Recycling of Apoprotein E Is Associated with Cholesterol Efflux and High Density Lipoprotein Internalization*

Joerg HeerenDagger §, Thomas GrewalDagger , Alexander LaatschDagger , Daniel RottkeDagger ||, Franz Rinninger**, Carlos EnrichDagger Dagger , and Ulrike BeisiegelDagger

From the Dagger  Institute for Medical Biochemistry and Molecular Biology, the Department of Molecular Cell Biology and the ** Department of Internal Medicine, University Hospital Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany and Dagger Dagger  Departament de Biologia Cellular, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain

Received for publication, September 4, 2002, and in revised form, February 10, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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After receptor-mediated endocytosis of triglyceride-rich lipoproteins (TRL) into the liver, TRL particles are immediately disintegrated in peripheral endosomal compartments. Whereas core lipids and apoprotein B are delivered for degradation into lysosomes, TRL-derived apoE is efficiently recycled back to the plasma membrane. This is followed by apoE re-secretion and association of apoE with high density lipoproteins (HDL). Because HDL and apoE can independently promote cholesterol efflux, we investigated whether recycling of TRL-derived apoE in human hepatoma cells and fibroblasts could be linked to intracellular cholesterol transport. In this study we demonstrate that HDL3 does not only act as an extracellular acceptor for recycled apoE but also stimulates the recycling of internalized TRL-derived apoE. Furthermore, radioactive pulse-chase experiments indicate that apoE recycling is accompanied by cholesterol efflux. Confocal imaging reveals co-localization of apoE and cholesterol in early endosome antigen 1-positive endosomes. During apoE re-secretion, HDL3-derived apoA-I is found in these early endosome antigen 1, cholesterol-containing endosomes. As shown by time-lapse fluorescence microscopy, apoE recycling involves the intracellular trafficking of apoA-I to pre-existing and TRL-derived apoE/cholesterol-containing endosomes in the periphery. Thus, these studies provide evidence for a new intracellular link between TRL-derived apoE, cellular cholesterol transport, and HDL metabolism.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Chylomicrons (CM)1 and very low density lipoproteins (VLDL) represent the two classes of triglyceride-rich lipoproteins (TRL) that are responsible for the transport of lipids to various cells of the body. After assembly in the intestine, CM mediate the transport of dietary lipids, whereas VLDL are synthesized in the liver to deliver endogenous lipids to peripheral tissues. In the bloodstream CM are hydrolyzed by lipoprotein lipase (LPL), resulting in the formation of chylomicron remnants (CR) (for review see Refs. 1-3). During lipolysis these remnants become enriched with high density lipoproteins (HDL)-derived apoprotein E (apoE) and are rapidly cleared from the plasma in a process known to depend on apoE and LPL via the LDL receptor-related protein and the low density lipoprotein (4-7).

After receptor-mediated endocytosis, the intracellular fate of TRL constituents is far more complex than the classical degradation pathway of low density lipoproteins (LDL) described by Brown and Goldstein (8). A number of studies revealed that the intracellular processing of TRL first involves disintegration in peripheral endosomal compartments, which is followed by a differential sorting of TRL components (9). Using beta -VLDL as a "TRL model particle," Maxfield and co-workers (10-12) identified beta -VLDL-derived lipids in lysosomal compartments of mouse macrophages, whereas apoE was present in peripheral vesicles. Consistent with these findings, lysosomal targeting of TRL proteins and apoE was markedly reduced compared with the degradation of cholesteryl oleate in mouse hepatocytes (13). In human hepatoma cells and fibroblasts, the majority of TRL lipids are targeted to the lysosomal compartment, whereas TRL-derived apoE is found in peripheral recycling endosomes, which are distinct from the perinuclear transferrin recycling compartment (14, 15). Subsequently substantial amounts of TRL-derived apoE are recycled back to the cell surface and re-secreted (14). Recently, several studies (13, 16-18) on the hepatic TRL metabolism in vivo confirmed the disintegration of TRL components within the endosomal compartment and the targeting of TRL lipids to lysosomes. Most remarkably, TRL-derived apoE is recycled and found associated with newly synthesized or exogenous lipoproteins (16). Fazio and co-workers (17) observed an association of recycled apoE with nascent VLDL particles in liver Golgi fractions. Alternatively, we recently identified (14, 16) that HDL serves as a potent extracellular acceptor for re-secreted apoE in hepatocytes in vitro and in vivo.

Because HDL is known to induce cholesterol efflux (19-21), we investigated whether recycling of TRL-derived apoE is accompanied by the mobilization of cellular cholesterol. In this study we demonstrate that HDL3-inducible apoE recycling is associated with cholesterol efflux. Life cell imaging and confocal microscopy revealed that this process involves the targeting of HDL3-derived apoA-I to EEA1-positive endosomes containing both TRL-derived apoE and cholesterol. After re-secretion, TRL-derived apoE and cholesterol are found associated with extracellular HDL particles. Taken together, these findings indicate that apoE recycling provides an efficient mechanism to re-supply plasma with apoE-containing HDL particles. This process would ultimately accelerate the enrichment of CR with apoE and stimulate the hepatic clearance of CR in the postprandial state.

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INTRODUCTION
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Antibodies and Reagents-- Paraformaldehyde (PFA), filipin, glycine, and BSA were purchased from Sigma. Mowiol®4-88 was purchased from Calbiochem. DMEM, PBS, fetal calf serum, trypsin, penicillin, and streptomycin were from Invitrogen. Iodogen was from Pierce. 125I and [3H]cholesterol ([3H]Chol) were from Amersham Biosciences. Heparin (Liquemin®) was purchased from Roche Molecular Biochemicals. Polyclonal antibody against human apoE was from Dako. Monoclonal antibody against early endosome antigen 1 (EEA1) was from BD Biosciences. Monoclonal antibody against adaptor protein 1 (AP1) was kindly provided by E. Ungewickell (22). Alexa 568 nm fluorescence labeling kit and NBD-cholesterol were from Molecular Probes. Cy3 and Cy5 fluorescence protein labeling kit were from Amersham Biosciences. Cy2-conjugated goat anti-rabbit F(ab')2 fragments and horseradish peroxidase-conjugated goat anti-rabbit F(ab')2 fragments were purchased from Jackson ImmunoResearch.

Cell Culture-- Human skin fibroblasts and hepatoma cells (HuH7) were grown in DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin at 37 °C in 5% CO2.

Ligand Preparation-- Human serum, LDL, and apoE-deprived HDL3 (d = 1.125-1.21 g/ml) from normal healthy donors (23, 24) and TRL from an apoCII-deficient patient were isolated as described (25). ApoE3 was isolated by preparative SDS-PAGE (14). TRL were radiolabeled by the iodine monochloride method, whereas apoE was radiolabeled with Iodogen (16). 100 µg of unlabeled or 125I-labeled apoE were associated with TRL (0.5 mg of protein) to prepare apoE-TRL (final concentration 0.28 mg of protein/ml) or 125I-apoE-TRL as described (16). The protein concentrations of the 125I-TRL and the 125I-apoE-TRL preparations were 0.2 ± 0.05 mg/ml, and the specific radioactivity was 100-180 cpm/ng protein, respectively. 125I-TRL and the 125I-apoE-TRL preparations were routinely separated by 10% SDS-PAGE, and radiolabeling of TRL apoproteins was confirmed by autoradiography. For the labeling of lipoproteins with [3H]Chol, 100 µl of [3H]Chol (3.7 MBq) was dried under liquid nitrogen, resuspended in 100 µl of DMEM + 2% BSA, and incubated overnight with 0.5-1 mg of 125I-apoE-TRL ([3H]Chol/125I-apoE-TRL) or 3-5 mg of LDL ([3H]Chol-LDL) in PBS + 10 mM EDTA at 37 °C. Non-incorporated [3H]Chol was removed by PD10 gel chromatography (Amersham Biosciences). For immunofluorescence experiments, apoE-TRL (0.2 mg) were labeled with Cy3 (Cy3-apoE-TRL), and HDL3 apoproteins (1 mg) were labeled with Alexa 568 nm (Alexa-HDL3) or Cy5 (Cy5-HDL3) according to the instructions of the manufacturers. Fluorescent label was found predominantly in apoE of Cy3-apoE-TRL (~80-90%) and apoA-I of Alexa-HDL3 or Cy5-HDL3 (~90-95%) as determined by SDS-PAGE, respectively (data not shown). To label Cy3-apoE-TRL with NBD-cholesterol, 0.2 mg of Cy3-apoE-TRL was incubated with 250 µg of NBD-cholesterol in PBS, 1 mM EDTA at 37 °C overnight. Non-incorporated NBD-cholesterol was removed by PD10 gel chromatography. For electron microscopy, HDL3 (600 µg; pI 5.5) and apoE3 (100 µg; pI 5.78) were labeled with colloidal gold of 5 (gold-HDL) and 12 nm gold, respectively, according to Handley et al. (26). Then apoE3 was associated with TRL (gold-apoE-TRL) as described above. Conjugations of gold-HDL3 and gold-labeled apoE3 were confirmed after staining with sodium phosphotungstate by electron microscopy (data not shown).

Uptake and Recycling Assays-- For radioactive pulse-chase experiments, cells were washed with PBS and incubated with 125I-TRL (2.5 µg/ml) for 60 min at 37 °C in 1 ml of DMEM containing 5% BSA (pH 7.4). The cells were then washed with ice-cold PBS, and surface-bound ligands were released with 770 units/ml heparin (14, 23). To promote recycling, 125I-TRL labeled cells were incubated for 60 min at 37 °C with DMEM + 0.1% BSA supplemented with 50 µg/ml HDL3, 100 µg/ml LDL, or 10% human serum. Then the content of trichloroacetic acid-precipitable, recycled radioactivity in the harvested media was determined. Cells were solubilized in 0.1 N NaOH to calculate the amount of internalized and recycled radioactivity (14). In one set of experiments, recycled and radiolabeled TRL apoproteins from cell culture media were subjected to density gradient ultracentrifugation (KBr density from 1.006 to 1.21 g/ml) (23). After gradient fractionation, the radioactivity in each fraction was determined. The positions of lipoproteins were identified according to their density and the cholesterol profile of the gradient (data not shown). In another set of experiments, the recycled radiolabeled TRL apoproteins were separated by FPLC analysis with a Superdex 200 column (Amersham Biosciences). Cholesterol and protein profiles from standards (TRL, LDL, HDL, albumin) allowed the identification of the fractions containing lipoproteins and albumin (lipoprotein-free fraction (LFF)).

Immunofluorescence and Electron Microscopy-- For immunofluorescence experiments, fibroblasts and hepatoma cells were incubated with apoE-TRL (1 µg/ml) in DMEM + 2% BSA for 60 min at 37 °C. Cells were washed in DMEM and treated with heparin at 4 °C for 15 min (see above). Then cells were incubated for an additional 0, 15, or 60 min at 37 °C in DMEM (0.1% BSA) ± 50 µg/ml unlabeled or fluorescent-labeled HDL3 (Alexa-HDL3, Cy5-HDL3). Cells were washed in PBS and fixed in 4% PFA, and indirect immunofluorescence against apoE, AP1, and EEA1 was performed (14). Cellular cholesterol was visualized using 50 µg/ml filipin (27). Full cell images were taken with an Axiovert 100 microscope equipped with a Zeiss Axiocam. Confocal images were collected using a Zeiss LSM 510 (version 3.0) equipped with an UV laser to detect filipin-stained cholesterol. For living cell microscopy, fibroblasts were incubated with double-labeled NBD-cholesterol/Cy3-apoE-TRL (1 µg/ml) in DMEM + 2% BSA for 0-30 min at 37 °C. Cells were washed in DMEM and incubated for an additional 10 min with 50 µg/ml Cy5-HDL3. Confocal images were taken every minute in the multitrack mode using optimized pinhole adjustment for each fluorochrome.

For electron microscopy, a pre-embedding procedure was performed. Hepatoma cells were incubated with 1 µg/ml gold-apoE-TRL (12 nm) in DMEM + 2% BSA for 60 min at 37 °C. Cells were washed, treated with heparin (see above), and incubated with 5 µg/ml gold-HDL3 (5 nm) at 37 °C for 15 min. Cells were rinsed with PBS and fixed in 2% PFA, 2.5% glutaraldehyde in PBS for 60 min at room temperature. Then fixed cells were scraped and post-fixed in 2% PFA in PBS for 2 days at 4 °C. Samples were washed, treated with 1% OsO4, 0.8% potassium ferricyanide (Sigma) in PBS, dehydrated with acetone, and embedded in Spurr resin (Sigma). Ultrathin sections were obtained and counterstained with uranyl acetate and lead citrate as described previously (28).

Western Blotting-- Fibroblasts and hepatoma cells were incubated with apoE-TRL (1 µg/ml) for 60 min at 37 °C, washed, and incubated for an additional 0 or 60 min at 37 °C ± 50 µg/ml HDL3 in DMEM + 0.1% BSA. The media were harvested, filtered (inner diameter, 0.45 µm), and cleared by centrifugation at 14,000 × g for 10 min. The supernatants were subjected to 10% SDS-PAGE and immunoblotted against apoE. After incubation with peroxidase-conjugated secondary antibodies, the reaction product was detected using the ECL system (Amersham Biosciences).

Cholesterol Efflux Experiments-- In the first set of experiments, cells were incubated with double-labeled [3H]Chol/125I-apoE-TRL (1 µg/ml) for 60 min at 37 °C and then washed with ice-cold PBS and heparin to remove surface-bound ligands (see above). [3H]Chol/125I-apoE-TRL labeled cells were incubated for additional 60 min at 37 °C in DMEM + 0.1% BSA in the presence or absence of 50 µg/ml HDL3. The media were harvested to quantify 125I-apoE recycling after trichloroacetic acid precipitation (see above), whereas [3H]Chol efflux was determined after Dole extraction (29). In the second set of experiments, intracellular cholesterol pools were radiolabeled overnight with [3H]Chol-LDL (100,000 cpm/ml) at 37 °C. Non-internalized [3H]Chol-LDL was removed by heparin treatment (see above). Then cells were incubated with or without apoE-TRL for 60 min at 37 °C. After heparin treatment (see above), cells were incubated in DMEM + 0.1% BSA in the presence or absence of 50 µg/ml HDL3 for an additional 60 min at 37 °C. The media were harvested, and cells were lysed in 0.1 N NaOH. Dole extraction of media and cell lysates was performed to determine [3H]Chol efflux.

    RESULTS
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INTRODUCTION
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DISCUSSION
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HDL3 Stimulates the Recycling of TRL-derived ApoE-- We have demonstrated previously (9, 14, 16) that the recycling of internalized TRL-derived apoE and LPL requires the presence of an extracellular lipoprotein acceptor. To compare the ability of different lipoproteins to mediate TRL recycling, human hepatoma cells were preincubated with 125I-TRL, and recycling of 125I-TRL proteins was induced with human serum, LDL, or HDL3 (Fig. 1A). Consistent with previous results (16), addition of 10% human serum stimulated recycling of internalized 125I-TRL proteins 2-fold compared with the lipoprotein-free control (Fig. 1A, w/o). Similarly, in the presence of HDL3 (50 µg/ml protein), a 1.8-fold increase of 125I-TRL recycling was observed. This stimulatory effect of HDL3 on 125I-TRL recycling was already detectable at very low levels of HDL3 (2.5 µg/ml), dose-dependent, and saturable in the presence of 50 µg/ml HDL3 (Fig. 1B). In contrast, no significant stimulation of 125I-TRL recycling was determined when LDL (100 µg/ml protein) was utilized (Fig. 1A). These findings indicated that HDL3 could stimulate 125I-TRL recycling and serve as an acceptor for recycled 125I-TRL proteins. To analyze the association of recycled 125I-TRL proteins with lipoproteins, the media containing the lipoprotein acceptors and the released 125I-TRL-derived radioactivity were harvested and separated either by density gradient centrifugation (Fig. 2A) or gel filtration chromatography (Fig. 2B). Recycled 125I-TRL proteins from the media of cells incubated with human serum identified ~60% of TRL radioactivity in the HDL fractions (fractions 7-11 in Fig. 2A), whereas 40% was found in the lipid-free bottom of the gradient (fractions 1-4). It is likely that lipid-free apoA-I in human serum is responsible for the association of 125I-TRL proteins in these bottom fractions, because lipid-free apoA-I also induced recycling of 125I-TRL proteins into these fractions (data not shown). When 125I-TRL-loaded cells were incubated with HDL3, a predominant association of recycled 125I-TRL proteins with HDL was observed (fractions 7-11, Fig. 2A). To confirm the association of recycled 125I-TRL with HDL, re-secreted 125I-TRL proteins were separated by Superdex 200 gel filtration (Fig. 2B), which is appropriate for the resolution of small sized lipoproteins (10-600 kDa). As expected HDL3-induced recycling of internalized 125I-TRL resulted in the association of 125I-labeled apoproteins with HDL (fractions 24-28; Fig. 2B). In addition 125I-TRL proteins were found in lipid-free fractions (LFF; fractions 29-31), which were representative of recycled 125I-TRL proteins when lipid-free apoA-I was used as an acceptor (data not shown). Independent of density gradient or gel filtration analysis, we never detected significant amounts of recycled 125I-TRL proteins in the fractions containing LDL (fractions 16-18 in Fig. 2A), TRL (fractions 21 and 22 in Fig. 2A), or both LDL and TRL (fractions 17-19 in Fig. 2B). These results demonstrate that TRL or LDL do not serve as acceptors for recycled 125I-TRL apoproteins. Taken together, these results indicate that HDL in human serum is capable of stimulating TRL protein recycling (Fig. 1, A and B) and serve as an acceptor for recycled TRL proteins (Fig. 2, A and B).


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Fig. 1.   HDL-induced recycling of TRL-associated apoproteins in hepatoma cells. Pulse-chase experiments were performed by preincubating human HuH7 hepatoma cells with 125I-TRL for 60 min at 37 °C (see "Experimental Procedures"). Cells were washed at 4 °C, and cell-bound radiolabeled material was removed by heparin. After incubation for additional 60 min at 37 °C, cells were incubated with media only (0.1% BSA in DMEM; w/o, white bar) or media supplemented with 10% human serum (hatched bar), 100 µg/ml LDL (black bar), or 50 µg/ml HDL3 (gray bar) (A); or with increasing amounts of HDL3 (0-250 µg/ml) (B). The media were collected to calculate the amount of recycled, intact (trichloroacetic acid-precipitable) 125I-TRL-derived apoproteins. The remaining cells were lysed, and protein content was determined. The data are given in ng of apoprotein recycling/mg of cell protein and represent the mean ± S.D. of five (A) and four (B) independent experiments with duplicate samples. Depending on the specific activity of 125I-TRL preparations (100-180 cpm/ng protein, see "Experimental Procedures"), background 125I-TRL recycling in the absence of HDL3 was in the range of ~20-60 ng/mg cell protein.


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Fig. 2.   Density gradient and FPLC analysis of recycled 125I-TRL-derived apoproteins. A, the non-degraded radioactivity of media from cells analyzed in Fig. 1A, which were incubated with media alone (open circle , w/o), with 50 µg/ml HDL3 (, HDL3) or 10% human serum (black-diamond , serum) were separated by KBr density gradient (see "Experimental Procedures"). Fractions from bottom to top (500 µl) were collected, and the radioactivity was determined in each fraction. The gradient distribution (mean values of duplicate samples) of recycled 125I-TRL apoproteins (counts/min) of a representative experiment is shown (n = 3). The fractions containing TRL (fractions 21 and 22), LDL (fractions 16-18), HDL (fractions 7-11), and the lipid-free bottom (fractions 1-4) are indicated and were identified by their respective density and cholesterol content. B, the non-degraded radioactivity of media from cells analyzed in Fig. 1A, which were incubated with media alone (open circle , w/o) or with 50 µg/ml HDL3 (, HDL3) were separated by a Superdex 200 column (see "Experimental Procedures"); 500-µl fractions were collected, and the radioactivity was determined in each fraction. The distribution of recycled 125I-TRL apoproteins (counts/min) for a representative experiment is shown (n = 3). The fractions containing TRL/LDL (fractions 17-19) and HDL (fractions 24-28) are indicated. The LFF (fractions 29-31) contain proteins in the range of 20-80 kDa. The positions of lipoproteins were identified by the comparison of cholesterol profiles from the analyzed samples with isolated preparations of TRL, LDL, and HDL, respectively.

Recycling of ApoE Is Associated with Cholesterol Efflux-- It is well known that HDL can stimulate cellular cholesterol efflux (19-21). Because HDL-dependent re-secretion of 125I-TRL (see above) mainly involves recycling of apoE (14), we investigated whether recycling of TRL-derived apoE could be linked to intracellular cholesterol transport. Therefore, the distribution of TRL-derived apoE and cholesterol in the presence or absence of HDL3 was first studied by immunocytochemistry. In initial experiments with human hepatoma cells, immunostaining against TRL-derived apoE interfered with endogenous apoE expression (data not shown). Thus, human fibroblasts with no detectable amounts of endogenous apoE were analyzed. These cells, similar to the HuH7 hepatoma cells described above, efficiently recycled TRL proteins through peripheral endosomal compartments (14). In the following set of experiments, fibroblasts were incubated with apoE-enriched TRL (apoE-TRL), which was followed by a 60-min chase ± HDL3 (Fig. 3). The cells were then fixed, immunostained against apoE to detect TRL-derived apoE, and incubated with filipin to visualize cellular cholesterol. In the absence of HDL3 (Fig. 3, A and B), significant amounts of internalized TRL-derived apoE remained intracellularly (Fig. 3A). In these cells large amounts of cholesterol were found in peripheral and perinuclear compartments (Fig. 3B), which appeared to co-localize with apoE mainly in the periphery. In contrast, when cells were incubated with HDL3, only residual amounts of internalized TRL-derived apoE were detected intracellularly (Fig. 3A, c). These findings suggested HDL3-induced apoE recycling, which was confirmed by apoE Western blot analysis of the chase media (Fig. 3B, compare lanes 3 and 4 which correspond to media from cells shown in Fig. 3A, a and b, and c and d, respectively). In addition, apoE recycling was accompanied by a strong reduction of filipin staining mainly in peripheral, most likely endosomal compartments (Fig. 3A, d).


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Fig. 3.   Recycling of TRL-derived apoE from cholesterol-containing endosomes. A, human fibroblasts were preincubated with apoE-TRL for 60 min at 37 °C. After removal of cell-bound material with heparin, cells were incubated ± HDL3 (50 µg/ml) for an additional 60 min at 37 °C as indicated. Media were collected to identify recycled apoE (see B). Cells were fixed and analyzed by fluorescence microscopy for apoE (green, a and c) and cholesterol (blue, b and d). Arrows point to apoE staining in cholesterol-rich endosomes labeled with filipin (see "Experimental Procedures"). Bar is 10 µm. B, pulse-chase experiments were performed by preincubating human fibroblasts (lanes 1-4) or hepatoma cells (lanes 5-8) with apoE-TRL for 60 min at 37 °C. Cell-bound material was removed with heparin, and cells were incubated ± HDL3 (50 µg/ml) for 0 or 60 min at 37 °C as indicated. Then cell culture media were harvested, and the presence of recycled, intact apoE was determined by Western blot analysis (see "Experimental Procedures"). The position of apoE is indicated. Molecular mass is given in kDa.

Although filipin stains endosomal cholesterol, it is well known that large amounts of cholesterol are found in perinuclear Golgi and lysosomal compartments (30). In addition, analysis of full thickness cell images could lead to the overlap of apoE and cholesterol signals by random chance. Thus, confocal microscopy was performed to characterize apoE and cholesterol-containing vesicles in detail (Fig. 4). First, human fibroblasts were incubated with apoE-TRL, fixed, and stained against apoE together with AP1, a protein predominantly found in the Golgi apparatus (22) (Fig. 4a), or EEA1, a marker for early endosomes (31) (Fig. 4b). These experiments identified large amounts of internalized TRL-derived apoE in EEA1-positive endosomes that did not co-localize with AP1. As shown in Fig. 4c, these apoE-containing and EEA1-positive endosomes contain cholesterol. In contrast, cholesterol in the perinuclear region that was not stained with EEA1 did not contain any TRL-derived apoE. These findings indicated that HDL3-induced apoE recycling is linked to cholesterol efflux from endosomal compartments. However, filipin staining is not a reliable method to estimate changes in endosomal cholesterol. Therefore, we first tested whether HDL3-induced recycling of apoE is accompanied by the mobilization of radiolabeled TRL-derived cholesterol from endosomes. Because internalization and disintegration of TRL particles could be associated with a substantial redistribution of TRL-derived cholesterol to multiple cellular compartments, control experiments with NBD-cholesterol-labeled TRL were performed. In these studies TRL-derived NBD-cholesterol and apoE co-localized in EEA1-positive endosomes even after 60 min at 37 °C (data not shown, see also Fig. 11). Then cells were incubated with double-labeled [3H]Chol/125I-apoE-TRL (Fig. 5). In these experiments HDL3 simultaneously stimulated recycling of internalized TRL-derived 125I-apoE (1.8-2.4-fold) and efflux of TRL-derived [3H]Chol (2.8-3.2-fold). Thus HDL3 stimulates apoE recycling and cholesterol efflux from internalized TRL particles in endosomal compartments.


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Fig. 4.   Internalized TRL-derived apoE co-localizes with cholesterol in EEA1-positive endosomes. Human fibroblasts were incubated with apoE-TRL for 60 min at 37 °C. After removal of cell-bound material with heparin, cells were fixed, and sections (<= 1 µm) were analyzed by confocal fluorescence microscopy. Fibroblasts were stained against apoE (red) and AP1 (green) in a or apoE (red) and EEA1 (green) in b as indicated in the lower panel. The merged images and co-localization of apoE and EEA1 (yellow) are shown in the upper panels. c, the merged image of fibroblasts stained against EEA1 (red), apoE (green), and cholesterol (blue) is shown in the upper panel (N = nucleus). In the lower panel the highlighted area is enlarged and shown separately for each channel. Arrowheads point to EEA1 and apoE staining in cholesterol-rich endosomes labeled with filipin. Arrows mark endosomes only containing EEA1, apoE or cholesterol, respectively. Bar is 10 µm.


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Fig. 5.   HDL3-induced recycling of TRL-derived apoE and cholesterol. Pulse-chase experiments were performed by preincubating human HuH7 hepatoma cells with [3H]Chol/125I-apoE-TRL for 60 min at 37 °C. Cells were washed with heparin and incubated for additional 60 min at 37 °C with media in the presence or absence of 50 µg/ml HDL3 as indicated. The media were harvested, and the amount of re-secreted 125I-apoE (gray bar) and [3H]Chol (white bar) was determined. The remaining cells were lysed, and protein content was determined. The radioactivity is given (cpm × 103/mg of cell protein) and represents the mean ± S.D. of four independent experiments with triplicate samples.

To determine whether apoE recycling is associated with cholesterol efflux from intracellular cholesterol pools in the absence of HDL3, hepatoma cells were pre-labeled overnight with [3H]Chol-LDL, and cell surface-bound [3H]Chol-LDL was removed by heparin. Then cells were incubated ± apoE-TRL for 60 min, followed by an additional incubation ± HDL3 for 60 min. Finally the amount of [3H]Chol in the media was determined (Fig. 6). Similar to results obtained from hepatoma cells (32), HDL3 stimulated [3H]Chol efflux 3-fold (compare columns 1 and 2). In the absence of HDL3, internalized TRL-derived apoE is concatenated by [3H]Chol efflux ~3-fold compared with controls (compare columns 1 and 3). In the presence of HDL3, a 6-fold induction of [3H]Chol efflux from apoE-TRL-loaded cells was observed (compare columns 1 and 4). These findings indicate that apoE recycling is linked to cholesterol efflux not only from TRL-associated pools but also from other intracellular cholesterol pools (Figs. 5 and 6). To study the possible association of secreted [3H]Chol with HDL, Superdex 200 gel filtration analysis of the chase media was performed (Fig. 7). The majority of released [3H]Chol (~80%) was found in HDL fractions, which were shown to contain recycled TRL-derived apoE in this study (see Fig. 2, A and B) and previous studies (16). In contrast, only ~20% of [3H]Chol was found in fractions smaller than HDL (LFF). Taken together, HDL3-dependent and -independent apoE recycling is accompanied by the mobilization of intracellular cholesterol leading to a concomitant association of TRL-derived apoE and cholesterol with HDL particles.


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Fig. 6.   Recycling of apoE is associated with efflux of cellular cholesterol. Human hepatoma cells were pre-loaded with [3H]Chol-LDL (100,000 cpm/ml) for 24 h at 37 °C. Cell surface-bound [3H]Chol-LDL was removed with heparin, and cells were incubated in the presence or absence of apoE-TRL for 60 min at 37 °C. After incubation with or without HDL3 (50 µg/ml) as indicated, aliquots of the media were harvested to determine [3H]Chol efflux. The remaining media were analyzed by FPLC (see Fig. 7). Cells were lysed, and the internalized amount of [3H]Chol was determined. Values of [3H]Chol efflux are given as a percentage of internalized radioactivity and represent the mean ± S.D. of four independent experiments with triplicates.


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Fig. 7.   FPLC analysis of re-secreted cellular [3H]cholesterol. [3H]Chol-containing media (see Fig. 5) obtained from cells incubated with (, HDL3) or without HDL3 (open circle , w/o) were separated by a Superdex 200 column; 500-µl fractions were collected, and the radioactivity was determined in each fraction. The association of re-secreted [3H]Chol (counts/min) with HDL and LFF for a representative experiment is shown. The fractions containing TRL/LDL (fractions 17-19), HDL (fractions 24-28) and LFF (fractions 29-31) are indicated. The positions of lipoproteins were determined as described above (Fig. 2B).

Recycling of ApoE Is Associated with the Internalization of HDL3-derived apoA-I-- Most current models favor the localization of HDL at the plasma membrane to promote cholesterol efflux. We suggested that HDL is located at the cell surface to serve as an acceptor for recycled apoE (9, 14). Although the pulse-chase experiments described above (Figs. 2 and 7) demonstrated the association of recycled apoE and cholesterol with HDL, these experiments did not provide information on the question whether complex formation of TRL-derived apoE and cholesterol with HDL could already occur intracellularly. To address this matter, apoE recycling in apoE-TRL-loaded cells was induced with fluorescent-labeled HDL3 proteins (Alexa-HDL3) containing predominantly labeled apoA-I (see "Experimental Procedures"). Cells were fixed and analyzed for the distribution of apoE, cholesterol, and Alexa-HDL3. Similar to the results described in Fig. 3, a and b, TRL-derived apoE accumulates in peripheral compartments (Fig. 8a), which could also be stained with filipin (Fig. 8c), indicating co-localization of apoE and cholesterol in endosomes. Because complete recycling of TRL-derived apoE is observed after 60 min (Fig. 3A, c and d), only a 15-min induction of apoE recycling with Alexa-HDL3 was performed (Fig. 8, d-f). This enabled us to detect simultaneously TRL-derived apoE, cholesterol, and Alexa-HDL3. As expected, Alexa-HDL3 stimulated apoE recycling and cholesterol efflux as shown by a reduced endosomal apoE and cholesterol staining (Fig. 8, d and f). In these experiments the reduction in cholesterol staining in the periphery was not as prominent as described in Fig. 3, which could in part be explained by the shorter exposure time to HDL3. In addition, filipin staining of full cell images is not suitable to detect minimal changes in endosomal cholesterol concentrations. Most strikingly, co-localization of apoE/cholesterol and Alexa-HDL3 was visible in peripheral compartments (compare arrows in Fig. 8, d-f) indicating the formation of endosomal apoE/cholesterol and apoA-I complexes. This observation was confirmed by confocal microscopy, which identified the co-localization of apoE and apoA-I in EEA1-positive endosomes during apoE recycling (Fig. 9).


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Fig. 8.   Internalized apoA-I co-localizes with TRL-derived apoE and cholesterol. Human fibroblasts were preincubated with apoE-TRL for 60 min at 37 °C. After removal of cell-bound material with heparin, cells were incubated with HDL3 (50 µg/ml) for 0 (a-c) or 15 min (d-f) at 37 °C as indicated. Cells were fixed and analyzed by fluorescence microscopy for apoE (green, a and d), HDL3 (red, b and e) and cholesterol (blue, c and f). Arrowheads point to apoE and cholesterol-containing endosomes (compare a and c). In the presence of fluorescent-labeled HDL3, HDL3-derived apoA-I is found almost exclusively in apoE-containing and cholesterol-rich endosomes (compare arrows, d-f). Bar is 10 µm.


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Fig. 9.   TRL-derived apoE co-localizes with apoA-I in EEA1-positive endosomes during apoE recycling. Human fibroblasts were incubated with apoE-TRL for 60 min at 37 °C. After removal of cell-bound material with heparin, cells were incubated for 15 min with 50 µg/ml Cy5-HDL3. Cells were fixed, and sections (<= 1 µm) of a representative area in the cellular periphery were analyzed by confocal fluorescence microscopy. Fibroblasts were analyzed for EEA1 (red) (a), apoE (green) (b), and apoA-I (blue) (c). The merged image is shown in d. Arrows point to EEA1-positive endosomes containing apoE and apoA-I. Arrowheads mark endosomes only containing EEA1, apoE, or apoA-I, respectively. Bar is 10 µm.

To support these findings, electron microscopy of apoE-TRL-loaded hepatoma cells ± HDL3 was performed. Incubation of cells with gold-labeled apoE-TRL (12 nm gold) resulted in the association of gold-apoE with endocytic structures (data not shown). To promote apoE recycling, gold-apoE-loaded cells were incubated with gold-labeled HDL3 (5 nm gold) for an additional 15 min (Fig. 10). Subsequent analysis of EM sections identified gold-labeled protein complexes consisting of apoE- and HDL3-derived apoA-I to be associated with peripheral endosomes and membranous structures at the plasma membrane.


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Fig. 10.   Co-localization of apoA-I with TRL-derived apoE. Human hepatoma cells were preincubated with gold-apoE-TRL (12 nm) for 60 min at 37 °C. After removal of cell-bound material with heparin, cells were incubated with gold-HDL3 (5 nm) for 15 min at 37 °C. Cells were fixed, prepared, and analyzed by electron microscopy for apoE (arrowheads) and HDL3 (arrows). In the presence of HDL3, complexes containing gold-labeled apoE and HDL3 are found associated with endosomal structures or at the plasma membrane. Bar is 200 nm.

These findings suggested that HDL3-derived apoA-I is internalized to pre-existing apoE/cholesterol-containing endosomes to promote apoE recycling and cholesterol efflux. To confirm this hypothesis we performed time-lapse confocal microscopy to study intracellular localization of apoA-I during early stages of apoE recycling (Fig. 11). Cells were first incubated for 30 min with double-labeled NBD-cholesterol/Cy3-apoE-TRL. Then Cy5-HDL3 was added for an additional 10 min. An example of a mobile NBD-cholesterol/apoE-containing endosome (frames 6-15) that interacts with another mobile apoA-I-containing endosome is shown (frames 16-20). The merged images (frames 1-5) revealed co-localization of NBD-cholesterol and apoE indicating that these TRL components remained associated even 30 min after TRL internalization (frames 2 and 3). Most importantly, immediately after addition of Cy5-HDL3, endosomal apoA-I is targeted to the NBD-cholesterol/apoE-containing vesicle (frames 4 and 5). Thus, the intracellular formation of apoE/cholesterol/apoA-I-containing complexes is involved in apoE recycling.


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Fig. 11.   Dynamic association of apoA-I with pre-existing apoE- and cholesterol-containing complexes. Human fibroblasts were incubated with double-labeled NBD-cholesterol/Cy3-apoE-TRL for 0-30 min at 37 °C. Cells were washed in DMEM and incubated for an additional 0-10 min with 50 µg/ml Cy5-HDL3. Time-lapse confocal image acquisition of apoE (red), NBD-cholesterol (green), and HDL3-protein (blue) in a representative area of the cellular periphery was performed at 37 °C (see "Experimental Procedures"). The merged image is shown in the upper panel (image numbers 1-5). Each channel is shown separately in the lower panels as indicated. Arrowheads mark and follow the movement of a complex in the merge (image numbers 2-5) and in each lower panel: apoE (image numbers 7-10), NBD-cholesterol (image numbers 12-15), and apoA-I (image numbers 19 and 20). Bar is 2 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to identify a possible intracellular link between the recycling of apoE, cholesterol efflux, and HDL metabolism. It is well documented that internalized TRL-derived apoE is efficiently recycled in liver cells (13, 14, 16-18), but relatively little is known about the intracellular events that accompany apoE recycling. Here we demonstrate that apoE recycling is linked to HDL3-dependent and -independent cholesterol efflux. In the presence of HDL the re-secretion of apoE and cholesterol leads to the formation of apoE/cholesterol-enriched HDL particles. Detailed confocal microscopy and live cell imaging revealed that after internalization TRL-derived apoE and cholesterol remain associated in EEA1-positive endosomes. Most importantly, apoE recycling involves the internalization of HDL3-derived apoA-I and its targeting to apoE/cholesterol-containing endosomes. These findings indicate that endosomal HDL3-derived apoA-I can mobilize pre-existing apoE/cholesterol complexes.

In the light of these findings, our previous model that HDL acts as an extracellular acceptor for recycled TRL-derived apoE (9) deserves to be reconsidered and extended. Concurrent cholesterol efflux and apoE recycling would be in agreement with models favoring the binding of HDL to the plasma membrane. At the cell surface, HDL could interact with ATP-binding cassette transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI), or other HDL-binding proteins (for reviews see Refs. 19-21 and 33-36). However, the evidence of HDL-derived apoA-I internalization during apoE/cholesterol re-secretion (Figs. 8-11) is in agreement with a number of reports describing the intracellular localization of HDL receptors during HDL-mediated cholesterol transport (for reviews see Refs. 20, 33, and 36). Similarly, results presented in this work identified HDL-derived apoA-I in EEA1-positive early endosomes. Oram (20) recently proposed that ABCA1 and apoA-I are internalized to intracellular lipid vesicles, where ABCA1 could deliver lipids into the lumen of exocytic vesicles before re-secretion. This is based on the following observations. First, ABCA1 recycles between the plasma membrane and endosomes (37). Second, ABCA1 promotes cholesterol efflux from endosomal compartments (38). Third, apoA-I is internalized and re-secreted during cAMP-inducible and ABCA1-dependent cholesterol efflux (39-42).

Although these observations would favor a role for ABCA1 in the HDL-induced and concurrent apoE recycling and cholesterol efflux, the potential contribution of SR-BI cannot be excluded. SR-BI is localized predominantly in caveolae and not in clathrin-coated pits (43, 44). However, detailed studies revealed that a significant proportion of SR-BI proteins are rapidly internalized (45). This is accompanied by endocytosis and re-secretion of HDL (46). The concept that SR-BI could mediate uptake and recycling of HDL particles is consistent with a punctate, endosomal localization of fluorescent-labeled HDL immediately (10 min) after cell surface ligand binding in SR-BI-overexpressing cells (47) (see also Figs. 8-11). In addition, Silver et al. (45) observed the internalization and recycling of HDL particles through an endocytic recycling compartment in non-polarized hepatocytes. These results are in agreement with earlier studies (48-51) describing receptor-mediated endocytosis and retroendocytosis of HDL.

Co-localization of apoA-I, apoE, and cholesterol in TRL-loaded cells indicates that apoE recycling and cholesterol efflux results in the intracellular assembly of apoA-I with apoE/cholesterol-containing complexes. Similarly, a number of reports (52, 53) hypothesized an interaction between apoA-I and apoE and concluded that apoA-I concurrently stimulates secretion of endogenous apoE and cholesterol efflux from lipid-loaded macrophages. In addition secretion of apoE from macrophages is at least in part associated with cell-derived cholesterol (54, 55). This stimulatory role of apoA-I and endogenous apoE for cholesterol efflux in peripheral cells is the presupposition for efficient reverse cholesterol transport to the liver for bile secretion (33, 56). In our study we identified the concomitant re-secretion of endosomal TRL-derived and/or intracellular cholesterol with internalized TRL-derived apoE in hepatoma cells. These findings suggest that intracellular association of cholesterol with TRL-derived apoE and concurrent re-secretion could provide a mechanism allowing dietary cholesterol to escape bile secretion.

Most importantly we demonstrate that the intracellular association of apoA-I with apoE-cholesterol complexes could lead to the formation of apoE-enriched HDL particles. Once secreted, these particles would represent a pool of apoE proteins associated with HDL. In the postprandial state HDL-derived apoE is the major source for apoE enrichment of CR to ensure the rapid clearance of CR into the liver (1, 2). To avoid a rapid decrease of HDL-derived apoE during the postprandial state, the recycling of TRL-derived apoE and subsequent generation of apoE-containing HDL particles would secure the maintenance of high HDL-associated apoE levels in plasma. Therefore, recycled apoE could serve as a continuous donor for intravascular apoE transfer from HDL to CR during lipolysis and hepatic clearance (9, 16). Future in vivo experiments will have to clarify whether newly formed HDL particles play a physiological role in CR enrichment with apoE.

    ACKNOWLEDGEMENTS

We thank D. Wendt for excellent technical assistance. We are most grateful to M. Calvo (Institut d'Investigacions Biomèdiques August Pi i Sunyer/Fons d'Investigació Sanitària) for assistance in the preparation of gold probes and to Serveis Científico-tècnics, Universitat de Barcelona, for the electron microscopy facilities.

    FOOTNOTES

* This work was supported in part by the Deutsche Forschungsgemeinschaft Grants Be 829/5-1, Ja 421/3-1, and Ri 436/8-1 and Ministerio de Ciencia y Tecnologia Grants PM99-0166, Acciones Integradas HA98-0007, and Generalitat de Catalunya BE2002 (to C. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Institute for Medical Biochemistry and Molecular Biology, Dept. of Molecular Cell Biology, University Hospital Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. Tel.: 49-40-42803-3917; Fax: 49-40-42803-4592; E-mail: heeren@uke.uni-hamburg.de.

Recipient of a fellowship from the Studienstiftung des Deutschen Volkes.

|| Recipient of Fellowship GRK 336 from the Deutsche Forschungsgemeinschaft-financed Graduiertenkolleg.

Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M209006200

    ABBREVIATIONS

The abbreviations used are: CM, chylomicrons; AP1, adaptor protein 1; apo, apoprotein; ABCA1, ATP-binding cassette transporter A1; BSA, bovine serum albumin; Chol, cholesterol; CR, chylomicron remnants; EEA1, early endosome antigen 1; FPLC, fast performance liquid chromatography; HDL, high density lipoprotein; HuH7, human hepatoma cell line 7; LDL, low density lipoprotein; LFF, lipoprotein-free fraction; LPL, lipoprotein lipase; NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)23,24-bisnor-5-cholen-3B-ol; TRL, triglyceride-rich lipoproteins; PBS, phosphate-buffered saline; PFA, paraformaldehyde; VLDL, very low density lipoproteins; DMEM, Dulbecco's modified Eagle's medium; SR-BI, scavenger receptor class B type I; w/o, without.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Olivecrona, G., and Olivecrona, T. (1995) Curr. Opin. Lipidol. 6, 291-305[Medline] [Order article via Infotrieve]
2. Mahley, R. W., and Ji, Z. S. (1999) J. Lipid Res. 40, 1-16[Abstract/Free Full Text]
3. Goldberg, I. J., and Merkel, M. (2001) Front. Biosci. 6, D388-D405[Medline] [Order article via Infotrieve]
4. Beisiegel, U., Weber, W., Ihrke, G., Herz, J., and Stanley, K. K. (1989) Nature 341, 162-164[CrossRef][Medline] [Order article via Infotrieve]
5. Beisiegel, U., Weber, W., and Bengtsson-Olivecrona, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8342-8346[Abstract]
6. Rohlmann, A., Gotthardt, M., Hammer, R. E., and Herz, J. (1998) J. Clin. Invest. 101, 689-695[Abstract/Free Full Text]
7. Zhang, S. H., Reddick, R. L., Piedrahita, J. A., and Maeda, N. (1992) Science 258, 468-471[Medline] [Order article via Infotrieve]
8. Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34-47[Medline] [Order article via Infotrieve]
9. Heeren, J., and Beisiegel, U. (2001) Curr. Opin. Lipidol. 12, 255-260[CrossRef][Medline] [Order article via Infotrieve]
10. Tabas, I., Lim, S., Xu, X. X., and Maxfield, F. R. (1990) J. Cell Biol. 111, 929-940[Abstract]
11. Tabas, I., Myers, J. N., Innerarity, T. L., Xu, X. X., Arnold, K., Boyles, J., and Maxfield, F. R. (1991) J. Cell Biol. 115, 1547-1560[Abstract]
12. Myers, J. N., Tabas, I., Jones, N. L., and Maxfield, F. R. (1993) J. Cell Biol. 123, 1389-1402[Abstract]
13. Rensen, P. C., Jong, M. C., van Vark, L. C., van der Boom, H., Hendriks, W. L., van Berkel, T. J., Biessen, E. A., and Havekes, L. M. (2000) J. Biol. Chem. 275, 8564-8571[Abstract/Free Full Text]
14. Heeren, J., Weber, W., and Beisiegel, U. (1999) J. Cell Sci. 112, 349-359[Abstract/Free Full Text]
15. Enrich, C., Pol, A., Calvo, M., Pons, M., and Jäckle, S. (1999) Hepatology 30, 1115-1120[Medline] [Order article via Infotrieve]
16. Heeren, J., Grewal, T., Jäckle, S., and Beisiegel, U. (2001) J. Biol. Chem. 276, 42333-42338[Abstract/Free Full Text]
17. Fazio, S., Linton, M. F., Hasty, A. H., and Swift, L. L. (1999) J. Biol. Chem. 274, 8247-8253[Abstract/Free Full Text]
18. 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]
19. von Eckardstein, A., Nofer, J. R., and Assmann, G. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 13-27[Abstract/Free Full Text]
20. Oram, J. F. (2002) Curr. Opin. Lipidol. 13, 373-381[CrossRef][Medline] [Order article via Infotrieve]
21. Fielding, C. J., and Fielding, P. E. (2001) Adv. Drug Delivery Rev. 49, 251-264[CrossRef][Medline] [Order article via Infotrieve]
22. Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929[Abstract]
23. Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260[Medline] [Order article via Infotrieve]
24. Rinninger, F., Brundert, M., Jäckle, S., Galle, P. R., Busch, C., Izbicki, J. R., Rogiers, X., Henne-Bruns, D., Kremer, B., Broelsch, C. E., and Greten, H. (1994) Hepatology 19, 1100-1114[Medline] [Order article via Infotrieve]
25. Niemeier, A., Gafvels, M., Heeren, J., Meyer, N., Angelin, B., and Beisiegel, U. (1996) J. Lipid Res. 37, 1733-1742[Abstract]
26. Handley, D. A., Arbeeny, C. M., Witte, L. D., and Chien, S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 368-371[Abstract]
27. De Diego, I., Schwartz, F., Siegfried, H., Dauterstedt, P., Heeren, J., Beisiegel, U., Enrich, C., and Grewal, T. (2002) J. Biol. Chem. 277, 32187-32194[Abstract/Free Full Text]
28. Ortega, D., Pol, A., Biermer, M., Jäckle, S., and Enrich, C. (1998) J. Cell Sci. 111, 261-269[Abstract/Free Full Text]
29. Merkel, M., Heeren, J., Dudeck, W., Rinninger, F., Radner, H., Breslow, J. L., Goldberg, I. J., Zechner, R., and Greten, H. (2002) J. Biol. Chem. 277, 7405-7411[Abstract/Free Full Text]
30. Holtta-Vuori, M., Tanhuanpaa, K., Mobius, W., Somerharju, P., and Ikonen, E. (2002) Mol. Biol. Cell 13, 3107-3122[Abstract/Free Full Text]
31. Christoforidis, S., McBride, H. M., Burgoyne, R. D., and Zerial, M. (1999) Nature 397, 621-625[CrossRef][Medline] [Order article via Infotrieve]
32. Mahlberg, F. H., and Rothblat, G. H. (1992) J. Biol. Chem. 267, 4541-4550[Abstract/Free Full Text]
33. Silver, D. L., and Tall, A. R. (2001) Curr. Opin. Lipidol. 12, 497-504[CrossRef][Medline] [Order article via Infotrieve]
34. Krieger, M. (2001) J. Clin. Invest. 108, 793-797[Free Full Text]
35. Schmitz, G., and Langmann, T. (2001) Curr. Opin. Lipidol. 12, 129-140[CrossRef][Medline] [Order article via Infotrieve]
36. Santamarina-Fojo, S., Remaley, A. T., Neufeld, E. B., and Brewer, H. B., Jr. (2001) J. Lipid Res. 42, 1339-1345[Abstract/Free Full Text]
37. Neufeld, E. B., Remaley, A. T., Demosky, S. J., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590[Abstract/Free Full Text]
38. Chen, W., Sun, Y., Welch, C., Gorelik, A., Leventhal, A. R., Tabas, I., and Tall, A. R. (2001) J. Biol. Chem. 276, 43564-43569[Abstract/Free Full Text]
39. Takahashi, Y., and Smith, J. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11358-11363[Abstract/Free Full Text]
40. Bortnick, A. E., Rothblat, G. H., Stoudt, G., Hoppe, K. L., Royer, L. J., McNeish, J., and Francone, O. L. (2000) J. Biol. Chem. 275, 28634-28640[Abstract/Free Full Text]
41. Zheng, P., Horwitz, A., Waelde, C. A., and Smith, J. D. (2001) Biochim. Biophys. Acta 1534, 121-128[Medline] [Order article via Infotrieve]
42. Oram, J. F., Lawn, R. M., Garvin, M. R., and Wade, D. P. (2000) J. Biol. Chem. 275, 34508-34511[Abstract/Free Full Text]
43. Babitt, J., Trigatti, B., Rigotti, A., Smart, E. J., Anderson, R. G., Xu, S., and Krieger, M. (1997) J. Biol. Chem. 272, 13242-13249[Abstract/Free Full Text]
44. Calvo, M., and Enrich, C. (2000) Electrophoresis 21, 3386-3395[CrossRef][Medline] [Order article via Infotrieve]
45. Silver, D. L., Wang, N., and Tall, A. R. (2000) J. Clin. Invest. 105, 151-159[Abstract/Free Full Text]
46. Silver, D. L., Wang, N., Xiao, X., and Tall, A. R. (2001) J. Biol. Chem. 276, 25287-25293[Abstract/Free Full Text]
47. Gu, X., Trigatti, B., Xu, S., Acton, S., Babitt, J., and Krieger, M. (1998) J. Biol. Chem. 273, 26338-26348[Abstract/Free Full Text]
48. Schmitz, G., Robenek, H., Lohmann, U., and Assmann, G. (1985) EMBO J. 4, 613-622[Abstract]
49. Garcia, A., Barbaras, R., Collet, X., Bogyo, A., Chap, H., and Perret, B. (1996) Biochemistry 35, 13064-13071[CrossRef][Medline] [Order article via Infotrieve]
50. Klinger, A., Reimann, F. M., Klinger, M. H., and Stange, E. F. (1997) Biochim. Biophys. Acta 1345, 65-70[Medline] [Order article via Infotrieve]
51. Rogler, G., Herold, G., and Stange, E. F. (1991) Biochim. Biophys. Acta 1095, 30-38[Medline] [Order article via Infotrieve]
52. Rees, D., Sloane, T., Jessup, W., Dean, R. T., and Kritharides, L. (1999) J. Biol. Chem. 274, 27925-27933[Abstract/Free Full Text]
53. Bielicki, J. K., McCall, M. R., and Forte, T. M. (1999) J. Lipid Res. 40, 85-92[Abstract/Free Full Text]
54. Basu, S. K., Ho, Y. K., Brown, M. S., Bilheimer, D. W., Anderson, R. G., and Goldstein, J. L. (1982) J. Biol. Chem. 257, 9788-9795[Free Full Text]
55. Herscovitz, H., Gantz, D., Tercyak, A. M., Zannis, V. I., and Small, D. M. (1992) J. Lipid Res. 33, 791-803[Abstract]
56. Schwartz, C. C., Halloran, L. G., Vlahcevic, Z. R., Gregory, D. H., and Swell, L. (1978) Science 200, 62-64[Medline] [Order article via Infotrieve]


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