© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Effect of Macrophage-Derived Mouse ApoE, Human ApoE3-Leiden, and Human ApoE2 (Arg158
Cys) on Cholesterol Levels and Atherosclerosis in ApoE-Deficient Mice
From the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research (M.V.E., N.H., T.J.C.V.B.), the Department of Human Genetics (K.W.V.D., M.H.H.), Sylvius Laboratories, Leiden University, and TNO Prevention and Health (L.M.H.), Leiden, The Netherlands; and SmithKline Beecham Pharmaceuticals (P.H.E.G.), NFSP(N), Coldharbour Road, Harlow, Essex CM19 5AD, UK.
Correspondence to M. Van Eck, MSc, Division of Biopharmaceutics, Sylvius Laboratories, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands. E-mail M.Eck{at}LACDR.LeidenUniv.nl
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Abstract—The effect of monocyte/macrophage-derived wild-type mouse apolipoprotein E (apoE), human apoE3-Leiden, and human apoE2 on serum cholesterol levels and the development of atherosclerosis in apoE-deficient (apoe–/–) mice was investigated by using bone marrow transplantation (BMT). At 4 weeks after BMT, murine apoe+/+ bone marrow reduced serum cholesterol levels by 87% in apoe–/– mice, whereas macrophage-derived human apoE3-Leiden and human apoE2 induced a maximal, transient reduction of 35% and 48%, respectively. At 4 months after BMT, atherosclerosis was 23-fold (P<0.001) reduced in apoe+/+
apoe–/– mice, whereas no
significant reduction in
apoE3-Leiden.apoe–/–apoe–/– and
apoE2.apoe–/–apoe–/– mice could be
demonstrated. A highly significant decrease in serum
cholesterol levels (78% reduction) and
atherosclerosis (21-fold, P<0.001) was
found in apoE3-Leiden.apoe–/– animals expressing high
levels of apoE in multiple tissues, whereas apoE2 was ineffective even
at high concentrations. Furthermore, in contrast to apoE-deficient
macrophages, cholesterol efflux from apoE2 or
apoE3-Leiden macrophages was not impaired. In conclusion,
apoE3-Leiden as well as apoE2 are less effective in reducing
cholesterol levels and atherosclerosis in
apoe–/– animals, compared with apoe+/+,
with apoE2<apoE3-Leiden<apoe+/+, irrespective of the
observed adequate efflux of cholesterol from
macrophages expressing apoE2 and apoE3-Leiden, indicating that
normalization of cholesterol efflux by macrophages
is not accompanied by measurable effects on lesion growth.
Key Words: apolipoprotein E • atherosclerosis • hyperlipidemia • macrophages • bone marrow transplantation
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Introduction |
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ApoE is a 34-kDa, arginine-rich protein that mediates the uptake of remnant lipoproteins by several receptor systems in the liver.1 2 3 Structural mutations in the apoE gene, including homozygosity for apoE2 (Arg158
Cys)4 5 6 7 or a
complete deficiency of apoE,8 9 10 11 may lead to the
development of type III hyperlipidemia. Type III
hyperlipidemia is associated with the accumulation of
VLDL and chylomicron remnants in the circulation due to impaired
clearance of these lipoproteins, leading to the premature development
of atherosclerosis.1 In 90% of cases, recessive inheritance of type III hyperlipidemia is associated with the apoE2 phenotype.12 Besides this recessive inheritance of type III hyperlipidemia, several other mutations of apoE are known that lead to dominant inheritance of this disease, including apoE3-Leiden.4 13 14 15 16 Compared with the recessive apoE2 variant, apoE3-Leiden demonstrates only mildly reduced in vitro binding efficiency to the LDL receptor apoE3-Leiden binding is 20% to 40% of normal apoE3 versus 1% to 2% for apoE2. Although other secondary genetic or environmental factors are necessary to induce type III hyperlipidemia in subjects homozygous for apoE2, almost all subjects with dominant apoE3-Leiden develop hyperlipidemia.12 13
Recently, Van den Maagdenberg et al14 and Van Vlijmen et al15 reported that transgenic mice expressing the apoE3-Leiden mutation develop hyperlipidemia in the presence of endogenous mouse apoE. In addition, atherosclerotic lesion development was observed to be dependent on the level of serum cholesterol.16 Huang et al17 demonstrated that in the presence of endogenous mouse apoE, low concentrations of apoE2 (<10 mg/dL) did not alter serum lipid levels, whereas intermediate concentrations of apoE2 (10 to 30 mg/dL) induced hypocholesterolemia, and high concentrations of apoE2 (>50 mg/dL) resulted in hyperlipidemia.17 Van Vlijmen et al18 additionally demonstrated that apoE2-transgenic mice develop hyperlipidemia only in the absence of the endogenous mouse apoE gene. As with humans, apoE2 and apoE3-Leiden in transgenic mice behave like a recessive and a dominant trait, respectively, in the expression of hyperlipidemia. Consequently, these transgenic mice can be considered suitable animal models for the further understanding of the development of type III hyperlipidemia. In addition, various groups have generated apoE-deficient mice by targeted inactivation of the apoE gene in embryonic stem cells.19 20 21 Inactivation of the apoE gene in these mice is also associated with a prominent increase in serum cholesterol levels and the development of atherosclerosis, further indicating the importance of apoE in the development of hyperlipidemia and atherosclerosis.
Although the liver is the major source of apoE synthesis, macrophages in different organs are also active in secreting large quantities of apoE.22 23 24 25 Because foam cells in atherosclerotic lesions are derived from macrophages, modulation of macrophage apoE synthesis might influence the development of atherosclerosis. Recently, we26 and others,27 28 29 using the technique of bone marrow transplantation (BMT), have demonstrated that macrophage-derived, wild-type mouse apoE nearly normalizes serum cholesterol levels in apoE-deficient mice, leading to a marked decrease in atherosclerosis.
In the present study, the effects of apoE3-Leiden and apoE2 expression in macrophages or by the liver on serum cholesterol levels and atherosclerosis development were studied against an endogenous mouse apoE-knockout background. Our results indicate that macrophage-derived human apoE3-Leiden and human apoE2 can only transiently reduce serum cholesterol levels in apoE-deficient mice without significantly reducing the susceptibility to atherosclerosis, irrespective of an adequate efflux of cholesterol from macrophages. Only high serum levels of apoE3-Leiden, achieved by production in the liver, reduced serum cholesterol levels and atherosclerosis development dramatically, whereas high levels of apoE2 were ineffective. In conclusion, apoE3-Leiden as well as apoE2 is less effective in reducing cholesterol levels and atherosclerosis in apoe–/– mice compared with wild-type apoe, irrespective of an adequate efflux of cholesterol. This finding indicates that normalization of cholesterol efflux from macrophages is not accompanied by measurable effects on lesion growth.
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Methods |
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Animals
ApoE-deficient (apoe–/–) mice and transgenic mice without endogenous mouse apoE but expressing either human apoE3-Leiden (apoE3-Leiden.apoe–/–) or human apoE2 (apoE2.apoe–/–) were generated as described previously.18 21 30 All mice were hybrids of the C57BL/6 and 129Sv strains. The animals were housed and bred at the animal facility of the Sylvius Laboratories in Leiden, The Netherlands. Mice used for BMT experiments were housed in sterilized, filter-top cages and fed a sterilized, regular chow diet (SRM-A) containing 5.7% fat (Hope Farms). Drinking water was supplied with antibiotics (83 mg/L ciprofloxacin and 67 mg/L polymyxin B sulfate) and 6.5 g/L sugar. Animal procedures were performed at the Sylvius Laboratories of the Leiden/Amsterdam Center for Drug Research in accordance with national laws. All experimental protocols were approved by the Ethics Committee for Animal Experiments of Leiden University.
Irradiation and BMT
To induce bone marrow aplasia, female (aged 5 to 6 weeks)
apoe–/– (nontransgenic littermates),
apoE3-Leiden.apoe–/–, and apoE2.apoe–/– mice
were exposed to a single dose of 13 Gy (0.28 Gy/min, 200 kV, 4 mA)
total-body irradiation by using an Andrex Smart 225 Röntgen
source (Andrex Radiation Products AS) with a 4-mm aluminum filter 1
day before transplantation. Bone marrow cell suspensions were isolated
by flushing the femurs and tibias from male apoe–/–,
apoE3-Leiden.apoe–/–, apoE2.apoe–/–, and
C57BL/6 mice with PBS. Single-cell suspensions were prepared by
passing the cells through a 30-µm nylon gauze. Irradiated recipients
received 107 bone marrow cells by
intravenous injection into the tail vein.
Serum Cholesterol and Triglyceride Analysis
After an overnight fast, 
100 µL of blood was drawn from
each individual mouse by tail bleeding. The concentrations of total
cholesterol, free cholesterol, and
triglycerides in serum were determined by using enzymatic
procedures (Boehringer Mannheim). Precipath (standardized
serum, Boehringer Mannheim) was used as an internal
standard.
The distribution of cholesterol and triglycerides over the different lipoprotein classes in serum was determined by loading 30 µL of serum from each mouse onto a Superose 6 column (3.2x30 mm, Smart system, Pharmacia). Serum was fractionated at a constant flow rate of 50 µL/min with PBS. Total cholesterol content in the effluent was determined enzymatically.
Quantitation of ApoE
Both human and murine apoEs were measured by using a
sandwich ELISA. For determination of human apoE, purified goat
anti-human apoE polyclonal antibody was used as a primary antibody,
whereas purified goat anti-human apoE polyclonal antibody, conjugated
to horseradish peroxidase, was used as a secondary antibody. For
determination of mouse apoE, a rabbit anti-mouse apoE polyclonal
antibody (SB rabbit 67-AH) was used as a primary antibody, biotinylated
rabbit anti-mouse apoE polyclonal antibody was used as a secondary
antibody (SB rabbit 67-AH–biotin), and finally biotinylated
horseradish peroxidase–conjugated streptavidin was
used.26 Horseradish peroxidase was detected by incubation
with 3,3',5,5'-tetramethylbenzidine (Pierce Chemical Co) for 30 minutes
at room temperature. The reaction was stopped with addition of 2 mol/L
H2SO4, and the absorbance
was read at 450 nm. Pooled serum from healthy, human volunteers and
C57BL mice with known apoE levels was used as the standard for the
human apoE ELISA and the murine apoE ELISA, respectively.
LDL Isolation, Acetylation, and Labeling
Human LDL was isolated from healthy volunteers as
described by Redgrave et al.31 After density
ultracentrifugation, LDL (1.019<d<1.063
g/mL) and lipoprotein-deficient serum (d>1.21 g/mL) were
collected and dialyzed against PBS/1 mmol/L EDTA. Protein content
was determined according to Lowry et al32 with BSA as
an internal standard. LDL was acetylated according to Basu et
al33 and subsequently labeled with 25 µCi of
[3H-1
,2(n)]cholesteryl oleate according
to Blomhoff et al34 in the presence of human
lipoprotein-deficient serum as the source of cholesteryl ester transfer
protein. Radiolabeled acetylated LDL was subsequently isolated
by density ultracentrifugation. Hydrolysis of the
cholesteryl ester label was tested by lipid extraction followed by
thin-layer chromatography, and the electrophoretic
mobility was examined by agarose gel electrophoresis. The specific
activity of the [3H]cholesteryl
oleate–radiolabeled acetylated LDL, after density
ultracentrifugation, was 37±7 disintegrations per
minute per nanogram protein (n=5, mean±SEM).
Histological Analysis of Hearts and Aortas
for Atherosclerosis
To analyze the development of
atherosclerosis throughout the aortic tree,
transplanted mice were killed 4 months after BMT. Hearts and aortas
were perfused in situ with oxygenated Krebs’ buffer
(37°C, 100 mm Hg) for 20 to 30 minutes via a cannula in the
left ventricle, followed by perfusion with 3.7% neutral buffered
formalin (Formal-fixx, Shandon Scientific Ltd) for 30 minutes. Hearts
and aortas were excised and stored in formalin.
To evaluate the development of atherosclerotic lesions, the aortas were separated from the hearts. Hearts were bisected at the level of the atria, and the base of the heart plus aortic root were taken for analysis. Cryostat 10-µm cross sections of the aortic root were made and stained with oil red O (BDH Ltd). The atherosclerotic lesion area in the sections was quantified by using a light microscope connected to a 24-bit, full-color video camera and Optimas 6.1 image analysis software (BioScan). Mean lesion area was calculated (in µm2) from 10 sections, starting at the appearance of the tricuspid valves as described previously.16 35
Isolation of Murine Peritoneal Macrophages
Five days after intraperitoneal injection of
3% Brewer’s thioglycollate medium, macrophages were harvested
from control apoe+/+, apoe–/–, and transgenic
apoE3-Leiden.apoe-/- and apoE2.apoe–/–
animals by lavage of the peritoneal cavity with 10 mL of PBS. The
isolated macrophages were washed 3 times with sterilized PBS
and plated in 24-well plates at a density of
0.5x106 cells/500 µL in Dulbecco’s modified
Eagle’s medium supplemented with 2% (wt/vol) BSA, 2 mmol/L
L-glutamine, 100 µg/mL streptomycin, and 100
IU/mL penicillin. After 4 hours, nonadhering cells were removed by
washing. At 1 day after isolation, the cells were loaded with 50
µg/mL acetylated LDL and labeled with 25 µCi of
[3H-1,2(n)]cholesteryl oleate according
to Blomhoff et al34 for 45 hours at 37°C.
Subsequently the supernatant was removed, and the cells were washed 2
times with washing buffer (0.9% wt/vol NaCl, 1 mmol/L EDTA, 0.05
mol/L Tris-HCl, 5 mmol/L CaCl2, and 0.2%
wt/vol BSA, pH 7.4) and 2 times with washing buffer without BSA to
determine the 100% value. Cells were lysed with 0.1N NaOH,
radioactivity was measured with a scintillation counter, and protein
content was determined according to Lowry et al.32 No
significant difference in cholesterol loading was observed
between the different groups. The specific activity of the cells after
being loaded with [3H-1,2(n)]cholesteryl
oleate–labeled acetylated LDL was 9.0±0.6, 9.4±1.1,
11.4±0.9, and 9.0±1.3 µg/mg cell protein (n=5 or 6, mean±SEM) for
apoe-/-, apoe+/+, apoE3-Leiden, and apoE2
macrophages, respectively. Cholesterol efflux was
studied in the absence of cholesterol acceptors in
Dulbecco’s modified Eagle’s medium supplemented with 2% wt/vol BSA
for 5 hours after extensive washing of the cells with Dulbecco’s
modified Eagle’s medium and 2% wt/vol BSA. After incubation,
cholesterol efflux was determined as described by Zhu et
al.36 In brief, the supernatant was removed and cell
debris was pelleted by centrifugation at
16 000g for 10 minutes. After removal of the supernatant, a
200-µL aliquot was counted for radioactivity with a scintillation
counter to determine the cholesterol efflux. The cells were
washed 2 times with washing buffer with BSA and 2 times with washing
buffer without BSA and lysed with 0.1N NaOH; radioactivity was measured
with a scintillation counter; and protein content was determined
according to Lowry et al.32 The percent
cholesterol efflux was calculated as
(dpm/ng)medium/[(dpm/ng)cell+(dpm/ng)medium].
Statistical Analysis
Statistically significant differences among the means of the
different populations were tested by ANOVA. The Student-Newman-Keuls
multiple comparison test was performed after ANOVA.
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Results |
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Peritoneal Macrophage ApoE Production in ApoE2.apoe-/- and ApoE3-Leiden.apoe-/- Transgenic Mice
Analysis of apoE secretion by thioglycollate-elicited macrophages demonstrated that apoE secretion was higher in wild-type macrophages (1059±204 ng of mouse apoE secreted per milligram of cell protein per 24 hours, n=5) compared with macrophages expressing human apoE (208±75, n=4 and 246±64, n=4 ng · mg-1 · 24 h-1 for apoE3-Leiden and apoE2, respectively).
Effect of Macrophage or Liver
ApoE2.apoe-/- and
ApoE3-Leiden.apoe-/- Production on Serum
Lipids and ApoE Levels
To study the effect of murine apoe, human apoE2, and human
apoE3-Leiden production by macrophages in an
apoE-deficient background, bone marrow from wild-type
(apoe+/+), apoE3-Leiden.apoe–/–, and
apoE2.apoe–/– animals was transplanted into apoE-deficient
recipients. Control groups of apoE3-Leiden.apoe–/–,
apoE2.apoe–/–, and apoe–/– mice receiving
homologous bone marrow were also included.
At 4 weeks after BMT, the concentration of apoE as measured in
the circulation of apoe–/– mice transplanted with
apoe+/+, apoE2.apoe–/–, or
apoE3-Leiden.apoe–/– bone marrow was 131±28, 25±2.9, and
19±1.6 µg/dL (n=6 or 7), respectively. High concentrations of
circulating apoE3-Leiden and apoE2 were found in the
apoE3-Leiden.apoe–/– and apoE2.apoe–/– mice
expressing the mutant apoE forms in multiple tissues
(macrophages, liver, brain, and muscles18 )
but mainly in the liver. The circulating human apoE levels in these
animals were 8392±386 and 657±38 µg/dL in the
apoE2.apoe–/–apoE2.apoe–/– and the
apoE3-Leiden.apoe–/–apoE3- Leiden.apoe–/–
mice, respectively. At 10 weeks after BMT, the human serum apoE
concentration was further increased to 39±4.9 and 83±4.1 µg/dL in
the apoe–/– mice transplanted with
apoE2.apoe–/– and apoE3-Leiden.apoe–/– bone
marrow, whereas apoE levels in the control transplanted
apoE2.apoe–/– and the apoE3-Leiden.apoe–/–
animals were 4408±277 and 436±86 µg/dL, respectively.
In the weeks after BMT, serum cholesterol levels were
analyzed (Figure 1
). As
previously reported,26 transplantation of wild-type bone
marrow to apoe–/– animals results in an almost-complete
correction of their hypercholesterolemia within
4 weeks after BMT (87% reduction, Figure 1A). Transplantation
of apoE2.apoe–/– or apoE3-Leiden.apoe–/– bone
marrow to apoe–/– animals resulted in an initial transient
decrease in total serum cholesterol levels (Figures 1B and 1C). Analysis of the effect of transplantation of
apoE2.apoe–/– or apoE3-Leiden.apoe–/– bone
marrow to mice lacking both apoE and the LDL receptor demonstrated that
this initial decrease was mediated by the LDL receptor (data not
shown). At 4 weeks after BMT, the distribution of
cholesterol over the different serum lipoproteins in
transplanted apoe–/– mice was determined by using liquid
chromatography. As indicated in Figure 2, the decrease in serum
cholesterol in the animals transplanted with
apoe+/+, apoE2.apoe–/–, or
apoE3-Leiden.apoe–/– bone marrow was mainly confined to
the VLDL-size lipoprotein fractions and, to a lesser extent, to the
LDL-size fractions.
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), apoe+/+ (•),
apoE3-Leiden.apoe–/– (
), and
apoE2.apoe–/– (
) bone marrow. Transplantation of
apoE3-Leiden.apoe–/– 
) and
apoE2.apoe–/– 
)
were used as high-expressing controls. Values are mean±SD of 6 or 7
animals. *P<0.05, **P<0.01,
***P<0.001 vs baseline levels.
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As demonstrated in Figure 1
, after an initial decrease the
serum cholesterol levels in the apoe–/–
animals transplanted with apoE2.apoe-/- or
apoE3-Leiden.apoe-/- bone marrow did not persist and
increased to a level slightly lower than that in control transplanted
apoe–/– animals. This result contrasts with that in
animals transplanted with wild-type bone marrow, indicating that the
secondary increase in serum cholesterol levels was not due
to a loss of apoE-producing bone marrow cells, which was also confirmed
by the increase in serum apoE2 and apoE3-Leiden levels at 10 weeks
after BMT. Fractionation of serum lipoproteins at 12 weeks after BMT
indicated that the secondary increase in serum cholesterol
levels was caused by an increase in cholesterol associated
with the VLDL fraction (data not shown). No significant effect of BMT
on serum triglyceride levels could be demonstrated.
As presented in the Table, in
animals expressing human apoE solely in macrophages, all
lipoprotein subclasses contained significantly less apoE compared with
those from the control apoE2.apoe–/– and
apoE3-Leiden.apoe–/– animals. Furthermore, a preferential
association of apoE2, compared with apoE3-Leiden, to HDL could be
demonstrated in the apoE2.apoe–/–apoe–/–
animals compared with
apoE3-Leiden.apoe–/–apoe–/– mice, which
is in accordance with data from Tsukamoto et
al.37
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Effect of Macrophage or Liver ApoE2 and ApoE3-Leiden
Production on Atherosclerosis
To investigate whether the ability of macrophages in the
arterial wall to produce human apoE2 or apoE3-Leiden would
influence lipid deposition and the formation of foam cells,
atherosclerosis was quantified in all transplanted
groups at 4 months after BMT. The measured mean lesion area for each
mouse is depicted in Figure 3, and representative photomicrographs of lesions in cross
sections are shown in Figure 4. Cross
sections of control apoe–/– animals transplanted with
apoe–/– bone marrow showed extensive lipid-rich lesions,
even 1 with an aneurysm in the vascular wall (Figure 4A). The mean lesion area measured in these animals was
5.85±0.91x105 µm2
(Figure 3A). Although atherosclerotic lesion development in the
apoe–/– animals transplanted with
apoE2.apoe–/– and apoE3-Leiden.apoe–/– bone
marrow appeared slightly less dramatic, no significant decrease in
lesion area could be demonstrated
(4.21±1.27x105 and
4.18±0.16x105µm2,
respectively). Also, no significant decrease in lesion area of control
transplanted apoE2.apoe–/– mice
(3.50±0.53x105
µm2) was found. In contrast,
apoe–/– animals transplanted with wild-type
apoe+/+ bone marrow
(0.25±0.16x105
µm2) and the control transplanted
apoE3-Leiden.apoe–/– animals
(0.27±0.17x105
µm2) did show a dramatic decrease in
atherosclerosis. The inability of
macrophage-derived apoE2 and apoE3-Leiden to reduce
atherosclerosis was confirmed by data from mice lacking
both apoE and the LDL receptor (apoe–/–.LDLr-/-; Figure 3B).
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Effect of Murine Wild-Type apoe, Human ApoE3-Leiden, and Human
ApoE2 on Cholesterol Efflux by Macrophages
Previous studies indicate that macrophage-derived apoE
may play a protective role in the development of foam cells in the
early atherosclerotic lesion by increasing cholesterol
efflux.22 25 Therefore, we compared the ability of
wild-type murine macrophages, apoE-deficient
macrophages, and macrophages expressing human
apoE3-Leiden or apoE2 to induce cholesterol efflux in the
absence of specific cholesterol acceptors.
Macrophages were loaded with 50 µg/mL
[3H-1,2(n)]cholesteryl oleate–labeled
acetylated LDL for 45 hours, and cholesterol efflux
was subsequently studied during a 5-hour period. As indicated in Figure 5, wild-type macrophages and
macrophages expressing either apoE3-Leiden or apoE2 were
equally effective in inducing cholesterol efflux, whereas
efflux in apoE-deficient macrophages was reduced 49%
compared with wild-type macrophages.
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Discussion |
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Inactivation of the gene for mouse apoE is associated with a prominent increase in serum cholesterol levels and the premature development of atherosclerosis.19 20 21 ApoE displays antiatherogenic properties, due not only to its role as a receptor ligand that mediates removal of cholesterol-rich lipoproteins by the liver but also to its contribution to reverse cholesterol transport. Although the liver produces the majority of apoE in the circulation, macrophages also secrete this important protein.25 Recently, we and others demonstrated that introduction of apoE-producing monocytes/macrophages into apoE-deficient mice by BMT reduces circulating cholesterol levels and atherosclerosis, even though the circulating apoE levels were only 3.5% of normal.26 27 28 29 Several isoforms of apoE, including apoE2 and apoE3-Leiden, differ in their affinity for several binding sites on the liver, like the LDL receptor, proteoglycans, and/or the remnant receptor.1 2 3 The purpose of this study was to compare the effects of macrophage-derived wild-type apoE, human apoE2, and human apoE3-Leiden on circulating cholesterol levels and atherosclerosis in the absence of endogenous mouse apoE by using the technique of BMT.
It appears that macrophage-derived human apoE3-Leiden and
human apoE2, in contrast to wild-type murine apoe, only transiently
reduced serum cholesterol levels. The reduction in serum
cholesterol levels on transplantation of wild-type murine
bone marrow into apoE-deficient recipients is caused by a reduction in
VLDL and LDL levels. Previously, we demonstrated that this reduction in
VLDL levels is induced by increased recognition by the liver owing to
the association of macrophage-derived apoe with these
lipoproteins.26 In contrast to wild-type apoe, the
reduction in circulating cholesterol levels on
transplantation of apoE3-Leiden or apoE2 bone marrow into
apoE-deficient animals, though mainly confined to VLDL, was only
transient. At 4 to 6 weeks after transplantation, a secondary increase
in serum cholesterol levels was observed, as reflected by
an increase in remnant lipoproteins compared with a sustained decrease
when wild-type murine apoe was used. This secondary increase in serum
cholesterol levels in
apoE3-Leiden.apoe–/–apoe–/– and
apoE2.apoe–/–apoe–/– animals was not
associated with a decrease in circulating apoE levels, because its
concentration even increased slightly over time. Furthermore, it is
unlikely that the increase in serum cholesterol was caused
by generation of antibodies against apoE, because the cells in the
donor bone marrow form the new immune system in the reconstituted
animal.38 Another mechanism underlying this
observation might be downregulation of the LDL receptor, a classic
sterol-responsive-gene.39 40 Srivastava et
al41 demonstrated in vivo that downregulation of the LDL
receptor occurs within 2 weeks of feeding the animals a
cholesterol-rich diet. Recently, it has also been
demonstrated in vivo that the LDL receptor on macrophages can
be downregulated after transplantation of wild-type murine bone marrow
to LDL receptor–deficient animals owing to a sustained increase in
serum cholesterol levels.42 Indeed, the
transient reduction in serum cholesterol levels on
transplantation of apoE3-Leiden or apoE2 bone marrow to apoE-deficient
mice appeared to be mediated by the LDL receptor, because no such
reduction was observed after transplantation to mice lacking both apoE
and the LDL receptor. However, at present it is not clear why this
effect is observed only with macrophage-derived human
apoE3-Leiden and human apoE2 and not with wild-type mouse apoE.
A permanent reduction of serum cholesterol levels in apoE-deficient mice could only be achieved by inducing high concentrations of circulating apoE3-Leiden (657±38 µg/dL) by cross-breeding apoE3-Leiden mice against an apoe–/– background, leading to the expression of mutant apoE3-Leiden in multiple tissues, including macrophages, liver, brain, and muscles. This is in accordance with the data of Bellosta et al,43 who demonstrated that levels of macrophage-derived human apoE3 must exceed 800 µg/dL to correct the hyperlipidemia in apoE-deficient mice.
Human apoE2 appears to be ineffective in reducing serum cholesterol concentrations, even at circulating concentrations of 8392±386 µg/dL. This difference in response is most likely caused by the fact that the in vivo binding of apoE2 to the LDL receptor is severely defective, whereas the binding of apoE3-Leiden is only mildly affected.20 Furthermore, the higher level of human apoE3 macrophage expression necessary to reduce serum cholesterol levels as demonstrated by Bellosta et al43 compared with murine wild-type apoE as found in our studies indicates that murine apoE more effectively facilitates the clearance of mouse lipoproteins than does human apoE3. A further increase in macrophage human apoE3-Leiden production may induce a permanent reduction of lipid levels in apoE-deficient mice. Because human apoE2 concentrations as extreme as 8392±386 µg/dL were unable to reduce atherosclerosis, no effect of a further increase of macrophage human apoE2 production on atherosclerosis is expected to be beneficial.
Macrophages are cells that actively secrete large quantities of apoE.25 44 The type of apoE secreted and the regulation of apoE synthesis by this cell type may be important in maintaining the balance between cholesterol influx and efflux. Macrophage foam cell formation and thus, atherosclerosis, will occur when either cholesterol influx is increased or cholesterol efflux is decreased or both. Several possible roles for macrophage-derived apoE in atherogenesis have been postulated. According to 1 hypothesis, apoE secretion by macrophages is proatherosclerotic by its association with lipoproteins in the extracellular space of the vessel wall, thereby enriching them with a high-affinity ligand for receptor-mediated uptake of lipoproteins and subsequently inducing foam cell formation.45 Ishibashi et al45 demonstrated that macrophage-derived apoE facilitates the uptake of VLDL by these macrophages. Furthermore, it appears that apoE3 macrophages incorporated more VLDL particles than did macrophages expressing the receptor binding–defective form apoE2. Alternatively, macrophage-derived apoE may facilitate reverse cholesterol transport, thereby reducing the formation of foam cells. The role of macrophage-derived apoE in reverse cholesterol transport was confirmed in vitro by the observation that J774 macrophages, which do not synthesize endogenous apoE, have impaired efflux in the presence of an exogenous cholesterol acceptor.46 Subsequent stable expression of human apoE3 cDNA in these macrophages restored cholesterol efflux. According to the first model, as described by Basu et al,47 apoE and free cholesterol are secreted from macrophages by 2 independent mechanisms, leading to apoE-mediated facilitation of HDL-induced cholesterol efflux. This model was recently confirmed by data from Hayek et al,48 who demonstrated that peritoneal macrophages from apoE-deficient mice have similar rates of cholesterol efflux compared with wild-type macrophages. However, the efflux of cholesterol from wild-type macrophages was 40% lower when induced by HDL from apoE-deficient mice compared with HDL from wild-type mice. Addition of exogenous apoE to the apoE-deficient HDL restored the cholesterol efflux capacity, suggesting that apoE on HDL, rather than macrophage apoE, is responsible for cholesterol efflux. In contrast, addition of exogenous apoE3 to J774 macrophages failed to facilitate cholesterol efflux to HDL, as does endogenous expression in transfected J774 cells.49 Furthermore, in human monocyte–derived macrophages, it was demonstrated that they can release cholesterol without exogenously added cholesterol acceptors.50 From these in vitro studies, it can be concluded that macrophage-derived apoE may facilitate reverse cholesterol transport by 2 major pathways: (1) HDL-independent secretion of cholesterol in apoE discoidal lipid particles and (2) an apoE-mediated facilitation of HDL-dependent release of cholesterol.
Recent in vivo data on the effect of selective expression of apoE in the vessel wall support the antiatherosclerotic function of locally produced apoE. To test whether apoE in the arterial wall has a local effect on atherogenesis, Shimano et al51 established transgenic mice expressing human apoE under control of the mouse H2 Ld promotor. These mice, with high expression of human apoE in the vessel wall, showed no difference in plasma cholesterol levels compared with controls when fed either a control or an atherogenic diet, whereas the formation of fatty streaks was markedly inhibited. In addition, Bellosta et al43 studied the effect of macrophage-specific expression of human apoE3 in apoE-deficient mice under control of the visna virus long-terminal repeat. Macrophage-specific expression of human apoE3 markedly reduced atherosclerotic lesion development even in the presence of high levels of atherogenic lipoproteins, indicating that macrophage-derived human apoE3 prevents atherosclerosis by promoting cholesterol efflux from the arterial wall.
BMT will lead to replacement of monocytes/macrophages in the
recipient by cells of donor origin, including arterial wall
macrophages. Using this technique, we studied the ability of
macrophage-derived human apoE2 and human apoE3-Leiden to
reduce atherosclerosis in apoE-deficient mice compared
with wild-type murine apoE at 4 months after BMT. Introduction of only
macrophage-derived wild-type mouse apoE and high systemic
levels of apoE3-Leiden were able to protect apoe–/– mice
from the spontaneous development of atherosclerosis.
Both macrophage-derived apoE3-Leiden and apoE2 were unable
to reduce atherosclerosis in apoe–/– mice.
High concentrations of systemic apoE2 were ineffective in significantly
reducing the severity of atherosclerosis, although
there might have been a tendency to less dramatic lesions. These
results are in accordance with recent ones of Tsukamoto et
al,37 who investigated the effect of human apoE3,
apoE2, and apoE4 on atherosclerosis by expressing these
different isoforms in the livers of apoe–/– mice.
Quantification of atherosclerosis indicated that apoE3,
but not apoE2 and apoE4, was able to induce regression of
atherosclerosis in apoe–/– animals.
However, Sullivan et al52 demonstrated, by using
targeted replacement of the mouse apoE gene by either human apoE3 or
human apoE2, that apoE2 could induce a 2-fold reduction in lesion area
compared with apoe–/– mice. Using this same technique of
targeted replacement of the mouse apoE gene, they also demonstrated
that mice expressing human apoE3 are more susceptible to diet-induced
atherosclerosis compared with wild-type
mice.53 In addition, comparison of our
transplantation data with the published data of Bellosta et
al43 indicated that higher levels of
macrophage-derived human apoE3 were required to reduce
serum cholesterol levels compared with wild-type murine
apoe. Nevertheless, macrophage-derived human apoE3
inhibited atherogenesis in these apoE-deficient animals despite high
serum cholesterol levels. In this study, we demonstrated
that macrophage-derived human apoE3-Leiden and human apoE2
were unable to reduce atherosclerosis at comparably
high levels of cholesterol. Because the accumulation of
cholesterol in macrophages and the subsequent
formation of foam cells are the net effects of cholesterol
influx and efflux, it may be expected that cholesterol
efflux from macrophages expressing these mutant forms of apoE
is impaired. To study cholesterol efflux induced by these
mutant human apoE forms compared with apoe+/+ and
apoe–/– macrophages, thioglycolate-elicited
macrophages were isolated and loaded with
[3H-1,2(n)]cholesteryl oleate–labeled
acetylated LDL. Cholesterol efflux was subsequently
studied in the absence of specific cholesterol acceptors to
deduce the direct effect of apoE secretion on cholesterol
efflux,50 instead of apoE-facilitated HDL-dependent
release. Compared with apoe+/+ macrophages, efflux
from apoe–/– macrophages was reduced by 50%.
This reduction of cholesterol efflux from
apoe–/– macrophages is in accordance with results
obtained by Mazzone et al,46 who demonstrated that
transfection of J774 macrophages with the gene for human apoE
enhances cholesterol efflux from these cells. Recently, we
(Van Eck et al, unpublished results, 1999) and Fazio et
al54 also found that transplantation of
apoe-/- bone marrow to wild-type animals increased the
sensitivity to atherosclerosis, indicating that
wild-type mouse apoe promotes cholesterol efflux in vitro
as well as in vivo.
Both human apoE2- and human apoE3-Leiden–expressing macrophages were equally capable of inducing cholesterol efflux compared with wild-type murine apoe. Because both apoE3-Leiden and apoE2 macrophages secrete low amounts of apoE compared with wild-type murine apoe and their efflux capacity is equal, it may be concluded that both apoE3-Leiden and apoE2 are very efficient in inducing cholesterol efflux. This view is in accordance with recent data from Cullen et al,55 who demonstrated that apoE2 macrophages secrete low amounts of apoE2, which is very efficient in inducing cholesterol efflux, thereby protecting these cells from cholesterol storage. In addition, apoE3 macrophages secrete very large amounts of apoE, thus protecting these cells from cholesterol accumulation despite their lower cholesterol efflux efficiency compared with that of apoE2 macrophages. In contrast, apoE4 macrophages were demonstrated to secrete the highest amounts of apoE, but they lack effective efflux capacity. Although human apoE3-Leiden and human apoE2 production by macrophages was very efficient in inducing cholesterol efflux in vitro, no significant effect was found on in vivo atherosclerotic lesion development. Thus, either normalization of cholesterol efflux in macrophages is not accompanied by measurable effects on arterial lesion growth under these conditions or the in vitro conditions do not completely reflect the in vivo situation with its complex interaction between various cell types and multiple factors.
In the present study, we demonstrated that the efficiency in
reducing atherosclerosis in apoe–/– mice
decreases in the order murine apoe > apoE3-Leiden > apoE2.
A similar order was demonstrated for the relation between the type of
apoE and serum cholesterol levels, indicating that
accumulation of atherogenic, cholesterol-rich lipoproteins
in the circulation is an important determinant for the development of
atherosclerosis in these mice. Because we did not
observe a difference in cholesterol efflux between
wild-type mouse apoe, human apoE2, and human apoE3-Leiden in isolated
peritoneal macrophages, we suggest that normalization of
cholesterol efflux in macrophages is not
accompanied by measurable effects on arterial lesion growth
under these conditions and that the higher level of
atherosclerosis in
apoE2.apoe–/–apoe–/– and
apoE3-Leiden.apoe–/–apoe–/– mice, compared
with apoe+/+apoe–/– animals, is caused by a
difference in cholesterol influx due to differences in
serum cholesterol levels. Under circumstances of high
circulating cholesterol levels, adequate efflux of
cholesterol from macrophages does not prevent foam
cell formation and the development of atherosclerosis,
and it appears that the efficiency in decreasing serum
cholesterol levels is a more important
parameter for apoe, apoE3-Leiden, and apoE2.
|
Acknowledgments |
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The PhD studentship of M.V.E. was supported by SmithKline Beecham Pharmaceuticals, United Kingdom. M.H. Hofker is an Established Investigator of the Netherlands Heart Foundation. The authors wish to thank M. Vidgeon-Hart for expert technical help with sectioning and Dr G.M. Benson for helpful assistance with image analysis of atherosclerotic lesions.
Received December 10, 1998; accepted April 28, 1999.
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References |
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