or without recombinant tPA (tissue-type plasminogen activator) immediately after recanalization. Primary outcome criteria were the infarct volume and hemorrhagic transformation rate measured at 24 hours. Platelets, coagulation, and neutrophil activation biomarkers were measured in brain homogenates and plasma. Additional in vitro experiments studied the effects of ApoA1-M on platelet aggregation and platelet-neutrophil interactions. Results- The infusion of ApoA1-M immediately or 4 hours after 120-minute transient middle cerebral artery occlusion significantly reduced the infarct volume compared with saline (P=0.034 and P=0.036, respectively). Compared with tPA alone, co-administration of ApoA1-M and tPA showed similar rates of hemorrhagic transformation. ApoA1-M had no significant inhibition effect on neutrophil activation biomarkers. Platelet activation was slightly decreased in rats treated with ApoA1-M compared with saline. In vitro, the incubation of human and rat platelet-rich plasma with ApoA1-M significantly reduced ADP-induced platelet aggregation (P=0.001 and P=0.02, respectively). Conclusions- ApoA1-Milano significantly decreased the infarct volume through an inhibition of platelet aggregation but did not reduce hemorrhagic transformation and neutrophils activation as expected after previous experimental studies with nascent HDL. Visual Overview- An online visual overview is available for this article.

Nascent HDL (High-Density Lipoprotein) Discs Carry Cholesterol to HDL Spheres: Effects of HDL Particle Remodeling on Cholesterol Efflux.

To characterize the fate of protein and lipid in nascent HDL (high-density lipoprotein) in plasma and explore the role of interaction between nascent HDL and mature HDL in promoting ABCA1 (ATP-binding cassette transporter 1)-dependent cholesterol efflux. Approach and Results: Two discoidal species, nascent HDL produced by RAW264.7 cells expressing ABCA1 (LpA-I [apo AI containing particles formed by incubating ABCA1-expressing cells with apo AI]), and CSL112, human apo AI (apolipoprotein AI) reconstituted with phospholipids, were used for in vitro incubations with human plasma or purified spherical plasma HDL. Fluorescent labeling and biotinylation of HDL were employed to follow the redistribution of cholesterol and apo AI, cholesterol efflux was measured using cholesterol-loaded cells. We show that both nascent LpA-I and CSL112 can rapidly fuse with spherical HDL. Redistribution of the apo AI molecules and cholesterol after particle fusion leads to the formation of (1) enlarged, remodeled, lipid-rich HDL particles carrying lipid and apo AI from LpA-I and (2) lipid-poor apo AI particles carrying apo AI from both discs and spheres. The interaction of discs and spheres led to a greater than additive elevation of ABCA1-dependent cholesterol efflux. These data demonstrate that although newly formed discs are relatively poor substrates for ABCA1, they can interact with spheres to produce lipid-poor apo AI, a much better substrate for ABCA1. Because the lipid-poor apo AI generated in this interaction can itself become discoid by the action of ABCA1, cycles of cholesterol efflux and disc-sphere fusion may result in net ABCA1-dependent transfer of cholesterol from cells to HDL spheres. This process may be of particular importance in atherosclerotic plaque where cholesterol acceptors may be limiting.

Apolipoprotein A-I improves hepatic autophagy through the AMPK pathway

Dysfunction in lipid metabolism may result in a decrease in hepatic autophagy, which contributes to the pathogenesis of non-alcoholic steatohepatitis. ATP-binding cassette transporter A1 transports free cholesterol and phospholipids to apolipoprotein A-I (apoA-I) to form nascent high-density lipoprotein particles. Results from previous studies showed that the overexpression of apoA-I significantly reduced levels of hepatic lipids and endoplasmic reticulum stress by modifying lipid transport. Here, we investigated the effects of apoA-I overexpression on hepatic autophagy in cultured hepatocytes and mice. The overexpression of apoA-I in HepG2 cells resulted in an increase in the levels of autophagy as well as the phosphorylation of AMP-activated protein kinase α (AMPKα) and ULK1 and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR). An AMPK inhibitor and siRNA eliminated this apoA-I effect. Consistently, apoA-I transgenic mice showed increased autophagy and AMPKα phosphorylation. These results suggest that apoA-I overexpression alleviates steatohepatitis by increasing hepatic autophagy through the AMPK-mTOR-ULK1 pathway.

Artificial High Density Lipoprotein Nanoparticles in Cardiovascular Research.

Lipoproteins are endogenous nanoparticles which are the major transporter of fats and cholesterol in the human body. They play a key role in the regulatory mechanisms of cardiovascular events. Lipoproteins can be modified and manipulated to act as drug delivery systems or nanocarriers for contrast agents. In particular, high density lipoproteins (HDL), which are the smallest class of lipoproteins, can be synthetically engineered either as nascent HDL nanodiscs or spherical HDL nanoparticles. Reconstituted HDL (rHDL) particles are formed by self-assembly of various lipids and apolipoprotein AI (apo-AI). A variety of substances including drugs, nucleic acids, signal emitting molecules, or dyes can be loaded, making them efficient nanocarriers for therapeutic applications or medical diagnostics. This review provides an overview about synthesis techniques, physicochemical properties of rHDL nanoparticles, and structural determinants for rHDL function. We discuss recent developments utilizing either apo-AI or apo-AI mimetic peptides for the design of pharmaceutical rHDL formulations. Advantages, limitations, challenges, and prospects for clinical translation are evaluated with a special focus on promising strategies for the treatment and diagnosis of atherosclerosis and cardiovascular diseases.