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Originally published In Press as doi:10.1074/jbc.M611754200 on March 6, 2007

J. Biol. Chem., Vol. 282, Issue 18, 13746-13753, May 4, 2007
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Impaired Secretion of Apolipoprotein E2 from Macrophages*Formula

Daping Fan{ddagger}, Shenfeng Qiu§, Cheryl D. Overton{ddagger}, Patricia G. Yancey{ddagger}, Larry L. Swift, W. Gray Jerome, MacRae F. Linton{ddagger}||, and Sergio Fazio{ddagger}1

From the {ddagger}Atherosclerosis Research Unit, Division of Cardiology, Department of Medicine, §Department of Molecular Physiology and Biophysics, Department of Pathology and ||Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6300

Received for publication, December 22, 2006 , and in revised form, March 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human apoE is a multifunctional and polymorphic protein synthesized and secreted by liver, brain, and tissue macrophages. Here we show that apoE isoforms and mutants expressed through lentiviral transduction display cell-specific differences in secretion efficiency. Whereas apoE3, apoE4, and a natural mutant of apoE4 (apoE-Cys142) were efficiently secreted from macrophages, apoE2 and a non-natural apoE mutant (apoE-Cys112/Cys142) were retained in the perinuclear region and only minimally secreted. The secretory block for apoE2 in macrophages was not affected by the ablation of LDLR (low density lipoprotein receptor), ABCA-1, or SR-BI (scavenger receptor class B type I) but was released in the absence of low density lipoprotein receptor related protein (LRP). In co-immunoprecipitation experiments, an anti-apoE antibody pulled down two times more LRP in apoE2-transduced macrophages than in apoE3-expressing macrophages. Non-reducing SDS-PAGE/Western blot analyses showed that macrophage apoE2 is mostly dimeric and multimeric, whereas apoE3 is predominantly monomeric. ApoE2 retention and multimer formation also occurred in human macrophages derived from the monocyte cell line THP-1. These results were specific for macrophages, as in transduced mouse primary hepatocytes: 1) ApoE2 was secreted as efficiently as apoE3 and apoE4; 2) all isoforms were exclusively in monomeric form; 3) there was no co-immunoprecipitation of apoE and LRP. A microsomal triglyceride transfer protein (MTP) inhibitor nearly deleted apoB100 secretion from hepatocytes without affecting apoE secretion. These data show that macrophages retain apoE2, a highly expressed protein carried by about 8% of the human population. Given the role of locally produced apoE in regulating cholesterol efflux, modulating inflammation, and controlling oxidative stress, this unique property of apoE2 may have important impacts on atherogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
ApoE,2 a 299-residue exchangeable plasma apolipoprotein, is a major player in lipoprotein metabolism and cardiovascular disease (1). It also plays critical roles in many other important biological processes, including Alzheimer disease and cognitive function, immunoregulation and response to infectious agents, intracellular cholesterol trafficking and control of oxidation, and apoptosis (2). ApoE is synthesized and secreted primarily by the liver but also by brain, skin, and tissue macrophages throughout the body (1, 3). Although most lipoprotein-associated apoE is of hepatic origin, macrophage apoE is a major regulator of cholesterol entry and exit within the atherosclerotic plaque. The atheroprotective role of macrophage apoE has been well documented. Macrophage-derived apoE has been shown to protect against atherosclerosis, both early (4-6) and late during plaque development (7, 8), and even when expressed in amounts too low to affect plasma lipid levels (9). Moreover, C57BL/6 mice reconstituted with apoE-/- bone marrow develop 10-fold more atherosclerosis than control mice fed a butter fat diet without any differences in plasma lipid levels (10). The atherogenic effect of apoE deletion from macrophages has also been confirmed in other mouse models, including apoAI-/- (11) and LDLR-/- (7) mice. Macrophage apoE exerts its atheroprotective effects not only through an influence on plasma and cellular cholesterol metabolism (12, 13) but also through non-lipid effects including control of oxidation and cell proliferation (14-16).

Experimental evidence suggests that hepatocyte-derived apoE and macrophage-derived apoE either are functionally different or operate at different physiologic thresholds. For example, we showed that macrophage apoE will not induce lipoprotein clearance in LDLR-/-apoE-/- mice even at plasma concentrations up to 17 times above normal (17). This indicates that hepatocyte apoE has a unique effect on lipoprotein capture and internalization by LRP when the LDLR is absent. Conversely, low plasma levels of apoE derived from the liver are able to maintain normolipidemia, whereas correspondingly low levels of apoE derived from the macrophage are associated with elevated cholesterol in the same murine model (18).

Human apoE is a polymorphic protein; three common isoforms, apoE2 (Cys112/Cys158), apoE3 (Cys112/Arg158), and apoE4 (Arg112/Arg158), have frequencies in the general population of about 8, 77, and 15%, respectively (19). The polymorphism of human apoE profoundly influences its functions due to the isoform-dependent differences in receptor binding activity and lipoprotein association preference. For example, apoE2 has drastically reduced LDLR binding activity compared with apoE3 and apoE4. Also, whereas apoE2 and apoE3 mostly associate with HDL, apoE4 preferentially binds to VLDL (2). In addition, apoE4 has been implicated in the development of Alzheimer disease (20). The structural basis underlying this functional variability has been thoroughly investigated (21-28), but the structure of full-length apoE has not yet been solved. Other than the three common isoforms, there are numerous apoE rare variants associated with abnormal lipid levels; among them, apoE-Cys142 causes dominant type III hyperlipidemia because of severely impaired heparin binding activity (29, 30).

To investigate the mechanisms of functional variation between apoE isoforms and mutants in different cells, we developed a highly efficient lentivirus-based transduction system for human apoE. Using this system, we introduced high level expression of the three human apoE isoforms and two mutants into mouse primary macrophages and hepatocytes to study apoE secretion patterns. We identified a unique LRP-mediated processing of apoE2 by macrophages leading to intracellular retention and impared secretion of this isoform, likely due to multimerization of the protein. Given the importance of macrophage apoE in the local regulation of atherogenesis, these findings may have relevance for the many individuals carrying the apoE2 isoform.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Reagents—Mice of different genotypes were maintained in microisolator cages on a rodent chow diet containing 4.5% fat (No. 5010; Purina Mills, Inc., St. Louis, MO) in the Vanderbilt University animal care facility. Animal care and experimental procedures were performed according to the regulations of the Institutional Animal Care and Usage Committee of Vanderbilt University. Cells of the human monocyte line THP-1 were obtained from ATCC (catalog number TIB-202). Dulbecco's modified Eagle's medium (DMEM) was purchased from Sigma. HDL (d = 1.063-1.121) was prepared from C57BL/6 mouse serum by ultracentrifugation on a table top centrifuge (TL120; Beckman Instruments, Inc.). MTP inhibitor BMS197636 was a gift from Dr. Richard Gregg, Bristol-Myers Squibb.

Lentiviral Constructs Generation—Human immunodeficiency virus-based lentiviral vectors PWPT-GFP and PWPIGFP were kindly provided by Dr. Dider Trono (Lausanne, Switzerland). A pGL3 plasmid containing human apoE multimer enhancer 1 (ME1) and human apoE promoter (apoE-p) was a gift from Dr. John Tayor (San Francisco); pET32a plasmids harboring human apoE2 and apoE4 cDNA were provided by Dr. Karl Weisgraber. First, ME1-apoE-p sequence was subcloned in a pBlueScript II (pBSII) vector, followed by human apoE cDNA (954 bps) and human growth hormone poly(A) tail as a transcriptional termination signal to generate pBSII-apoE2 and pBSII-apoE4; second, pBSII-apoE3, pBSII-apoE-Cys142, and pBSII-apoE-Cys112/Cys142 were generated by site-directed mutagenesis using pBSII-apoE2 or pBSII-apoE4 as template; last, the fragments of ME1-apoE-p-apoE cDNA were amplified by PCR from pBSII constructs and subcloned in PWPT-GFP vector in place of EF-1 short promoter and GFP sequence. The final lentiviral constructs were designated as PWPT-apoE2, PWPT-apoE3, PWPT-apoE4, PWPT-apoEC (apoE-Cys-142), and PWPT-apoECC (apoE-Cys112/Cys142). Bi-cistronic expression constructs were also generated for human apoE2, apoE3 and apoE4, designated PWPI-apoE-GFP. Briefly, apoE coding sequence (954 bp) was subcloned into PWPI-GFP vector using single enzyme (PmeI) insertion and direction screening. Supplemental Fig. 1 shows a schematic drawing of the lentiviral apoE constructs. The sequence of all constructs was verified by DNA sequencing.

Transfection and Viral Production—Lentiviral expression plasmid for human apoE, along with packaging plasmid pCMV{Delta}R8.91 and envelope plasmid pMD2.G (both courtesy of Dr. Dider Trono) were co-transferred with a mass ratio of 3:2:1 into 30-40% confluent human embryonic kidney epithelical cell line (HEK) 293T cells using ProFection® mammalian transfection system (Promega, catalog number E1200) following the manufacture's instructions. Six to eight hours later the medium was changed to new DMEM medium with 10% FBS to collect viral particles for 48 h. The viral particle suspension was filtered and subjected to ultracentrifugation at 26,000 rpm for 2 h on a Beckman L-80 ultracentrifuge and to remove medium; and the viral particles were re-suspended in a small volume of PBS. The titer of concentrated viral particle suspension is determined by standard HeLa titer procedure using GFP-lentiviral suspension prepared from a parallel transfection and PWPI-apoE-GFP lentiviral suspensions.

Peritoneal Macrophage Preparation and Transduction—Peritoneal macrophages were elicited from mice by injection of 3 ml 3% thioglycollate and harvested 3 days later by injecting 10 ml PBS into peritoneal cavity and collecting the exudate cells (8). Exudate cells were allowed to adhere for 1 h and nonadherent cells were washed from plates. The remaining macrophage-enriched adherent cells were scraped off plates and suspended in DMEM with 10% FBS at desired cell density. For macrophage transduction, 1 x 106 cells in 1 ml medium was plated to each well of 24-well plate, proper amount of viral concentrate (m.o.i. = 15 ~ 30) was added to each well and mixed and the plate was spun at 1000 x g for 1 h at 25°C. Sixteen to twenty-four hours later, the medium was changed to DMEM with 1% FBS. Another 24-48 h later, the medium and cells were collected for Western blot analysis of apoE production and secretion or for total RNA extraction.

Transduction of THP-1 Cells—THP-1 cells were cultured in DMEM with 1% FBS at 106/well in 12-well plate and were induced to differentiate by phorbol ester 12-O-tetradecanoylphobol-13-acetate (15 ng/ml). After cell differentiation for 72 h, the attached mature macrophages were transduced with lentiviruses as described above for primary mouse macrophages.

Primary Hepatocyte Isolation and Transduction—Hepatocytes were isolated as described previously (31). The desired number of viral particles (m.o.i. = 30) were coated by Retronectin (TaKaRa Bio Inc.) to 12-well poly-D-lysine-coated dishes (BD Biosciences) following the manufacture's instructions. Cells at a density of 5 x 105 viable cells/ml (1 x 106 cells/well in 12-well culture dish) were plated onto human apoE or control lentivirus coated dish and cultured as described (32). Four hours after plating, the medium along with nonadherent cells was removed, and fresh medium was added. Forty-eight hours later, the medium and cell lysates were collected for Western blot analysis. For MTP inhibitor treatment, 200 nM BMS197636 was added to the fresh medium when the medium was changed 4 h after plating.

Western Blot—Culture medium and cell lysates proteins were separated by NuPAGE® 4-12% BisTris gels (Invitrogen) and transferred to nitrocellulose membranes (Amersham Bioscience). The primary antibody against human apoE was a polyclonal goat anti-human apoE (Academy Bio-Medical Co., catalog number 50A-G1b); the primary antibody against mouse LRP was a polyclonal rabbit anti-mouse LRP (Dr. Joachim Herz, UTSW, Dallas), which specifically recognizes the 85-kDa fragment of LRP (33, 34), and has no cross-reactivity with any receptors in LRP-/- macrophage (35); the primary antibody against mouse apoB48/100 was a polyclonal rabbit anti-mouse apoB (BioDesign, catalog number K23300R). The secondary antibodies were horseradish peroxidase-conjugated rabbit anti-goat IgG and goat anti-rabbit IgG (Amersham Biosciences). Signal was detected using the ECL kit (Amersham Biosciences).

Co-immunoprecipitation—Transduced macrophages or hepatocytes were lysed with cell lysis buffer containing 20 mM Tris pH 7.5, 100 mM NaCl, 0.5% Nonidet P-40, 0.5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5% protease inhibitor mixture (Sigma). The cell extracts were spun at top speed at 4 °C to remove cell debris and checked for the protein concentration using a Lowry assay (36). Ten micrograms of monoclonal antibody against human apoE (BioDesign, catalog number H11004M) was added to 500 µg of cell protein in 500-µl volume. The mixture was incubated for 2 h at 4°C with constant shaking; 30 µl of washed protein G beads (New England Biolabs) was added, and the mixture was further incubated for 2 h at 4°C with shaking. The tubes were put on a Magnetic Separation Rack (New England Biolabs), the solution was removed, and the beads were washed four times with cell lysis buffer. Sixty microliters 3x SDS-PAGE sample buffer with beta-mecaptomethanol were added to the beads and the beads were boiled for 5 min. The tubes were put on the rack again and the solution was loaded to SDS-PAGE for detecting human apoE and mouse LRP by Western blots.

Immunofluorescence Staining and Confocal Microscopy—Confocal microscopy was used to determine the cellular localization of apoE. Macrophages were transduced in Lab-Tek® Chamber SlideTM (Nalge Nunc International, Rochester, NY). Twenty-four hours after transduction, medium was changed to DMEM with 1% FBS. After further incubation for 24 h, macrophages were washed once with ice-cold PBS before fixation in 2% paraformaldehyde for 30 min on ice and permeabilized in 0.1% Triton X-100 in PBS before addition of polyclonal rabbit anti-human apoE antibody (Dako). To differentiate cell surface apoE from internal apoE, we stained nonpermeabilized cells; macrophages were fixed as above and then incubated in 5% normal goat serum in PBS for 30 min on ice before antibody addition. Antibodies were diluted in 5% normal goat serum in PBS and incubated for 1 h at room temperature; samples were washed with PBS after antibody incubation. Cells then were incubated with goat-anti-rabbit F(ab) fragment-TRITC (Jackson ImmunoResearch Laboratories) in 5% normal goat serum PBS for 1 h and washed twice with PBS before mounting. Digital images were acquired with a Zeiss LSM510 confocal microscope using a 40x oil immersion lens (numerical aperture 1.3). For comparison of the fluorescent intensity of cell labeling, all images from the same experiment were acquired with identical settings for laser power, photomultiplier gain, offset, and pinhole sizes. Both single and stack of images were taken across the focal planes that showed maximal fluorescent intensity for each sample.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ApoE2 Secretion in Macrophages—The lentiviral apoE transduction system resulted in an efficiency of transduction higher than 90% for a m.o.i. of 30 in mouse primary macrophages (data not shown). Twenty-four hours after transduction, PCR showed very similar apoE mRNA levels among macrophages transduced with the different apoE isoforms and mutants (Fig. 1A). Interestingly, Western blot analyses showed that whereas apoE3, apoE4, and apoE-Cys142 (apoEC) were almost completely secreted to the cell culture medium by 24 h after transduction, only trace amounts of apoE2 were detected in the medium, and the protein was retained intact inside the macrophages (Fig. 1A). After incubation with normal mouse HDL (50 µg of protein/ml of medium), the secretion of both apoE3 and apoE2 was significantly increased above control conditions, 1.8- and 2.2-fold, respectively (Fig. 1B), indicating that the secretory block for apoE2 can be partially released by HDL. Support to the notion that the blocked apoE2 secretion is not absolute was also given by the observation that by 3 days after transduction, the apoE2 in the medium of transduced macrophages reached a level close to that of apoE3 at 24 h post-transduction (Fig. 1C). However, even with prolonged incubations (up to 9 days) apoE2 continued to be mostly retained in macrophages and did not accumulate in the medium (Fig. 1D).

To explore whether the presence of two cysteines in apoE2 explains its intracellular retention and secretory block in macrophages, we generated a non-natural double-cysteine apoE mutant construct (apoE-Cys112/Cys142, defined as apoECC). Fig. 1E shows that the double cysteine mutant, like apoE2, was retained intact and inefficiently secreted by macrophages.

Fig. 2 shows a stronger immunofluorescence signal for apoE2 compared with apoE3 in macrophages transduced with either PWPT (A and B) or PWPI (C and D) apoE lentiviruses. However, there were no differences in localization, with both apoE2 (A and C) and apoE3 (B and D) predominantly in the perinuclear region.

To identify the involvement of lipoprotein receptors or cholesterol transporters in the apoE2 secretion block, we transduced macrophages of different genotypes, including wildtype, ABCA1-/-, apoE-/-/LDLR-/-, and apoE-/-/SR-BI-/-. The results show that the repressed secretion of apoE2 is not sensitive to or modifiable by endogenous apoE, ABCA1, LDLR, or SR-BI (Fig. 3). Interestingly, the apoE2 secretory block was not observed in LRP-/- macrophages (Fig. 4A). To exclude the possibility that the apparent apoE2 retention may result from a preferential LRP-mediated apoE2 re-uptake by macrophages, we treated transduced apoE-/- macrophages with RAP protein (250 nM) to block cell surface LRP. Fig. 4B shows that RAP had no effect on apoE2, apoE3, and apoE4 secretion and intracellular retention, suggesting that LRP interacts with apoE2 inside the cell to block its secretion.


Figure 1
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  		FIGURE 1.
Human apoE2 is retained by primary macrophages. Thioglycollate-elicited apoE-/- mouse peritoneal macrophages were transduced with human apoE lentiviruses. A, 24 h after transduction, apoE in media and cell lysate were analyzed by Western blot; some cells were used to extract total mRNA, which was reversely transcribed to cDNA as template to measure the apoE transcript by PCR. EC, apoE-Cys142. B, 24 h after transduction, the culture media were changed to DMEM alone or with 50 µg (protein) of HDL/ml for another 24 h before the media were collected for Western blot. C, the medium samples collected at day 1 and day 3 were analyzed for apoE.D, the secretion and retention of apoE isoforms after prolonged macrophage post-transduction incubation were compared by Western blot. E, secretion of a non-natural double cysteine mutant, apoE-Cys112/Cys142 (ECC), was compared with apoE2 and apoE3.

 


Figure 2
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  		FIGURE 2.
Intracellular location of human apoE in macrophages. Confocal microscopic immunofluorescence images of transduced apoE-/- mouse macrophages. Human apoE stains red. GFP shows green. A, PWPT-apoE2-transduced macrophages. B, PWPT-apoE3-transduced macrophages. C, PWPI-apoE2 transduced macrophages. D, PWPI-apoE3 transduced macrophages.

 


Figure 3
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  		FIGURE 3.
The secretory block of apoE2 in macrophages is not affected by endogenous apoE, LDLR, ABCA1, and SR-BI. Primary macrophages of different genotypes were transduced with apoE lentiviruses. The conditioned media and cell lysates were analyzed by Western blot for human apoE. The cross-reaction with mouse apoE is indicated by a black arrow.

 


Figure 4
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  		FIGURE 4.
The secretory block of apoE2 is significantly attenuated by LRP deletion in macrophages. A, LRP-/- macrophages were transduced with apoE lentiviruses; cell lysate and conditioned media were blotted by antihuman apoE antibody. B, the addition of 250 nM recombinant RAP protein to the medium did not affect secretion of apoE isoforms from transduced apoE-/- macrophages.

 
ApoE2 Forms Disulfide Bond-linked Multimers in Macrophages and Binds to LRP—As observed above, because both double cysteine apoE (apoE2 and apoECC) were retained in macrophages, we reasoned that formation of intermolecular disulfide bonds could explain the retention phenomenon. Therefore, we used non-reducing conditions to check whether apoE2 forms intracellular disulfide-bond linked multimers. Fig. 5A shows that apoE2 was in the form of dimer, trimer, tetramer, and higher multimers inside the macrophage while accumulating mainly as a dimer in the medium. On the other hand, apoE3 was mostly monomeric both inside macrophages and in the medium.

To investigate whether apoE2 retention is caused by binding of this isoform to LRP, we performed co-immunoprecipitation experiments with a monoclonal anti-human apoE antibody to precipitate human apoE from the cell lysate and culture medium of transduced macrophages. Fig. 5B shows that coimmunoprecipitation of LRP was two times higher in apoE2 macrophages than in apoE3 macrophages, in a manner roughly proportional to the amounts of precipitated apoE (1.8-fold more apoE2 than apoE3).


Figure 5
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  		FIGURE 5.
ApoE2 forms disulfide bond-linked multimers in macrophages and binds to LRP. A, non-reducing SDS-PAGE and Western blot of cell lysates and conditioned media from transduced apoE-/- macrophages. B, LRP was co-immunoprecipitated with apoE from cell lysates of transduced macrophages. The cell lysate and culture medium were precipitated by anti-human apoE monoclonal antibody under native condition; the precipitate then was blotted by goat anti-human apoE antibody and rabbit anti-mouse LRP antibody.

 


Figure 6
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  		FIGURE 6.
Effect of co-transduction of apoE2 with other isoforms on the secretion of apoE from macrophages. ApoE-/- macrophages were transduced with apoE2, E3 or E4 alone, or co-transduced with E2 and E3 or E2 and E4. The conditioned medium and cell lysates were subjected to non-reducing SDS-PAGE and Western blot.

 


Figure 7
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  		FIGURE 7.
ApoE2 secretion is impaired in THP-1-derived human macrophages. THP-1-derived macrophages were transduced with lentiviruses; 24 h after transduction, culture media and cell lysates were analyzed for apoE by non-reducing SDS-PAGE and Western blot. Note: THP-1-derived macrophages are apoE3-expressing cells; they synthesize and secrete human apoE3 (as indicated in the lanes with GFP transduction).

 
Taking advantage of the ability to distinguish macrophage apoE2 from apoE3 and apoE4 by conformation via non-reducing SDS-PAGE/Western blot, we co-transduced macrophages with apoE2 and either apoE3 or apoE4 to determine whether the apoE2 retention would have a dominant-negative effect on other isoforms, causing either increased apoE3 retention or enhanced apoE2 secretion. Fig. 6 shows that the distribution of bands in doubly transduced macrophages is equal to the sum of the bands in singly transduced cells, suggesting that apoE isoforms do not cross-influence conformation and trafficking pathway in a dominant-negative loop.

To rule out the possibility that overexpressed human apoE2 could be artificially retained in mouse macrophages and to test whether the secretory block of apoE2 also occurs in human macrophages, we transduced macrophages derived from the human monocyte THP-1 cell line. Since THP-1-derived macrophages produce endogenous human apoE3, we adjusted the lentiviral MOI so that synthesis of the transduced apoE would occur at comparable levels to endogenous apoE3. Non-reducing SDS-PAGE/Western blot analysis showed that apoE2 was mainly retained in macrophages in dimers and trimers, with minimal secretion into the culture medium, whereas apoE3 and apoE4 were efficiently secreted, apoE3 as monomers and dimers and apoE4 as monomers (Fig. 7).

ApoE Secretion from Primary Hepatocytes—We next studied whether the impaired secretion of apoE2 also occurs in hepatocytes. Primary hepatocytes from apoE-/- mice were transduced with apoE2, apoE3, apoE4, and apoEC lentiviruses. Nonreducing SDS-PAGE/Western blots showed that all of the apoE isoforms were efficiently secreted by the hepatocytes. Also, in striking contrast with the results obtained with transduced macrophages, all apoE isoforms in hepatocytes were exclusively monomeric (Fig. 8A). The anti-human apoE antibody precipitated comparable amounts of apoE from apoE2 or apoE3 transduced hepatocytes (Fig. 8B), but no LRP was co-precipitated (Fig. 8C).

To study whether the different secretory trajectory shown by apoE in hepatocytes was linked to its association and exocytosis with newly formed apoB-containing lipoproteins, we treated transduced hepatocytes with the MTP inhibitor BMS-197636 to block VLDL assembly. Fig. 9 shows that even though the apoB100 secretion was nearly completely abolished by MTP inhibition, the secretion efficiency of apoE2 as well as apoB48 was not affected.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we took advantage of a highly efficient lentiviral gene delivery system to induce high level expression of human apoE isoforms in primary mouse macrophages and hepatocytes. We found that apoE molecules with two cysteines (apoE2 and apoE-Cys112/Cys142) were inefficiently secreted from primary macrophages, and apoE2 was mostly present in the form of dimers and higher multimers linked by intermolecular disulfide bonds. The secretory block and multimer formation of apoE2 were also found true in human THP-1 macrophages even when apoE2 was at a 1:1 ratio with endogenous human apoE3. The apoE multimers were bound to LRP and retained in the secretion pathway possibly because of this association. On the other hand, primary hepatocytes secreted double cysteine apoE molecules as efficiently as the other apoE variants, and all of apoE forms were exclusively in monomeric form and not associated with LRP.


Figure 8
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  		FIGURE 8.
Human apoE is secreted from hepatocytes as monomers without isoform-dependent differences and does not bind to LRP. ApoE-/- mouse primary hepatocytes were transduced with human apoE isoforms and a variant apoE-Cys142 (EC). A, non-reducing SDS-PAGE and Western blot of cell lysate and conditioned medium of transduced hepatocytes. B and C, the cell lysates of apoE2- and apoE3-transduced hepatocytes were precipitated by anti-human apoE monoclonal antibody; the precipitate was then blotted for human apoE (B) or mouse LRP (C) under reducing conditions.

 


Figure 9
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  		FIGURE 9.
ApoE secretion from hepatocytes is not dependent on apoB100 secretion. ApoE-/- mouse primary hepatocytes were transduced with human apoE2 or apoE3 lentiviruses, with or without MTP inhibitor added in the culture medium. The cell lysate and culture medium were blotted for human apoE and mouse apoB.

 
The most interesting finding of this study is the previously unknown secretion block of apoE2 from macrophages (Fig. 1). Secretion of apoE from macrophages is believed to influence cholesterol efflux, thus protecting against the development of atherosclerosis. In this sense, rapid secretion of apoE from macrophages may be seen as beneficial (5). On the other hand, intracellular retention of apoE in some other cell types, for example neurons, may positively correlate to cellular functions including neurite outgrowth and stabilization of the cytoskeleton (37) and therefore may be more desirable than efficient secretion. Macrophage apoE secretion is regulated by multiple mechanisms, including post-transcriptional glycosylation (38), intracellular sterol milieu (39), cholesterol loading (40, 41), cell surface receptor expression (42-44), and presence of apoAI/HDL in the culture medium (45). Whether the secretory block of apoE2 affects cholesterol homeostasis or other aspects of macrophage biology (i.e. anti-oxidation) is currently under investigation. Studies on monocyte-derived macrophages isolated from E2/E2, E3/E3, or E4/E4 subjects showed that E2 cells secreted less apoE than E3 or E4 cells but were more protected from cholesterol accumulation than E3 macrophages (40). In our study the apoE2 secretion block was not absolute, and the secretion was improved by HDL stimulation and with prolonged culture time (Fig. 1, B and C), suggesting that under certain stimuli macrophage apoE2 may be secreted for an extracellular effect.

The mechanism underlying the apoE2 secretory block in macrophages may be related to the formation of disulfide bondlinked apoE2 dimers and higher multimers which bind to LRP intracellularly. The evidence includes: 1) LRP-/- macrophages, but not ABCA1-/-, LDLR-/-, and SR-B1-/- macrophages, secrete apoE2 efficiently (Figs. 3 and 4); 2) apoE2 binds to LRP in macrophages (Fig. 5B); 3) apoE2 forms dimers and higher multimers in macrophages (Fig. 5A); 4) another multimerprone mutant (apoE-Cys112/Cys142) is also retained inside macrophages (Fig. 1D); 5) incubation with RAP does not increase apoE2 in the medium (Fig. 4B); 6) the intracellular location of apoE2 is compatible with a block in its secretory routing (Fig. 2). Even though the existence of a bridging protein cannot be excluded, direct apoE-LRP interaction is likely accountable for this phenomenon. LRP prefers lipid-bound apoE but also binds avidly to lipid-free apoE aggregates or disulfide-linked dimers (46-48). It is known that chaperone molecules like RAP bind to LRP during its maturation and intracellular transport and prevent ligands like apoE from binding to LRP (49, 50). Whether the binding of the apoE2 multimers to LRP and the retention of apoE2-LRP complex in macrophages leads to dysfunction of LRP remains to be explored.

This study revealed a striking difference between hepatocyte apoE and macrophage apoE. Whereas macrophages retained apoE2 as dimers and higher multimers bound to LRP, apoE2 in hepatocytes was efficiently secreted, was primarily monomeric both inside the cell and upon secretion, and did not bind to LRP (Fig. 8). These differences could result from the different lipidation status and conformation of apoE in the two cell types, as macrophage apoE is lipid-poor, whereas hepatocyte apoE is secreted as a component of lipoprotein particles. Hepatocytes recruit newly synthesized apoE into nascent VLDL during transit through the endoplasmic reticulum and therefore can potentially rescue apoE2 from the LRP-mediated trapping observed in macrophages. To test whether this was indeed the cause for the difference in apoE2 handling between hepatocytes and macrophages, we used an MTP inhibitor to block VLDL secretion, with the intent of allowing hepatocyte apoE to move along the secretory pathway as a lipid-poor protein. The MTP inhibitor completely blocked apoB100 secretion but had no effect on apoE2 release (Fig. 9). Although secretion of apoB48 lipoproteins was not inhibited, we believe the experiment provides data strongly suggestive of the possibility that the normal processing of apoE2 in hepatocytes is not due to its being embedded with the newly forming apoB-containing lipoproteins.

A likely explanation for the difference in hepatocyte and macrophage apoE handling is that the lipidation of hepatocyte apoE molecules prevents the formation of intermolecular disulfide bonds. Additionally, the concentration of apoE on the lipoprotein particle surface may be lower than the threshold for intracellular binding to LRP, since it is known that native VLDL particles need to be enriched with large amounts of apoE extracellularly to display sufficient LRP/HSPG binding on hepatocyte surface (51, 52).

Experimental evidence suggests that hepatocyte-derived apoE and macrophage-derived apoE may be either functionally different or operate at different physiologic thresholds (10, 17, 18, 53-60). The difference in their conformation suggested in this study may underlie the functional difference between hepatocyte and macrophage apoE.

ApoE isoforms have profound functional implications relevant to many human diseases. While apoE4 is associated with increased risk for both cardiovascular disease and Alzheimer disease (20), apoE2 has been suggested to be protective despite the fact that 10% of the homozygous apoE2 subjects develop the atherogenic type III hyperlipidemia (61). Whether secretion block of apoE2 from macrophages contributes to its specific role in the development of atherosclerosis warrants further investigation.

In addition, apoE3 has been shown in human plasma to form either homodimers (62) or heterodimers with apoAII (63); and both of the dimer forms displayed a marked preference for HDL (62, 64). By column chromatography apoE3-AII heterodimer and apoE3-apoE3 homodimer were found to account for ~26 and ~28% of total apoE3 in plasma (62), but it is not clear how and where the dimerization takes place. We showed that, in macrophages apoE2 and apoE3 formed dimers or higher multimers while secreted (Fig. 5A), whereas both apoE2 and apoE3 were secreted from hepatocytes exclusively as monomers (Fig. 8A). These results suggest that the dimers of apoE3 and multimers of apoE2 in plasma may come from macrophages.

In conclusion, our data suggest that apoE adopts a unique conformation in macrophages, leading to poor secretion and intracellular retention of the apoE2 isoform, which accumulates in multimeric forms and binds to LRP. Given the large biologic effect of macrophage apoE in atherogenesis and the high prevalence of the apoE2 isoform in human populations, these results may have wide relevance to the pathogenesis of the arterial plaque.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL-057986 (to S. F.) and an American Heart Association Postdoctoral Fellowship (to D. F.). This study was also supported in part by the Lipid, Lipoprotein, and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Centers (National Institutes of Health Grant DK59637). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. Back

1 To whom correspondence should be addressed: 2220 Pierce Ave., 383 PRB, Vanderbilt University, Nashville, TN 37232. Tel.: 615-936-1450; Fax: 615-936-3486; E-mail: sergio.fazio{at}vanderbilt.edu.

2 The abbreviations used are: apoE, apolipoprotein E; LDLR, low density lipoprotein receptor; LRP, low density lipoprotein receptor-related protein; RAP, receptor-associated protein; MTP, microsomal triglyceride transfer protein; ABCA1, ATP-binding cassette transporter A-1; SR-BI, scavenger receptor class B type I; m.o.i., multiplicity of infection; VLDL, very low density lipoprotein; DMEM, Dulbecco's modified Eagle's medium; HDL, high density lipoprotein; GFP, green fluorescent protein; FBS, fetal bovine serum; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We thank John Blakemore, Dr. Yan Ru Su, and Lei Ding for their assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mahley, R. W. (1988) Science 240, 622-630[Abstract/Free Full Text]
  2. Mahley, R. W., and Rall, S. C., Jr. (2000) Annu. Rev. Genomics Hum. Genet. 1, 507-537[CrossRef][Medline] [Order article via Infotrieve]
  3. Mahley, R. W., and Huang, Y. (1999) Curr. Opin. Lipidol. 10, 207-217[CrossRef][Medline] [Order article via Infotrieve]
  4. Shimano, H., Ohsuga, J., Shimada, M., Namba, Y., Gotoda, T., Harada, K., Katsuki, M., Yazaki, Y., and Yamada, N. (1995) J. Clin. Invest. 95, 469-476[Medline] [Order article via Infotrieve]
  5. Bellosta, S., Mahley, R. W., Sanan, D. A., Murata, J., Newland, D. L., Taylor, J. M., and Pitas, R. E. (1995) J. Clin. Invest. 96, 2170-2179[Medline] [Order article via Infotrieve]
  6. Hasty, A. H., Linton, M. F., Brandt, S. J., Babaev, V. R., Gleaves, L. A., and Fazio, S. (1999) Circulation 99, 2571-2576[Abstract/Free Full Text]
  7. Fazio, S., Babaev, V. R., Burleigh, M. E., Major, A. S., Hasty, A. H., and Linton, M. F. (2002) J. Lipid Res. 43, 1602-1609[Abstract/Free Full Text]
  8. Linton, M. F., Atkinson, J. B., and Fazio, S. (1995) Science 267, 1034-1037[Abstract/Free Full Text]
  9. Yoshida, H., Hasty, A. H., Major, A. S., Ishiguro, H., Su, Y. R., Gleaves, L. A., Babaev, V. R., Linton, M. F., and Fazio, S. (2001) Circulation 104, 2820-2825[Abstract/Free Full Text]
  10. Fazio, S., Babaev, V. R., Murray, A. B., Hasty, A. H., Carter, K. J., Gleaves, L. A., Atkinson, J. B., and Linton, M. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4647-4652[Abstract/Free Full Text]
  11. Ishiguro, H., Yoshida, H., Major, A. S., Zhu, T., Babaev, V. R., Linton, M. F., and Fazio, S. (2001) J. Biol. Chem. 276, 36742-36748[Abstract/Free Full Text]
  12. Lin, C. Y., Duan, H., and Mazzone, T. (1999) J. Lipid Res. 40, 1618-1627[Abstract/Free Full Text]
  13. Zhu, Y., Bellosta, S., Langer, C., Bernini, F., Pitas, R. E., Mahley, R. W., Assmann, G., and von Eckardstein, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7585-7590[Abstract/Free Full Text]
  14. Vogel, T., Guo, N. H., Guy, R., Drezlich, N., Krutzsch, H. C., Blake, D. A., Panet, A., and Roberts, D. D. (1994) J. Cell. Biochem. 54, 299-308[CrossRef][Medline] [Order article via Infotrieve]
  15. Mazzone, T. (1996) Curr. Opin. Lipidol. 7, 303-307[Medline] [Order article via Infotrieve]
  16. Mabile, L., Lefebvre, C., Lavigne, J., Boulet, L., Davignon, J., Lussier-Cacan, S., and Bernier, L. (2003) J. Cell. Biochem. 90, 766-776[CrossRef][Medline] [Order article via Infotrieve]
  17. Linton, M. F., Hasty, A. H., Babaev, V. R., and Fazio, S. (1998) J. Clin. Invest. 101, 1726-1736[Medline] [Order article via Infotrieve]
  18. Raffai, R. L., Hasty, A. H., Wang, Y., Mettler, S. E., Sanan, D. A., Linton, M. F., Fazio, S., and Weisgraber, K. H. (2003) J. Biol. Chem. 278, 11670-11675[Abstract/Free Full Text]
  19. Weisgraber, K. H. (1994) Adv. Protein Chem. 45, 249-302[Medline] [Order article via Infotrieve]
  20. Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small, G. W., Roses, A. D., Haines, J. L., and Pericak-Vance, M. A. (1993) Science 261, 921-923[Abstract/Free Full Text]
  21. Dong, L. M., and Weisgraber, K. H. (1996) J. Biol. Chem. 271, 19053-19057[Abstract/Free Full Text]
  22. Dong, L. M., Wilson, C., Wardell, M. R., Simmons, T., Mahley, R. W., Weisgraber, K. H., and Agard, D. A. (1994) J. Biol. Chem. 269, 22358-22365[Abstract/Free Full Text]
  23. Huang, Y., Weisgraber, K. H., Mucke, L., and Mahley, R. W. (2004) J. Mol. Neurosci. 23, 189-204[CrossRef][Medline] [Order article via Infotrieve]
  24. Lund-Katz, S., Wehrli, S., Zaiou, M., Newhouse, Y., Weisgraber, K. H., and Phillips, M. C. (2001) J. Lipid Res. 42, 894-901[Abstract/Free Full Text]
  25. Acharya, P., Segall, M. L., Zaiou, M., Morrow, J., Weisgraber, K. H., Phillips, M. C., Lund-Katz, S., and Snow, J. (2002) Biochim. Biophys. Acta 1584, 9-19[Medline] [Order article via Infotrieve]
  26. Morrow, J. A., Segall, M. L., Lund-Katz, S., Phillips, M. C., Knapp, M., Rupp, B., and Weisgraber, K. H. (2000) Biochemistry 39, 11657-11666[CrossRef][Medline] [Order article via Infotrieve]
  27. Morrow, J. A., Hatters, D. M., Lu, B., Hochtl, P., Oberg, K. A., Rupp, B., and Weisgraber, K. H. (2002) J. Biol. Chem. 277, 50380-50385[Abstract/Free Full Text]
  28. Hatters, D. M., Peters-Libeu, C. A., and Weisgraber, K. H. (2006) Trends Biochem. Sci. 31, 445-454[CrossRef][Medline] [Order article via Infotrieve]
  29. Ji, Z. S., Fazio, S., and Mahley, R. W. (1994) J. Biol. Chem. 269, 13421-13428[Abstract/Free Full Text]
  30. Fazio, S., Lee, Y. L., Ji, Z. S., and Rall, S. C., Jr. (1993) J. Clin. Invest. 92, 1497-1503[Medline] [Order article via Infotrieve]
  31. Swift, L. L., Farkas, M. H., Major, A. S., Valyi-Nagy, K., Linton, M. F., and Fazio, S. (2001) J. Biol. Chem. 276, 22965-22970[Abstract/Free Full Text]
  32. Braun, N. A., Mohler, P. J., Weisgraber, K. H., Hasty, A. H., Linton, M. F., Yancey, P. G., Su, Y. R., Fazio, S., and Swift, L. L. (2006) J. Lipid Res. 47, 1176-1186[Abstract/Free Full Text]
  33. Herz, J., Kowal, R. C., Ho, Y. K., Brown, M. S., and Goldstein, J. L. (1990) J. Biol. Chem. 265, 21355-21362[Abstract/Free Full Text]
  34. Willnow, T. E., Moehring, J. M., Inocencio, N. M., Moehring, T. J., and Herz, J. (1996) Biochem. J. 313, 71-76[Medline] [Order article via Infotrieve]
  35. Overton, C. D., Yancey, P. G., Major, A. S., Linton, M. F., and Fazio, S. (2007) Circ. Res. 100, 670-677[Abstract/Free Full Text]
  36. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  37. Ji, Z. S., Pitas, R. E., and Mahley, R. W. (1998) J. Biol. Chem. 273, 13452-13460[Abstract/Free Full Text]
  38. Huang, Z. H., Gu, D., and Mazzone, T. (2004) J. Biol. Chem. 279, 29195-29201[Abstract/Free Full Text]
  39. Duan, H., Lin, C. Y., and Mazzone, T. (1997) J. Biol. Chem. 272, 31156-31162[Abstract/Free Full Text]
  40. Cullen, P., Cignarella, A., Brennhausen, B., Mohr, S., Assmann, G., and von Eckardstein, A. (1998) J. Clin. Invest. 101, 1670-1677[Medline] [Order article via Infotrieve]
  41. Mazzone, T., Basheeruddin, K., and Poulos, C. (1989) J. Lipid Res. 30, 1055-1064[Abstract]
  42. Lucas, M., and Mazzone, T. (1996) J. Biol. Chem. 271, 13454-13460[Abstract/Free Full Text]
  43. Zhao, Y., and Mazzone, T. (1999) J. Lipid Res. 40, 1029-1035[Abstract/Free Full Text]
  44. Lucic, D., Huang, Z. H., Gu, D. S., Altenburg, M. K., Maeda, N., and Mazzone, T. (2006) J. Lipid Res. 48, 366-372[CrossRef][Medline] [Order article via Infotrieve]
  45. Kockx, M., Rye, K. A., Gaus, K., Quinn, C. M., Wright, J., Sloane, T., Sviridov, D., Fu, Y., Sullivan, D., Burnett, J. R., Rust, S., Assmann, G., Anantharamaiah, G., Palgunachari, M. N., Lund-Katz, S., Phillips, M. C., Dean, R. T., Jessup, W., and Kritharides, L. (2004) J. Biol. Chem. 279, 25966-25977[Abstract/Free Full Text]
  46. Narita, M., Holtzman, D. M., Fagan, A. M., LaDu, M. J., Yu, L., Han, X., Gross, R. W., Bu, G., and Schwartz, A. L. (2002) J Biochem. (Tokyo) 132, 743-749[Abstract/Free Full Text]
  47. Ruiz, J., Kouiavskaia, D., Migliorini, M., Robinson, S., Saenko, E. L., Gorlatova, N., Li, D., Lawrence, D., Hyman, B. T., Weisgraber, K. H., and Strickland, D. K. (2005) J. Lipid Res. 46, 1721-1731[Abstract/Free Full Text]
  48. LaDu, M. J., Stine, W. B., Jr., Narita, M., Getz, G. S., Reardon, C. A., and Bu, G. (2006) Biochemistry 45, 381-390[CrossRef][Medline] [Order article via Infotrieve]
  49. Bu, G., Geuze, H. J., Strous, G. J., and Schwartz, A. L. (1995) EMBO J. 14, 2269-2280[Medline] [Order article via Infotrieve]
  50. Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238[Abstract/Free Full Text]
  51. Kowal, R. C., Herz, J., Goldstein, J. L., Esser, V., and Brown, M. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5810-5814[Abstract/Free Full Text]
  52. Ishibashi, S., Herz, J., Maeda, N., Goldstein, J. L., and Brown, M. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4431-4435[Abstract/Free Full Text]
  53. Linton, M. F., Gish, R., Hubl, S. T., Butler, E., Esquivel, C., Bry, W. I., Boyles, J. K., Wardell, M. R., and Young, S. G. (1991) J. Clin. Invest. 88, 270-281[Medline] [Order article via Infotrieve]
  54. Tsukamoto, K., Tangirala, R. K., Chun, S., Usher, D., Pure, E., and Rader, D. J. (2000) Mol. Ther. 1, 189-194[CrossRef][Medline] [Order article via Infotrieve]
  55. Tangirala, R. K., Pratico, D., FitzGerald, G. A., Chun, S., Tsukamoto, K., Maugeais, C., Usher, D. C., Pure, E., and Rader, D. J. (2001) J. Biol. Chem. 276, 261-266[Abstract/Free Full Text]
  56. Tsukamoto, K., Smith, P., Glick, J. M., and Rader, D. J. (1997) J. Clin. Invest. 100, 107-114[Medline] [Order article via Infotrieve]
  57. Kitajima, K., Marchadier, D. H., Miller, G. C., Gao, G. P., Wilson, J. M., and Rader, D. J. (2006) Arterioscler. Thromb. Vasc. Biol. 26, 1852-1857[Abstract/Free Full Text]
  58. Hamilton, R. L., Wong, J. S., Guo, L. S., Krisans, S., and Havel, R. J. (1990) J. Lipid Res. 31, 1589-1603[Abstract]
  59. Weisgraber, K. H., Mahley, R. W., Kowal, R. C., Herz, J., Goldstein, J. L., and Brown, M. S. (1990) J. Biol. Chem. 265, 22453-22459[Abstract/Free Full Text]
  60. Ji, Z. S., Fazio, S., Lee, Y. L., and Mahley, R. W. (1994) J. Biol. Chem. 269, 2764-2772[Abstract/Free Full Text]
  61. Mahley, R. W., Huang, Y., and Rall, S. C., Jr. (1999) J. Lipid Res. 40, 1933-1949[Abstract/Free Full Text]
  62. Weisgraber, K. H., and Shinto, L. H. (1991) J. Biol. Chem. 266, 12029-12034[Abstract/Free Full Text]
  63. Weisgraber, K. H., and Mahley, R. W. (1978) J. Biol. Chem. 253, 6281-6288[Free Full Text]
  64. Weisgraber, K. H. (1990) J. Lipid Res. 31, 1503-1511[Abstract]

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