© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Effect of Macrophage-Derived Apolipoprotein E on Established Atherosclerosis in Apolipoprotein E–Deficient Mice
From the Department of Medicine (W.S., X.W., N.J.W., A.J.L.), Department of Microbiology and Molecular Genetics, and the Department of Radiation Oncology (W.H.M.), School of Medicine, University of California, Los Angeles.
Correspondence to Aldons J. Lusis, Department of Medicine, UCLA School of Medicine, 47-123 CHS, Los Angeles, CA 90095-1679. E-mail jlusis{at}medicine.medsch.ucla.edu
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Abstract |
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Abstract—Apolipoprotein E–deficient (apoE-/-) mice have hyperlipidemia and develop spontaneous atherosclerosis in a time-dependent manner. Although macrophage-derived apoE has been shown to prevent the development of atherosclerosis in apoE-/- mice, whether it would induce regression of established atherosclerosis is unknown. To determine this, 8-week-old apoE-/- mice were transplanted with apoE+/+ bone marrow. Four weeks after transplantation, when plasma cholesterol levels had reached normal levels, a group of mice (n=12) were killed and their aortic lesions were measured and used as a baseline to judge regression. Twelve and 20 weeks after transplantation, aortic lesion areas of the mice were 9340±2184 µm2 (mean±SEM, n=8) and 12 211±1433 µm2 (n=9), respectively, values not significantly different from the lesion areas of the baseline mice (12 347±2487 µm2; n=12, P>0.05). In contrast, apoE-/- mice reconstituted with apoE-/- bone marrow developed severe atherosclerotic lesions (453 036±29 767 µm2, n=7) 20 weeks after transplantation. These data suggest that macrophage-derived apoE was insufficient to induce significant regression of established atherosclerotic lesions in apoE-/- mice, although it was sufficient to eliminate hypercholesterolemia and prevent progression of aortic lesions.
Key Words: atherosclerosis • macrophages • apolipoprotein E • regression • bone marrow transplantation
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Introduction |
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Previous studies have provided evidence that atherosclerosis can undergo regression in both humans and animal models,1 2 3 4 but results have varied greatly, depending on the animal models used or the population investigated.5 6 7 Mice are increasingly used in the study of atherosclerosis, and certain mice such as apoE-deficient (apoE-/-) mice develop all phases of lesions found in humans.8 9 Recently, several studies have reported that atherosclerotic lesions of mice can undergo considerable regression.10 11 12 13 14 15 16
ApoE is a 34-kD glycoprotein that plays an important role in lipoprotein metabolism.17 It mediates uptake and degradation of chylomicron and VLDL remnants by acting as a ligand for the LDL receptor and the LDL receptor–related protein.17 18 Although the vast majority of plasma apoE is derived from the liver,19 20 21 22 apoE is also synthesized by macrophages in various organs.23 24 ApoE deficiency results in severe hypercholesterolemia and diffuse atherosclerotic disease in humans25 and in gene-targeted mice.8 9 Targeted mice develop foam cell–rich fatty streaks in the aortic sinus and proximal aorta by the age of 3 months, and after 5 months, fibrous lesions are present.26 27 Because apoE is synthesized by monocytes/macrophages23 24 but not by granulocytes and lymphocytes,28 bone marrow transplantation (BMT) has been used to examine the role of macrophage-derived apoE in atherosclerosis in vivo. Recent BMT studies have shown that macrophage-derived apoE results in the normalization of serum cholesterol levels and prevents the development of atherosclerosis in apoE-/- mice.29 30 31 However, it is unknown whether macrophage-derived apoE can induce regression of established atherosclerotic lesions. The purpose of the present study was to determine the effect of macrophage-derived apoE on established atherosclerosis in apoE-/- mice by BMT.
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Methods |
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Mice
ApoE-/- C57BL/6J mice and wild-type C57BL/6J mice were purchased from the Jackson Laboratory, Bar Harbor, Me. Mice were fed with a regular chow diet and maintained in a temperature-controlled room with a 12-hour light/dark cycle. All procedures were in accordance with current National Institutes of Health guidelines and were approved by the UCLA Animal Research Committee.
Bone Marrow Transplantation
Female recipient apoE-/- mice were
lethally irradiated with a dose of 1000 rads from a cobalt source. Bone
marrow cells were harvested by flushing the femurs and tibias of male
donor mice with Dulbecco’s modified Eagle’s medium containing 10%
fetal bovine serum and 5 U/mL heparin. Red blood cells were lysed with
ACK buffer (150 mmol/L NH4Cl, 61 mmol/L
KHCO3, and 1 mmol/L
Na2EDTA, pH 7.3). The remaining cells were washed
and suspended in Dulbecco’s modified Eagle’s medium with 1% bovine
albumin. Each recipient mouse was injected with
107 bone marrow cells in 0.3 mL through the tail
vein.
Experimental Protocols
At 8 weeks of age, female apoE-/- mice
(n=29) were reconstituted with bone marrow cells from wild-type male
C57BL/6J mice
(apoE+/+
apoE-/-) and
maintained on a chow diet. Four weeks after transplantation, a group of
mice (n=12) were killed and served as the baseline group by which to
judge regression of atherosclerotic lesions. The remaining mice were
killed at 12 (n=8) or 20 (n=9) weeks after transplantation for lesion
analyses. In addition, a group of age-matched, female
apoE-/- mice were transplanted with bone marrow
from male apoE-/- mice
(apoE-/-apoE-/-) and
maintained on a chow diet for 20 weeks (n=7).
Western Blot Analysis for ApoE
The presence of apoE in plasma was determined by Western blot
analysis. In brief, 1 µL of plasma was separated by
electrophoresis on 12% SDS polyacrylamide gels and
electrophoretically transferred to nitrocellulose membranes. The
membranes were incubated with a polyclonal rabbit anti-mouse apoE
antibody (BioDesign International) for 1 hour and then incubated for
0.5 hour with a horseradish peroxidase–conjugated anti-rabbit
secondary antibody. The signals were detected by the enhanced
chemiluminescent detection method according to the manufacturer’s
instructions (ECL Western blotting, Amersham).
DNA Preparation and PCR of Male-Specific Sequences
Overnight-fasted mice were bled from the retro-orbital vein
under isoflurane anesthesia. After
centrifugation, the plasma was collected and used for
lipid analyses as indicated below. The blood cell pellet was
lysed in ACK buffer to remove red blood cells. DNA was prepared by
adding polymerase chain reaction (PCR) solution (10 mmol/L Tris,
pH 8.0; 2.5 mmol/L MgCl2; 1% Tween-20; and
0.4 mg/mL proteinase K) to each leukocyte pellet and incubating the
resulting mixture at 60°C for 2 hours followed by a 95°C incubation
for 20 minutes. PCR was performed to amplify a 250-bp sequence of the Y
chromosome.32 The upstream primer was 5'-GAG GGC CAT GTC
AAG CGC CCC ATG AATG-3' and the downstream primer was 5'-AGA CAC TGT
GAA ATC GGG AGG CT-3'. The cycling conditions were denaturing for 1
minute at 94°C, annealing for 1 minute at 62°C, and extension for 1
minute at 72°C.
Aortic Lesion Analysis
Methods for the quantification of atherosclerotic lesions in the
aorta were done as previously reported.33 In brief, the
heart and proximal aorta were excised and embedded in OCT compound.
Serial 10-µm-thick cryosections from the middle portion of the
ventricle to the aortic arch were collected and mounted on
poly-D-lysine–coated slides. In the region from the
appearance to the disappearance of the aortic valves, every other
section was collected. In all other regions, every fifth section was
collected. Sections were stained with oil red O and hematoxylin,
counterstained with fast green, and examined by light microscopy.
Immunohistochemical Analyses of Atherosclerotic
Lesions
Immunohistochemical analyses of atherosclerotic lesions
in the aortic root were performed as previously
described.33 In brief, 10-µm-thick cryosections were
fixed in acetone and incubated with a rabbit polyclonal antibody to
mouse apoE or a rat monoclonal antibody to mouse macrophages,
MOMA-2 (Accurate Chemicals), followed by incubation with biotinylated
anti-rabbit or anti-rat secondary antibodies. Signals were detected
with peroxidase chromogen kits (Vector Laboratories). We used an
FITC-labeled polyclonal antibody to human smooth muscle cell actin
(Sigma) to detect smooth muscle cells in the lesions.
Plasma Lipid Measurements
Enzymatic assays for total cholesterol, HDL
cholesterol, and triglyceride were performed in
96-well plates on a Biomek 2000 automated laboratory workstation
(Beckman Instruments, Inc) as described.34 Measurements on
plasma samples were performed in triplicate with known control samples
on each plate to ensure accuracy.
Statistical Analysis
Plasma lipid levels were expressed as mean±SEM, with n
indicating the number of mice. Atherosclerotic lesion areas were
expressed as values of individual mice. ANOVA was used to compare
differences in atherosclerotic lesions and lipid levels among different
groups of mice over time. Differences were considered statistically
significant at P<0.05.
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Results |
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Reconstitution of Recipient Bone Marrow
ApoE was detected by Western blot analysis in the plasma of apoE-/- mice reconstituted with wild-type bone marrow as early as 2 weeks after transplantation (Figure 1A
). In contrast, apoE was absent in
apoE-/- mice reconstituted with
apoE-/- bone marrow. Moreover, because male
mice containing XY chromosomes were used as donors for the female
recipients, we designed primers to genotype a segment of the Y
chromosome. As shown in Figure 1B, 2 weeks after
transplantation, the Y chromosome was detected by PCR in
peripheral leukocytes of recipient mice.
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Effect of BMT on Plasma Lipids
Reconstitution of apoE-/- mice with
wild-type bone marrow resulted in dramatic changes in plasma
cholesterol and triglyceride levels (Figure 2
). Two weeks after transplantation,
plasma total cholesterol and triglyceride
levels were significantly reduced and HDL cholesterol
levels increased (P<0.05). By 4 weeks
after transplantation, plasma total cholesterol,
triglyceride, and HDL cholesterol levels had
reached normal levels. In contrast, in
apoE-/-apoE-/- mice,
plasma total cholesterol levels gradually increased, from
334±11 mg/L (mean±SEM) before transplantation to 556±24 mg/L 20
weeks after transplantation. There was a significant decrease in
triglyceride levels 2 weeks after transplantation
(P<0.05). Plasma HDL cholesterol levels were
not significantly altered after transplantation.
Effect on Aortic Atherosclerotic Lesion Areas
The size of atherosclerotic lesions at the aortic root was
quantified by light microscopy. The
apoE+/+apoE-/- mice
that were killed 4 weeks after transplantation had an average lesion
area per section of 12 347±2487 µm2
(mean±SEM, n=12; Figure 3). Twelve and
20 weeks after transplantation, the average lesion areas of
apoE+/+apoE-/- mice
were 9340±2185 µm2 (n=8) and
12 211±1433 µm2 (n=9), respectively.
Compared with mice that were killed 4 weeks after transplantation,
aortic lesion areas did not show a significant increase or decrease in
mice that were killed at 12 and 20 weeks after transplantation
(P>0.05). In contrast,
apoE-/-apoE-/- mice
developed severe atherosclerosis 20 weeks after
transplantation, with an average lesion area of 453 036±29 767
µm2 per section (n=7).
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Effect on Morphology of Atherosclerotic Lesions
In mice that were killed 4 weeks after transplantation, aortic
lesions consisted primarily of macrophage-derived foam
cells (Figure 4). Smooth muscle cells
were undetectable in the lesions, and there were no fibrous caps. In
mice that were killed 12 weeks and 20 weeks after transplantation,
aortic lesions were flatter and had developed thin, fibrous caps in
about half of the mice. Macrophage-derived foam cells were
still the main cellular component of the lesions. Smooth muscle cells
were observed in the fibrous caps. In contrast, mice reconstituted with
apoE-/- bone marrow developed advanced lesions
containing numerous smooth muscle cells, calcification, and necrotic
areas.
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ApoE Expression in Atherosclerotic Lesions
Immunohistochemistry analysis showed that apoE was
abundantly expressed in atherosclerotic lesions of the
apoE-/- mice reconstituted with wild-type bone
marrow (Figure 5). In contrast, apoE was
not detected in lesions of those apoE-/- mice
reconstituted with apoE-/- bone marrow.
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Discussion |
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The principal aim of the present study was to evaluate whether macrophage-derived apoE was sufficient to induce significant regression of established atherosclerotic lesions in apoE-deficient mice by BMT. Wild-type and apoE-deficient mice were used as bone marrow donors for apoE-deficient mice. We found that repopulation of cells in BMT-treated mice with normal hematopoietic cells eliminated hypercholesterolemia and prevented progression of the lesions but did not induce significant regression of established atherosclerosis in apoE-deficient mice.
BMT leads to the replacement of recipient tissue macrophages by macrophages of donor origin.35 Bone marrow cells accumulate in the liver, spleen, and bone marrow several hours after injection.36 37 By 4 weeks after transplantation, >95% of macrophages in the bone marrow were of donor origin.29 In the present study, we observed that apoE was present in the peripheral circulation as early as 2 weeks after transplantation, and by 4 weeks, macrophage-derived apoE was sufficient to normalize plasma lipid levels of apoE-deficient recipient mice. As in previous studies,38 39 we used male mice as bone marrow donors for female recipients so that engraftment of bone marrow could be verified by genotyping male-specific makers of the Y chromosome. Because donor and recipient mice were derived from the same inbred strain, graft-versus-host disease would not occur. Moreover, all recipient mice were healthy throughout the experiment.
Although macrophages produce only a small percentage (
10%)
of plasma apoE, this amount is sufficient to reverse
hypercholesterolemia and elevate plasma HDL
levels.29 30 Our present findings are
consistent with this observation. The finding that BMT induced
a decrease of plasma triglyceride levels in
apoE+/+apoE-/- mice is
consistent with that of a previous study.31 The
temporary decrease at 2 weeks was probably caused by the
transplantation procedure, because it was also observed in
apoE-/-apoE-/-
mice.
Previous studies29 30 31 and our present study have indicated that after transplantation, a period of 4 weeks is required for macrophage-derived apoE to achieve its full therapeutic effect in apoE-deficient recipients. During this period, as peripheral tissues are reconstituted with apoE-expressing macrophages, plasma cholesterol levels gradually fall but atherosclerotic lesions continue to progress. We observed a significant increase in atherosclerotic lesion size, from 2613±781 µm2 at the time of transplantation to 12 347±2487 µm2 4 weeks after transplantation. Therefore, selecting atherosclerotic lesions at 4 weeks after transplantation as the baseline seems an appropriate standard by which to judge regression in apoE-deficient mice. In the present experiment, apoE-deficient mice received transplantation at 8 weeks of age, and by the time their bone marrow was replaced with donor marrow, they were 12 weeks of age. Nakashima et al27 reported that apoE-deficient mice of this age develop fatty streak lesions. Indeed, we observed that atherosclerotic lesions of these mice consisted primarily of macrophage-derived foam cells and that smooth muscle cells and fibrous caps were absent in the lesions.
One important finding of the present study is that 16 weeks after plasma lipid levels were normalized, the size of the atherosclerotic lesions was not significantly reduced in apoE-deficient mice. This finding is consistent with the notion that the regression of atherosclerotic lesions is a slow process. Indeed, in the rhesus monkey model, Tucker et al5 did not find definite evidence of regression after 4 months on a low-fat diet. Kokatnur et al40 reported that experimental atherosclerosis in rhesus monkeys showed evidence of regression only after treatment with a low-fat diet for 64 weeks. However, Tsukamoto et al16 recently reported that liver-directed gene transfer and hepatic expression of human apoE3 in chow-fed, apoE-deficient mice resulted in an almost complete regression of fatty streaks within 6 weeks, whereas expression of human apoE4 reduced cholesterol levels to the same extent as apoE3 but did not induce significant regression. That study suggests that effects beyond the reduction of plasma cholesterol levels are required to induce regression. Mouse apoE is similar to human apoE3 in term of the 2 polymorphic amino acids.41 The reasons for the discrepancy between the data of Tsukamoto et al16 and ours are unclear. One possible explanation is that gene transfer resulted in more apoE production than did BMT. Indeed, Desurmont et al11 observed that regression of fatty streak lesions in apoE-/- mice 6 months after injection of the adenovirus encoding human apoE cDNA was dependent on plasma apoE concentration.
Hyperlipidemia plays an important role in the progression of atherosclerosis. However, elimination of hyperlipidemia alone seems insufficient to induce regression of atherosclerosis. Indeed, our failure to observe a significant reduction of atherosclerotic lesions 16 weeks after normalization of plasma lipid levels suggests that treatments other than normalizing plasma lipid levels are necessary to induce significant regression of atherosclerosis.
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Acknowledgments |
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This work was supported by National Institutes of Health grant HL-30568. The authors thank Yishou Shi for technical help.
Received June 8, 2000; accepted July 17, 2000.
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W. Shi, X. Wang, K. Tangchitpiyanond, J. Wong, Y. Shi, and A. J. Lusis Atherosclerosis in C3H/HeJ Mice Reconstituted With Apolipoprotein E-Null Bone Marrow Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 650 - 655. [Abstract] [Full Text] [PDF]
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