prepared by passing the cells through a 30-mm nylon gauze. Irradiated recipients received 1.0 × 10
7
bone marrow cells by intravenous injection into the tail
vein.
2
.
3
. Serum cholesterol and triglyceride analysis After an overnight fasting-period, approximately 100
ml blood was drawn from each individual mouse by tail bleeding. The concentrations of total cholesterol in
serum were determined using enzymatic procedures Boehringer Mannheim, Germany. Precipath stan-
dardized serum; Boehringer Mannheim, Germany was used as an internal standard.
The distribution of cholesterol over the different lipoproteins in serum was determined by loading of 30
ml serum of each mouse onto a Superose six column 3.2 × 30 mm, Smart-system, Pharmacia, Uppsala, Swe-
den. Serum was fractionated at a constant flow rate of 50 mlmin, using phosphate-buffered saline. Total
cholesterol content in the effluent was determined enzymatically.
2
.
4
. Quantification of apoE ApoE was measured using a sandwich ELISA spe-
cific for mouse apoE, as described earlier [25]. Briefly, for determination of apoE, a rabbit-anti-mouse apoE
polyclonal antibody SB Rabbit 67-AH, SmithKline Beecham, Harlow, UK was used as primary antibody,
biotinylated rabbit-anti-mouse apoE polyclonal anti- body was used as secondary antibody SB Rabbit 67-
AH-biotin, SmithKline Beecham, Harlow, UK, and finally biotinylated HRP conjugated streptavidin was
applied. HRP was detected by incubation with 3,3,5,5- tetramethylbenzidin TMB; Pierce, USA for 30 min at
room temperature. The reaction was stopped with 2 moll H
2
SO
4
and the absorbance was read at 450 nm. Pooled serum from C57Bl6 mice, with known apoE
level, was used as standard.
2
.
5
. b
VLDL isolation and characterization bVLDL was isolated from pooled serum of three
mice from each transplantation group at 4 weeks after BMT by discontinuous KBr gradient ultracentrifuga-
tion at 250 000 × g for 18 h, as described by Redgrave et al. [30]. The fraction of d B 1.006 gml was isolated,
dialyzed against PBS1 mmoll EDTA, and subse- quently characterized with respect to the free choles-
terol, cholesteryl ester, phospholipid, and triglyceride content,
using enzymatic
procedures Boehringer
Mannheim, Germany. The protein content was deter- mined according to Lowry et al. [31].
2
.
6
. Histological analysis of hearts and aortas for atherosclerosis
To analyze the development of atherosclerosis, mice were sacrificed at 4 months after BMT. Hearts and
aortas were perfused in situ with oxygenated Krebs buffer 37°C, 100 mm Hg for 20 – 30 min via a cannula
in the left ventricle, followed by a post-perfusion fixa- tion with 3.7 neutral-buffered formalin Formal-fixx,
Shandon Scientific, England and subsequent storage in formalin. To evaluate the development of atheroscle-
rotic 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 mm cross sections of the aortic
root were made and stained with oil red O BDH, England. The atherosclerotic lesion area in the sections
was quantified using a light microscope connected with a 24-bits full color video camera and Optimas 6.1 image
analysis software BioScan, Edmonds, WA. Mean le- sion area was calculated in mm
2
from 10 sections, starting at the appearance of the tricuspid valves as
described previously [32].
2
.
7
. Statistical analysis Statistically significant differences among the means
of the different populations were tested by ANOVA. To compare pairs of groups, the Student-Newman-
Keuls multiple comparison test was performed after ANOVA.
3. Results
3
.
1
. Effect of reconstitution of macrophage apoE synthesis in apoE − − . LDLr
+ +
and apoE − − .LDLr − − mice on serum cholesterol
le6els To examine the role of the hepatic LDL receptor in
the macrophage apoE-induced reduction in serum cholesterol levels and atherosclerosis, apoE knockout
apoE − − .LDLr + + and
apoE.LDLr double
knockout apoE − − .LDLr − − mice were trans- planted with wild-type apoE + + .LDLr + + and
LDLr knockout apoE + + .LDLr − − bone mar- row, respectively. By using apoE − − .LDLr + +
mice as recipients, the LDL receptor is expressed in a wide variety of cell types and tissues, including muscle,
adipose tissue, parenchymal cells and Kupffer cells of the liver, and macrophages. The majority of the LDL
receptor activity which is responsible for serum LDL turnover, however, is located in the liver, the only
organ by which cholesterol can be irreversibly removed from the body via secretion into the bile [33]. Since, the
majority of the LDL receptor activity is found in the liver we will refer to the absence or presence of the
hepatic LDL receptor for functional simplicity. The effect on serum cholesterol levels and atheroscle-
rosis were determined in time after bone marrow trans- plantation.
The effect
of reconstitution
of apoE − − .LDLr + + and apoE − − .LDLr − −
mice with apoE + + .LDLr + + and apoE + + .LDLr − − bone marrow on serum cholesterol levels
is depicted in Fig. 1. As previously reported [25], pro- duction of apoE by macrophages induced a 86 P B
0.001 reduction in serum cholesterol levels at both 4 and 16 weeks after BMT, indicating that macrophage-
derived apoE can promote the clearance of cholesterol- rich lipoproteins in the presence of the LDL receptor
Fig. 1A. Interestingly, in the absence of a functional hepatic
LDL receptor
apoE + + .LDLr − − apoE − − .LDLr − − , serum cholesterol levels were
only reduced 40 P B 0.05 by macrophage-derived apoE at 6 weeks after BMT and 31 P B 0.01 at 16
weeks after BMT as compared to control transplanted mice apoE − − .LDLr − − apoE − − .LDLr −
− ; Fig. 1B. Strikingly, both apoE − − .LDLr − −
mice, transplanted with apoE − − .LDLr − − bone marrow, and apoE − − .LDLr − −
mice, trans- planted with apoE + + .LDLr − − bone marrow,
show a gradual increase in serum cholesterol levels after bone marrow transplantation. Although the exact
mechanism for this increase in both groups is unknown, a possible explanation might be down regulation of an
alternative uptake route in the absence of the LDL receptor.
At 16 weeks after BMT, the distribution of choles- terol over the different lipoprotein fractions was deter-
mined by liquid chromatography of serum of each individual animal. In control transplanted apoE − −
.LDLr + + apoE − − .LDLr + + mice the ma- jority of cholesterol is transported by VLDL and LDL.
Introduction
of apoE-producing
macrophages in
apoE − − .LDLr + + animals induced a large de- crease in VLDL 12-fold; P B 0.001 and LDL four-
fold; P B 0.001 cholesterol levels, thereby reducing the total serum cholesterol levels dramatically Fig. 2A. In
contrast, introduction of apoE producing macrophages in apoE − − .LDLr − − mice resulted only in a two-
fold P B 0.001 decrease in serum VLDL cholesterol, whereas no effect could be demonstrated on LDL
cholesterol levels Fig. 2B. Serum VLDL cholesterol levels in control transplanted apoE − − .LDLr − −
apoE − − .LDLr − − mice were approximately twofold higher as compared to apoE − − .LDLr + +
apoE − − .LDLr + + mice. In Table 1, a sum-
mary is given of the data on the effect of macrophage apoE synthesis in the absence or presence of the hep-
atic LDL receptor on cholesterol levels. The effect of BMT on the composition and mean
diameter of VLDL, the major cholesterol transporting lipoprotein in apoE − − .LDLr + + and apoE − −
.LDLr − − mice was analyzed at 4 weeks after BMT. VLDL from both apoE − − .LDLr + + and apoE −
−
.LDLr − − mice is enriched in cholesteryl esters as compared to triglycerides, due to the impaired clear-
ance of these lipoproteins by the liver and the thereby increased circulation time and exposure to lipoprotein
lipase. As indicated in Table 2, introduction of apoE- producing
monocytesmacrophages in
apoE − − .LDLr + + mice induced a decrease in cholesteryl
esters in the VLDL core, while the relative proportion of core triglycerides was increased. Replacement of
cholesteryl esters in the VLDL core was found to be associated with an increase in the mean diameter of
these particles. These compositional changes of the VLDL core from cholesteryl ester-rich to triglyceride-
rich indicate that the circulation time is largely de- creased. In the absence of a functional LDL receptor,
Fig. 1. Effect of reconstitution of macrophage apoE-synthesis in apoE − − .LDLr + + and apoE − − .LDLr − − mice on serum
cholesterol levels.
After transplantation
of either
apoE − − .LDLr + + panel A or apoE − − .LDLr − − panel B mice
with apoE − − hatched bars or apoE + + closed bars bone marrow, serum cholesterol levels were followed in time. Values are
means 9 SEM of 5 – 7 animals. Statistically significant difference P B 0.05, P B 0.01, P B 0.001 versus control transplanted mice.
Fig. 2. Distribution of serum cholesterol over the different lipoprotein fractions in apoE − − .LDLr + + panel A and apoE − −
.LDLr − − panel B mice, transplanted with apoE − − or apoE + + bone marrow at 16 weeks after BMT. Blood samples
were drawn by tail bleeding after an overnight fasting period. A 30 ml aliquot of serum of each individual mouse was loaded onto a
Superose six column and fractions were collected. Fractions 3 – 7 represent VLDL and chylomicrons, fractions 8 – 14 LDL, and frac-
tions 15 – 19 HDL. Open circles represent transplantation with apoE − − bone marrow and closed circles transplantation with
apoE + + bone marrow. Values are means 9 SEM of 4 – 7 mice. Statistically significant difference P B 0.001 versus control trans-
planted mice. Table 2
Percentual lipid composition of dB1.006 lipoproteins, isolated at 4 weeks after BMT
a
ApoE−−.LDLr++ ApoE−−.LDLr−−
Recipient ApoE−−
ApoE++ ApoE−−
ApoE++ Donor
of total composition Free
9.2 9.3
5.2 7.9
cholesterol 5.2
25.6 32.4
23.3 Cholesterol
esters 68.6
32.4 Triglycerides
44.1 38.1
Phospholipids 15.9
17.3 16.0
16.4 9.7
5.2 11.0
Proteins 7.2
mean particle diameter nm
47 9 10.5 74.7 9 20.1 Diameter
49.0 9 10.6 57.7 9 12.2
a
At 4 weeks after BMT, the effect of reconstitution of macrophage apoE synthesis in apoE−−.LDLr++ and apoE−−.LDLr−−
mice on the composition and mean diameter was determined of bVLDL isolated from pooled serum of six mice.
however, no change in either the ratio of triglycerides and cholesteryl esters or mean particle diameter was
observed.
3
.
2
. Effect of reconstitution of macrophage apoE synthesis in apoE − − . LDLr
+ +
and apoE − − .LDLr − − mice on serum apoE le6els
Since the availability of macrophage-derived apoE to lipoproteins in the circulation may be of critical impor-
tance for uptake via several receptor systems in the liver, serum apoE levels were measured at 4 weeks after
transplantation in the different transplantation groups using an ELISA, specific for mouse apoE. In apoE +
+
.LDLr + + apoE − − .LDLr + + mice serum apoE levels reached a steady state concentration of
0.13 9 0.028 mgdl, which is : 2 of the concentration
Table 1 Summary of the effects of macrophage versus hepatic LDL receptor expression or apoE synthesis on cholesterol levels
ApoE−−.LDLr++ apoE++.LDLr−−
ApoE++.LDLr++ ApoE−−.LDLr−− apoE++.LDLr−− [29]
Donor ApoE−−.LDLr++ ApoE−−.LDLr++ ApoE−−.LDLr−− apoE−−.LDLr−−
Recipient apoE++.LDLr−−
+ −
+ +
− Macrophage apoE
− −
+ −
Hepatic apoE −
+ +
Macrophage LDLr −
− −
+ +
Hepatic LDLr −
− −
926 9 66 85 9 17
Total cholesterol mgdl 1444 9 100
763 9 277 365 9 16
VLDL 70.0
74.8 3.9
37.9 85.0
24.8 45.1
LDL 22.4
35.7 13.4
41.4 5.2
1.5 26.4
HDL 2.8
in wild-type C57Bl6 mice. However, in the absence of a functional hepatic LDL receptor in apoE + +
.LDLr − − apoE − − .LDLr − − mice,
serum apoE levels increased to 6.67 9 0.47 mgdl, which is
: 93 of the concentration in wild-type C57Bl6 mice.
No further increase in serum apoE concentrations was observed at later time points after transplantation, indi-
cating that no further accumulation of lipoproteins is occurring, which correlates with the steady cholesterol
levels in the circulation.
3
.
3
. Effect of reconstitution of macrophage apoE synthesis in apoE − − . LDLr
+ +
and apoE − − .LDLr − − mice on atherosclerosis
Macrophage-derived apoE may not only function as a high affinity ligand for uptake by several receptor
systems in the liver; local enrichment of lipoproteins with extrahepatic apoE in the vessel wall may also
influence the uptake by macrophages, thereby influenc- ing the atherosclerotic process [34,35]. To investigate
Fig. 3. Photomicrographs of atherosclerotic lesions in cross sections of the aortic root, illustrating the effect of reconstitution of macrophage apoE production in apoE − − .LDLr + + and apoE − − .LDLr − − mice. Sections are stained with oil red O to visualize lipid- rich lesions and
counterstained with hematoxylin. Representative cross sections of apoE − − .LDLr + + mice and apoE − − .LDLr − − animals, trans- planted with either apoE − − A,C or apoE + + bone marrow B,D are shown. Magnification × 40.
Fig. 4. Effect of reconstitution of macrophage apoE-synthesis in apoE − − .LDLr + + and apoE − − .LDLr − − mice on sus-
ceptibility to atherosclerosis. The mean atherosclerotic lesion area was calculated from oil red O-stained cross sections of the aortic root
at the level of the tricuspid valves. Values indicate the mean atherosclerotic lesion area of 10 cross sections of the aortic root in
each mouse. Statistically significant difference P B 0.001 versus control transplanted mice.
ing that hepatic production of apoE and subsequent local enrichment of lipoproteins with apoE is crucial for
the LRP-mediated uptake of lipoprotein remnants [36]. The present study demonstrates that macrophage-
derived apoE is unable to normalize hypercholes- terolemia and significantly decrease the susceptibility to
atherosclerosis in apoE-deficient mice, lacking the LDL receptor, despite high levels of apoE in the circulation.
Therefore, it is concluded that for efficient clearance of remnant lipoproteins, associated with extrahepatically
produced apoE, expression of functional LDL recep- tors in the liver is crucial.
The role of the hepatic LDL receptor in the macrophage apoE-induced reduction in serum choles-
terol levels and atherosclerosis was investigated by com- parison of the effect of reconstitution of macrophage
apoE production
in apoE − − .LDLr + +
and apoE − − .LDLr − − mice by bone marrow trans-
plantation. Bone marrow transplantation is a powerful tool to investigate the role of apoE production in
hematopoietic stem cell-derived cells. ApoE is produced by monocytes and macrophages, but not by other
hematopoietic cells [23,24]. Therefore, using bone mar- row transplantation, apoE production can be specifi-
cally
modified in
monocytes and
macrophages. Although several recent reports have proposed that
endothelial cells [37,38], muscle cells [39], and hepatic oval cells [40] can also be bone marrow-derived, these
processes do not play a role in our studies, as induction of differentiation of bone marrow to these cell types
requires acute, severe injury to the tissues involved.
As previously
reported [25],
reconstitution of
macrophage apoE production in apoE − − .LDLr + +
mice resulted in normalization of hypercholes- terolemia and protection against atherosclerosis, at
serum apoE levels of only 2 of the concentration in wild-type mice. Recent data of Thorngate and Williams
demonstrated that this ability is not restricted to macrophage-derived apoE, as apoE production by the
adrenal gland was also able to normalize serum choles- terol levels in apoE-deficient mice [41]. However, in the
absence
of functional
hepatic LDL
receptors, macrophage-derived apoE was unable to normalize hy-
percholesterolemia and prevent atherosclerosis develop- ment in these mice, although serum apoE levels were
increased to 93 of the level in C57Bl6 mice. From these results it can be concluded that expression of
LDL receptors in the liver is crucial for the efficient clearance of remnant lipoproteins, associated with ex-
trahepatically produced apoE. In these apoE + + .LDLr − − apoE − − .LDLr − − mice a small
decrease in serum cholesterol levels due to a reduction in VLDL cholesterol was observed, indicating that in
addition to the LDL receptor, although less important, a non-LDL receptor system also can recognize lipo-
protein remnants loaded with macrophage-derived apoE.
the effect
of macrophage
apoE production
on atherosclerotic lesion development in the absence or
presence of the LDL receptor, the mean atherosclerotic lesion area was determined in the transplanted animals.
The hearts and aortas were perfused, fixed, and exam- ined histologically at 4 months after BMT. Representa-
tive photomicrographs of lesions in cross sections of the different transplantation groups stained for lipid with
oil red O are shown in Fig. 3. Quantification of the mean atherosclerotic lesion area in the aortic root
revealed that macrophage-derived apoE induced a 17- fold reduction in the mean atherosclerotic lesion area
from 437 9 61 × 10
3
mm
2
n = 11; mean 9 SEM to 25 9 16 × 10
3
mm
2
n = 4; mean 9 SEM, P B 0.001 in apoE − − .LDLr + + mice Fig. 4. In the absence
of functional LDL receptors, however, no significant reduction in the mean atherosclerotic lesion area was
observed in
apoE + + .LDLr − − apoE − − .LDLr − −
mice 147 9 11 × 10
3
mm
2
; n = 4;
mean 9 SEM, as compared to apoE − − .LDLr − −
apoE − − .LDLr − − 293 9 98 × 10
3
mm
2
; n = 7; mean 9 SEM animals. Although there was a
tendency to slightly smaller lesions, which correlates with the observed small decrease in serum cholesterol
levels, the effect is not comparable to the 17-fold reduc- tion in the presence of functional hepatic LDL recep-
tors.
4. Discussion
