An apolipoprotein A-I mimetic dose-dependently increases the formation of prebeta1 HDL in human plasma

Prebeta1 HDL is the initial plasma acceptor of cell-derived cholesterol in reverse cholesterol transport. Recently, small amphipathic peptides composed of D-amino acids have been shown to mimic apolipoprotein A-I (apoA-I) as a precursor for HDL formation. ApoA-I mimetic peptides have been proposed to stimulate the formation of prebeta1 HDL and increase reverse cholesterol transport in apoE-null mice. The existence of a monoclonal antibody (MAb 55201) and a corresponding ELISA method that is selective for the detection of the prebeta(1) subclass of HDL provides a means of establishing a correlation between apoA-I mimetic dose and prebeta1 HDL formation in human plasma. Using this prebeta1 HDL ELISA, we demonstrate marked apoA-I mimetic dose-dependent prebeta1 HDL formation in human plasma. These results correlated with increases in band density of the plasma prebeta1 HDL, when observed by Western blotting, as a function of increased apoA-I mimetic concentration. Increased prebeta1 HDL formation was observed after as little as 1 min and was maximal within 1 h. Together, these data suggest that a high-throughput prebeta1 HDL ELISA provides a way to quantitatively measure a key component of the reverse cholesterol transport pathway in human plasma, thus providing a possible method for the identification of apoA-I mimetic molecules.     

Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I

Carriers of the apolipoprotein A-I_Milano (apoA-I_M) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I_M mutation: R173S apoA-I, similar to apoA-I_M but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild type≈R173K>R173S>R173C. Compared with wild-type apoA-I, apoA-I_M had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I_M. The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding characteristics.     

Naturally occurring variant of mouse apolipoprotein A-I alters the lipid and HDL association properties of the protein

Plasma HDL levels are inversely associated with atherosclerosis. Inbred mouse strains differ in plasma HDL levels and susceptibility to atherosclerosis. Atherosclerosis-susceptible C57BL/6J mice possess plasma HDL levels 2-fold lower than atherosclerosis-resistant FVB/NJ mice. Polymorphisms have been previously identified between the two mouse strains in the major HDL apolipoproteins, ApoA-I and ApoA-II, which may affect their function on HDL. To begin to understand the HDL differences, we here report on a detailed comparison of the lipid-associated functions of the two mouse ApoA-I proteins. We demonstrate that these polymorphisms significantly alter the protein self-association properties, the ability of the proteins to clear lipid micelles from solution, and their binding affinity for mature mouse HDL. The changes in lipid binding do not appear to alter the ability of the protein to promote cholesterol efflux from cells or the formation of nascent HDL from primary hepatocytes. These apolipoprotein polymorphisms do not change the rate at which HDL protein or cholesterol are catabolized in vivo. Although the presence of the polymorphisms in ApoA-I alters important factors in HDL formation, the basis for the differences in the HDL plasma levels observed in the various mouse strains is more complex and requires additional investigation.     

Protein unfolding allows use of commercial antibodies in an apolipoprotein M sandwich ELISA

apoM is a member of the lipocalin superfamily and circulates in plasma attached to HDL particles. apoM plays a role in cholesterol metabolism and has recently been identified as transporter for the signaling lipid, sphingosine-1-phosphate (S1P), in plasma. S1P is implicated in several inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. The ability to accurately measure apoM is crucial for investigating its biological functions and possible clinical implications. However, reliable commercial methods have been lacking so far. Therefore, we have developed an assay that specifically recognizes human apoM in plasma using commercially available reagents. Commercial apoM antibodies were screened for compatibility in a sandwich ELISA-based assay. One optimal pair of antibodies was chosen, and sample preparation, buffers, and incubation times were optimized to generate a simple and reproducible method. Validation and comparison to a previously described ELISA for apoM confirmed that the assay displays a high degree of sensitivity, specificity, and precision. Our results show that commercially available antibodies can be used to accurately measure human plasma apoM. This method can be implemented in every laboratory and will help promote high quality research.     

Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.     

The recycling of apolipoprotein E in macrophages: influence of HDL and apolipoprotein A-I

The ability of apolipoprotein E (apoE) to be spared degradation in lysosomes and to recycle to the cell surface has been demonstrated by our group and others, but its physiologic relevance is unknown. In this study, we characterized apoE recycling in primary murine macrophages and probed the effects of HDL and apoA-I on this process. In cells pulsed with (125)I.apoE bound to VLDL, intact apoE was found in the chase medium for up to 24 h after the pulse. Approximately 27 +/- 5% of the apoE internalized during the pulse was recycled after 4 h of chase. Addition of apoA-I and HDL increased apoE recycling to 45 +/- 3% and 46 +/- 3%, respectively, similar to the amount of apoE recycled after pulsing the cells with (125)I.apoE.HDL. In addition, apoA-I-producing macrophages from transgenic mice showed increased apoE recycling at 4 h (38 +/- 3%). Increased ABCA1 expression potentiated apoE recycling, suggesting that recycling occurs via ABCA1. Finally, in the presence of apoA-I, recycled apoE exited the cells on HDL-like particles. These results suggest that apoE recycling in macrophages may be part of a larger signaling loop activated by HDL and directed at maximizing cholesterol losses from the cell.     

Anti-inflammatory and cholesterol-reducing properties of apolipoprotein mimetics: a review

Reduced levels of HDL cholesterol (HDL-C) are a strong independent predictor of coronary artery disease (CAD) risk. The major anti-atherogenic function of HDL is to mediate reverse cholesterol transport. This response is highly dependent on apoA-I and apoE, protein components of HDL. Randomized clinical trials have assessed effects of several classes of drugs on plasma cholesterol levels in CAD patients. Agents including cholestyramine, fibrates, niacin, and statins significantly lower LDL cholesterol (LDL-C) and induce modest increases in HDL-C, but tolerance issues and undesirable side effects are common. Additionally, residual risk may be present in patients with persistently low HDL-C and other complications despite a reduction in LDL-C. These observations have fueled interest in the development of new pharmacotherapies that positively impact circulating lipoproteins. The goal of this review is to discuss the therapeutic potential of synthetic apolipoprotein mimetic peptides. These include apoA-I mimetic peptides that have undergone initial clinical assessment. We also discuss newer apoE mimetics that mediate the clearance of atherogenic lipids from the circulation and possess anti-inflammatory properties. One of these (AEM-28) has recently been given orphan drug status and is undergoing clinical trials.     

Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides

Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders.     

ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin

ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-β-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.     

Loss of liver FA binding protein significantly alters hepatocyte plasma membrane microdomains

Although lipid-rich microdomains of hepatocyte plasma membranes serve as the major scaffolding regions for cholesterol transport proteins important in cholesterol disposition, little is known regarding intracellular factors regulating cholesterol distribution therein. On the basis of its ability to bind cholesterol and alter hepatic cholesterol accumulation, the cytosolic liver type FA binding protein (L-FABP) was hypothesized to be a candidate protein regulating these microdomains. Compared with wild-type hepatocyte plasma membranes, L-FABP gene ablation significantly increased the proportion of cholesterol-rich microdomains. Lack of L-FABP selectively increased cholesterol, phospholipid (especially phosphatidylcholine), and branched-chain FA accumulation in the cholesterol-rich microdomains. These cholesterol-rich microdomains are important, owing to enrichment therein of significant amounts of key transport proteins involved in uptake of cholesterol [SR-B1, ABCA-1, P-glycoprotein (P-gp), sterol carrier binding protein (SCP-2)], FA transport protein (FATP), and glucose transporters 1 and 2 (GLUT1, GLUT2) insulin receptor. L-FABP gene ablation enhanced the concentration of SCP-2, SR-B1, FATP4, and GLUT1 in the cholesterol-poor microdomains, with functional implications in HDL-mediated uptake and efflux of cholesterol. Thus L-FABP gene ablation significantly impacted the proportion of cholesterol-rich versus -poor microdomains in the hepatocyte plasma membrane and altered the distribution of lipids and proteins involved in cholesterol uptake therein.     

Prevalence of coronary heart disease risk factors in a Cambridge, UK study

This paper examines the distribution and prevalence of risk factors for coronary heart disease in a sample of 165 men and 202 women over 40 years of age who had earlier participated in a coronary prevention trial from a general practice in Cambridge, UK. No significant differences were observed in total cholesterol levels between men and women, and a quarter of the sample had concentrations above 6.5 mmol/l which is 250 mg/dl. There were significant sex differences in a number of risk factors with males having significantly higher prevalence of low high density lipoprotein, systolic and diastolic blood pressures, obesity, and smoking than women. About 8% of men and women were obese (as defined by a body mass index > 30), while 47% of men and 35% of women were mildly overweight (body mass index > 25). Two or more risk factors for coronary heart disease (high total cholesterol and/or hypertension and/or obesity) were present in 4% and 9% of older men and women respectively. Furthermore, about half the subjects had more than one risk factor for coronary heart disease.     

Levels of apolipoprotein M are not associated with the risk of coronary heart disease in two independent case-control studies

Apolipoprotein M (apoM), a 25 kDa plasma protein belonging to the lipocalin protein family, is predominantly associated with HDL. Studies in mice have suggested apoM to be important for the formation of pre-beta-HDL and to increase cholesterol efflux from macrophage foam cells. Overexpression of human apoM in LDL receptor-deficient mice reduced the atherogenic effect of a cholesterol-rich diet. The aim of the present study was to investigate whether the apoM levels in man predict the risk for coronary heart disease (CHD). ApoM was measured in samples from two separate case-control studies. FINRISK '92 consisted of 255 individuals, of whom 80 developed CHD during follow-up and 175 were controls. The Copenhagen City Heart Study included 1,865 individuals, of whom 921 developed CHD during follow-up and 944 were controls. Correlation studies of apoM concentration with several analytes showed a marked positive correlation with HDL and total cholesterol as well as with apoA-I and apoB. There was no significant difference in mean apoM level between CHD and control subjects in either study. In conditional logistic regression analyses, apoM was not a predictor of CHD events, [odds ratio (95% CI) 0.97 (0.74-1.27) and 0.92 (0.84-1.02), respectively]. In conclusion, no association between apoM and CHD could be found in this study.     

The effects of apolipoprotein B depletion on HDL subspecies composition and function

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl₂), heparin sodium/manganese chloride (MnCl₂), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl₂, heparin sodium/MnCl₂, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.     

An ELISA for apolipoprotein M reveals a strong correlation to total cholesterol in human plasma

Apolipoprotein M (apoM) is a 188 amino acid, 25 kDa protein belonging to the lipocalin protein superfamily. Although predominantly associated with high density lipoprotein, apoM is found in all major lipoprotein classes. To facilitate clinical studies of apoM, we have developed a sandwich ELISA for the measurement of apoM in human plasma. This method has been used to investigate normal apoM variation and to establish reference values for healthy individuals through the measurement of 598 samples from the Nordic Reference Interval Project Bio-bank and Database (NOBIDA) biobank. For women 18-49 years old, the reference interval for apoM was 0.58-1.18 micromol/l, whereas for women 50+ years and for men, the reference range was 0.61-1.30 micromol/l. Correlation studies of apoM with 26 common clinical chemical analytes from the NOBIDA database revealed a marked positive correlation with plasma total cholesterol (r = 0.52) and LDL and HDL cholesterol (r = 0.43 and 0.36, respectively). There was no statistically significant correlation with HDL/total cholesterol ratio or body mass index. In conclusion, a sandwich ELISA for the measurement of apoM in human plasma shows that apoM concentration is strongly correlated to total cholesterol in healthy individuals.     

Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has gained attention as a key regulator of serum low density lipoprotein cholesterol (LDL-C) levels. This novel protease causes the degradation of hepatic low density lipoprotein receptors. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDL-C levels and cardiovascular risk. Previous studies have demonstrated that statins upregulate PCSK9 mRNA expression in cultured cells and animal models. In light of these observations, we studied the effect of atorvastatin on circulating PCSK9 protein levels in humans using a sandwich ELISA to quantitate serum PCSK9 levels in patients treated with atorvastatin or placebo for 16 weeks. We observed that atorvastatin (40 mg/day) significantly increased circulating PCSK9 levels by 34% compared with baseline and placebo and decreased LDL-C levels by 42%. These results suggest that the addition of a PCSK9 inhibitor to statin therapy may result in even further LDL-C decreases.     

The high-resolution crystal structure of human LCAT

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.     

Binding characteristics of a panel of monoclonal antibodies against the ligand binding domain of the human LDLr

To obtain a panel of monoclonal antibodies (MAbs) to study the folding and conformation of the low density lipoprotein receptor (LDLr), we have generated hybridomas from LDLr-deficient mice that had been immunized with the extracellular domain of the human LDLr. The 12 MAbs were specific for the ligand binding domain of the LDLr, with individual MAbs recognizing epitopes in ligand binding repeats 1, 2, 3, 5, and 7. A subset of the MAbs failed to react with the LDLr when disulfide bonds were reduced, and one MAb, specific for an epitope that spans ligand binding repeats 1 and 2, recognized two conformational forms of the LDLr with different affinities. Antibodies specific for ligand binding repeats 3, 5, and 7 completely blocked the binding of LDL particles to the LDLr on cultured human fibroblasts, whereas MAbs with epitopes in ligand binding repeats 1 and 2 partially blocked the binding of LDL to the LDLr. These anti-LDLr MAbs will serve as useful probes for further analysis of LDLr conformation and LDLr-mediated lipoprotein binding.     

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.     

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.