Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2010-2019

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, Y.
Right arrow Articles by von Eckardstein, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, Y.
Right arrow Articles by von Eckardstein, A.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2010-2019.)
© 1997 American Heart Association, Inc.

Articles

Effects of Genotype and Diet on Cholesterol Efflux into Plasma and Lipoproteins of Normal, Apolipoprotein A-I-, and Apolipoprotein E-Deficient Mice

Yadong Huang; Yanhong Zhu; Claus Langer; Martin Raabe; Shili Wu; Bernd Wiesenhütter; Udo Seedorf; Nobuyo Maeda; Gerd Assmann; ; Arnold von Eckardstein

From the Institut für Arterioskleroseforschung an der Universität Münster, Münster, Germany (Y.H., Y.Z., M.R., S.W., B.W., U.S., G.A., A.v.E.); the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster, Münster, Germany (C.L., G.A., A.v.E.); and the Department of Pathology and Curriculum in Genetics and of the Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina (N.M.).

Correspondence to Dr. Arnold von Eckardstein, Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, Germany.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract We investigated the contribution of apoE to cholesterol efflux into plasmas of normal, apoA-I-, and apoE-deficient mice, which were fed with chow- and cholesterol-rich diets. Plasmas of normal and apoA-I-deficient mice contain apoE in pre-ß-migrating VLDL as well as in HDL-like lipoproteins, which have either electrophoretic {alpha}- or {gamma}-mobilities. The latter particle resembled {gamma}-LpE in human plasma also by its mobility on nondenaturing two-dimensional electrophoresis. No apoE-containing lipoproteins were found in plasmas of apoE-deficient mice. When apoA-I- and apoE-deficient mice received both chow- and fat-rich diets, their plasmas released significantly less 3H-cholesterol from radiolabeled fibroblasts than did plasma of normal mice. Removal of apoE from plasmas of normal and apoA-I-deficient mice by anti-apoE immunoaffinity chromatography decreased their cholesterol efflux capacities (per 1 minute/per 1 hour) by 26%/40% (P=0.0092/0.0007) and 30%/26% (P=0.0092/0.0003), respectively. Net cholesterol efflux from fibroblasts into apoA-I-deficient plasma was 45% lower compared with plasma of normal mice. Incubation of fibroblasts with apoE-deficient plasma caused net influx of cholesterol. Prior addition of human apoE to or removal of apoB-containing lipoproteins from apoE-deficient plasma restored its ability to cause net cholesterol efflux to 50% of normal plasma. Some of the differences between cholesterol efflux into normal and apoE-deficient plasmas were attributable to the failure of apoE-deficient plasmas to take up cell-derived 3H-cholesterol into {gamma}-LpE. Compared with normal plasma, both apoA-I-deficient and apoE-deficient plasmas were significantly decreased in their activity to esterify cell-derived 3H-cholesterol. Anti-apoE chromatography decreased significantly cholesterol esterification in normal plasma and apoA-I-deficient plasma but not in apoE-deficient plasma. Taken together, the data provide evidence that apoE is an important contributor to reverse cholesterol transport, partially because of initial uptake of cell-derived cholesterol by {gamma}-LpE and partially because of the contribution of apoE-containing lipoproteins to esterification of cholesterol in plasma.


Key Words: reverse cholesterol transport • HDL subclasses • atherosclerosis • animal models


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
It is widely assumed that high-density lipoproteins (HDL) exert their antiatherogenic role by their involvement in the reverse transport of excess cholesterol from peripheral cells to the liver and steroidogenic organs (reviewed in Reference 11 ). In the plasma compartment, reverse cholesterol transport is initiated by the uptake of cholesterol from plasma membranes through HDL. HDL represents a very heterogenous class of lipoproteins, most of which are lipid-rich, exhibit electrophoretic {alpha}-mobility, and contain apoA-I as the predominant protein constitutent. Quantitatively minor subgroups are lipid-poor or even lipid-free, exhibit electrophoretic pre-ß- or {gamma}-mobilities, and contain apoA-I, apoA-IV, or apoE as the only protein constituents (reviewed in References 11 –3). Lipid-rich and lipid-poor HDL subclasses take up cholesterol from different cellular pools by different kinetics. Cholesterol flux between plasma membranes and lipid-rich {alpha}-HDL is slow and bidirectional. Net cholesterol efflux is caused by the esterification of cholesterol through lecithin:cholesterol acyltransferase (LCAT). By contrast, lipid-free apoA-I and lipid-poor pre-ß1-LpA-I cause fast and unidirectional cholesterol efflux, which is independent of LCAT (References 44 –6; reviewed in Reference 77 ). By various experimental approaches, Fielding and coworkers1 8 9 indicated that it is likely that uptake of cell-derived cholesterol through pre-ß1-LpA-I requires specific interactions of this particle with specific, protease-sensitive domains on cell surfaces. From experiments with apoA-I-depleted plasmas, these authors concluded that pre-ß1-LpA-I is responsible for 50% of the cholesterol efflux promoting activity of plasma. They attributed the residual, unspecific cholesterol efflux activity of plasma to albumin.1 8 10

The absence of premature atherosclerosis in some patients with apoA-I-deficiency (Reference 1111 ; reviewed in Reference 1212 ) stimulated us to search for HDL-subclasses, which are free of apoA-I but fulfill important functions in reverse cholesterol transport. These experiments led to the identification of plasma lipoproteins, which contain either apoE or apoA-IV as their only apolipoproteins and which also serve as initial acceptors of cell-derived cholesterol in the plasma compartment.13 14 Because of its electrophoretic {gamma}-mobility, we termed the apoE-containing particle as {gamma}-LpE. Like pre-ß1-LpA-I, this particle is relatively rich in sphingomyelin and phosphatidylcholine but poor in cholesterol. A lipoprotein with similar characteristics has recently been identified in media of cultured hepatocytes.15 Pulse-chase experiments with the plasma of an apoA-I-deficient patient revealed that, in addition to apoB-containing lipoproteins, {gamma}-LpE is responsible for a significant proportion of the residual cholesterol efflux-promoting activity of this patient's plasma.16

In the study presented herein, we compared the cholesterol efflux from fibroblasts into plasmas and lipoproteins of normal, apoA-I-, and apoE-deficient mice17 18 to further investigate the importance of apoE in general and {gamma}-LpE in particular for reverse cholesterol transport. In this context, we also analyzed the influence of dietary fat on cholesterol efflux into plasma and lipoproteins.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animals and Sample Acquisition
Normal C57BL/6J mice were purchased from Harlan (Borchen, Germany). ApoA-I- and apoE-deficient mice were previously generated by gene targeting in embryonic stem cells and have been described previously.17 18 The mice were entered into the study when they were 8 to 10 weeks old. Three animals were caged together in a temperature-controlled room with 14-hour light/10-hour dark cycles. The regular chow diet (CD; Furina Altromin 1034, Lage, Germany) contained 4% fat from grain and soya as well as 0.015% cholesterol and 0.015% various other sterols, mainly ß-sitosterol, campesterol, and dihydrobrassicasterol (all weight per weight; Kannenberg, Seedorf, Assmann; unpublished results). For some experiments, a high-fat/high-cholesterol diet (FD) with 16% coconut oil, 1% cholesterol, and 0.5% sodium cholate (all weight per weight; ratio polyunsaturated/saturated fatty acids, 1:45) was fed for 4 weeks to groups of nine to ten male mice per genotype. In these experiments, equally sized control groups continued with CD.

Blood was collected from anesthetized animals by intracardial or intra-aortal puncture. Streptokinase was used as the anticoagulant at a final concentration of 1000 units/mL of blood. Plasma samples were obtained by centrifugation at 4°C (2000 rounds per minute, 15 minutes).

Quantification of Lipids, Protein, and Apolipoproteins
Concentrations of cholesterol and triglycerides were measured by enzymatic methods using commercially available tests from Sigma (Deisenhofen, Germany). HDL cholesterol was measured after precipitation of apoB-containing lipoproteins with phosphotungstic acid/MgCl2 (Boehringer Mannheim). The effects of different diets on levels of apoA-I and apoE were analyzed by a semiquantitative method. Aliquots with 5 µL of plasma of the differently fed mice were separated by SDS-polyacrylamide gel electrophoresis together in one gel.19 Plasma proteins were then electroblotted onto a nitrocellulose membrane. The membrane was incubated with goat antibodies against human apoA-I and human apoE, respectively (Boehringer Mannheim), which had been biotinylated according to the recommendations of the manufacturer of this kit (Sigma). The antigen-antibody complexes were visualized with a streptavidin-biotinylated horseradish peroxidase complex (Amersham). The intensity of bands was measured by densitometry using the Ultrascan (Pharmacia, Uppsala, Sweden).

Preparation of apoB-Depleted Plasma, apoE-Depleted Plasma, and apoE
ApoB-free mouse plasma was used in some experiments to determine net cholesterol efflux. ApoB-containing lipoproteins were precipitated by the addition of phosphotungstic acid/MgCl2 as recommended by the manufacturer (Boehringer Mannheim). The apoB-free supernatant was subsequently dialyzed against PBS (pH 7.4). Complete removal of apoB-containing lipoproteins was ascertained by immunoblotting.

For some experiments on the role of apoE for cholesterol efflux capacity of plasma, apoE was removed from plasma by anti-apoE-immunochromatography. ApoE antisera were produced by immunization of rabbits with recombinant mouse apoE (done by Dr. Y. Huang at the Gladstone Institute of Cardiovascular Disease, San Francisco, Calif). A total of 3.6 mg of IgG of a rabbit anti-mouse apoE-antiserum was coupled to 2 mL of NHS-activated Sepharose (Pharmacia), following the recommendations of the manufacturer. Aliquots of 100 µL of mouse plasma in 1 mL of PBS (pH 7.4) were circulated through the column for 18 hours. The bound fraction was eluted with 3 M sodium thiocyanate. The unbound fraction was dialyzed against PBS and concentrated by ultrafiltration (Amicon) to a final volume of 2.5 mL (equivalent to a 4% dilution of plasma). Immunoaffinity chromatography, dialysis, and ultrafiltration were performed at 4°C. Removal of apoE and apoA-I was ascertained by immunodotblot analysis of 100 µL of this diluted material using the biotinylated antibodies against human apoE and apoA-I described below. Densitometry of the dot blots revealed that anti-apoE immunoaffinity chromatography decreased the content of apoE by 71±4% in normal and apoA-I-deficient plasmas. The content of apoA-I was reduced by 6±2% in normal plasma and insignificantly in apoE-deficient plasma. The concentration of cholesterol decreased by 2 to 6% in normal plasma, by 10 to 16% in apoA-I-deficient plasma, and by 8 to 11% in apoE-deficient plasma. Until use for incubations with 3H-cholesterol-labeled cells (see below), aliquots with 1000 µL of apoE-depleted fractions were stored at -70°C.

Human apoE was isolated from delipidated VLDL by gel filtration using the FPLC system of Pharmacia and a Hiload HR16/60 column filled with Sephacryl S100HR as the stable phase and a buffer with 10 mM sodium phosphate, 150 mM sodium chloride, and 0.05% (w/v) sodium acid (pH 7.4) as the mobile phase. The apoE-containing fraction was then separated by reversed phase high-performance liquid chromatography.20 Purity of apoE was verified by SDS-polyacrylamide gel electrophoresis.19

Demonstration of Lipoproteins by Agarose Gel Electrophoresis and Nondenaturing Two-Dimensional Electrophoresis
Aliquots of 1 µL of plasma were electrophoresed in precasted 1% agarose gels (Ciba Corning, Palo Alto, Calif). Lipoproteins were stained for neutral lipids by Fat Red 7B according to the manufacturer's instructions. Alternatively, proteins were electroblotted onto nitrocellulose membranes, which were then incubated with biotinylated sheep antibodies against human apoA-I (Boehringer Mannheim) or biotinylated rabbit antibodies from the anti-mouse apoE antiserum mentioned previously. The antigen-antibody complexes were visualized with a streptavidin-biotinylated horseradish peroxidase complex (Amersham).

ApoE-containing lipoproteins were also demonstrated by separation of 20 µL of mouse plasma by nondenaturing two-dimensional polyacrylamide gradient gel electrophoresis (2D-PAGGE) in lab-made gels and subsequent anti-apoE immunoblotting as described previously.16 20 21 Briefly, in the first dimension, 20 µL of plasma samples were separated by electrophoresis at 4°C in a 0.75% agarose gel using a 50-mM merbital buffer (pH 8.7, Serva, Heidelberg, Germany). Bromphenol blue was added to a standard sample to visualize albumin in the native gel. The electrophoresis was stopped when the albumin/bromphenol blue marker had migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins were then transferred to a 3 to 20% polyacrylamide gradient gel. Separation in the second dimension was performed at 40 mA for 4 to 5 hours at 10°C. During this time, the endogenous plasma albumin, which because of bromphenol blue added to the cathodic buffer (300 µL per liter of buffer) was visible in the native gel as a faint blue band, had migrated 10 cm. The proteins separated in the PAGGE gel were electroblotted onto a nitrocellulose membrane. ApoE-containing lipoproteins were detected by the use of sheep antibodies against human apoE (Boehringer Mannheim), which had been biotinylated following the manufacturer's recommendations (Sigma), and streptavidin-biotinylated horseradish peroxidase complex (Amersham).

Determination of 3H-Cholesterol Efflux From Fibroblasts into Mouse Plasma or Lipoproteins
To investigate the role of apoA-I and apoE for cholesterol efflux into plasma and lipoprotein fractions, native or apoE-depleted mouse plasmas were incubated with radiolabeled fibroblasts. Normal human skin fibroblasts (3x105) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in either dishes of 3.5-cm diameter or in plates with 12 wells of 2.5-cm diameter. At the state of near confluence, cells were labeled in the presence of fetal calf serum with 0.5 (3.5-cm dishes) or 0.2 mCi (2.5-cm wells) (1,2-3H)-cholesterol (3H-UC, New England Nuclear, Boston, Mass, 51.7 Ci/mmol) for 72 hours at 37°C. After washing six times with PBS, pH 7.4, the specific radioactivity in the cells was 5.7±2.1x108 counts per minute per microgram of protein.

To measure the effects of apoE removal on efflux and esterification of cell-derived cholesterol, radiolabeled fibroblasts of 2.5-cm wells were incubated for 1 minute or 1 hour with native plasmas or apoE-depleted plasmas (see above), which were diluted in PBS to a final concentration of 4% (vol/vol). Thereafter, the medium was removed into microliter tubes. Eventual cell debris in the medium was pelleted by centrifugation at 30 000 revolutions per minute for 15 minutes at 4°C. After removal of the supernatant, an aliquot of 50 µL was directly used for determination of radioactivity by scintillation spectrometry. Lipids of another aliquot with 375 µL were extracted by chloroform/methanol (2:1) for 72 hours22 to subsequently separate UC and cholesteryl esters by thin layer chromatography in silica gel plates (Merck, Darmstadt, Germany) as the immobile phase and hexane:ether 6:4 (vol/vol) as the mobile phase. Cells were lysed with 1.5 mL of 0.5 M NaOH, and their lipids were extracted by incubation with hexane:isopropanol 3:2. The associated radioactivity was counted by scintillation spectrometry. Fractional cholesterol efflux was calculated as cpmmedium/(cpmmedium+cpmcells)x100%. Fractional esterification rate (FCR) was calculated as cpmcholesteryl esters/(cpmcholesteryl esters+cpmUC).

To measure the uptake of cell-derived 3H-UC into lipoprotein subfractions, radiolabeled cells of 3.5-cm dishes were incubated for 1 minute with 0.5 mL of mouse plasma.13 16 21 23 After incubation, the medium was removed and cell debris sedimented by centrifugation at 4°C (see above). Thereafter, aliquots of 20 µL of plasma were separated by agarose gel electrophoresis under the conditions described above for 2D-PAGGE.13 20 21 Every lane of the gel was cut into segments of 0.5 cm in length and transferred to a vial with 2 mL of scintillation buffer (Instant Scint Gel PL, Packard Instruments BV, Groningen, The Netherlands). This buffer completely solubilized the agarose gel so that the radioactivity of 3H-cholesterol could be measured directly.

Determination of Cholesterol Net Mass Transfer
Net cholesterol mass transfer from fibroblasts into mouse plasmas was determined as described previously.13 20 21 Briefly, confluently growing fibroblasts were preloaded with cholesterol for 48 hours.24 A total of 2 mL of PBS containing 5% mouse plasma or apoB-free mouse plasma (vol/vol) was then added to these cells and incubated for 2 hours at 37°C. In parallel, another 2-mL aliquot of the same plasma solution was incubated in a dish without cells. After removal, media were incubated with chloroform/methanol (2:1, vol/vol) three times for 6 hours to extract lipids.22 The extracts were disolved in 1 mL of ethanol. Cholesterol concentration was determined in both solutions by the use of a fluoroenzymatic assay as reported previously.25 The difference in the concentration of UC between the two aliquots gives the net cholesterol mass transfer rate.

Statistics
All data are presented as mean values±standard deviations. The level of significance for differences in cholesterol efflux because of diet, genotype, and/or apoE-removal was calculated by the ANOVA test using Excel (Microsoft) and an add-in program for Excel (Astute, 1993 DDU Software, The University of Leeds, Leeds, United Kingdom).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Plasma Levels of Lipids and Apolipoproteins
Table 1Down summarizes the levels of lipids and apolipoproteins in different mouse plasmas after 4 weeks CD or FD feeding. Feeding of FD increased the level of total cholesterol by factors 2, 3, and 2.5 in normal, apoA-I-deficient, and apoE-deficient mice, respectively. Triglycerides changed insignificantly in normal and apoE-deficient mice and increased by factor 2 in apoA-I-deficient mice. In all three kinds of animals, levels of HDL cholesterol did not change significantly, indicating that the rise in total cholesterol was attributable to an increase of cholesterol in apoB-containing lipoproteins. Moreover, feeding of FD slightly decreased apoA-I levels in plasmas of both normal and apoE-deficient mice but increased apoE levels significantly by 30% and 70% in plasmas of normal and apoA-I-deficient mice, respectively.


View this table:
[in this window]
[in a new window]
  		Table 1. Genotypic and Dietary Effects on Serum Levels of Lipids and Apolipoproteins in Mice

Electrophoretic Appearance of Lipoproteins
Fig. 1ADown shows fat red-stained electrophoretograms of lipoproteins in the different mouse plasmas. Feeding of FD increased the staining of neutral lipids in ß- and pre-ß-migrating lipoproteins of all animals (Fig. 1ADown, lanes 2, 4, and 6). By contrast the staining of {alpha}-migrating lipoproteins became less intense in plasmas of apoA-I-deficient mice (Fig. 1ADown, lane 4) and more pronounced in plasmas of apoE-deficient mice (Fig. 1ADown, lane 6).



View larger version (71K):
[in this window]
[in a new window]
  		Figure 1. Electrophoretic demonstration of lipoproteins in plasmas of normal, apoA-I- and apoE-deficient mice. Aliquots of 1 µL of mouse plasma were electrophoresed in a 1% agarose gel. Lipoproteins were then demonstrated by staining with Fat Red 7B (A), by anti-apoA-I immunoblotting (B), or anti-apoE immunoblotting (C). Lanes 1 and 2 contain normal mouse plasma, lanes 3 and 4 contain apoA-I-deficient plasma, lanes 5 and 6 contain apoE-deficient plasma. Lanes 1, 3, and 5 present lipoproteins in plasmas of CD-fed animals and lanes 2, 4, and 6 lipoproteins in plasmas of FD-fed animals.

Anti-apoA-I immunoblotting of electrophoretograms of normal mouse plasmas identified one quantitatively major particle with {alpha}-mobility and one quantitatively minor particle with slow ß-mobility, which both were present after feeding of either CD or FD (Fig. 1BUp, lanes 1 and 2). In plasmas of CD-fed, apoE-deficient mice, apoA-I was immunodetectable predominantly in an {alpha}-migrating lipoprotein and, less pronouncedly, in a ß-migrating lipoprotein (Fig. 1BUp, lane 5). After feeding of FD to apoE-deficient mice, apoA-I was only detectable in a particle with broad ß-mobility (Fig. 1BUp, lane 6). As expected, no anti-apoA-I immunoreactive particle was found in the plasmas of apoA-I-deficient mice (Fig. 1BUp, lanes 3 and 4).

In plasmas of normal and apoA-I-deficient mice fed with CD, anti-apoE antibodies immunoreacted with four particles, which exhibited {alpha}-, ß-, pre-ß-, and {gamma}-mobilities (Fig. 1CUp, lanes 1 and 3). After feeding of FD, the anti-apoE immunoreactivity of lipoproteins in normal mouse plasma did not change significantly (Fig. 1CUp, lanes 1 and 2). Feeding of FD to apoA-I-deficient mice decreased the anti-apoE immunoreactivity of the {alpha}-migrating fraction (Fig. 1CUp, lanes 3 and 4). As expected, apoE was undetectable in apoE-deficient mice (Fig. 1CUp, lanes 5 and 6).

To further differentiate apoE-containing lipoproteins, plasmas were separated by 2D-PAGGE. In plasmas of normal or apoA-I-deficient mice fed with chow diet, subsequent anti-apoE immunoblotting identified two differently sized apoE-containing particles with {gamma}-mobility, one particle with ß-mobility and one particle with {alpha}-mobility (Fig. 2aDown and b). The larger {gamma}-migrating particle was as large as {gamma}-LpE in human plasma (apparent Stokes diameter, 14 to 16 nm). After fat feeding, the intensity of immunostaining of the smaller {gamma}-migrating lipoprotein (Fig. 2dDown and e) increased. The pre-ß-migrating particle detected after agarose gel electrophoresis (Fig. 1CUp) was not detectable on 2D-PAGGE, presumably because this particle (VLDL) is too big to migrate into the polyacrylamide gel. No apoE-containing particle was found in apoE-deficient mouse plasmas (Fig. 2cDown and 2fDown).



View larger version (113K):
[in this window]
[in a new window]
  		Figure 2. 2D-PAGGE and immunoblotting of apoE-containing lipoproteins in different mouse plasmas. Nondenaturing 2D-PAGGE was performed in the sequence agarose gel electrophoresis->nondena-turing polyacrylamide gel electrophoresis. After electroblotting to nitrocellulose membranes, apoE-containing lipoproteins were detected with biotinylated polyclonal goat antibodies against human apoE and streptavidin-biotinylated horseradish peroxidase complex. Parts a and b present electrophoretograms of normal mouse plasma, parts c and d electrophoretograms of apoA-I-deficient plasma, parts e and f electrophoretograms of apoE-deficient plasma. Plasmas of CD-fed animals have been separated for parts a, c, and e and plasmas of FD-fed animals for parts b, d, and f. ApoE-containing lipoproteins of normal and apoA-I-deficient mice with pre-ß mobility in agarose gels (cf. Fig. 1Up, lanes 2 and 4) are not detectable in the polyacrylamide gel. They probably correspond to VLDL, which because of their size do not migrate in polyacrylamide gels.

Genotypic and Dietary Effects on Efflux of Radioactive Cholesterol from Fibroblasts into Mouse Plasmas
During short, 1-minute incubations with radiolabeled fibroblasts, plasmas of CD-fed apoA-I-deficient or apoE-deficient mice released 24% (P<0.05) and 45% (P<0.01) less 3H-cholesterol from cells than normal mouse plasmas, respectively (Table 2Down). Feeding of FD increased 3H-cholesterol efflux from fibroblasts into plasmas of normal and apoA-I-deficient mice by 21% and 25% (both P<0.05), respectively, compared with the plasmas of CD-fed animals (Table 2Down). By contrast, feeding of FD did not significantly affect 3H-cholesterol efflux into apoE-deficient plasma (Table 2Down).


View this table:
[in this window]
[in a new window]
  		Table 2. Dietary and Genotypic Effects on the Release of 3H-Cholesterol From Fibroblasts by Mouse Plasmas

ApoE-deficient plasma differs from normal and apoA-I-deficient mouse plasmas not only by the absence of apoE but also by the concentration and composition of all lipoprotein classes (Table 1Up, Fig. 1Up). We, therefore, investigated the contribution of apoE to cholesterol efflux by comparing the cholesterol efflux capacity of the various plasmas before and after removal of apoE-containing lipoproteins by anti-apoE immunoaffinity chromatography (Tables 3Down and 4Down). Native plasmas of both apoA-I-deficient and apoE-deficient mice took up significantly less cellular 3H-UC than normal mouse plasma during incubations with radiolabeled fibroblasts for either 1 minute (Table 3Down, -26% and 28%, respectively) or 1 hour (Table 4Down, -34%). Removal of apoE by anti-apoE immunoaffinity chromatography significantly decreased cholesterol efflux capacity of normal plasma (1 minute, -26%; 1 hour, -40%) and apoA-I-deficient plasma (1 minute, -30%; 1 hour, -26%; Tables 3Down and 4Down). In control experiments, anti-apoE immunoaffinity chromatography of apoE-deficient plasma did not change its cholesterol efflux capacity (Tables 3Down and 4Down). Moreover and interestingly, cholesterol efflux capacities of apoE-depleted plasmas of normal mice and plasmas of apoE-deficient mice did not differ. By contrast, apoE-depleted plasmas of apoA-I-deficient mice were significantly less efficient in promoting cholesterol efflux than apoE-depleted plasmas of either normal or apoE-deficient mice (Tables 3Down and 4Down). Taken together, these data indicate that both apoA-I- and apoE-containing lipoproteins contribute to cholesterol efflux into mouse plasmas.


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of Genotype, Diet, and apoE on Cholesterol Efflux Capacity of Murine Plasmas (1-Minute Incubation)


View this table:
[in this window]
[in a new window]
  		Table 4. Effect of Genotype, Diet, and apoE on Cholesterol Efflux Capacity of Murine Plasmas (1 Hour Incubation)

Effects of apoA-I, apoE, and apoB-Containing Lipoproteins on Net Cholesterol Efflux from Fibroblasts into Mouse Plasmas
Net cholesterol efflux from fibroblasts into apoA-I-deficient plasma was 45% lower than into normal mouse plasma (Table 5Down). This difference was even more pronounced after depletion of plasma from apoB-containing particles. Incubation of fibroblasts with apoE-deficient plasma rather caused influx of cholesterol from plasma into the cells. Both, removal of apoB-containing lipoproteins or addition of 20 µg/mL human apoE to apoE-deficient mouse plasma before its incubation with cells partially restored the ability of apoE-deficient plasma to promote net cholesterol efflux (Table 5Down). Taken together, these data indicated that the promotion of net influx rather than efflux of cholesterol from cells by apoE-deficient plasma was the result of both hypercholesterolemia and lack of apoE. Similar to apoA-I-deficiency, deficiency of apoE appeared to reduce cholesterol efflux into plasma by approximately 50%.


View this table:
[in this window]
[in a new window]
  		Table 5. Effect of Genotype, Diet, and apoE on Esterification of Cell-Derived Cholesterol in Mouse Plasma

Genotypic and Dietary Effects on Esterification of Cell-Derived Radioactive Cholesterol in Mouse Plasmas
During 1 hour of incubation with radiolabeled fibroblasts, normal mouse plasma esterified a significantly higher percentage of cell-derived 3H-UC than plasmas of either apoA-I-deficient or apoE-deficient mice (Table 6Down). Feeding of FD did not cause significant changes in the FCR of mouse plasmas (not shown). Removal of apoE by immunoaffinity chromatography significantly decreased FCR in both normal plasma (-62%) and apoA-I-deficient plasma (-37%). Anti-apoE immunoaffinity chromatography, however, did not alter FCR of apoE-deficient plasma (Table 6Down).


View this table:
[in this window]
[in a new window]
  		Table 6. Dietary and Genotypic Effects on Net Cholesterol Efflux from Cholesterol-Loaded Fibroblasts into Murine Plasmas

Genotypic and Dietary Effects on Efflux of Radioactive Cholesterol from Fibroblasts into Lipoproteins of Mouse Plasmas
Next, we characterized the lipoproteins that are responsible for the increased uptake of cell-derived 3H-cholesterol by plasmas of FD-fed normal and apoA-I-deficient mice. Plasmas were pulsed with cellular 3H-cholesterol. After removal and sedimentation of cell debris, one aliquot was separated by agarose gel electrophoresis to determine the radioactivity in different lipoprotein fractions (Fig. 3Down). Plasmas of normal CD-fed mice took up cell-derived 3H-cholesterol predominantly in fractions with either {gamma}- or {alpha}-mobility, namely 26% and 53%, respectively (Fig. 3aDown). In apoA-I-deficient plasma, radioactivity was found in both {gamma}-migrating (23%) and {alpha}-migrating lipoproteins (41%), although at lower amounts than in normal plasma (Fig. 3bDown). ApoE-deficient plasma accumulated radioactivity in {alpha}-migrating and pre-ß-migrating lipoproteins but not in the {gamma}-migrating fraction (Fig. 3cDown). Feeding of FD to normal and apoA-I-deficient mice increased the uptake of cell-derived 3H-cholesterol by the {gamma}-migrating lipoprotein by 31% and 60%, respectively (Fig. 3dDown and e). This enhanced accumulation of radioactivity in the {gamma}-migrating fraction corresponded to 55% and 66% of the increased 3H-cholesterol efflux into normal and apoA-I-deficient plasma, respectively. By contrast, feeding of FD to apoE-deficient mice did not affect the uptake of 3H-cholesterol by either total plasma or the {gamma}-migrating fraction (Fig. 3fDown).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 3. Dietary effects on the uptake of cell-derived 3H-cholesterol by various lipoproteins in plasmas of normal, apoA-I-deficient, and apoE-deficient mice. Mouse plasmas were incubated for 1 minute with 3H-cholesterol-labeled normal human fibroblasts. Aliquots of 20 µL of plasma were then electrophoresed in 0.75% agarose gels. After electrophoresis, each lane of the gel was cut into 5-mm-long segments. The lipids in each slice were extracted with chloroform/methanol (2:1), and their radioactivity was determined by liquid scintillation spectrometry. The values present means and standard deviations of three experiments.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
ApoE-deficient mice develop severe atherosclerosis.26 27 The implicated anti-atherogenic properties of apoE have mostly been attributed to its role as a receptor ligand that mediates the hepatic removal of cholesterol-rich and atherogenic lipoproteins.26 27 28 29 30 Other in vivo animal studies point to additional anti-atherogenic mechanisms exerted by apoE. Infusion of apoE into Watanabe Heritable Hyperlipidemic rabbits31 as well as specific expression of apoE either in cells of the arterial wall in apoE-transgenic mice32 or in macrophages of apoE-deficient mice33 inhibited the formation of atherosclerotic lesions without changing plasma lipid levels. The authors of the latter studies hypothesized that apoE may prevent atherosclerosis by its contribution to reverse cholesterol transport.31 32 33 Several in vitro properties have been described by which apoE may contribute to reverse cholesterol transport. For example, apoE is produced by macrophages that have been loaded with cholesterol. In the presence of HDL, apoE secretion by macrophages enhances cholesterol efflux.34 35 Kruth et al36 described the secretion of apoE/lipid complexes by human macrophage foam cells, which does not depend on the extracellular presence of acceptor particles. Our laboratory has previously identified {gamma}-LpE, in which apoE constitutes the only apolipoprotein and which serves as an initial acceptor of cell-derived cholesterol in the plasma compartment.13 Finally, apoE is present in some HDL that can, therefore, be eliminated by apoE and apoB,E receptors.37 38 39 In the study presented herein, we have provided further evidence that apoE, in addition to apoA-I, contributes to reverse cholesterol transport by facilitating cholesterol efflux from cells.

As apoA-I-deficient mouse plasmas, apoE-deficient mouse plasmas had a significantly lower capacity than normal mouse plasmas to release cholesterol from human fibroblasts, independent of whether we measured efflux of radiolabeled cholesterol during a very short incubation for 1 minute (Tables 2Up and 3Up) or during a longer incubation for 1 hour (Table 4Up) or of whether we assayed net cholesterol mass efflux (Table 5Up). Interestingly, the decreases in cholesterol efflux capacity of apoE-deficient and apoA-I-deficient plasmas were similar. One may argue that the measurement of both 3H-cholesterol efflux and net cholesterol efflux from fibroblasts into plasma is confounded with different cholesterol levels among the different mouse plasmas. On the one hand, accumulation of radioactivity in mouse plasmas may have resulted from unproductive equilibration between labeled cholesterol present in cell membranes and unlabeled cholesterol present in plasma lipoproteins rather than productive cholesterol efflux. However, if such unproductive equilibration played some role during incubation of plasma with radiolabeled cells, the hypercholesterolemic apoE-deficient plasma should rather lead to an apparent increase in cholesterol efflux. In this situation, the data of our isotope experiments would rather underestimate the importance of apoE-containing lipoproteins.

On the other hand, flux of cholesterol between plasma membrane and the majority of plasma lipoproteins is bidirectional5 6 7 and may be changed to influx in the presence of hypercholesterolemic plasmas of apoE-deficient mice. Actually, we observed net cholesterol influx from apoE-deficient plasmas into fibroblasts instead of net cholesterol efflux from cells into normocholesterolemic plasmas of either normal or apoA-I-deficient mice. Whether mediated by lipoprotein receptors or by receptor-independent diffusion of cholesterol from lipoproteins into the cell membrane,23 40 this net influx of cholesterol may well occur in vivo and then contribute to the atherogenicity of apoE-deficiency. Both removal of cholesterol-rich lipoproteins by precipitation of apoB-containing particles and prior addition of physiologic amounts of human apoE to apoE-deficient plasmas (20 µg/mL final concentration) partially restored the ability of these plasmas to promote cholesterol efflux to 50% of normal plasma (Table 5Up). Together the data indicate that the presence of cholesterol-rich remnants in apoE-deficient plasma enhances cholesterol influx into fibroblasts and that absence of apoE significantly impairs cholesterol efflux into plasma. In agreement with the latter interpretation, we found that removal of apoE by immunoaffinity chromatography decreased the cholesterol efflux capacity of plasmas from either normal or apoA-I-deficient mice (Tables 3Up and 4Up). The decrease in cholesterol efflux capacity of mouse plasma by removal of apoE is in contrast with the similar experiments of Fielding and Fielding41 who did not observe any effect of apoE removal on cholesterol efflux capacity of human plasma.41

Net cholesterol efflux rates are calculated from the different levels of unesterified cholesterol found after incubation of plasma either in the presence or in the absence of cells. Because this difference is not only influenced by the flux of cholesterol between cells and plasma but also by esterification, decreased net cholesterol efflux rates in apoA-I- and apoE-deficient plasma can also reflect increased cholesterol esterification. However, esterification of cell-derived 3H-cholesterol was found to be reduced in both apoA-I- and apoE-deficient mouse plasmas (Table 6Up). Thus, if differences in LCAT activity affect net cholesterol efflux rates, we would have rather underestimated the net cholesterol efflux rates of apoA-I- and apoE-deficient plasmas.

What are the mechanisms by which apoE may facilitate cholesterol efflux? One reason for the decreased cholesterol efflux capacity of apoE-deficient plasma may be the quantitative and qualitative changes in the composition of lipoproteins in the plasma of apoE-deficient mice. Thus, apoE-deficient mice were reported to have lower plasma levels of apoA-I and higher plasma levels of apoA-IV.42 However, the decreased cholesterol efflux capacities of apoE-depleted plasmas from normal and apoA-I-deficient mice make the relevance of these explanations unlikely (Table 3Up). Basu et al34 35 showed previously that enrichment of HDL with apoE increases their activity to remove cholesterol from macrophages. In agreement with these pioneering studies, Hayek and colleagues43 reported previously that HDL of apoE-deficient mice are reduced in their activity to promote cholesterol efflux from mouse peritoneal macrophages and that this decreased capacity can be restored by the addition of apoE to HDL.43 In our experiments, we observed that removal of apoE decreases the cholesterol efflux capacity not only of normal but also apoA-I-deficient plasma. This points to a direct involvement of apoE-containing lipoproteins in cholesterol efflux. Our studies provide two explanations by which apoE may regulate the ability of plasma to take up cholesterol from cells.

Plasmas of both normal and apoA-I-deficient mice but not plasmas of apoE-deficient mice contain an apoE-containing lipoprotein, which by its electrophoretic mobility in agarose and nondenaturing polyacrylamide gels resembles {gamma}-LpE in human plasma (Fig. 1Up and 2Up). As in human plasma,13 21 murine {gamma}-LpE serves as an initial acceptor of cell-derived cholesterol into plasmas of either normal or apoA-I-deficient mice (Fig. 3aUp and b), where it takes up 25% of the radiolabel effluxed into plasma. The absence of radioactive cholesterol in this fraction after 1 minute of incubation of radiolabeled cells with apoE-deficient plasma (Fig. 3cUp) explained 50% of the difference between 3H-cholesterol efflux into normal and apoE-deficient plasmas. The importance of {gamma}-LpE for cholesterol efflux promotion can also be estimated from our data on the dietary effects on 3H-cholesterol efflux into both total plasma and {gamma}-LpE during a 1-minute incubation with radiolabeled cells (Table 2Up and Fig. 3dUp-f). Four weeks of feeding of FD increased 3H-cholesterol efflux into plasmas of normal and apoA-I-deficient mice by 21% and 25%, respectively. A total of 55% and 66% of the increased 3H-cholesterol efflux into normal and apoA-I-deficient plasma, respectively, was attributable to the enhanced uptake of radioactivity by {gamma}-LpE. By contrast, feeding of FD to apoE-deficient mice neither led to the occurrence of radioactivity in the {gamma}-migrating fraction of their plasmas nor to any increase in the uptake of cell-derived 3H-cholesterol by total plasma. Together with our previous findings that {gamma}-LpE accounts for a significant proportion of the 50% residual cholesterol efflux capacity of apoA-I-deficient human plasma16 and that severely decreased levels of {gamma}-LpE in plasmas of homozygotes for apoE2/2 or apoE4/4 are associated with a 30% lower cholesterol efflux capacity compared with plasmas of apoE3/3 homozygotes,21 the study presented herein further supports the importance of {gamma}-LpE for the activity of plasma to release cholesterol from cells.

As plasmas of apoA-I-deficient mice, plasmas of apoE-deficient mice are reduced by 30% to 50% in their activity to esterify cell-derived cholesterol (Table 5Up). As in plasmas of human patients with familial HDL deficiency, the decreased FCR in apoA-I-deficient mouse plasma probably reflects both decreased LCAT mass and the absence of lipoproteins where cholesterol esterification usually takes place.44 45 46 47 Which of these reasons accounts for the decreased FCR in apoE-deficient plasma is unknown. One has also to consider changes in the composition of apoB-containing lipoproteins as another reason for the decreased FCR in apoE-deficient plasma, because these lipoproteins serve as the most efficient donors of cholesterol to the LCAT reaction at least in normolipidemic human plasmas.23 48 49 Whatever the reason, the decreased activity of apoE-deficient mouse plasma to esterify cell-derived cholesterol may be causally linked to decreased cholesterol efflux. Czarnecka and Yokoyama5 6 demonstrated previously that esterification of cell-derived cholesterol by LCAT is a prerequisite for net cholesterol efflux into lipid-rich, mature HDL. Otherwise, the flux of cholesterol between plasma membranes and lipoproteins would be bidirectional and unproductive.5 6

Taking together with the data of these and previous in vitro studies of our laboratory,13 16 21 we conclude that apoE-containing lipoproteins play an important role in regulating cholesterol efflux from cells into plasma. We hypothesize that the lack of these functions contribute to the development of complicated atherosclerotic lesions in apoE-deficient mice26 27 and that the gain of these functions interferes with the progression of atherosclerosis in susceptible animals.31 32 33


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
CD = chow-diet
FCR = fractional esterification rate
FD = fat- and cholesterol-rich diet
LCAT = lecithin:cholesterol acyltransferase
Lp = lipoprotein
2D-PAGGE = two-dimensional polyacrylamide gradient gel electrophoresis
UC = unesterified cholesterol


*    Acknowledgments
 
The project was supported by grants from Deutsche Forschungsgemeinschaft (Ec 116, 3-1 Ec 116, 3-2) to Dr. von Eckardstein. This work contains parts of the thesis work of Claus Langer performed in partial fulfillment of the requirements of the Westfälische Wilhelms-Universität. We thank Martina Plüster for excellent technical assistance. We are grateful to Prof. Dr. Galla (Institut für Biochemie, Westfälische Wilhelms-Universität Münster) for supervision of the thesis work of Claus Langer.

Received October 13, 1995; accepted February 10, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res.. 1995;36:211-228.[Abstract]
  2. von Eckardstein A, Huang Y, Assmann G. Physiological role and clinical relevance of high-density lipoprotein subclasses. Curr Opin Lipidol.. 1994;5:404-416.[Medline] [Order article via Infotrieve]
  3. Barrans A, Jaspard B, Barbaras R, Chap H, Perret B, Collet X. Pre-ß-HDL: structure and metabolism. Biochim Biophys Acta.. 1996;1300:73-85.[Medline] [Order article via Infotrieve]
  4. Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre-ß-migrating high density lipoprotein. Biochemistry. 1988;27:25-29.[Medline] [Order article via Infotrieve]
  5. Czarnecka H, Yokoyama S. Regulation of cellular cholesterol efflux by lecithin: cholesterol acyltransferase reaction through nonspecific lipid exchange. J Biol Chem.. 1996;266:2023-2028.
  6. Czarnecka H, Yokoyama S. Lecithin: cholesterol acyltransferase reaction on cellular lipid released by free apolipoprotein-mediated efflux. Biochemistry. 1995;34:4385-4392.[Medline] [Order article via Infotrieve]
  7. von Eckardstein A. Cholesterol efflux from macrophages and other cells. Curr Opin Lipidol.. 1996;7:311-320.
  8. Kawano M, Miida T, Fielding CJ, Fielding PE. Quantitation of preß-HDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid. Biochemistry. 1993;32:5025-5028.[Medline] [Order article via Infotrieve]
  9. Fielding PE, Kawano M, Catapano A, Zoppo A, Marcovina S, Fielding CJ. Unique epitope of apolipoprotein A-I in pre-ß-1 high density lipoprotein and its role in the catalyzed efflux of cellular cholesterol. Biochemistry. 1994;33:6981-6985.[Medline] [Order article via Infotrieve]
  10. Fielding CJ, Moser A. Evidence for the separation of albumin- and apo A-I-dependent mechanisms of cholesterol efflux from cultured fibroblasts into human plasma. J Biol Chem.. 1982;257:10955-10960.[Abstract/Free Full Text]
  11. Römling R, von Eckardstein A, Funke H, Motti C, Fragiacomo G, Noseda G, Assmann G. A nonsense mutation in the apolipoprotein A-I gene is associated with high density lipoprotein deficiency but not with coronary heart disease. Arterioscler Thromb.. 1994;14:1915-1923.[Abstract/Free Full Text]
  12. Assmann G, von Eckardstein A, Funke H. High density lipoproteins, reverse transport of cholesterol, and coronary heart disease: insights from mutants. Circulation 1993;87(suppl. III):28-34.
  13. Huang Y, von Eckardstein A, Wu S, Maeda N, Assmann G. A plasma lipoprotein containing only apolipoprotein E and with {gamma} mobility on electrophoresis releases cholesterol from cells. Proc Natl Acad Sci U S A.. 1994;91:1834-1838.[Abstract/Free Full Text]
  14. von Eckardstein A, Huang Y, Wu S, SaadatSarmadi S, Schwarz S, Steinmetz A, Assmann G. High density lipoproteins containing apolipoprotein A-IV but not apolipoprotein A-I take up and esterify cell-derived cholesterol in plasma. Arterioscler Thromb Vasc Biol.. 1995;15:1755-1763.[Abstract/Free Full Text]
  15. Fazio S, Yao Z. The enhanced association of apolipoprotein E with apolipoprotein B-containing lipoproteins in serum-stimulated hepatocytes occurs intracellularly. Arterioscler Thromb Vasc Biol.. 1995;15:593-600.[Abstract/Free Full Text]
  16. von Eckardstein A, Huang Y, Wu S, Funke H, Noseda G, Assmann G. Reverse cholesterol transport in plasmas of patients with different forms of familial high density lipoprotein deficiency. Arterioscler Thromb Vasc Biol.. 1995;15:691-703.[Abstract/Free Full Text]
  17. Piedrahita, Zhang SH, Hagamann J, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci U S A.. 1992;89:4471-4475.[Abstract/Free Full Text]
  18. Williamson R, Lee D, Hagamann J, Maeda N. Marked reduction of high density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I. Proc Natl Acad Sci U S A.. 1992;89:7134-7138.[Abstract/Free Full Text]
  19. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.[Medline] [Order article via Infotrieve]
  20. von Eckardstein A, Walter M, Benninghoven A, Assmann G. Site specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity in apolipoproteins A-I, C-II, and C-III. J Lipid Res.. 1991;32:1465-1476.[Abstract]
  21. Huang Y, von Eckardstein A, Wu S, Assmann G. Effects of the apolipoprotein E-polymorphism on uptake and transfer of cell-derived cholesterol in plasma. J Clin Invest.. 1995;96:2693-2701.
  22. Folch JM, Lees M, Sloane GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem.. 1957;226:497-509.[Free Full Text]
  23. Huang Y, von Eckardstein A, Assmann G. Cell-derived cholesterol cycles between different HDLs and LDL for its effective esterification in plasma. Arterioscler Thromb.. 1993;13:445-458.[Abstract/Free Full Text]
  24. Johnson WI, Mahlberg FH, Chacko GK, Phillips MC, Rothblat GH. The influence of cellular and lipoprotein cholesterol cholesterol contents on the flux of cholesterol between fibroblasts and high density lipoproteins. J Biol Chem.. 1988;263:14099-14106.[Abstract/Free Full Text]
  25. Heider JG, Boyett RL. The picomole determination of free and total cholesterol in cells in culture. J Lipid Res.. 1978;19:514-518.[Abstract]
  26. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992;258:468-471.[Abstract/Free Full Text]
  27. Plump AS, Smith JD, Hayek T, Aalto-Setälä K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992;71:343-353.[Medline] [Order article via Infotrieve]
  28. Linton MF, Atkinson JB, Fazio S. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation. Science. 1995;267:1034-1037.[Abstract/Free Full Text]
  29. Bolsvert WA, Spangenberg J, Curtiss LK. Treatment of severe hyperlipidemia in apolipoprotein E-deficient mice by bone marrow transplantation. J Clin Invest.. 1995;96:1118-1124.
  30. Kashyap VS, Santamarian-Fojo S, Brown DR, Parrott CL, Applebaum-Bowden, Meyn S, Talley G, Paigen B, Maeda N, Brewer HB. Apolipoprotein E-deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vecots. J Clin Invest.. 1995;96:1612-1620.
  31. Yamada N, Inoue I, Kawamura M, Harada M, Watanabe Y, Shimano H, Gotoda T, Shimada M, Kohzaki M, Tsukada T, Shiomini M, Watanabe Y, Yazaki Y. Apolipoprotein E prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits. J Clin Invest.. 1992;89:706-711.
  32. Shimano H, Ohsuga J, Shimada M, Namba Y, Gotoda T, Harada Y, Katsuki M, Yazaki Y, Yamada N. Inhibition of dietinduced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall. J Clin Invest.. 1995;95:469-476.
  33. Bellosta S, Mahley RW, Sanan DA, Newland DL, Taylor JM, Pitas RE. Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein-E-null mice. J Clin Invest.. 1995;96:2170-2179.
  34. Basu SK, Ho Y, Brown MS, Bilheimer DW, Anderson RGW, Goldstein J. Biochemical and genetic studies of the apoprotein E secreted by mouse macrophages and human monocytes. J Biol Chem.. 1982;257:9788-9795.[Free Full Text]
  35. Basu S, Goldstein JL, Brown MS. Independent pathways for secretion of free cholesterol and apolipoprotein E by macrophages. Science. 1983;219:871-873.[Abstract/Free Full Text]
  36. Kruth HS, Skarlatos SI, Gaynor PM, Gamble W. Production of cholesterol-enriched nascent high density lipoproteins by human monocyte-derived macrophages is a mechanism that contributes to macrophage cholesterol efflux. J Biol Chem.. 1994;269:24511-24518.[Abstract/Free Full Text]
  37. Koo C, Innerarity TL, Mahley RW. Obligatory role of cholesterol and apolipoprotein E in the formation of large cholesterol enriched and receptor-active high density lipoproteins. J Biol Chem.. 1985;260:11934-11943.[Abstract/Free Full Text]
  38. Gordon V, Innerarity TL, Mahley RW. Formation of cholesterol- and apoprotein E enriched high density lipoproteins in vitro. J Biol Chem.. 1983;258:6202-6212.[Abstract/Free Full Text]
  39. Funke H, Boyles J, Weisgraber KH, Ludwig EH, Hui DY, Mahley RW. Uptake of apolipoprotein E containing high density lipoproteins by hepatic parenchymal cells. Arteriosclerosis. 1984;4:452-461.[Abstract/Free Full Text]
  40. Fielding CJ, Fielding PE. Role of an N-ethylmaleimide-sensitive factor in the selective cellular uptake of low density lipoprotein free cholesterol. Biochemistry. 1995;34:14237-14244.[Medline] [Order article via Infotrieve]
  41. Fielding CJ, Fielding PE. Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and ist relationship to lecithin:cholesterol acyltransferase. Proc Natl Acad Sci U S A.. 1981;78:3911-3914.[Abstract/Free Full Text]
  42. Ishibashi S, Herz J, Maeda N, Goldstein JL, Brown MS. The two-receptor model of lipoprotein clearance: tests of the hypothesis in `knockout' mice lacking the low density lipoprotein receptor, apolipoprotein E, or both proteins. Proc Natl Acad Sci U S A.. 1994;91:4431-4435.[Abstract/Free Full Text]
  43. Hayek T, Oiknine J, Brook JG, Aviram M. Role of HDL apolipoprotein E in cellular cholesterol efflux: studies in apoE knock out transgenic mice. Biochem Biophys Res Commun.. 1994;205:1072-1078.[Medline] [Order article via Infotrieve]
  44. Parks JS, Li H, Gebre AK, Smith TL, Maeda N. Effect of apolipoprotein A-I deficiency on lecithin:cholesterol acyltransferase activation in mouse plasma. J Lipid Res.. 1995;36:349-355.[Abstract]
  45. Forte TM, Nichols AV, Krauss RM, Norum RA. Familial apolipoprotein A-I and C-III deficiency: subclass distribution, composition and morphology of lipoproteins in a disorder associated with premature atherosclerosis. J Clin Invest.. 1984;74:1601-1613.
  46. Pritchard PH, McLeod R, Frohlich J, Park MC, Kudchodkar BJ, Lacko AG. Lecithin:cholesterol acyltransferase in familial HDL-deficiency. Biochim Biophys Acta.. 1988;958:227-234.[Medline] [Order article via Infotrieve]
  47. Cheung MC, Wang D, Lum KD, Albers JJ. Cholesterol esterification by lecithin:cholesterol acyltransferase in A-I-free plasma. Biochim Biophys Acta.. 1988;962:258-264.[Medline] [Order article via Infotrieve]
  48. Miida T, Fielding CJ, Fielding PE. Mechanism of transfer of LDL-derived free cholesterol to HDL subfractions in human plasma. Biochemistry. 1990;29:10469-10474.[Medline] [Order article via Infotrieve]
  49. Subbaiah PV, Banerij B, Gregg RE, Bagdade JD. Molecular species of cholesteryl esters formed in abetalipoproteinemia: effect of apoprotein B containing lipoproteins. J Lipid Res.. 1990;31:927-932.[Abstract]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. E. Kypreos, K. W. van Dijk, L. M. Havekes, and V. I. Zannis
Generation of a Recombinant Apolipoprotein E Variant with Improved Biological Functions: HYDROPHOBIC RESIDUES (LEU-261, TRP-264, PHE-265, LEU-268, VAL-269) OF apoE CAN ACCOUNT FOR THE apoE-INDUCED HYPERTRIGLYCERIDEMIA
J. Biol. Chem., February 25, 2005; 280(8): 6276 - 6284.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
A. M. Fagan, E. Christopher, J. W. Taylor, M. Parsadanian, M. Spinner, M. Watson, J. D. Fryer, S. Wahrle, K. R. Bales, S. M. Paul, et al.
ApoAI Deficiency Results in Marked Reductions in Plasma Cholesterol But No Alterations in Amyloid-{beta} Pathology in a Mouse Model of Alzheimer's Disease-Like Cerebral Amyloidosis
Am. J. Pathol., October 1, 2004; 165(4): 1413 - 1422.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
A. von Eckardstein, J.-R. Nofer, and G. Assmann
High Density Lipoproteins and Arteriosclerosis : Role of Cholesterol Efflux and Reverse Cholesterol Transport
Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 13 - 27.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
R. K. Tangirala, K. Tsukamoto, S. H. Chun, D. Usher, E. Pure, and D. J. Rader
Regression of Atherosclerosis Induced by Liver-Directed Gene Transfer of Apolipoprotein A-I in Mice
Circulation, October 26, 1999; 100(17): 1816 - 1822.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
K. Tsukamoto, R. Tangirala, S. H. Chun, E. Pure, and D. J. Rader
Rapid Regression of Atherosclerosis Induced by Liver-Directed Gene Transfer of ApoE in ApoE-Deficient Mice
Arterioscler. Thromb. Vasc. Biol., September 1, 1999; 19(9): 2162 - 2170.
[Abstract] [Full Text] [PDF]