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© 1997 American Heart Association, Inc.
Articles |
Bone Marrow Transplantation in Apolipoprotein E–Deficient Mice
Effect of ApoE Gene Dosage on Serum Lipid Concentrations, (ß)VLDL Catabolism, and Atherosclerosis
From the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Leiden University, Leiden, The Netherlands (M. Van E., N.H., Th.J.C. Van B.); SmithKline Beecham Pharmaceuticals, Harlow, Essex, UK (J.Y., N.J.P., P.H.E.G.); and University Hospital Leiden, Department of Pediatrics, Leiden, The Netherlands (P.M.H.).
Correspondence to M. Van Eck (MSc), Division of Biopharmaceutics, Sylvius Laboratories, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, PO Box 9503, 2300 RA Leiden, The Netherlands. E-mail M.Eck{at}LACDR.LeidenUniv.nl
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Abstract Apolipoprotein E (apoE), a high-affinity ligand for lipoprotein receptors, is synthesized by the liver and extrahepatic tissues, including cells of the monocyte/macrophage lineage. Inactivation of the apoE gene in mice leads to a prominent increase in serum cholesterol and triglyceride levels and the development of premature atherosclerosis. In this study, the role of monocyte/macrophage-derived apoE in lipoprotein remnant metabolism and atherogenesis was assessed. The influence of apoE gene dosage on serum lipid concentrations was determined by transplantation of homozygous apoE-deficient (apoE-/-), heterozygous apoE-deficient (apoE+/-), and wild-type (apoE+/+) bone marrow in homozygous apoE-deficient mice. The concentration of apoE detected in serum was found to be gene dosage dependent, being 3.52±0.30%, 1.87±0.17%, and 0% of normal in transplanted mice receiving either apoE+/+, apoE+/-, or apoE-/- bone marrow, respectively. These low concentrations of apoE nevertheless dramatically reduced serum cholesterol levels owing to a reduction of VLDL and, to a lesser extent, LDL, while HDL levels were slightly raised. After 4 months on a "Western-type" diet, atherosclerosis was evidently reduced in mice transplanted with apoE+/+ bone marrow, compared with control transplanted mice. To study the mechanism of the lipoprotein changes on bone marrow transplantation, the in vivo turnover of autologous serum (ß)VLDL was studied. The serum half-life of (ß)VLDL in transplanted mice, compared with control apoE-deficient mice, was shortened mainly as a consequence of an increased recognition and uptake by the liver. Analysis of the relative contribution of the liver parenchymal cells, endothelial cells, and Kupffer cells (liver tissue macrophages) indicated an increased uptake by parenchymal cells, while the relative contribution of Kupffer cells was decreased. In conclusion, macrophage-derived apoE can dose-dependently reduce hypercholesterolemia in apoE-deficient mice owing to increased recognition and uptake of (ß)VLDL by parenchymal liver cells, leading to a decreased susceptibility to atherosclerosis.
Key Words: apoE deficiency • atherosclerosis • gene transfer • macrophages • VLDL
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Introduction |
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Abnormalities in plasma lipoprotein metabolism, including defects in the gene encoding for apoE, form a well-defined risk factor for atherosclerosis. Apo E, a 34-kDa arginine-rich protein, plays an important role in lipoprotein metabolism. It serves as a high-affinity ligand for several receptor systems in the liver, including the low-density lipoprotein (LDL) receptor, LDL receptor–related protein (LRP), remnant receptor, and/or proteoglycans.1–3 Structural mutations in the apoE gene,4–6 associated with a loss in binding affinity for lipoprotein receptors or even complete deficiency in apoE,7–10 can result in the development of type III hyperlipoproteinemia (HLP). Recently, apoE-deficient mice have been generated by targeted inactivation of the apoE gene in embryonic stem cells.11–13 Inactivation of the apoE gene in these mice is associated with a prominent increase in serum cholesterol levels and the development of premature atherosclerosis.
Although the liver is the major source of apoE synthesis, 20% to 40% of the mRNA for apoE is found extrahepatically.14–16 ApoE mRNA has been demonstrated in brain, spleen, lungs, adrenals, ovaries, kidneys, and muscles. In addition, macrophages are active in secreting large quantities of apoE (first described by Basu et al17). Since apoE synthesis and secretion by macrophages are influenced by the cellular cholesterol content, regulation of apoE secretion may be important in maintaining the balance between cholesterol influx and efflux in this cell type.18–20 Foam cells in atherosclerotic lesions are mainly derived from macrophages, and regulation of apoE secretion by this cell type thus might have direct consequences for the atherosclerotic process. In human and rabbit atherosclerotic lesions, it was demonstrated that most of the apoE is synthesized by arterial wall macrophages.21 Several possible roles of apoE secretion in the development of atherosclerotic lesions have been postulated. A proatherosclerotic response can be expected when secreted apoE stimulates the receptor-mediated uptake of lipoproteins by macrophages via the LDL receptor or LRP.3,22 Alternatively, macrophage-derived apoE may facilitate reverse cholesterol transport.23,24 The role of apoE in reverse cholesterol transport was confirmed in vitro by the observation that transfection of J774 macrophages, which do not express endogenous apoE, with the gene for human apoE enhances cholesterol efflux to HDL3, whereas addition of exogenous apoE did not enhance cholesterol efflux.25–26 Furthermore, Shimano et al27 and Bellosta et al28 demonstrated in vivo that expression of human apoE in the arterial wall of transgenic mice results in a marked inhibition in atherosclerotic lesion development without influencing systemic cholesterol levels. These data suggest that a locally increased concentration of apoE in the arterial wall enhances cholesterol efflux from the vessel wall into plasma.
The role of macrophage apoE synthesis in the clearance of cholesterol from the circulation can also be studied, using bone marrow transplantation (BMT), as performed by Linton et al29,30 and Boisvert et al31 BMT can be used to introduce wild-type hematopoietic stem cells into an apoE-deficient recipient. This highly specialized technique provides a unique model to specifically study the role of monocyte/macrophage-derived apoE in lipoprotein metabolism and atherogenesis, since apoE synthesis will then be limited to hematopoietic cells of the monocyte/macrophage lineage.32 Linton et al29,30 and Boisvert et al31 found that transplantation of wild-type bone marrow into apoE-deficient mice resulted in a large decrease in serum cholesterol levels and reduced susceptibility to diet-induced atherosclerosis.
The aim of the present study was to determine the effect of apoE gene dosage in cells of the monocyte/macrophage cell lineage on hypercholesterolemia and to delineate the mechanism of the highly efficient decrease of serum cholesterol levels after BMT. To attain this, bone marrow of apoE+/+, apoE+/-, and apoE-/- donors was transplanted into irradiated apoE-deficient mice. Our results indicate that macrophage-derived apoE can dose-dependently reduce cholesterol and triglyceride levels by increasing the uptake of (ß)VLDL by parenchymal cells of the liver, resulting in a decreased susceptibility to atherosclerosis.
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Methods |
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Chemicals
Collagenase (type I), precipath, cholesterol esterase, cholesterol oxidase, cholesterol peroxidase, triglyceride analysis kits, and phospholipid analysis kits were obtained from Boehringer Mannheim, Germany. Human lactoferrin was supplied by Serva, Heidelberg, Germany. Na125I in NaOH and Streptavidin biotinylated horseradish peroxidase was from Amersham International Plc., England. Formal-fixx was from Shandon Scientific Ltd., England. OCT compound was purchased from Miles, Inc., Elkhart, Indiana, USA, and Oil red O was from BDH Ltd., Poole, England. Tetramethylbenzidine (TMB) was supplied by Pierce, Rockford, Illinois, USA. All other chemicals were of analytical grade.
Animals
ApoE-deficient mice,13 hybrids between
C57Bl/6 (haplotype H-2b) and 129 Sv
(haplotype H-2b) strains (F3 generation of
backcrosses to C57Bl/6), and control C57Bl/6 mice were
used. Mice were housed and bred under specific pathogen-free conditions
at the animal facility at TNO-PG Gaubius Institute and Sylvius
Laboratories in Leiden, The Netherlands. Mice used for BMT experiments
were housed in sterilized filter-top cages and fed sterilized regular
chow diet (SRM-A), containing 5.7% fat, or a sucrose-based
semisynthetic "Western-type" diet, containing 0.25%
cholesterol and 15.0% fat, composed according to Nishina
et al.33 All diets were purchased from Hope
Farms, Woerden, The Netherlands. 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 the national laws. All experimental protocols were approved by the University Animal Care and Use Committee.
Irradiation and BMT
To induce bone marrow aplasia, female apoE-deficient mice (age 5
to 6 weeks) were exposed to a single dose of 13 Gy (0.28 Gy/min, 200
kV, 4 mA) x-ray total body irradiation, using an Andrex Smart 225
(Andrex Radiation Products AS, Copenhagen, Denmark) with a 4-mm
aluminum filter, one day before the transplantation. Bone marrow cell
suspensions were isolated by flushing the femurs and tibias from either
male homozygous or heterozygous apoE-deficient mice or C57Bl/6
mice with phosphate-buffered saline. Single-cell suspensions were
prepared by passing the cells through a 30-µm nylon gauze. Irradiated
recipients received 1.5x107 bone marrow cells by
intravenous injection into the tail vein.
Serum Cholesterol and Triglyceride Analysis
After an overnight fasting period, approximately 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,
Germany). Precipath (standardized serum) was used as an internal
standard.
The distribution of cholesterol and triglycerides over the different lipoproteins in serum was determined by loading 30 µL of serum of each mouse onto a Superose 6 column (3.2x 30 mm, Smart-system, Pharmacia, Uppsala, Sweden). Serum was fractionated at a constant flow rate of 80 µL/min, using a buffer containing 150 mmol/L NaCl and 1 mmol/L EDTA, pH 8.0. First, two fractions of 400 µL were collected, followed by 30 fractions with a volume of 40 µL. Total cholesterol and triglyceride contents in the effluent were determined enzymatically.
Quantitation of ApoE
Murine apoE concentrations were measured by using a sandwich
ELISA. Plates were coated with rabbit-anti-mouse apoE polyclonal
antibody (SB Rabbit 67-AH; 2.5 µg/ml) overnight at 4°C.
Unbound antibody was removed, and nonspecific binding sites were
blocked with 1% (w/v) BSA in blocking buffer (50 mmol/L
Tris, 150 mmol/L NaCl, 1 mmol/L
MgCl2, pH 7.4) at 37°C for 1 hour. After four
washing steps with washing buffer (10 mmol/L Tris, 150
mmol/L NaCl, 0.05% (w/v) Tween-20, pH 7.4), 50 ng biotinylated
secondary antibody (SB Rabbit 67-AH-biotin) diluted in assay buffer
(0.5% (w/v) BSA, 1% (v/v) normal goat serum, 0.05% (w/v) gamma
globulin, 50 mmol/L Tris, 150 mmol/L NaCl,
0.01% (v/v) Tween-40, 40 mmol/L CHAPS, pH 7.4) and
standard (wild-type mouse serum) or samples were added and incubated at
room temperature for 1 hour on an orbital shaker. Thereafter, plates
were washed and incubated with biotinylated horseradish peroxidase
conjugated streptavidin in assay buffer without normal goat serum (at
room temperature for 30 minutes). Finally, after washing, plates were
incubated with 3,3',5,5'-tetramethylbenzidine (TMB) for 30 minutes at
room temperature on an orbital shaker, the reaction was stopped with 2
mol/L H2SO4, and the
absorbance was read at 450 nm. Pooled serum of control C57Bl/6
mice (n=5) was used as standard. Control C57Bl/6 mice were
housed in filter-top cages and fed sterilized regular chow diet (SRM-A)
containing 5.7% fat. Drinking water was supplied with antibiotics (83
mg/L ciprofloxacin and 67 mg/L polymyxin B sulfate) and
6.5 g/L sugar.
Isolation and Characterization of (ß)VLDL
For the isolation of (ß)VLDL, blood was drawn from
transplanted and control apoE-deficient mice by tail bleeding after an
overnight fasting period. Serum was pooled and adjusted to 3 mL with
phosphate buffered saline. By using a discontinuous KBr gradient, the
top fraction of d<1.006 g/mL, containing (ß)VLDL,
was isolated after 18 hours of centrifugation at 40,000
rpm as described by Redgrave et al.34
The isolated (ß)VLDL was characterized with respect to the free cholesterol, cholesterol ester, phospholipid, and triglyceride contents, using enzymatic procedures as described above. Protein content was determined according to Lowry et al35 with BSA as an internal standard.
Lipoproteins were labeled with 125I at pH 10.0 according to McFarlane,36 as modified by Van Tol et al.37 Free 125I was removed by dialysis at 4°C against PBS, containing 1 mmol/L EDTA, with repeated changes of buffer.
Serum Decay, Liver Uptake, and Organ Distribution
Mice were anesthetized by
intraperitoneal injection of sodium pentobarbital
(70 to 90 mg/kg). The abdomen was opened, and 10 µg of
radiolabeled ligand was injected into the vena cava
inferior. Where indicated, human lactoferrin (70
mg/kg) was injected 1 minute before (ß)VLDL administration. At
the indicated times, blood samples (75 µL) were drawn from the vena
cava inferior, and liver lobules were tied off and excised.
Blood samples were collected in heparinized reaction tubes and
centrifuged for 10 minutes at 4000 rpm. Plasma samples of 10
µL were counted for radioactivity to calculate the total amount of
radioactivity in plasma. The total plasma volume was read from a
standard curve, describing the relation between body weight and plasma
volume, as determined by the distribution of
125I-BSA in C57Bl and apoE-deficient mice with
body weights between 15 and 30 g. At 30 minutes after injection,
the mice were killed, and the organs were excised and weighed. The
radioactivity in the different organs was corrected for serum
present at the time of sampling as determined by the distribution
of 125I-BSA.
Liver Cell Distribution
To determine the liver cell type responsible for the uptake of
the investigated ligands, 10 µg of ligand was injected, and at 30
minutes after injection, a linear liver perfusion was performed at
4°C. The liver was perfused with oxygenated Hanks'
buffer, containing 5.6 mmol/L glucose and 6.7
mmol/L HEPES, at a flow rate of 10 mL/min. After 10 minutes of
perfusion, 0.05% (w/v) collagenase type I in Hanks'
buffer was flushed through the liver for 10 minutes. Parenchymal,
endothelial, and Kupffer cells were separated by
differential centrifugation and counterflow elutriation
at 4°C, according to a modification of the method as described by Van
Berkel et al.38 Nonparenchymal cells were
purified by counterflow elutriation; endothelial cells
were obtained at a flow rate of 18 mL/min, and Kupffer cells at 50
mL/min. The contribution of the different cell types to the uptake of
the injected lipoproteins by the liver is calculated with the
assumption that parenchymal, endothelial, and Kupffer
cells form 92.5%, 3.3%, and 2.5%, respectively, of the total amount
of liver protein.39,40
Histological Analysis of Hearts and Aortas
for Atherosclerosis
To analyze the development of
atherosclerosis throughout the aortic tree,
apoE-deficient mice, transplanted with either wild-type or knock-out
bone marrow, were fed a "Western-type" diet. After 4 months, mice
were killed, and 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 for 30 minutes. Hearts
and aortas were excised and stored in formalin as described
previously.41 To evaluate the development of
atherosclerotic lesions in the aortic tree, aortas were stained with
1% (w/v) Sudan IV for 30 minutes and stored in formalin.
Statistical Analysis
Statistically significant differences among the means of
different populations were tested by using ANOVA. To compare pairs of
groups, the Student-Newman-Keuls multiple comparison test was performed
after ANOVA.
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Results |
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Effect of BMT on Serum Cholesterol and Triglyceride Levels
The effect of BMT of apoE-deficient mice with marrow from wild-type mice (apoE+/+
-/-), heterozygous
apoE-deficient mice (apoE+/- -/-), or
apoE-deficient animals (apoE-/- -/-) on
serum lipids is shown in Fig 1
. Even on a
normal chow diet, apoE-deficient mice demonstrate a marked
hyperlipidemia, and transplantation with either
apoE+/- or apoE+/+ bone
marrow efficiently decreases serum cholesterol levels in
these animals. At 4 weeks after transplantation, the control
transplantation apoE-/- -/- induced a
decrease of 13.4% and 35.8% for total cholesterol and
triglyceride levels, respectively.
ApoE+/- bone marrow induced decreases of 81.6%
and 51.9%, respectively, while apoE+/+ bone
marrow induced decreases of 90.8% and 72.3%, respectively. Serum
cholesterol and triglyceride levels remained
lowered in the period following the transplantation, indicating that
the chimeric status in the transplanted animals is stable at least up
to 10 weeks after transplantation.
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Effect of BMT on Serum Lipoproteins
The effect of BMT on serum lipoprotein profiles was
analyzed at 5 weeks after transplantation. Serum lipoproteins
were fractionated for each animal by liquid
chromatography. The lowering of serum
cholesterol levels on BMT was mainly due to a dramatic
decrease in cholesterol, associated with the VLDL fraction,
while LDL levels were lowered to a lesser extent (Fig 2). This effect was accompanied by a
small increase in HDL levels. The lowering of triglyceride
levels was caused solely by a decrease in VLDL levels (data not
shown).
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; n=4) and C57Bl/6 (
; n=2) mice. Panel B shows the
distribution in transplanted apoE-deficient mice (n=5 to 6) 5 weeks
after BMT. Open circles represent transplantation with
apoE-/- bone marrow (
), and closed triangles
apoE+/+ bone marrow (
).
At 4 weeks after BMT, the concentration of apoE measured in serum of
apoE+/+ -/- mice was 3.52±0.30% of the
concentration in wild-type mice (99±8%), whereas the concentration
was approximately twofold lower (1.87±0.17%) if
apoE+/- bone marrow was used (mean±s.e.m.;
n=5 to 6). To further illustrate the effect of gene dosage,
the apoE levels in the three transplantation groups are plotted against
the corresponding cholesterol concentration in VLDL, LDL,
and HDL for each individual mouse (Fig 3). A marked reciprocal lowering of
cholesterol, associated with VLDL and LDL, with increasing
serum apoE concentrations was apparent, while HDL demonstrated a
dose-dependent increase. A serum apoE concentration of 3.52±0.30% of
that from normal C57Bl/6 mice, induced approximately a 25-fold
decrease in VLDL cholesterol and a 5-fold decrease in LDL
cholesterol. In addition, HDL cholesterol
increased 2.5-fold.
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The effect of BMT on the composition of the VLDL fraction, the major
cholesterol transporting fraction in apoE-deficient mice,
was analyzed at 6 and 12 weeks after transplantation. As can be
seen in Fig 4, BMT induced an apoE gene
dosage–dependent decrease in cholesterol esters, while the
relative amount of free cholesterol remained constant. As
may be expected, the relative proportion of triglycerides
was found to be inversely related to the amount of
cholesterol esters, while no effect could be demonstrated
on phospholipids and the relative amount of protein. Thus, on BMT, the
composition of the VLDL core changes (replacement of
cholesterol esters by triglycerides), while the
composition of the surface film remained essentially unaltered. The
replacement of cholesterol esters by
triglycerides is accompanied by a shift in mobility on an
agarose gel from a ß to a pre-ß position (data not shown).
Therefore, the VLDL-fraction, isolated from serum from apoE-deficient
mice transplanted with wild-type bone marrow should be classified as
VLDL.
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Effect of BMT on Serum Decay and Organ Uptake of (ß)VLDL
The mechanism of the change in metabolic behavior of
the (ß)VLDL on BMT was investigated by labeling the isolated
(ß)VLDL with 125I and following the kinetics of
serum decay and liver uptake. A comparison was also made between the
uptake of (ß)VLDL by hepatic and extrahepatic tissues. Fig 5A depicts the serum decay and liver
uptake of iodinated (ß)VLDL, isolated from apoE-deficient
mice in apoE-deficient and C57Bl/6 mice. The serum half-life of
apoE-/- (ß)VLDL is greatly shortened, and the
liver uptake is increased approximately 20-fold on injection into
control C57Bl/6 mice as compared to apoE-deficient mice.
Preinjection of lactoferrin, a specific inhibitor of apoE
recognition by the remnant receptor on the
liver,42,43 revealed that the induced liver
uptake of apoE-/- (ß)VLDL in C57Bl/6
mice can be largely inhibited. These results demonstrate that apoE from
either the circulation or the Kupffer cells in the liver can rapidly
associate with apoE-/- (ß)VLDL and thus
induce apoE-mediated liver uptake.
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Subsequently, (ß)VLDL was isolated from mice transplanted with either
apoE+/- or apoE+/+ bone
marrow. On injection into mice of the identical group, a shortened
serum half-life and an increased liver uptake was observed (Fig 5B
).
These data indicate that the increase in liver uptake does depend on
the serum apoE concentration. The liver uptake of the injected ligands
is compared to the uptake by the spleen, lungs, and adrenals at 30
minutes after injection (Fig 6). Although
there may be a tendency to an increased uptake by lungs and adrenals
after BMT, no statistical significance was achieved.
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Effect of BMT on Uptake of (ß)VLDL in Different Liver
Cell Types
In Fig 5, it is demonstrated that BMT results in an
increased recognition of (ß)VLDL by the liver. It is therefore of
interest to analyze the extent to which the increased liver
uptake is mediated by the apoE-producing liver tissue
macrophages, that is, Kupffer cells. Therefore, we
analyzed the relative contribution of the various liver cells
to the uptake of iodinated
125I-apoE-/- (ß)VLDL at
30 minutes after injection (Fig 7). In
C57Bl/6, the uptake of apoE-/- (ß)VLDL
by parenchymal cells was responsible for 97.9±1.7% of the total liver
uptake; consequently, the relative contribution by nonparenchymal cells
(endothelial and Kupffer cells) was low. In
apoE-/- mice, parenchymal cells were
responsible for 66.1±4.9% of the total liver uptake, and liver
endothelial and Kupffer cells contributed approximately
9.5±4.6% and 24.1±6.9% to the liver uptake, respectively. In
transplanted mice, injection of apoE-/- (ß)VLDL led to
an increased relative contribution by parenchymal cells (92.1±4.6%)
and a significantly lower contribution by the Kupffer cells. This
indicates that BMT increases the recognition of (ß)VLDL by
parenchymal cells and thus restores the normal uptake route.
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g of
125I-apoE-/- (ß)VLDL was injected into
C57Bl/6 mice (open bars), apoE-/- mice (hatched bars), or
apoE+/+
Effect of BMT on Atherosclerosis
To assess the effect of transplantation of wild-type bone marrow
into apoE-/- mice on
atherosclerosis, apoE-deficient mice, transplanted with
apoE-/- or apoE+/+ bone
marrow, were challenged with a "Western-type" diet (0.25%
cholesterol, 15% fat). After 6 weeks on this diet, serum
cholesterol levels rose 40.0±5.9% in
apoE-/- mice (n=3), 39.2±2.0% in
apoE-/- -/- mice (n=2), and
26.6±8.7% in apoE+/+ -/- mice
(n=4). No significant effect could be demonstrated on serum
triglyceride levels. Fractionation of serum lipoproteins by
liquid chromatography revealed that the increase in
serum cholesterol in both apoE-/-
and apoE-/- -/- mice was mainly due to an
increase in the LDL fraction and, to a lesser extent, the VLDL
fraction, whereas the increase in serum cholesterol in
apoE+/+ -/- mice was mainly due to
elevations in HDL.
After 4 months on the "Western-type" diet, the hearts and
aortae were perfused, fixed, and examined
histologically. Representative
photomicrographs of lipid-rich atherosclerotic lesions throughout the
aortic tree of the transplanted mice are shown in Fig 8. In apoE-/-
-/- mice, lesions were observed in the aortic arch and
coronary arteries, as well as at branch sites along the whole
aorta, while in the apoE+/+ -/- mice, these
sites were relatively unaffected. In agreement with Linton et
al29,30 and Boisvert et
al,31 the mean lesion area in apoE+/+
-/- mice was greatly reduced in size in comparison
to those in control apoE-/- and
apoE-/- -/- mice (data not shown).
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Discussion |
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Several functions have been proposed for the role of macrophage apoE synthesis in cholesterol homeostasis and its relation to atherosclerosis, including either proatherosclerotic or antiatherosclerotic functions.18–31 The present study confirms that hypercholesterolemia in apoE-deficient mice can be markedly reduced by transplantation with wild-type bone marrow, while heterozygous apoE-deficient bone marrow appeared to be nearly equally effective. We demonstrate that this reduction in cholesterol levels is accompanied by an increased recognition and uptake of (ß)VLDL by parenchymal liver cells.
Following total body irradiation, aplasia of bone marrow develops
within 2 to 3 days. BMT will lead to recovery of hematopoietic tissues
after two distinct phases of engraftment. In the first, unsustained
phase, monocytes and macrophages are derived from committed
progenitors. In the second, sustained phase, these cells are derived
from pluripotent stem cells in the bone marrow.44
Recently, it was demonstrated by transplantation with
fluorescently labeled bone marrow cells that these cells
accumulate in liver, spleen, and bone marrow within 4 hours after
injection.45,46 However, the
peripheral blood cell count remained depressed for a longer
period of time; it was restored to normal by the end of the fourth
week. In addition, the number of mononuclear cells, the apoE-producing
cells, remained depressed the longest.47
Transplantation of apoE-deficient mice with either heterozygous
apoE-deficient (apoE+/- -/-) or wild-type
(apoE+/+ -/-) bone marrow resulted in a
dramatic drop of cholesterol by 4 weeks after
transplantation. It is thus the initial phase of engraftment by
committed progenitors and the subsequent appearance of apoE-producing
mononuclear cells in the circulation by 4 weeks after transplantation
that are responsible for this effect. The time between transplantation
and the decrease in cholesterol levels is in accordance
with the results obtained by Linton et al29,30
and Boisvert et al.31
ApoE was found to be extremely efficient in lowering serum cholesterol levels, as at a steady state concentration as low as 1.89±0.17% of the apoE concentration in normal C57Bl/6 mice, an almost complete normalization of cholesterol levels was found. This decrease in cholesterol levels in transplanted mice is mainly caused by a decrease in (ß)VLDL (25-fold) and to a lesser extent in LDL (fivefold), implying that apoE is more important for the clearance of VLDL than for that of LDL. This can be explained by the fact that the affinity of apoE for the LDL receptor is higher than the affinity of apoB100, the apolipoprotein responsible for the recognition of LDL by the LDL receptor.48 In addition, the decrease in LDL can be explained by the fact that VLDL is a precursor for LDL and a reduction of the VLDL pool will lead to reduced competition with LDL for the LDL receptor, resulting in lowering of LDL levels. The reduction of VLDL and LDL and the increase in HDL are apoE concentration dependent. However, with only a small increase in the apoE concentration, already a dramatic decrease in VLDL and LDL levels is achieved. Lipid levels remained constant in the weeks following transplantation, a result suggesting that the chimeric status in the transplanted animals is stable at least up to 10 weeks after transplantation.
Analysis of the change in composition of (ß)VLDL from apoE-deficient and transplanted mice revealed that the inner core cholesterol esters are partially replaced by triglycerides, the degree of replacement depending on the apoE gene dosage used. Surface film components, such as phospholipids and free cholesterol, were not influenced. In apoE-deficient mice, serum cholesterol levels are increased enormously, whereas triglycerides levels are increased only slightly.13 VLDL of these mice contained very little triglycerides, a result suggesting either an increased lipolysis, decreased lipogenesis, or both. In vitro, it was demonstrated that apoE induces a dose-dependent inhibition of the enzyme lipoprotein lipase.50 So the relative increase in triglycerides in (ß)VLDL in transplanted mice may be directly related to inhibition of this enzyme by an increased serum concentration of apoE.
To analyze whether apoE can induce an increase in clearance
rate and uptake by specific organs, apoE-deficient (ß)VLDL was
injected into C57Bl/6 mice and control
apoE-/- animals. The presence of apoE in
C57Bl/6 mice indeed led to an increase in serum decay and liver
uptake of the originally apoE-deficient (ß)VLDL. The apoE-mediated
increased liver uptake could be greatly inhibited by human lactoferrin,
a glycoprotein that blocks binding of apoE to the remnant
receptor and proteoglycans on parenchymal liver
cells.43,51 Human lactoferrin resembles apoE in a
positively charged arginine/lysine-rich
sequence.51 Therefore, the inhibition by
lactoferrin indicates that the increased liver uptake of
apoE-/- (ß)VLDL in C57Bl is mediated by an
enrichement with apoE, either in the circulation or in the space of
Disse of the liver. Also the increase in serum apoE concentration after
transplantation with apoE+/- or
apoE+/+ bone marrow induced an apoE-mediated
increase in liver uptake of autologous (ß)VLDL. The observed fivefold
increase in liver uptake of autologous ßVLDL in apoE+/+
-/- mice compared to apoE-/- mice
may explain the apoE gene dosage–dependent decrease in serum
cholesterol levels in apoE-/- mice
after transplantation. This is the first evidence that transplantation
of apoE+/+ bone marrow leads to an increase in
the liver uptake of circulating (ß)VLDL. Uptake of (ß)VLDL by the
liver involves several receptor systems, including the LDL receptor,
the putative remnant receptor, LRP, and/or proteoglycans. According to
the "secretion-recapture" model, uptake via the LRP requires
enrichement of (ß)VLDL with parenchymal liver cell-derived apoE in
the space of Disse of the liver.52,53 In our
model, parenchymal cells are not able to synthesize apoE. Therefore, it
is not likely that the increased liver uptake after transplantation is
mediated by the LRP. However, sequestration of Kupffer cell–derived
apoE in the space of Disse cannot be excluded.
Since BMT induces an increased uptake by the liver, it is of interest to know the extent to which the increased liver uptake is mediated by either the apoE-producing liver tissue macrophages, that is, Kupffer cells, or the apoE-deficient parenchymal liver cells. The liver cell distribution of apoE-/- ßVLDL at 30 minutes after injection in apoE-/- and C57Bl/6 mice revealed that the relative contribution of parenchymal cells to the liver uptake is larger in C57Bl/6 than in apoE-/- mice. BMT restored the major contribution of the parenchymal cells to the liver uptake, indicating that apoE, derived from macrophages, including liver Kupffer cells, does lead to increased (ß)VLDL uptake by adjoining parenchymal cells. Thus, macrophage-derived apoE does not induce increased uptake in the apoE-producing Kupffer cells.
In general agreement with earlier results from Linton et al29 and Boisvert et al,31 transplantation of wild-type bone marrow into apoE-deficient mice protected these mice from developing atherosclerosis on a "Western-type" diet. This protection against atherogenesis after transplantation is probably a combined effect of both the dramatic lowering of the serum cholesterol levels and an apoE-mediated enhanced efflux of cholesterol from the arterial wall.28
The present data indicate that macrophage-derived apoE normalizes hypercholesterolemia by an apoE gene dosage–dependent reduction of cholesterol, due to an increased recognition and uptake of cholesterol-rich lipoproteins by parenchymal liver cells. It is suggested that pharmacological approaches to increase macrophage apoE synthesis in the arterial wall may be specifically useful in the treatment of atherosclerosis.
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Acknowledgments |
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Ph.D. studentship of M.V.E. was supported by SmithKline Beecham Pharmaceuticals, United Kingdom. The authors wish to thank Dr L.M. Havekes, Dr M.H. Hofker, and Dr J. van Ree for providing us with apoE-deficient mice; M. Vidgeon-Hart for expert technical help with sectioning; and Dr G.M. Bensen for helpful assistance with image analysis of atherosclerotic lesions.
Received October 25, 1996; accepted February 7, 1997.
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