© 1999 American Heart Association, Inc.
Original Contributions |
ApoA1 Reduces Free Cholesterol Accumulation in Atherosclerotic Lesions of ApoE–Deficient Mice Transplanted With ApoE–Expressing Macrophages
From The Scripps Research Institute, Departments of Immunology (W.A.B., A.S.B., L.K.C.) and Vascular Biology (L.K.C.), La Jolla, Calif.
Correspondence to Linda K. Curtiss, Departments of Immunology and Vascular Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037. E-mail lcurtiss{at}scripps.edu
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Abstract |
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Abstract—Along with apolipoprotein (apo) E, which promotes cholesterol efflux from foam cells, apoA1–containing high density lipoprotein (HDL) is thought to facilitate the transport of cholesterol from lesions. This role for apoA1 was tested in vivo by lethally irradiating apoE–deficient and apoE– plus apoA1–deficient mice and reconstituting them with bone marrow cells isolated from wild-type (WT) mice. ApoE, but not apoA1, was synthesized by the transplanted bone marrow–derived cells. Therefore, this transplantation procedure generated apoE–deficient animals with atherosclerotic lesions that contained both apoE and apoA1 (E/A1 mice) and apoE–deficient animals with lesions that contained apoE but no apoA1 (E/A1o mice). As shown previously, the transplanted WT macrophage–derived apoE dramatically lowered the plasma hypercholesterolemia in both groups. On feeding with an atherogenic diet after transplantation, plasma cholesterol levels were raised in both groups of mice, but the levels in the E/A1 mice at 20 weeks were 2- to 3-fold higher than in E/A1o mice. Immunohistochemical staining verified that apoE was abundant in lesions of both groups, whereas apoA1 was detected in the lesions of E/A1 mice only. Despite a 2- to 3-fold lower total plasma cholesterol in the E/A1o mice, the free cholesterol recovered from isolated aortas was
60%
higher and the mean lesion area in serial sections of the aortic valves
45% larger. Therefore, apoA1 reduces free cholesterol
accumulation in vivo in atherosclerotic lesions.
Key Words: atherosclerosis • bone marrow transplantation • apoA1 • apoE
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Introduction |
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The inverse relationship between plasma HDL and coronary heart disease (CHD) is well documented.1 2 3 4 5 6 Compilation of multiple epidemiological studies indicates a 2% to 3% increase in CHD risk for every 1% decrease in HDL levels.7 The importance of HDL in protection against CHD also is evident in patients with congenital apoA1 deficiency who have low HDL levels and are prone to premature CHD.8 9 Despite this abundance of carefully conducted studies documenting HDL to be antiatherogenic, exactly how HDL protects against CHD remains enigmatic.
One widely accepted and intensely studied proposed mechanism to explain the protective effects of HDL against CHD is its role in reverse cholesterol transport. This mechanism, first proposed by Glomset in 1968,10 involves the movement of excess cholesterol from peripheral tissues to HDL for subsequent delivery to the liver for catabolism. This delivery involves lecithin:cholesterol acyltransferase (LCAT), an enzyme that esterifies free cholesterol in plasma and is activated by apoA1. In 1983, Brown and Goldstein11 proposed that both HDL and apoE play a direct role in the removal of excess cellular cholesterol. They showed that macrophage-derived foam cells, a well-known hallmark of atherosclerosis, release free cholesterol along with apoE, which can be transferred to HDL where it is esterified by LCAT. Moreover, Gordon et al12 showed that the capacity of the HDL to carry cholesteryl esters is increased in the presence of apoE.
Both apoE and apoA1 are found within atherosclerotic lesions.13 Lesion apoE is made predominantly by resident macrophage-derived foam cells,14 15 whereas apoA1 is plasma derived.16 Although several studies have provided evidence of a role for apoA1–containing HDL in cholesterol efflux from lipid-loaded cells in vitro,17 18 19 20 21 there is little direct evidence of a role for apoA1–containing HDL in lesion cholesterol in vivo. Therefore, we examined in vivo whether apoA1–containing HDL could influence the cholesterol content of atherosclerotic lesions. The introduction of wild-type (WT) bone marrow into apoE–deficient mice results in a dramatic reduction in hypercholesterolemia and atherosclerosis due to the introduction of macrophage-derived apoE.22 23 However, when these mice are fed an atherogenic diet, they become hypercholesterolemic and develop atherosclerosis. For this study, we used apoE–deficient as well as apoE– plus apoA1–deficient mice to investigate the role of apoA1 and apoE in lesion cholesterol homeostasis. All mice were irradiated, transplanted with WT bone marrow, and fed an atherogenic diet to induce atherosclerotic lesions in both experimental groups. We verified that the lesions of all mice contained macrophage-derived apoE and found that apoA1–containing HDL decreased aortic cholesterol accumulation.
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Methods |
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Animals
ApoE–deficient and apoA1–deficient mice on a C57BL/6J background were purchased from Jackson Laboratories (Bar Harbor, Me). The WT C57BL/6J mice were from the rodent breeding colony of The Scripps Research Institute. ApoE/apoA1 double-knockout mice were generated by breeding male apoE–deficient mice with female apoA1–deficient mice. The resulting apoE–deficient and apoA1–deficient heterozygous offspring were bred, and double homozygotes were identified by Western blotting for plasma apoE and apoA1. Animals used in this study were the offspring of male and female confirmed homozygous apoE–deficient plus apoA1–deficient mice.
All mice studied were weaned at 4 weeks and were fed either a standard chow diet (diet 5015, Harlan Teklad) or an atherogenic diet (henceforth referred to as the high-fat diet [HFD]) containing 15.8% (wt/wt) fat and 1.25% (wt/wt) cholesterol (without sodium cholate; diet 94059, Harlan Teklad). The mice were housed 4 per cage in autoclaved, filter-top cages with autoclaved water and kept on a 12-hour light/dark cycle. All procedures employed were in accordance with institutional guidelines. Euthanasia was accomplished with an inhalational anesthetic overdose of Halothane.
Irradiation and Bone Marrow Transplantation
Eight 6-week-old male apoE–deficient and 8 age-matched, male
apoE– plus apoA1–deficient mice were subjected to 1000 rad of
total-body irradiation to eliminate their endogenous bone
marrow stem cells and bone marrow–derived cells. Bone marrow cells
were extracted from the femur and tibia of WT mice as described
previously,22 and 3x106 cells were
injected intravenously into the tail vein of all irradiated
mice. Four weeks after bone marrow transplantation (BMT), all animals
were fed the HFD for an additional 16 weeks.
Plasma Lipoprotein and Lipid Analyses
Blood was obtained by retro-orbital puncture under
methoxyflurane-induced anesthesia after a 6-hour fast.
Plasma cholesterol was measured enzymatically using a kit
from Sigma Chemical Co. Separation of plasma lipoproteins was achieved
by pooling equal volumes of individual plasma samples and injecting 0.1
mL onto a Superose 6 column connected to a fast protein liquid
chromatography (FPLC) apparatus from
Pharmacia (LKB Biotechnology). The relative cholesterol
content of each fraction was measured with an ultrasensitive enzymatic
assay.22 Pooled plasma also was separated by
SDS–polyacrylamide gel electrophoresis on an 8% to 25%
polyacrylamide gel using the PhastGel system (Pharmacia LKB
Biotechnology). The apolipoproteins were electroblotted onto nylon
membranes (Schleicher and Schuell) with the use of a semidry blotting
apparatus (Bio-Rad Laboratories), and the membranes were
incubated for 2 hours at room temperature with a rabbit anti-mouse apoE
antibody (a generous gift of Dr K. Weisgraber, Gladstone Institute, San
Francisco, Calif) and a rabbit anti-mouse apoA1 antibody (Biodesign,
Kennebunk, Me). Antibody binding was detected by chemiluminescence with
a rabbit IgG-specific horseradish peroxidase conjugate according to the
manufacturer's recommendations (Amersham).
Aortic Cholesterol
Immediately after they were humanely killed, the mice were
perfused with PBS and then with 4% paraformaldehyde.
After the heart was excised at its attachment site to the aortic arch,
the aorta was stripped of its adventitial fat and dissected from the
right common carotid artery to the superior mesenteric artery. The
aortas were weighed and subjected to lipid extraction by a modified
method of Folch et al.24 In brief, the aortas were
homogenized in 2 mL of chloroform/methanol (2:1, vol/vol)
and centrifuged at 1700g for 5 minutes. Two
milliliters of supernatant was transferred to a clean tube, and 0.4 mL
of 0.88% KCl was added to separate the aqueous and lipid layers. The
lipid layer was transferred to another tube, completely dried under
N2 at 37°C, and redissolved in 0.2 mL of 100%
ethanol. The total and free cholesterol contents in 15 µL
were assayed enzymatically as previously described25
in the absence or presence of 0.1 U/mL cholesteryl ester hydrolase,
respectively. Fluorescence was measured in a
fluorescence spectrophotometer with excitation at 325 nm
and emission at 415 nm.
The extracts also were used to measure triglycerides, free cholesterol, and cholesteryl esters after thin-layer chromatography (TLC) as described,26 with a few minor modifications. In brief, TLC was performed on 10x20-cm silica gel 60A plates (Whatman). The plates were developed twice in methanol and dried for 15 minutes at 100°C. Samples (0.01 mL each) were applied to the plates 1 cm apart and 1 cm from the bottom of the plate. Cholesterol and cholesteryl ester standards also were applied at 20, 10, 5, 2.5, and 1.25 µg per lane. The plates were developed to 7 cm from the origin in a system of 75:23:2 (vol/vol/vol) hexane/diethyl ether/acetic acid and then to the top with hexane. The separated lipids were visualized by spraying the plates with 10% (wt/vol) cupric sulfate in 8% (wt/vol) phosphoric acid and charring them at 180°C until the bands were visible. The free cholesterol, cholesteryl ester, and triglyceride bands were scanned using a laser scanning densitometer (Molecular Dynamics) and quantified using ImageQuant software (Molecular Dynamics).
Atherosclerotic Lesion Analysis
The top half of the heart excised from each animal was embedded
in OCT, snap-frozen, and kept at -70°C until sectioning. Serial
sections of 10-µm thickness were cut through a 250-µm segment of
the aortic valve, and 5 sections, each separated by 40 µm and
encompassing 200 µm of the valve, were examined from each mouse.
The sections were stained with oil red O to reveal the bright red
staining of the lesions and counterstained with hematoxylin. The oil
red O–stained areas of each section were quantified using a
computer-assisted video imaging system as described in
detail.27 The mean lesion areas of the 5 sections from
each mouse were used to calculate the mean of all mice in each group
(n=8).
Immunohistochemical Detection of Apolipoproteins in
Lesions
Serial aortic valve sections with similar lesion morphology were
selected for immunohistochemical detection of apoB, apoE, and apoA1.
After the sections were fixed for 2 minutes in acetone at -20°C,
they were incubated successively at room temperature as follows: 5%
normal goat serum for 30 minutes; 1:100 anti–apoB (Biodesign),
anti–apoE (a gift of Dr K. Weisgraber), or anti–apoA1 (Biodesign)
antibody (all made in the rabbit) in PBS containing 0.1% BSA
and 0.015% Triton X-100 for 2 hours; 5 µg/mL biotinylated
anti-rabbit IgG (Vector Laboratories, Burlingame, Calif) for 1 hour;
and 1:200 FITC-conjugated streptavidin (PharMingen) for 1 hour. The
slides were mounted in fluorescence mounting medium (Dako
Corp), viewed with a fluorescence microscope, and
photographed.
Statistical Analysis
Lesion areas were quantified by averaging the 5 sections from
each mouse heart and then calculating the mean lesion area of all the
mice in each group (8 mice per group). The mean lesion areas of the 2
groups were compared by the Mann-Whitney U test. Linear
regression analysis was used to determine the correlation
between lesion area and plasma cholesterol values as well
as the correlation between lesion area and cholesterol
content of the aortas. Statmost was used for data analysis.
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Results |
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Circulating plasma cholesterol levels before and after BMT are shown in Figure 1
. We
reported previously22 that repopulation of chow-fed
apoE–deficient mice with WT bone marrow gives rise to apoE–secreting
macrophages that lower plasma cholesterol to
near-normal levels within 4 weeks. After beginning the HFD at week 4,
plasma cholesterol increased in both groups of BMT mice at
weeks 8, 12, and 20. However, at all time points,
cholesterol levels were lower in the E/A-Io mice (mice with
lesions containing apoE but no apoA1) compared with the E/A-I mice
(mice with lesions with both apoE and apoA1), especially when the mice
were given the HFD 4 weeks after BMT. In contrast, fasting
triglycerides were similar at all time points between the 2
groups and remained between 32.3±4.3 and 53.5±14.3 mg/dL.
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The lipoprotein distribution of this plasma cholesterol is
shown in Figure 2. These profiles
were obtained by chromatographing 0.1 mL of pooled plasma drawn
at week 0 (pre-BMT), week 4 (post-BMT, chow), and week 8 (post-BMT,
HFD). As expected, the plasmas of the E/A-Io mice contained negligible
cholesterol in the HDL fraction. Although VLDL
cholesterol was fairly similar between the 2 groups at week
0, IDL+LDL cholesterol was consistently lower in
the E/A-Io mice at all time points. When the HFD was fed, VLDL and
IDL+LDL cholesterol levels did not increase in the E/A-Io
mice, whereas the increase in IDL+LDL cholesterol was
nearly 2-fold in the E/A-I mice (a comparison of weeks 4 and 8).
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A Western blot for plasma apoE and apoA1 is shown in Figure 3. Equal volumes of pooled plasmas
from weeks 0, 4, and 8 were electrophoresed on the SDS
polyacrylamide gel. For comparison, a comparable volume of
plasma from a WT mouse was analyzed as well. As expected, there
was no apoA1 detected at any time in the plasmas of the E/A-Io mice.
ApoE also was absent in the plasmas of both groups at week 0 before BMT
but appeared after BMT by week 4 due to its production by the
transplanted WT macrophages.22 Perhaps as a result
of the absence of apoA1, it appeared that the apoE levels were
consistently higher in the plasmas of E/A-Io mice compared with
their E/A-I counterparts. In both groups of mice, the HFD also appeared
to have raised the apoE levels in the plasma after BMT (a comparison of
weeks 4 and 8). Nevertheless, in both groups, the level of circulating
apoE was <10% of the amount of apoE in the plasma of fasting WT mice
consuming a chow diet (Figure 3, left lane).
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Atherosclerosis in the animals was examined with serial
sections of the aortic valves taken from the frozen hearts.
Apolipoproteins in these aortic valve lesions were visualized by
immunofluorescence (Figure 4). ApoB and apoE were readily visible in
the lesions of both experimental groups, whereas apoA1 staining was
apparent only in the E/A-I lesions. This indicated that both LDL (apoB)
and HDL (apoA1) had entered the arterial wall from the
plasma. In contrast, most of the apoE was probably made locally by the
foam cells, because apoE levels in the plasmas of these apoE–deficient
mice transplanted with WT bone marrow were quite low (Figure 3
).
Moreover, WT mice transplanted with apoE–deficient bone marrow and
placed on an atherogenic diet have high levels of plasma apoE, yet
their lesions do not contain appreciable amounts of
apoE.28 Importantly, these
immunofluorescence results confirmed that the E/A-I
mice had both apoE and apoA1 in their aortic valve lesions,
whereas the E/A-Io mice had lesions with apoE but no apoA1.
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Representative examples of the oil red O–stained valve
sections that were used to quantify lesion areas are shown in Figure 5. Major fatty streak lesions were
present in all animals studied. However, the staining intensity of
the lesions was distinct. Visual inspection suggested that the lesions
of E/A-Io mice stained brighter with oil red O. Because oil red O
stains neutral lipids, this suggested that more neutral lipid was
present in the lesions of the E/A-Io mice. Therefore, aortic lipids
of all mice were examined after lipid extraction of individual
dissected aortas. Direct analysis of total
cholesterol in the lipid-extracted mouse aortas revealed
that the mean cholesterol content of the E/A-Io mice was
38% higher than in the aortas of the E/A-I mice (P<0.05;
Figure 6). After TLC separation of
the lipids to remove the triglycerides, free
cholesterol and cholesteryl ester measurements revealed
that the difference in total cholesterol was due entirely
to differences in the free cholesterol content of the
aortas, and there was a positive and significant correlation between
aortic total cholesterol (or free cholesterol)
and lesion area (r2=0.58,
P=0.002). No differences in triglyceride levels
were revealed.
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To examine this more thoroughly, the lesion area
(represented as the mean of all mice in each group) was
calculated from the mean lesion area of 5 sections from each mouse. The
mean±SDs of the lesion areas were 264 447±64 225 and
384 341±69 139 µm2 for E/A-I and E/A-Io
mice, respectively. It is noteworthy that despite the
consistently lower plasma cholesterol levels in the
E/A-Io group (Figure 1), the mean lesion area was 45% higher
(P<0.01) in the E/A-Io mice compared with the E/A-I mice.
Importantly, no correlation was found between aortic valve lesion areas
of individual mice and their averaged post-HFD plasma
cholesterol levels
(r2=0.17, P=0.15).
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Discussion |
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The classic model of reverse cholesterol transport dictates that a cholesterol acceptor such as HDL participates in the transport of cholesterol from the peripheral tissues back to the liver. In atherosclerosis, this model is relevant to the removal of cholesterol from lipid-rich foam cells within lesions of the vessel wall. Brown and Goldstein11 proposed that cholesterol-loaded foam cells secrete both apoE and free cholesterol when there is an appropriate cholesterol acceptor in the vicinity. ApoA1 is a prototypical cholesterol acceptor. It is thought that free cholesterol effluxed by macrophage foam cells is transferred to the surface of HDL, where it can be esterified by LCAT and incorporated into the HDL core. At the same time, apoE secreted by the same macrophage foam cells in association with phospholipids is transferred to the HDL surface, where it can increase the capacity of HDL to accept additional cholesterol12 and ultimately facilitate its uptake by the liver. Although this is an attractive model to explain the beneficial role of HDL in atherosclerosis, the effect of apoA1–containing HDL on an accumulation of cholesterol in lesions in the presence or absence of locally produced apoE has not been examined in vivo. We therefore tested the hypothesis that apoA1–containing HDL plays a key role in decreasing the cholesterol content of lipid-rich lesions that contain macrophage-derived apoE. Using atherosclerosis-prone apoE–deficient and apoE–deficient plus apoA1–deficient mice, we used BMT to achieve macrophage apoE expression in lesions.
Throughout this study, plasma cholesterol levels of the
E/A-Io mice were lower. At week 8, when the animals had received the
HFD for 4 weeks, the plasma cholesterol in E/A-I mice was
more than double that of the E/A-Io mice. After 16 weeks on the HFD,
cholesterol was nearly 3-fold higher in the E/A-I mice.
Aside from the obvious discrepancy in HDL cholesterol
levels due to the presence or absence of apoA1, most of the remaining
differences in circulating cholesterol were due to
differences in IDL+LDL cholesterol (Figure 2).
Although the reason for this discrepancy is not known, it may be that
cholesterol clearance of IDL and LDL is delayed in E/A-I
mice compared with the E/A-Io mice, or that these same lipoproteins are
cleared more efficiently in E/A-Io mice. Alternatively, it is highly
likely that cholesterol absorption was hampered in the
E/A-Io mice owing to the lack of intestinal apoA1, and this may have
contributed to the lower plasma cholesterol levels. In any
case, further work will be needed to determine the reason for this
apparent difference in the plasma cholesterol levels.
The expected phenotype of the 2 BMT experimental groups was confirmed by 2 separate methods. First, Western blot analysis of the plasmas revealed that apoA1 was present as expected in the E/A-I mice but absent in the E/A-Io mice, regardless of treatment. ApoE was absent in all mice before BMT. However, on reconstitution with WT bone marrow cells, apoE was detected in the plasmas of all mice at <10% of WT levels. As reported previously,22 23 this amount of apoE was sufficient to lower the plasma cholesterol to near WT levels in all chow-fed mice. However, in animals fed an HFD, this amount of apoE is insufficient to reduce cholesterol levels to normal. Second, immunohistochemical examination of the aortic valve lesions revealed that apoB and apoE were present in all raised lesions. As expected, apoA1 was present in the lesions of the E/A-I mice but absent in the lesions of the E/A-Io mice. Thus, the transplanted WT macrophages that had migrated into the lesions expressed apoE. Importantly, this study also confirmed that (1) macrophages do not synthesize appreciable amounts of apoA1 and (2) as observed by others,13 lipoproteins containing apoB and apoA1 are capable of entering the arterial wall because they were detected in the lesions of the E/A-I mice.
The aortas of the E/A-Io mice contained 40% more total
cholesterol than those of E/A-I mice (P<0.05),
and this increase was due to accumulation of free rather than
esterified cholesterol. Compatible with the reverse
cholesterol transport model, this could imply that the lack
of apoA1–containing HDL prevented the efflux of
macrophage-derived free cholesterol from the
lesions. The lipid staining and cholesterol
analysis methods that we employed did not allow us to
distinguish between macrophage-derived and
lipoprotein-derived cholesterol in the lesions. Although
oil red O does not stain free cholesterol very well, the
pattern of staining in the aortic valve sections suggested that at
least the total lipid in lesions of the E/A-Io mice was more evenly
distributed and more abundant. Moreover, we recovered greater amounts
of free cholesterol than cholesteryl esters from the aortas
of both experimental groups (Figure 6). Therefore, although the
origin of the lesion free cholesterol is
controversial,29 the differences in lesion size
paralleled the differences in aortic free cholesterol.
It is feasible that this difference in free cholesterol was
due to differences in cholesterol influx into the vessel
wall. However, it is more likely that the free cholesterol,
either excreted by the foam cells or accumulated from modified or
aggregated lipoproteins, could not be removed from the vessel wall
owing to the lack of apoA1. This idea is supported by the finding that
the majority of the cholesterol entering the vessel wall is
a component of the apoB–containing lipoproteins, such as the
LDL.30 Thus, differences in HDL levels probably did not
cause any discrepancy in cholesterol entry into the vessel
wall. Clearly, more detailed studies are needed to elucidate the
mechanism behind the selective accumulation of free
cholesterol when apoA1 is absent.
Several in vivo studies have affirmed that apoA1 is antiatherogenic. Injection of either purified apoA1 or HDL into cholesterol-fed rabbits inhibits the formation of fatty streak lesions.31 32 33 When high levels of human apoA1 are expressed in transgenic mice, there is significant inhibition of fatty streak lesion formation,34 and the overexpression of human apoA1 in apoE–deficient mice results in a dramatic reduction in atherosclerosis.35 36 Similarly, atherosclerosis is reduced in cholesterol-fed transgenic rabbits that express human apoA1,37 and a deficiency of apoA1 can exacerbate atherosclerosis in human apoB–transgenic mice.38 Although "lesion" apoA1 was not measured in any of these other studies, it is reasonable to assume that higher plasma HDL levels would facilitate higher HDL penetration into the aortic wall, which could in turn lead to enhanced cholesterol removal from lesions. A delay in cholesterol removal has been reported in apoA1–deficient mice.39 Although HDL also contains apoA2 and A4, the function of these other apolipoproteins remains obscure. Whereas apoA2 is a poor acceptor of cholesterol and can inhibit the cholesterol acceptor role of apoA1,40 41 apoA4 is a reasonably good acceptor of cellular cholesterol in vitro.42 Nevertheless, our studies confirm that apoA1 is a key determinant of the free cholesterol content of lesions in vivo in the presence of apoE.
Despite their 2- to 3-fold lower levels of plasma cholesterol, the E/A-Io mice had a 45% larger mean lesion area than did the E/A-I mice. This confirms that apoA1–containing HDL and apoE participated in protection of the vessel wall from the effects of hypercholesterolemia. In addition, our estimates of cholesterol deposition in the aortas support the notion that apoA1 can enter the lesion and thereby participate with apoE in this protection. This may explain, at least in part, the antiatherogenic effects of HDL that have been reported in epidemiological studies as well as in vivo studies utilizing animal models.
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Acknowledgments |
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This work was supported by National Institutes of Health (Bethesda, Md) grants HL-35297 and HL-43815 to L.K.C. W.A.B. was supported by a fellowship from the American Heart Association, California Affiliate. The authors thank Kathi Richards for breeding and care of the animals.
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Footnotes |
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A portion of this work was presented at the 11th International Atherosclerosis Society meeting in Paris, France, October 5–9, 1997 and published in abstract form Atherosclerosis. 1997;134:365.
Received June 9, 1998; accepted July 31, 1998.
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