HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 25, 25966-25977, June 18, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/25/25966    most recent M401177200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kockx, M. Articles by Kritharides, L. Search for Related Content PubMed PubMed Citation Articles by Kockx, M. Articles by Kritharides, L. Social Bookmarking                      What's this?

Apolipoprotein A-I-stimulated Apolipoprotein E Secretion from Human Macrophages Is Independent of Cholesterol Efflux^*

From the ^a Macrophage Biology Group, Centre for Vascular Research, University of New South Wales, Sydney 2052, Australia, ^b The Heart Research Institute, Sydney 2050, Australia, the ^c Baker Heart Research Institute, Melbourne 8008, Australia, the ^d Department of Clinical Biochemistry, Royal Prince Alfred Hospital, University of Sydney, Sydney 2050, Australia, the ^e Department of Core Clinical Pathology and Biochemistry, Royal Perth Hospital, School of Medicine & Pharmacology, University of Western Australia, Perth 6847, Australia, the ^f Institut für Arterioskleroseforschung an der Westfälischen Wilhelms-Universität Münster, Münster 48149, Germany, the ^g University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294, the ^hChildren's Hospital of Philadelphia, Stokes Research Institute, Philadelphia, Pennsylvania 19104-4318, the ⁱ University of Canberra, Australian Capital Territory 2601, Australia, and the ^j Department of Cardiology Concord Hospital, University of Sydney, Sydney 2139, Australia

Received for publication, February 3, 2004 , and in revised form, March 29, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using ³⁵S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E_m) and stable (E_s) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer -helical peptides representing amphipathic -helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal -helix (domains 220–241) stimulates cholesterol efflux. Other -helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220–5222delTCT; and mutations A1046D and c.4629–4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by -helix-containing molecules including apoA-I and apoE.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The anti-atherogenic effects of high density lipoprotein (HDL)¹ (1) are at least in part attributed to the ability of HDL to stimulate cholesterol and phospholipid efflux from lipid-loaded macrophages, providing the initial step of reverse cholesterol transport (RCT). Apolipoprotein A-I (apoA-I), the major protein component of HDL, is understood to play an important role in this process (1).

Lipid removal by apoA-I involves a cAMP-inducible active transport pathway (2) and is known to be mediated by the ATP-binding cassette transporter-1 (ABCA1/ABC-1) (3). These transporters are membrane proteins that utilize ATP hydrolysis to transport substrates across membranes (4). Mutations in ABCA1/ABC-1 cause Tangier disease (TD), a severe HDL deficiency syndrome characterized by very low plasma levels of HDL and apoA-I, accumulation of cholesterol in tissue macrophages, and a predisposition to atherosclerosis (5–7). Fibroblasts from subjects with Tangier disease show impaired cholesterol and phospholipid release to lipid-free apolipoproteins (8). In addition, inhibition of ABCA1 expression or activity inhibits lipid efflux to apoA-I in normal fibroblasts (3, 6).

Properties additional to induction of cholesterol efflux may contribute to the anti-atherogenic effect of apoA-1. Human atherosclerotic lesions contain apoE protein and mRNA, especially in association with macrophage foam cells (9). Secretion of apoE by macrophages may protect against atherosclerosis, as indicated by the effects of transplantation of apoE-expressing bone marrow into apoE-null mice (10, 11). This may depend upon enhanced local clearance of cellular lipids and/or inhibition of local inflammatory responses, in addition to lowering plasma concentrations of atherogenic plasma lipoproteins. Recent studies demonstrated that apoA-I stimulates both apoE secretion and cholesterol efflux from human and mouse foam cell macrophages (12, 13). Stimulation of apoE secretion by apoA-I and by HDL is most likely post-transcriptionally regulated and is observed for both endogenous apoE (macrophages) and recycled apoE (hepatocytes) (12–15). Recent studies indicate that ABCA1 may be involved in basal apoE secretion (16). However, the requirement for ABCA1 activity in apoA-I-induced apoE secretion has never been reported.

In this study we provide evidence that apoA-I-stimulated apoE secretion is mechanistically distinct from its stimulation of ABCA1-mediated cholesterol efflux. We demonstrate that apoA-I promotes secretion of a mobile pool of apoE, which is otherwise destined for intracellular degradation, that this process does not require interactions between the C terminus of apoA-I and the plasma membrane and occurs normally in macrophages of patients with Tangier disease, implying independence from ABCA1 activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents—All solvents were high performance liquid chromatography (HPLC) grade (Mallinckrodt). Polyclonal goat anti-human antibodies to human apoE were obtained from Chemicon International Inc. For immunoblotting of nitrocellulose membranes after non-denaturing gel electrophoresis, polyclonal goat antibodies to human apoA-I and polyclonal sheep antibodies to human apoA-II (Roche Applied Science) were used. Secondary rabbit polyclonal anti-goat or anti-sheep horseradish peroxidase-linked antibodies, nitrocellulose membranes (0.45 µm), and enhanced chemiluminescence (ECL) reagents and Hyperfilm were obtained from Amersham Biosciences. Human apoE standard was from Biodesign. [1,2(n)-³H]cholesterol and [methyl-³H]choline chloride were supplied by Amersham Biosciences (specific activity: 44 Ci/mmol and 79 Ci/mmol, respectively). LDL, acetylated LDL (AcLDL), [³H]cholesterol-labeled AcLDL were prepared as described (17, 18). 1-Palmitoyl-2-oleoyl phosphatidylcholine (POPC), egg yolk sphingomyelin (SPM), and sodium cholate were purchased from Sigma. Bicinchoninic acid (BCA) reagent for protein determination was also supplied by Sigma-Aldrich. For pulse-chase studies, methionine-free medium was supplied by Invitrogen and [³⁵S]TRAN-label (1175 Ci/mmol) from ICN.

Apolipoproteins—Human apoA-I and apoA-II were isolated from HDL by ultracentrifugation and anion exchange chromatography (17, 19). Human apoA-IV was isolated from the density >1.25 g/ml fraction of plasma by adsorption onto synthetic triglyceride-rich microemulsions. The microemulsions were delipidated and the apoA-IV purified by anion exchange chromatography on a Q-Sepharose fast flow column (20, 21). Human apoE2, apoE3, and apoE4 were expressed by BL21 Escherichia coli (provided by Dr. Karl Weisgraber, Gladstone Institute) and purified by gel filtration chromatography.

ApoA-I Peptides and Recombinant Proteins—Pure peptides representing peptides of apoA-I were synthesized using an automated solid phase peptide synthesizer as described previously (22–24). To promote the -helical stability of the peptide molecules, the N and C termini were acetylated and amidated (except for peptide 1–33) respectively (25). Truncated apoA-I propeptide mutants were expressed in an E. coli/PGEX expression system (26). Cholesterol efflux to these proteins has previously been published (27). ApoA-I deletion mutants were constructed as described previously (28, 29). Wild-type apoA-I and engineered variants of apoA-I were expressed in expression host E. coli strain Bl21-DE3 and isolated by gel filtration and anion exchange chromatography (29). Identical stimulation of apoE secretion from human macrophage foam cells was observed in control experiments using recombinant wild-type apoA-I and human apoA-I prepared from human plasma. In previous studies we established that 5–10 µg/ml apoA-I was saturating for cholesterol efflux and apoE secretion from human and murine macrophages (13). In the present studies, peptides and proteins were added to cells at a final concentration of 25 µg/ml culture medium after first establishing that this was in excess of saturating concentrations for induction of apoE secretion for all molecules.

Preparation of Phospholipid ApoA-I and Phospholipid ApoA-II Discs—Discs were prepared as described previously (19) using the cholate dialysis method (30). After preparation, all discs were dialyzed extensively against 0.01 M Tris-buffered saline containing 0.15 M NaCl, 0.005% (w/v) EDTA-Na₂, 0,006% (w/v) NaN₃, and stored under argon. Lipid-free apoA-I and apoA-II were not present in the preparations as judged by electrophoresis on non-denaturing 3–40% gradient gels. Phospholipid composition was varied by altering the proportions of phospholipids added at the time of preparation. All chemical analyses of reconstituted particles were carried out on a Roche/Hitachi 902 analyzer (Roche Diagnostics, Zurich, Switzerland). Phospholipid concentrations were determined enzymatically (31, 32). The Stokes' diameter and surface charge of the particles was measured by non-denaturing PAGE and agarose gel electrophoresis (19, 33). In some experiments, after cell efflux incubations, media containing apoA-I phospholipid or apoA-II phospholipid discs underwent non-denaturing polyacrylamide gel electrophoresis, followed by electrophoretic transfer nitrocellulose membranes and immunoblotting for apoE, apoA-I, or apoA-II to investigate possible association of apoE with phospholipid-containing particles.

Isolation and Culture of Human Monocyte-derived Macrophages (HMDM)—Human monocytes (apoE3/E3 phenotype) were isolated from white cell concentrates from healthy donors using centrifugal elutriation as described (13). Genotyping was completed by Taq polymerase chain reaction amplification of a 232-base pair fragment of the apoE gene followed by cutting with the restriction endonuclease Cfo1 by the Department of Biochemistry, Royal Prince Alfred Hospital. White cell buffy coat concentrates (<24 h, ex vivo) and human serum were provided by the New South Wales Red Cross blood transfusion service, Sydney, Australia. Purified monocytes (>95% purity by nonspecific esterase staining) were differentiated by plating at 1.5 x 10⁶ cells per 22-mm diameter culture dish (Costar) in RPMI 1640 containing penicillin G and streptomycin (50 units/ml and 50 µg/ml, respectively), L-glutamine (2 mM), and 10% (v/v) heat-inactivated whole human serum for 6 days. Following differentiation, the cells were washed and enriched with unesterified cholesterol (FC) and cholesteryl ester (CE) by incubation in RPMI 1640 containing 10% LPDS (v/v) and AcLDL (50 µg protein/ml) for 4 days (13).

Mononuclear cells from individual donors (Tangier disease subjects and their controls) were isolated by minor modification of a previously described density separation method (34, 35) using OptiPrep^TM solution of density 1.068 g/ml and a solution of density 1.063 g/ml to remove platelets and lymphocytes, respectively. Total cells were plated at a density of 2 x 10⁶ cells/22-mm diameter culture well (Falcon). Cells were differentiated to macrophages in RPMI 1640-containing antibiotics, glutamine, 50 ng/ml M-CSF (36), and heat-inactivated whole human serum (13, 37) for 6 days. At this time cells were washed prior to cholesterol enrichment (see above). Direct comparisons between HMDM isolated from normal donors by density centrifugation and centrifugal elutriation confirmed identical cholesterol efflux and apoE secretion by either method.

Lipid Efflux and ApoE Secretion—To achieve cholesterol loading of macrophages, 2 µCi/ml [³H]cholesterol in ethanol was incorporated into AcLDL before incubating with cells for 4 days as described (18). Triplicate cultures were harvested after loading to confirm efficient enrichment with FC and CE by HPLC. Cells were subsequently washed, equilibrated overnight in RPMI 1640 containing 0.1% (w/v) bovine serum albumin, to allow equilibration of [³H]cholesterol in FC and CE pools. In some experiments, cells were loaded with AcLDL without [³H]cholesterol, and phospholipids were labeled at the end of the 4-day cholesterol-loading period by incubating cells with RPMI 1640 containing 3 µCi/ml [³H]choline chloride in ethanol (final 2% (v/v)) and 0.1% bovine serum albumin (w/v) for 22 h, followed by equilibration for 1 h in RPMI 1640 containing 0.1% bovine serum albumin.

Equilibrated, cholesterol-enriched cells were washed with warm PBS and incubated in 1.0 ml of efflux medium comprising RPMI 1640 with or without 25 µg/ml apoA-I or the appropriate additions. After 1–8 h, media were removed, mixed with Complete© protease inhibitor (Roche Applied Science) and 0.02 TIU of aprotinin (Sigma) and spun for 2 min at 16,000 x g to remove any detached cells. The cultures were washed twice with ice-cold PBS and then scraped in 0.8 ml of cold PBS containing Complete© protease inhibitor and aprotinin.

Quantitation of Cholesterol and Cholesteryl Ester in Cells and Media—40-µl media aliquots and 5-µl cell aliquots were analyzed by scintillation counting to quantify efflux of [³H]cholesterol. The proportions of label in cell [³H]FC and [³H]CE were determined after separation by TLC in heptane/ethyl acetate (1:1, v/v). Standards of FC and CE were identified by charring at 100 °C in 10% CuSO₄, 8.5%H₃PO₄. The masses of FC and CE in cells and media were determined by reverse-phase HPLC after extraction into methanol/hexane (17, 38). Radiochemical equilibrium was determined by calculating the specific activity of free and esterified cholesterol (dpm per nmol) (18) and was reproducibly achieved after 4 days of cholesterol enrichment and overnight equilibration.

Quantitation of Phospholipids in Cells and Media—Phospholipids were extracted from 150-µl aliquots of cell lysates and media samples using the method of Bligh and Dyer including two backwashes with methanol/water (10:9, v/v) as described previously (39). After evaporation of the chloroform phase, lipids were dissolved in 200 µl of chloroform/methanol (2:1, v/v), and 50-µl aliquots were counted by scintillation counting to determine phospholipid efflux. Total phospholipid mass was determined using a modification of the Bartlett assay as described (40) and was used to determine specific activity (dpm per nmol phospholipid).

Quantitation of ApoE in Cell Lysates and Efflux Media by Western Blot and by ELISA—Aliquots (55 µl) of cell culture medium were mixed with sample buffer (27.5 µl) containing 10 mM dithiothreitol, heated to 100 °C for 5 min, and separated by SDS-PAGE using a 4% stacking gel, and 12.5% polyacrylamide resolving gel under reducing conditions in Tris-glycine buffer as described (13). Electrophoretic Western blot transfer onto 0.45-µM nitrocellulose membranes was performed in Trisglycine buffer for 1 h at 25–30 V, blocked before incubating with primary antibody to apoE (goat anti-human polyclonal, 1:5000 dilution), and washing and incubating with 1:5000 dilution of secondary antibody (rabbit anti-goat IgG) conjugated with horseradish peroxidase (13). Membrane chemiluminescence signal was quantified using Kodak Digital Science 1D and expressed as arbitrary units (AU) per mg of cell protein. In most experiments, a serial dilution of authentic human apoE standard in sample buffer was separated and blotted in parallel to allow calculation of secreted apoE mass as microgram per culture or µg per mg of cell protein. The linear response range of chemiluminescence signal to mass of authentic human apoE standard was defined for each Western blot in each experiment.

In addition, apoE secretion was measured in some experiments using ELISA. NUNC 96 well immunoplates were coated with 1.5 µg/ml mouse monoclonal anti-human apoE antibody (Biodesign) in 0.2 M Na₂CO₃/NaHCO₃ buffer. Standards were prepared from purified human apoE (Biodesign), whereas antibodies used in Western analysis were used as tagging antibodies (goat anti-human polyclonal, 1:3750 and rabbit antigoat horseradish peroxidase, 1:2500).

Metabolic Labeling of Cell Proteins with [³⁵S]Methionine/Cysteine— To study the fate of newly synthesized apoE, cholesterol-enriched HMDM were labeled in methionine-free medium (Invitrogen) containing 250 µCi/ml [³⁵S]TRAN-label (1175 Ci/mmol, ICN) for 3 h, then washed and chased in RPMI 1640 media containing 2 mM methionine (Sigma) and cysteine (Sigma). ³⁵S-labeled apoE in cell lysates and culture medium were determined by immunoprecipitation using 1:10,000 dilution of the polyclonal goat antibody to human apoE and protein A-Sepharose (Amersham Biosciences) after separation by SDS-PAGE, quantified by phosphorimaging (Photostimulated Luminescence, Fujix BAS 1000) and expressed as AU of apoE/mg of cell protein (13). Where indicated, in experiments detecting small amounts of secreted apoE requiring increased sensitivity, immunoprecipitated ³⁵S-labeled apoE was directly quantified by radiometric detection, after confirming a single band on SDS-PAGE and phosphorimaging. [³⁵S]Methionine/cysteine labeling was also used to discriminate between cell derived (secreted) and exogenous apoE in experiments investigating the secretion of apoE by recombinant apoE2, E3, and E4.

Biotinylation and Isolation of Biotinylated ApoE—Cholesterol-enriched HMDM were incubated with 200 µCi/ml [³⁵S]TRAN-label in methionine-free Dulbecco's modified Eagle's medium (DMEM) containing 10% DMEM for 16 h. Surface proteins were then biotinylated by placing the cells on ice for 5 min, followed by incubation with 1 mg/ml sulfo-NHS-SS-Biotin (Pierce) in PBS at 4 °C for 45 min. Cells were washed twice with ice-cold PBS and subsequently incubated with or without 25 µg/ml apoA-I for 30 min at 37 °C. ApoE was immunoprecipitated as described above, and biotinylated apoE separated from total immunoprecipitated apoE as described (41). In short, total apoE was released from protein A-Sepharose beads by boiling in 100 µl of HEPES-buffered saline containing 1% SDS at 90 °C for 3 min. Biotinylated apoE was separated from unbiotinylated apoE using ImmunoPure Immobilized Streptavidin (Pierce) and both were quantified using scintillation counting and SDS-PAGE/phosphorimaging. Biotinylated apoE was quantified both as AU of apoE/mg of cell protein and as percent of total immunoprecipitated, secreted apoE. In these experiments, 3.5 ± 0.5% of total cellular apoE was biotinylated, and 34.0 ± 9.7% of cellular apoE protein was in plasma membrane fractions isolated by sucrose density gradient (42). As only cell surface proteins are biotinylated at 4 °C (41), it is estimated that 11.2 ± 3.2% of the plasma membrane pool of apoE was biotinylated in these experiments.

Analysis of apoE mRNA by Real Time PCR—Cells were harvested for total RNA using Tri Reagent (Sigma) according to the manufacturer's instructions with the exception that 1-bromo-3-chloropropane (Sigma) was substituted for chloroform. For each reverse-transcription reaction, 1 µg of total RNA was reverse-transcribed using oligo(dT) primers (Invitrogen) and Superscript II reverse transcriptase (Invitrogen). Relative real-time RT-PCR was performed using IQ SYBR Green Supermix (Bio-Rad) on an ABI 7700 Sequence Detector (PE Biosystems) and analyzed using ABI Prism sequence detector software v1.6.3 (PE Biosystems). Gene-specific primers were used as follows: ApoE, 5'-CTGCTCAGCTCCCAGGTCACCCAGG-3'(forward), 5'-CTTCTGCAGGTCATCGGCATCGCGG-3'(reverse), annealing at 60 °C, 30 cycles, product size 321 base pairs. ABCA1, 5'-GCACTGAGGAAGATGCTGAAA-3'(forward), 5'-AGTTCCTGGAAGGTCTTGTTCAC-3' (reverse), annealing at 60 °C, 28 cycles, product size 205 base pairs. Results were normalized to that of a housekeeping gene, PBGD, 5'-GAGTGATTCGCGTGGGTACC-3' (forward), 5'-GGCTCCGATGGTGAAGCC-3' (reverse), annealing at 55–60 °C. Melting curve analysis was performed to confirm production of a single product in these reactions. Results after exposure to apoA-I were related to those without apoA-I (RPMI 1640 medium control) in each experiment, and the results of three experiments averaged.

Sucrose Density Ultracentrifugation—To determine the buoyant density of secreted apoE, media samples were subjected to sucrose density ultracentrifugation using a minor modification of a published protocol (43). Following efflux, 625-µl aliquots of media were centrifuged at 16,000 x g for 2 min to remove cellular debris, and the supernatant underlayed. Underlay was completed using 2 ml of 1.33% sucrose solution, 1.23 ml of 35% sucrose solution, and 1.23 ml of 65% sucrose solution in 5.1 ml of Beckman quick-seal tubes, and samples were centrifuged at 100,000 rpm (543,000 x g), for 20 h at 16 °C, using a TL100.4 rotor in Beckman TLX-Ultracentrifuge. 15 x 250 µl aliquots were taken from above and analyzed for [³H]cholesterol and apoE (Western blot).

Tangier Disease Patients (TD1 and TD2)—A previously unreported 56-year-old male with clinical features consistent with Tangier Disease (TD1) was identified. The patient had premature coronary artery disease, very low plasma HDL-cholesterol (3.1–7.7 mg/dl, normal range 30–70 mg/dl) and apoA-I concentrations (0.002–0.006 mg/dl, normal 110–140 mg/dl), low total cholesterol (61.9–104.4 mg/dl, normal range 116–225 mg/dl) and LDL-cholesterol (19.3 mg/dl, normal range 50–190 mg/dl) level. He also had elevated plasma triglycerides (177.1–336.6 mg/dl, normal <200 mg/dl), bilateral cataracts and hepatomegaly. There was no splenomegaly, and tonsils had been removed in childhood. The patient was genotyped (S. Rust, G. Assmann, Institut für Arterioskleroseforschung an der Westfälischen Wilhelms-Universität Münster, Germany). Two novel amino acid mutations within the ABCA1 gene were found: C733R in exon 16 (c.2197C>T) and a deletion of three nucleotides in exon 38 (c.5220–5222delTCT) on different alleles as shown by segregation analysis (numbering follows recommendations of the international nomenclature working group). Both mutations affect amino acids conserved between human and mice. Neither of the mutations has been previously described. A healthy subject with normal lipoprotein profile and no history of vascular disease was used as a source of control macrophages (HC1). Since completion of this study, TD1 has died.

A second TD patient (TD2), his healthy normal brother (HC2), and his heterozygous parents (HZ2A (mother) and HZ2B (father)) have been previously described (44, 45). The ABCA1 mutations in this TD patient were published as a missense mutation in exon 21 (A986D) and a frameshift mutation in exon 33 (4570insA, A1484' X1492) (45). Using current nomenclature, these are respectively equivalent to a missense mutation in exon 22 (c.3137C>A; A1046D) and a frameshift mutation in exon 34 (c.4629–4630insA; A1544 fs X1552). HC2 has neither ABCA1 mutation, and HZ2A and HZ2B contain the 4629insA and the A1046D mutations, respectively. Plasma HDL-cholesterol concentrations for HC2, TD2, HZ2A, and HZ2B were respectively 43.0, 2.7, 25.8, and 38.6 mg/dl and apoA-I levels were respectively 129, <4.5, 95.7, and 115.8 mg/dl. Plasma triglyceride concentrations were elevated in all subjects, being 507.7 mg/dl for HC2, 246.3 mg/dl for TD2, 337.6 mg/dl for HZ2A, and 396.9 mg/dl for HZ2B.

Protein Estimation—Protein contents of LDL samples and cell lysates were determined in duplicate or triplicate for each sample measured, using the BCA method with bovine serum albumin as standard (17).

Cell Viability—Cell viability following various treatments was routinely assessed by light microscopic morphology, by estimation of cell protein and by measuring leakage of lactate dehydrogenase into the medium (46). Cell viability was routinely between 85 and 100% after incubation in efflux media.

Data Analysis—The degradation and secretion of cellular apoE from pulse-chase experiments were simultaneously fitted to a first order rate equation with k₁ and k₂ describing the rate constants for secretion and degradation, respectively. The first order rate equation shown in Equation 1,

(Eq. 1)

was fitted to the experimental secretion and degradation data using a non-linear least-squares fitting program (Solver in Microsoft Excel). The quality of the fit was evaluated by an error function as previously described (42).

Efflux was calculated as [³H]lipids (e.g. cholesterol or phospholipid) released to the medium expressed as a percentage of the total ³H cellular lipids at T₀ (17, 18). Unless otherwise stated, data presented are mean ± S.D. of triplicate cultures from single experiments and are representative of at least 2–3 independent experiments. The significance of results was assessed by unpaired Student's t test for simple comparisons of two groups, and one-way analysis of variance using Tukey's test as post-hoc correction for multiple comparisons. Differences were considered significant at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
ApoA-I Stimulates the Secretion of ApoE—We have previously shown that apoA-I stimulates the secretion of murine macrophage apoE in a time- and concentration-dependent manner and that this is not inhibited by the transcription inhibitor actinomycin D (13). Here we confirm that apoA-I stimulates apoE secretion from primary human macrophages in a time-dependent manner, without affecting net cell-associated apoE protein or cellular apoE mRNA levels (Fig. 1). Secreted apoE was always >34 kDa, consistent with the known extensive glycosylation of secreted apoE, whereas cell-associated apoE was of multiple masses consistent with variable degrees of glycosylation during apoE processing within cells.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Post-transcriptional stimulation of apoE secretion by apoA-I. HMDM were incubated for 4 days with 50 µg/ml AcLDL and then washed and incubated for the times indicated in RPMI 1640 with (apoA-I, ) or without (Control, ) 25 µg/ml apoA-I. ApoE protein was determined by Western blotting at 8 h (A, three independent cell cultures, media above, cell lysates below) or ELISA at variable times (B). ApoE mRNA levels (C) were determined at 8 h by real-time RT-PCR relative to the housekeeping gene PBGD, as described under "Experimental Procedures." *, p < 0.01 relative to control.

View larger version (15K): [in this window] [in a new window]   FIG. 2. ApoA-I stimulates secretion of preformed apoE and decreases net apoE degradation. HMDM were incubated for 4 days with 50 µg/ml AcLDL and then washed and incubated in methionine-free Dulbecco's modified Eagle's medium with 200 µCi/ml [35S]methionine/cysteine for 3 h. Cells were then washed and chased without () or with () 25 µg/ml apoA-I. At indicated times 35S-labeled apoE was immunoprecipitated from media and cell lysates, separated and detected by SDS-PAGE and phosphorimaging of a single 34-kDa band, and measured by scintillation counting of the excised band, as described under "Experimental Procedures". At the start of the chase (T0), total apoE was 2.78 ± 0.21 x 106 cpm/mg cell protein. A, secreted apoE; B, cell-associated apoE; C, net degradation of apoE, calculated by subtracting residual apoE in cells and media from apoE in cells at T0. All data are expressed as percent of total apoE at T0.

View larger version (16K): [in this window] [in a new window]   FIG. 3. ApoA-I-diverts apoE from degradation to secretion. The degradation and secretion of 35S-labeled cellular apoE pulse-chase experiments were simultaneously fitted to a first order rate equation as described under "Experimental Procedures." A, model for apoE turnover with Es and Em representing stable and mobile cellular apoE pools, respectively, and k1 and k2, respectively, describing the rate constants for secretion and degradation. B, experimental (open symbols, dashed line) and calculated (closed symbols, solid line) kinetic data for secreted (, ), cellular (, ), and degraded (, ) apoE in the presence of apoA-I. C, summary of modeling parameters, Es representing the percent of cellular apoE in the stable pool, and ERF representing the error function of modeling parameters.

View larger version (11K): [in this window] [in a new window]   FIG. 4. ApoA-I stimulates release of cell surface pools of apoE. HMDM were cholesterol-enriched with AcLDL and radiolabeled with 35S-methionine/cysteine for 16 h. A, [35S]methionine-labeled HMDM were placed on ice (4 °C) and cell surface proteins biotinylated as described under "Experimental Procedures." Cells were then incubated in medium without (Control) or with 25 µg/ml apoA-I (apoA-I) for 30 min at 37 °C. Total 35S-labeled apoE was immunoprecipitated, the biotinylated pool further immunoprecipitated from this pool, and quantified. Total 35S-labeled apoE immunoprecipitated from cell cultures at T0 was 838,768 ± 33,266 dpm/mg cell protein. B, [35S]methionine-labeled HMDM were placed on ice and incubated with or without apoA-I for 30 min at 4 °C. 35S-labeled apoE released to medium was immunoprecipitated and quantified, and represented 3.2 ± 0.4% and 6.1 ± 1.5% of total cellular apoE at T0 for Control and apoA-I, respectively. *, significantly different versus Control (p < 0.05).

C-terminal Truncations of pro-ApoA-I Differentially Affect Cholesterol Efflux and ApoE Secretion—Previous studies have indicated that the C terminus of apoA-I is particularly important for the stimulation of cholesterol efflux and binding of apoA-I to the cell surface (49), and this is related to the lipid binding ability of the C terminus (27, 50). We investigated the role of the C terminus in inducing apoE secretion from primary human foam cell macrophages using recombinant pro-apoA-I-(–6–243) with truncations of the C terminus (–6–150) and (–6–222)) (Fig. 5). Initial studies confirmed that pro-apoA-I and native human apoA-I induced similar cholesterol efflux and apoE secretion (data not shown). Consistent with previous studies in HepG2 cells (27), deletion of the C-terminal 21 amino acids (–6–222) decreased cholesterol efflux relative to whole apoA-I-(–6–243), whereas deletion of the C-terminal 93 amino acids (–6–150) had no effect. This correlated with the relative abilities of the proteins to stimulate phospholipid efflux (Fig. 5B). In contrast, both C-terminal deletions increased apoE secretion relative to full-length pro-apoA-I. The discrepant effects of C-terminal deletion on lipid efflux and apoE secretion suggest that induction of apoE secretion is not linked to apoA-I lipid affinity or its stimulation of cholesterol efflux via ABCA1.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Truncation of the C-terminal domain of recombinant pro-apoA-I increases apoE secretion from human macrophages. Monocytes were differentiated, incubated for 4 days with 50 µg/ml AcLDL-[3H]cholesterol (A), or AcLDL then [3H]choline (B), as described under "Experimental Procedures." Cells were then washed, equilibrated, and incubated with RPMI 1640 (Control) or 25 µg/ml of indicated recombinant whole pro-apoA-I-(–6–243), or pro-apoA-I with varying C-terminal truncations (–6–222 and –6 –150). Aliquots of media were removed after 8 h, and cholesterol efflux (A), phospholipid efflux (B), and apoE secretion (C, Western blot inset) were measured as described under "Experimental Procedures." Lipid composition after loading (at T0): FC 38.6 ± 10.7 nmol/mg cell protein, CE 54.9 ± 5.5 nmol/mg cell protein, specific activity FC 17856 ± 3188 dpm/nmol, specific activity CE 22771 ± 6928 dpm/nmol. B, specific activity PL 45817 ± 6110 dpm/nmol. *, significantly different versus control values (p < 0.05); ¶, significantly different versus –6 –243 (pro-apoA-I, p < 0.05).

View larger version (22K): [in this window] [in a new window]   FIG. 6. Recombinant ApoA-I with selected deletions differentially promote apoE secretion from human macrophages. Monocytes were differentiated and cholesterol-enriched and incubated with RPMI 1640 (Control), 25 µg/ml whole apoA-I, or 25 µg/ml recombinant apoA-I proteins with deletions of C-terminal (d190–243), N-terminal (d44–126), or central (d123–166) domains. Aliquots of media were removed after 8 h, and cholesterol efflux (A) and apoE secretion (B) were measured as described under "Experimental Procedures." Lipid composition after loading (at t = 0): FC 79.4 ± 2.1 nmol/mg of cell protein, CE 26.0 ± 7.3 nmol/mg of cell protein, specific activity FC 63622 ± 3234 dpm/nmol, specific activity CE 74321 ± 15523 dpm/nmol. Recombinant whole apoA-I and isolated human apoA-I induced identical apoE secretion and cholesterol efflux (data not shown). *, significantly different versus apoA-I (p < 0.05).

Synthetic Peptides Representing -Helical Segments of ApoA-I Induce ApoE Secretion—ApoA-I contains 10 -helical segments of 11- and 22-amino acids in length (24). Peptides representing each of the segments induce cholesterol efflux approximately in proportion to the lipid bilayer binding (lipid affinity) of each segment (50). To investigate whether the apparent differences between recombinant apoA-I molecules in their stimulation of apoE secretion were attributable to specific -helical domains within apoA-I, particularly in the N-terminal or central domains, we compared apoE secretion to 11- and 22-mer -helical peptides representing specific amphipathic -helical segments of apoA-I (Table I). Only the C-terminal peptide 220–241 stimulated cholesterol efflux significantly above basal (control medium) level. Although significant, cholesterol efflux to peptide 220–241 was less than that to intact apoA-I (6.2 versus 8.1% p < 0.05), consistent with previous literature (50). In contrast to cholesterol efflux, all -helical peptides stimulated apoE secretion. This ranged from 2.0- to 6.0-fold that of control medium, and many peptides achieved stimulation of apoE secretion equivalent to that achieved by apoA-I (e.g. peptides 1–33, 44–87, 88–120, 143–186, 220–241). There was no correlation between the known -helical content of these peptides (24) and stimulation of apoE secretion (R² = 0.002, p = 0.87, data not shown). The peptides studied represent segments of the N terminus, central region, and C terminus of apoA-I, many of which display low lipid affinity (50). This indicates that stimulation of apoE secretion by whole apoA-I or recombinant apoA-I molecules was not attributable to individual -helical segments or isolated domains within apoA-I.

View this table: [in this window] [in a new window]   TABLE I Cholesterol efflux and apoE secretion to -helical segments of apoA-I HMDM were incubated for 8 h with RPMI 1640 medium alone (control), control medium containing 25 µg/ml apoA-I, or control medium containing 25 µg/ml -helical peptides representing segments of apoA-I. Values are mean ± S.D. from triplicate cultures of one experiment, representative of three experiments. Percent cholesterol efflux was calculated by dividing [3H]cholesterol released to the medium by the total [3H]cholesterol in the cells after equilibration (T0), and apoE was determined by Western blotting.

View larger version (32K): [in this window] [in a new window]   FIG. 7. Density of secreted apoE with or without stimulation by apoA-I. Monocytes were differentiated, loaded for 4 days with 50 µg/ml AcLDL-[3H]cholesterol and then washed, equilibrated, and incubated with control medium ± 25 µg/ml apoA-I. Aliquots of media were subjected to ultracentrifugation on 1.33–65% sucrose gradients and analyzed for [3H]cholesterol, and apoE as described under "Experimental Procedures." A, cholesterol efflux without (open circles) and with apoA-I (closed circles); B, apoE secretion without (open circles) and with apoA-I (closed circles). Density of fractions is indicated (open diamonds). Total cholesterol efflux for control and apoA-I were 3.6 ± 0.4, and 8.9 ± 2.0%, respectively, and total apoE secretion for control and apoA-I were 0.4 ± 0.1 and 3.1 ± 0.3 µg/ml, respectively. In C, fractions analyzed by Western blot, after diluting media from cultures exposed to apoA-I 4.0-fold to facilitate comparison of relative distribution of apoE among fractions. Data are representative of three independent experiments.

View larger version (19K): [in this window] [in a new window]   FIG. 8. ApoE secretion is stimulated by apolipoproteins other than apoA-I. Monocytes were differentiated, cholesterol-enriched with AcLDL, and radiolabeled with [35S]methionine, before incubation with 25 µg/ml of each indicated apolipoprotein. A, aliquots of media were removed after 8 h, apoE immunoprecipitated, and secretion to media containing apoA-I, apoA-II, apoA-IV, apoE3, determined as AU of [35S]apoE per mg of cell protein by phosphorimaging of 34-kDa band as described under "Experimental Procedures." ApoE secretion to apoA-I, A-II, and A-IV was confirmed by Western blot as well as by phosphorimaging. In B, all data are expressed as AU of [35S]methionine-labeled apoE secreted/mg cell protein. Cholesterol efflux to all apolipoproteins was 2–3-fold that of control, and did not differ between apolipoproteins (data not shown). *, significantly different versus apoE3 (p < 0.05).

The three apoE isoforms (e.g. E2, E3, E4) differ in their effects on the risk of atherosclerosis and differ substantially in -helix organization, N- and C-terminal domain organization (55), and in the respective lipid binding properties of the C terminus (56). We therefore investigated whether these structural differences affected their stimulation of apoE secretion. All three isoforms stimulated macrophage apoE secretion above basal levels. However, apoE3 stimulated more apoE secretion than did apoE4 (p < 0.01). In contrast, cholesterol efflux to the three apoE isoforms did not differ (data not shown), consistent with a previous study (2). On non-reducing SDS-PAGE, apoE3 and E2, which contain one and two cysteine molecules respectively, self-associated into trimers and tetramers which were reducible with dithiothreitol, whereas apoE4 did not (data not shown). Thus it is possible that either the N- and C-terminal organization of exogenous apoE4, or its lesser self-association, may contribute to lesser apoE secretion to this isoform.

ApoA-I Maintains the Ability to Stimulate ApoE Secretion in Its Lipid-bound Conformation—When complexed with phospholipids (e.g. nascent HDL discs) apoA-I undergoes conformational change, which increases the -helicity of the molecule (54, 57), and potentially increases the exposure of polar residues to the cell surface. Such changes may affect the efficacy of apoA-I-mediated apoE secretion in lipidated particles. ApoE secretion may thus be diminished if critical domains of apoA-I are obscured by lipidation or may be enhanced if greater exposure of polar domains enhances apoE secretion. There is also potential for synergy between phospholipid and apoA-I, as previous studies have identified a lipid-soluble pool of apoE associated with the extracellular matrix of HepG2 cells and macrophages (13, 41, 48, 58).

ApoA-I-phospholipid discs containing POPC (A-I-POPC), POPC/SPM (A-I-POPC/SPM) were compared with POPC liposomes and lipid-free apoA-I for their ability to induce cholesterol efflux and apoE secretion (Table II). Acceptors were added at concentrations matched for apoA-I content to allow accurate comparisons of efficiency in stimulating apoE secretion. Lipidation of apoA-I with POPC significantly increased apoE secretion 2–3-fold relative to apoA-I alone, whereas POPC alone did not significantly stimulate apoE secretion. These data suggest that lipidation of apoA-I promotes apoE secretion, and this might be caused by secondary conformational changes, such as increased exposure of polar residues of apoA-I to the cell surface.

ApoA-II has higher average residue hydrophobicity than apoA-I (54). Consequently, its conformation in a POPC disc may differ from that of apoA-I. Unlike secretion to apoA-I, apoA-II-stimulated apoE secretion was not increased significantly by its lipidation with POPC, even though cholesterol efflux was enhanced (Table II, Part B). This suggests that a relatively specific conformation change in apoA-I, which is not seen in apoA-II, is important for the enhanced stimulation of apoE secretion by apoA-I following its lipidation.

To investigate whether secreted apoE associated with apoA-I-POPC particles, density gradient analysis, and non-denaturing gel electrophoresis was performed on efflux media. ApoE shared the same buoyant density as effluxed cholesterol and apoA-I in media containing apoA-I-POPC discs (density range 1.042–1.141). This differed from media containing lipid-free apoA-I, where effluxed cholesterol was of a more buoyant density than secreted apoE, and was consistent with the possibility that apoE associated with apoA-I-POPC discs. This was directly investigated by non-denaturing gel electrophoresis of media containing apoA-I-POPC and apoA-II-POPC discs, followed by electrophoretic transfer to nitrocellulose membranes and immunoblotting for apoA-I, apoA-II, and apoE (33). After efflux, apoA-I and apoA-II migrated as POPC discs (9.5–10.8-nm diameter and 9.5–10.6-nm diameter, respectively), without evidence of displacement of lipid-free apoA-I or apoA-II from these discs (data not shown). Most secreted apoE migrated as large diameter (17.1 nm) particles, and a small proportion migrated as particles of the same diameter as apoA-I- or apoA-II-POPC-containing discs (9.5–10.8-nm diameter). The association of apoE with apoA-I-POPC or apoA-II-POPC discs would be expected to increase the size of these particles. As the size of apoA-I-POPC or apoA-II-POPC particles was unaltered by secretion of apoE, these data suggest that secreted apoE does not significantly associate with apoA-I- or apoA-II-POPC discs, and does not displace apoA-I or apoA-II from these particles. These results also imply that the amount of apoE secreted to apoA-I-POPC or apoA-II-POPC particles is not explained by differential displacement of apoA-I and apoA-II from POPC particles.

ApoA-I-mediated ApoE Secretion Is Independent of ABCA1 Activity—ApoA-I-mediated cholesterol efflux is known to be critically dependent upon ABCA1 activity. Our data dissociating the structural requirements of apoA-I-stimulated apoE secretion and cholesterol efflux imply that apoA-I-mediated apoE secretion may be independent of ABCA1. Patients with Tangier disease have impaired ABCA1 activity, and, although a previous study indicated that basal apoE secretion was impaired in TD macrophages (16), apoA-I-stimulated apoE secretion was not investigated. To directly investigate the role of ABCA1 in apoE secretion from human macrophages, we studied macrophages of two unrelated patients with molecular, clinical, and cellular phenotypes of TD.

The first subject with TD (TD1, see "Experimental Procedures"), was a compound heterozygote for the novel ABCA1 mutations C733R in exon 16 and a deletion of three nucleotides (c.5220–5222 delTCT) in exon 38 (TD1-HMDM). ApoA-I-mediated cholesterol efflux from TD1-HMDM was severely impaired relative to an unrelated healthy control (HC1)-HMDM, confirming the cellular TD phenotype (Table III, A). Basal apoE secretion was lower from TD1-HMDM than from HC1-macrophages, consistent with an earlier study (16). However, apoA-I strongly stimulated apoE secretion from both TD1-HMDM (11-fold increase, p < 0.01 relative to control media) and control HMDM (3.4-fold increase, p < 0.01 relative to control media), achieving the same total apoE secretion from TD1-HMDM and HC1-HMDM. Thus, despite severely impaired ABCA-1-mediated cholesterol efflux, apoA-I stimulated apoE secretion normally from TD macrophages.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The secretion of apoE by macrophages has been shown to be anti-atherogenic (11), and factors that enhance its secretion may be important targets for the treatment or prevention of atherosclerosis. We have demonstrated that apoA-I stimulates the secretion of a mobile pool of apoE which is only in part derived from the cell surface, and which is otherwise destined for intracellular degradation. We have also identified for the first time a biological activity of apoA-I that is independent of its lipid binding properties, by demonstrating that apoA-I-stimulated apoE secretion is enhanced by C-terminal deletions and is independent of ABCA-1-mediated cholesterol efflux. These data establish that non-ABCA1-mediated processes contribute to prosecretory roles and potentially anti-atherogenic effects of apoA-I.

In previous studies, including our own, the secretion of apoE after exposure of cells to HDL or apoA-I has been concurrent with the efflux of cholesterol (12–15,60). However, cyclodextrin-stimulated cholesterol efflux in itself does not induce apoE secretion, and acute inhibition of apoE synthesis and secretion with cycloheximide does not inhibit cholesterol efflux to apoA-I (13). The latter observations dissociate stimulation of apoA-I-mediated cholesterol efflux and apoE secretion. A similar dissociation was earlier reported for HDL-mediated cholesterol efflux and apoE secretion (61). We have investigated in detail the structural and functional requirements for apoA-I-induced apoE secretion and identify these as being quite distinct from those required for apoA-I-induced cholesterol efflux.

ApoA-I does not increase macrophage apoE mRNA levels, and pulse-chase experiments demonstrate that apoA-I stimulates secretion of newly synthesized apoE, confirming that apoA-I-stimulates apoE secretion post-transcriptionally. Modeling of pulse-chase data indicated that human macrophages contain a larger mobile pool of apoE (E_m), and a lesser, more stable pool of apoE (E_s). Previous studies indicate that such heterogeneous pools of apoE may include apoE on the cell surface (47). Pronase treatment of human macrophages under our experimental conditions indicates that only a small proportion of E_s is on the cell surface.² E_m is, on the basis of our biotin-labeling and 4 °C displacement studies, in part derived from the cell surface pool of apoE, and apoA-I increases secretion and concurrently diminishes degradation of apoE from this pool. However, as the biotin-labeled pool represents only a minor proportion of total apoE secreted (<2%), its quantitative significance is unclear. These data are consistent with either continual displacement of apoE from the cell surface with its recycling and ongoing replenishment from intracellular pools (41), or is consistent with two sources of secreted apoE, a smaller cell surface pool and a larger intracellular pool, both of which are mobilized by apoA-I.

It is clear that the structural requirements for apoA-I-mediated apoE secretion are distinct from those required for cholesterol efflux. Our studies of recombinant (pro-)apoA-I molecules demonstrate that deletion of the C terminus of apoA-I enhances apoE secretion whereas it impairs cholesterol efflux and phospholipid efflux. This is remarkable as all previous studies of apoA-I function have emphasized the importance of its lipid binding C-terminal domain for functional integrity and for binding to the extracellular matrix of the cell surface (62). That C-terminal deletion enhances apoE secretion suggests that apoA-I does not bind to extracellular matrix to achieve this (62). This is consistent with our previous studies indicating that heparinase pretreatment does not inhibit apoA-I-mediated apoE secretion from primary macrophages (13).

The central domain of apoA-I regulates LCAT activity (63) and HDL-mediated inhibition of macrophage homing to atherosclerotic plaque (64). Deletion of this central region of apoA-I (d123–166) was the only modification that decreased the stimulation of apoE secretion by apoA-I. As this domain affects apoA-I flexibility (29), it suggests that for whole apoA-I, flexibility is more important than lipid affinity in inducing apoE secretion. It is unlikely to contain an epitope specific for apoA-I-induced apoE secretion given that -helical peptides away from this domain, and other apolipoproteins, also induced apoE secretion. Whether apolipoprotein flexibility is also important for other apolipoproteins, e.g. apoA-II, apoA-IV, and apoEs, is unknown.

Systematic comparison of individual -helical peptides of apoA-I identified that only the C-terminal -helical peptide was capable of independently inducing cholesterol efflux from primary human macrophages, consistent with earlier studies (50). In contrast, all -helical peptides stimulated apoE secretion. As cholesterol efflux to peptides is related to their lipid affinity (50), this property does not appear to determine induction of apoE secretion. Stimulation of apoE secretion appears to be a general structural property of -helical proteins and peptides, which, in the case of whole apoA-I, can be enhanced by overall protein flexibility and inhibited by lipid binding to the plasma membrane.

One of our most striking observations was the enhanced secretion of apoE to apoA-I-POPC discs relative to apoA-I. This was not observed with apoA-II, which stimulated apoE secretion to the same extent in free or lipid-bound state. The difference in response to lipidation of apoA-I and apoA-II is interesting given that cholesterol efflux to both apolipoproteins increased with lipidation. Apolipoprotein-mediated apoE secretion may allow subsequent binding of secreted apoE to exogenous phospholipids, and this could explain some apparent synergy between apoA-I and POPC. However, association of apoE with apoA-I- or apoA-II-POPC discs would be expected to require displacement of apoA-I or apoA-II and/or to increase the apparent size of POPC-containing particles, neither of which were found on non-denaturing gel electrophoresis. The absence of synergy between apoA-II and POPC therefore suggests conformational changes unique to apoA-I occur during its lipidation. We suggest that apoA-I exposes more polar, non-lipid bound residues as a POPC disc than does apoA-II, and that this enhances interaction with exposed residues of apoE on the cell surface, or with undefined plasma membrane sites critical for stimulating vesicular trafficking of apoE to the cell surface.

ApoA-I, A-II, A-IV, E3, and apolipoprotein-phospholipid discs all stimulated apoE secretion efficiently, indicating stimulation of apoE secretion is of broad relevance in vivo. The ability of apoE to stimulate apoE secretion is highly significant, as local (autocrine) secretion of apoE in the artery wall could mediate further apoE secretion from neighboring cells, independently of the requirement for plasma-derived HDL species. ApoE4 increases the risk of atherosclerosis and Alzheimer's disease relative to apoE3 (65). Reduced local stimulation of apoE secretion by apoE4 in the artery or in the CNS might contribute to disease states associated with the apoE4 phenotype. ApoE4 differs from apoE3 in a single amino acid substitution, arginine instead of cysteine in position 112. This confers a more positive apoE charge, and causes conformational change to the N-terminal helix of apoE4, as a salt bridge forms between Arg¹¹² and Glu¹⁰⁹, and this alters the conformation of Arg⁶¹, which interacts with Glu²⁵⁵ (