© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Low Levels of Extrahepatic Nonmacrophage ApoE Inhibit Atherosclerosis Without Correcting Hypercholesterolemia in ApoE-Deficient Mice
From the Department of Pharmacological Sciences (F.E.T., D.L.W.), University Medical Center, State University of New York at Stony Brook; the Department of Pathology (Comparative Medicine) (L.L.R.), Wake Forest University School of Medicine, Winston-Salem, NC; and the Poultry Science Department (R.L.W.), Texas A&M University, College Station.
Correspondence to David L. Williams, PhD, University Medical Center, State University of New York at Stony Brook, Stony Brook, NY 11794. E-maildave{at}pharm.sunysb.edu
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Abstract |
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Abstract—The prevention of atherosclerosis by apolipoprotein E (apoE) is generally attributed to the removal of plasma lipoprotein remnant particles. We developed transgenic apoE-knockout mice expressing apoE specifically in the adrenal gland and found that only 3% of the wild-type plasma level of apoE was sufficient to normalize plasma cholesterol levels in the apoE-deficient mouse. As expected, mice expressing apoE at levels that correct hypercholesterolemia had almost no cholesteryl ester deposition in their aortas. In contrast, their nontransgenic siblings had significant atherosclerosis. Unexpectedly, we found that atherosclerosis was also reduced in 2 transgenic lines expressing too little apoE (<1% to 2% of wild type) to correct their hypercholesterolemia. Gel exclusion chromatographic profiles of plasma lipoproteins and the size distributions of lipoproteins with density <1.063 (low density and very low density lipoproteins), as determined by dynamic laser light scattering, were the same in mice expressing <2 µg/mL plasma apoE and their nontransgenic littermates. We conclude that the antiatherogenic action of low levels of plasma apoE is not due to the clearance of remnant lipoproteins. Thus, low levels of apoE provided systemically, but not made in the liver or in macrophages, can block atherogenesis in the vascular wall independently of normalizing the plasma concentration of atherogenic remnant lipoprotein particles.
Key Words: apoE • atherosclerosis • hypercholesterolemia • transgenic apoE-knockout mice
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Introduction |
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In spite of our expanding knowledge of the causes of coronary heart disease, it remains the leading cause of death in the developed world.1 Elevated levels of atherogenic cholesteryl ester (CE)-rich lipoproteins are a major risk factor for atherosclerosis. These lipoproteins are believed to act in the artery wall to initiate or propagate complex inflammatory reactions leading to intimal accumulation of cholesterol-laden macrophage foam cells and the progressive development of atherosclerotic plaque.2 ApoE plays a critical role in plasma cholesterol homeostasis by mediating the hepatic uptake of CE-rich remnant lipoproteins by members of the LDL receptor family.3 Type III hyperlipoproteinemia and premature atherosclerosis occur in individuals lacking apoE4 and in a subset of individuals with apoE variants that are defective in binding to receptors.5 The apoE-knockout (ApoE-/-) mouse was the first mouse model to develop massive hypercholesterolemia and the full range of atherosclerotic lesions, even when maintained on a low-fat chow diet.6 7 ApoE-/- mice also have increased oxidation-specific autoantibodies8 and develop stenosis in peripheral arteries,9 as may result from atherosclerosis in humans.
Previous studies suggest that apoE may have a role in preventing atherosclerosis other than its role in remnant clearance. Watanabe LDL receptor–deficient rabbits given sustained intravenous apoE had reduced progression of lesions without a reduction in total plasma cholesterol (TPC).10 Also, transgenic C57BL/6 mice overexpressing apoE in the arterial wall had reduced atherosclerosis in response to a high-fat diet, although their plasma cholesterol concentrations and lipoprotein profiles were the same as those of the nontransgenic control mice.11 However, each of these cases reflects the effects of excess exogenous apoE in addition to the normal endogenous level of apoE.
In apoE-/- mice, a low level of apoE that was expressed either in the liver12 or in macrophages derived from wild-type bone marrow transplants13 14 15 was sufficient to normalize plasma cholesterol levels and prevent atherosclerosis. Bellosta et al16 showed that apoE expressed by macrophages, at levels too low to correct hypercholesterolemia in apoE-/- mice, significantly reduced atherosclerosis. They suggested that this protection was due to apoE production by macrophages in the arterial wall, leading to increased efflux of cholesterol from arterial macrophages, thereby inhibiting foam cell development.17
In the present study, we examined apoE-/- mice that express transgenic apoE selectively in the adrenal gland and have very low levels of circulating apoE. ApoE is normally expressed at relatively high levels in the adrenal glands of humans,18 nonhuman primates,19 and rodents.20 Hence, adrenal-specific expression provides a means to test the ability of extrahepatic nonmacrophage apoE to alter the systemic metabolism of plasma lipoprotein particles and the development of atherosclerosis.
In these transgenic apoE-/- mice, plasma apoE concentrations >2% to 4% of the wild-type level were sufficient to normalize plasma cholesterol levels and, as expected, prevent atherosclerosis. Unexpectedly, atherosclerosis was also dramatically reduced in 2 transgenic lines expressing too little apoE (<1% to 2% of wild type) to correct their plasma cholesterol levels. These data indicate that low levels of apoE in the plasma, not derived from the liver or macrophages, can prevent atherosclerosis independently of a role in maintaining plasma cholesterol homeostasis.
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Methods |
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Transgenic Mice Expressing ApoE in the Adrenal Gland
Mice were maintained on a 12-hour light/12-hour dark cycle with standard rodent chow and water ad libitum. Housing and experimental procedures were approved by the State University of New York at Stony Brook Committee on Laboratory Animal Resources. The DNA fragment used for oocyte microinjections contained
6 kb of the mouse
21-ß-hydroxylase promoter,21 attached to the mouse apoE
gene as described in Results. This construct was made by use of
standard molecular biology techniques22 and was grown in
Escherichia coli strain STBL2 (Life Technologies). We
coinjected a 2-fold molar excess of the 3-kb chicken lysozyme matrix
attachment region23 to improve specificity of
transgene expression.24 Transgenic mice were prepared in
the University Transgenic Mouse Facility, with FVB/N mice used as
donors and recipients. Positive founder mice were identified by triplex
polymerase chain reaction (PCR) analysis of DNA prepared from
tail biopsies with the use of QIAamp tissue kits (Qiagen Inc). Ten
independent founder lines were crossed onto apoE-/- mice
(C57BL/6x129), kindly provided by N. Maeda (Department of
Pathology, University of North Carolina, Chapel Hill).25
Mice carrying the transgene were crossed for 2 generations with
apoE-/- mice to generate mixed litters of mice either hemizygous for
the transgene on apoE-/- background or apoE -/- littermates.
Nontransgenic siblings were used as controls in all experiments to
minimize the effects of the mixed genetic background.
Western Blots
Tissues were ground in 20 mmol/L Tris-HCl (pH 7.4), 2
mmol/L MgCl2, 0.2 mol/L sucrose, 10 µg/mL
leupeptin, 20 µg/mL aprotinin, 5 µg/mL pepstatin, and 5 mmol/L
phenylmethylsulfonyl fluoride. Proteins26 (60
µg) were resolved on 10% polyacrylamide-SDS gels; 0.5 µL
of C57BL/6 plasma and 2.5 µL of the transgenic mouse plasma were also
analyzed. Samples were electrophoretically transferred to
nitrocellulose and immunostained as
described27 by use of an affinity-purified goat anti-rat
apoE (from P. Roheim, Department of Physiology, Louisiana State
University Medical Center). The secondary antibody was a
peroxidase-coupled anti-goat IgG (Sigma Chemical Co). Bands were
visualized by enhanced chemiluminescence (Amersham).
mRNA Measurement
RNA was prepared by grinding tissues in RNA STAT-60 (Tel-Test,
Inc) following the manufacturer’s protocol. Peritoneal
macrophages were elicited with thioglycolate broth and plated,
and adherent cells were maintained for 2 days.28 RNA was
made as described above, and RNA concentration was measured by
absorbance at 260 nm or with the RiboGreen fluorescent assay
kit (Molecular Probes). Tissue-specific expression was determined by
use of an RNA excess solution hybridization assay29 with a
mouse apoE–specific probe transcribed from pBSMAE7.30 A
reverse transcriptase–PCR protocol based on that of Rexin and
Feussner31 was developed for the macrophage RNA
with mouse apoE–specific primers.
Lipoprotein Analyses
Mice were fasted for 6 hours, anesthetized with inhaled
methoxyflurane, and bled from the tail into heparinized capillaries.
Plasma was obtained by centrifugation at
2000g, 4°C, for 20 minutes. TPC was assayed by use of the
Cholesterol CII assay (Wako Chemicals USA). Lipoprotein
profiles were analyzed by running 200 µL of plasma over a
25-mL Superose 6 column (Pharmacia) in PBS and 1 mmol/L EDTA at
0.5 mL/min; cholesterol was measured in each fraction.
Determination of LDL+VLDL Particle Size Distributions
Mice from line 619 were fasted, tail vein blood was collected,
and plasma was prepared as described above. Plasma from 9 male mice
carrying the transgene was pooled, as was plasma from 8 of their
apoE-/- siblings. The LDL+VLDL lipoprotein fraction (density <1.063)
was isolated by KBr density
ultracentrifugation.32 The isolated
fractions were dialyzed against 0.15 mol/L NaCl, 5 mmol/L EDTA,
and 0.05% sodium azide, and lipoprotein particle diameters were
determined by dynamic light scattering.33
Mouse ApoE ELISA
A mouse apoE sandwich ELISA, as previously described for human
apoE,34 used the affinity-purified goat anti-rat apoE
described above as the capture antibody. For the secondary antibody, an
anti-mouse apoE antiserum (BioDesign International) was
affinity-purified and biotinylated with sulfo-NHS-LC-biotin (Pierce).
This was followed by a streptavidin–horseradish peroxidase conjugate
(Life Technologies). An apoE–glutathione-S-transferase fusion protein,
expressed from pGEX-4T-1 (Pharmacia Biotech) containing the coding
region for amino acids 72 to 311 of mouse apoE, was used as the
standard in the ELISA. ApoE-/- plasma was added to the standard in
equal volume to the plasma samples. The final mass determination was
corrected for the percentage of the fusion protein that is apoE.
Measurement of CE Deposition in the Aorta
After 1 year on a standard chow diet, mice were
anesthetized with ketamine HCl (80 mg/kg) and xylazine
(1.2 mg/kg) administered intraperitoneally. Blood
was drawn by cardiac puncture into a syringe rinsed with 0.2 mol/L
EDTA, and plasma was obtained by centrifugation at
4°C. With the heart attached, the aorta was removed down to the ileal
bifurcation and fixed in 10% neutral buffered formalin and 5% sucrose
at 4°C. After removing the adventitia, aorta CE concentration was
determined as previously described.35 For visual
evaluation, aortas were fixed, cleaned, opened longitudinally, and
stained with Sudan IV.13 Aortas were pinned out en face
and photographed with a Leaf digital camera attached to a dissecting
microscope. Pin shadows were removed by using the airbrush function of
Adobe PhotoShop set to adjacent tissue color.
Statistical Analysis
Statistical significance was determined by the Student
t test if the data distributions were gaussian or a
Mann-Whitney U test if not. A value of P<0.05 was
considered significant. All values are presented as
mean±SEM.
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Results |
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Transgenic Mice Expressing ApoE in the Adrenal Gland
An adrenal-specific transgene was made with the promoter for 21-ß-hydroxylase, followed by the mouse genomic apoE sequence. 21-ß-Hydroxylase expression is unique to the adrenal gland and is highly tissue specific in transgenic mice when driving ß-galactosidase expression.21 We removed a 1.2-kb mouse retroviral remnant, containing a putative androgen response element, from the promoter region by PCR-mediated mutagenesis to prevent a sex bias in expression from the transgene.36 Microinjected oocytes were implanted in 2 FVB/N females, resulting in 10 pups with the transgene in the germ line. These founders were crossed for 2 generations with apoE-/- mice (C57BL/6x129) to generate mice producing apoE only from the transgene.
Detection of ApoE Protein in the Adrenal Gland and Plasma of the
Transgenic Knockout Mice
Western blotting of adrenal lysates showed wide variation in apoE
expression among the transgenic lines (Figure 1A
). Line 621 had only a trace of signal.
Line 624 also had low adrenal expression, whereas most of the other
lines made more apoE in the adrenal than did wild-type C57BL/6. Because
the transgenic lines had robust adrenal expression, we assayed plasma
samples for apoE by Western blotting (Figure 1B). The lines had
various levels of plasma apoE that were all considerably less than the
levels in C57BL/6. Except for line 616, the females had dramatically
lower levels of plasma apoE than did the males (and lower levels of
adrenal apoE, data not shown), suggesting that there is still an active
androgen response element in the 21-ß-hydroxylase promoter of the
transgene. Because of this difference, males were used in subsequent
studies.
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Tissue Specificity of Transgenic ApoE Expression
Lysates (100 µg protein) of kidney, liver, lung, spleen, adrenal
gland, and testis were assayed for apoE by Western blot. In the 2 lines
having the highest plasma levels of apoE, lines 614 and 616, we
observed a very faint signal in the liver and the testis in males. We
did not detect apoE in the liver or ovary in females from these lines,
only adrenal apoE (data not shown). The other lines had detectable apoE
expression only in the adrenal gland. To determine the tissue
specificity of expression from the transgene with more sensitivity, we
used an RNase protection assay to determine the amount of apoE mRNA in
the liver, adrenal gland, brain, and testes. We did not detect brain
apoE expression in any of the lines. Lines 619, 620, 621, and 630 had
no detectable apoE mRNA expression in the liver or the testes. The
levels of apoE mRNA in the adrenal glands in the various lines roughly
correlated with the observed protein levels in the Western blots. Line
614 had the highest apoE expression (494 pg apoE mRNA per microgram
total RNA), and line 619 had the lowest adrenal expression. For
transgenic lines 619 and 620, in which apoE expression was detected
only in the adrenal gland, line 619 males had 1.6 pg apoE mRNA per
microgram total RNA, and line 620 males had 36.9 pg apoE mRNA per
microgram total RNA. Wild-type mice averaged 10 pg apoE mRNA per
microgram total adrenal RNA, similar to the level in the adrenal glands
of rats.37 We did not detect apoE mRNA in peritoneal
macrophages from even the high apoE–expressing lines with the
use of a very sensitive reverse transcriptase–PCR assay. In contrast,
a control housekeeping message, cyclophilin, was readily amplified from
the same reverse transcription reactions (data not shown).
Correlation of Plasma ApoE and Plasma Cholesterol Levels
Although the levels of apoE in the plasma of the transgenic
knockout mice were low, we determined TPC levels in 2 of the highest
expressing lines to determine whether there was any reduction relative
to the nontransgenic knockout littermates. As an estimation of
wild-type levels, we used littermates without the transgene but
heterozygous for the normal mouse apoE allele. For line 614 with
the transgene, TPC was 123.2±12.1 mg/dL (plasma apoE 2.20±0.15
µg/mL, n=5). Line 614 without the transgene had a TPC of 843.1±137.4
mg/dL (n=6, P=0.0005), and mice heterozygous for normal
mouse apoE had a TPC of 83.8±5.9 mg/dL (n=4). Similarly, for line 615
with the transgene, TPC was 132.2±12.3 mg/dL (plasma apoE 1.96±0.28
µg/mL, n=5), and without the transgene, TPC was 792.5±164.1 mg/dL
(n=4, P=0.0013). The knockout mice carrying the transgene
have plasma cholesterol levels that are almost normalized
to the levels of their littermates heterozygous for wild-type mouse
apoE. (C57BL/6 mice typically have TPC levels ranging from 70 to 100
mg/dL.)
Because the various transgenic lines showed a gradient of plasma apoE,
the relationship between TPC and plasma apoE concentration was
determined with age-matched males from each line, and from the parental
apoE-/- mice, by using an ELISA for mouse apoE. The data shown in
Figure 2 demonstrate an inflection point
in the curve at 2 µg/mL of apoE; above this concentration, TPC was
similar to that in wild-type mice. Between 2 µg/mL and 0 µg/mL of
plasma apoE, an inverse linear correlation between apoE concentration
and cholesterol (r=-0.819,
P<0.0001) was seen. These data show that only 3% of
wild-type plasma apoE was sufficient to correct the
hypercholesterolemia of apoE-/- mice.
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Reduction in Atherosclerosis
With such a dramatic effect of low levels of apoE on plasma
cholesterol concentration, we tested whether a similar
reduction would be seen in atherosclerosis in the
transgenic knockout lines. For these experiments, we used lines of mice
that had almost normalized TPC, lines 614, 615, and 616. They were
maintained on a standard chow diet for 9 months to 1 year. (Complex
fibrous lesions are observed in apoE-/- mice by 8
months.6 ) We used age-matched knockout siblings as
controls. Aortic CE deposition was used as a measure of
atherosclerosis, because studies with apoE-deficient
mice have shown that aortic CE deposition provides as good a measure of
aortic atherosclerosis as measurements of surface area
covered by lesions.38 We have also observed a strong
correlation (r2=0.8528,
P<0.0001) between aortic CE and lesion surface area in the
aortas of LDL receptor–deficient human apoB transgenic mice (J.K.
Sawyer, L.L. Rudel, unpublished data, 1999). As seen in Figure 3, there was an almost complete
suppression of aortic CE deposition in the transgenic mice, in striking
contrast to their knockout siblings (P<0.0001), who showed
a range of atherosclerosis typically seen in apoE-/-
mice.39 40 Thus, plasma apoE concentrations as low as
2 µg/mL were sufficient to normalize plasma cholesterol
and prevent atherosclerosis.
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Having observed nearly complete protection from
atherosclerosis development in mice with >2 µg/mL
apoE, we measured atherosclerosis in transgenic lines
that have too little apoE to normalize plasma cholesterol
(0.5 to 1.5 µg/mL apoE). These mice all are
hypercholesterolemic and have plasma lipoprotein
profiles indistinguishable from those of their apoE-/- siblings on
Superose 6 gel exclusion chromatography (Figure 4A and 4B). In addition, as seen in
Figure 4C, we observed no significant differences in LDL+VLDL
particle sizes or distributions between line 619 versus apoE-/-
littermates as assayed by dynamic light scattering. The mean particle
diameter was 40.67±1.66 nm for the line 619 transgenic particles and
40.03±0.78 nm for the nontransgenic particles. There were no
significant differences between the sets in any of the size divisions.
Thus, the level of apoE in line 619 was too low to alter the
distribution of plasma lipoprotein remnants.
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Atherosclerosis in these very low apoE–expressing mice
was evaluated by en face examination after staining with Sudan IV to
reveal lipid-laden lesions (Figure 5).
Unexpectedly, we saw almost no lesion staining in the transgenic mice,
in spite of their persistent
hypercholesterolemia. One small lesion in the
line 620 transgenic male is evident in the aortic arch, but there are
no lesions in the 619 transgenic male. This is in marked contrast to
line 620 nontransgenic males, who have lesions throughout the aorta.
Aortas from line 619 mice not carrying the transgene also have lesions
throughout the aorta (data not shown). CE deposition was measured in
aortas of chow-fed males of transgenic lines 619 and 620 compared with
their nontransgenic-knockout siblings (Figure 6A). In both transgenic lines, there was
an almost complete suppression of lesion development in the mice over
time, from 7.5 months up to >2 years of age. There was no correlation
between age and the amount of aorta CE in these mice. However, we saw a
typical spread in aortic CE deposition and an increase with age in
their apoE-/- siblings. Line 619 has an 86% reduction in CE
deposition in the transgenic mice relative to their nontransgenic
siblings, and line 620 has a 95% reduction. The difference in aorta CE
between the transgenic mice (n=22) and nontransgenic mice (n=15) was
significant by a Mann-Whitney U test (P<0.0001).
As shown in Figure 6B, there was no significant change in TPC
with age for either group.
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For the line 619 mice, there was no significant difference in the
plasma cholesterol concentrations between the transgenic
mice and their nontransgenic siblings (for transgenic mice, TPC
580.7±73.4 mg/dL, n=9; for nontransgenic mice, TPC 646.6±54.4 mg/dL,
n=6; P=0.53). In spite of this high TPC, there is a striking
difference in the aortic CE accumulation (for transgenic mice,
6.91±2.01 µg/mg protein; for nontransgenic mice, 50.33±14.02
µg/mg protein; P=0.0037, by Mann-Whitney U
test). There was a significant difference in the mean TPC for
transgenic and nontransgenic line 620 mice; TPC levels were 302.4±48.1
mg/dL for transgenic mice (n=13) and 662.2±63.3 for nontransgenic mice
(n=9, P=0.0002). However, there was overlap between the 2
groups, and the transgenic mice were all
hypercholesterolemic. When we chose groups of
transgenic and nontransgenic mice from both lines that were matched for
TPC, as shown in the Table, we
still observed a dramatic and significant reduction in aortic CE
deposition that was due to low levels of apoE.
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Discussion |
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Our results showed that mice with levels of plasma apoE too low to correct the hypercholesterolemia of their apoE-/- background had almost complete suppression of atherosclerotic lesion development. The apoE in our mice came from neither the liver nor macrophages and seems to be protective by a mechanism independent of plasma cholesterol concentration or remnant particle clearance.
The mechanism by which low levels of systemic apoE protect against
atherosclerosis is unknown. One possibility is that the
low levels of apoE allow for the selective clearance of a minor, but
highly atherogenic, remnant lipoprotein subfraction. To test this
possibility, we examined the size distribution of remnant lipoproteins
in mice expressing low levels of apoE compared with their nontransgenic
littermates. Gel exclusion chromatography showed no
difference in the size profile due to low-level apoE expression (Figure
4A and 4B). A more sensitive analysis of size
differences was provided by dynamic laser light scattering of remnant
particles in the LDL+VLDL size range (density <1.063 g/mL). This
analysis also failed to detect a difference in the LDL+VLVL
particle size distribution due to low-level apoE expression (Figure
4C). This method would detect a change of as little as 2.5% of
the total remnant population if it occurred at a specific size range,
because the maximum analyzed in any sample bin, in the midrange
of particle sizes, is 10% of the total population. The sensitivity
of the analysis is increased at the extremes of particle size.
Nevertheless, we detected no significant difference due to low-level
apoE expression in any bin within the entire profile. These data argue
strongly that low-level apoE expression did not perturb remnant
metabolism. The mean particle diameters observed
(40.67±1.66 nm for line 619 plus the transgene and 40.03±0.78 nm
without the transgene) were very similar to those previously found for
VLDL in apoE-/- mice (41.1±11.0 nm, mean±SD41 ). These
results strongly suggest that the antiatherogenic action of low-level
apoE expression in line 619 is not due to the removal of a minor
fraction of highly atherogenic remnant lipoproteins.
In agreement with our results, Hasty et al42 observed no
differences in the gel exclusion chromatographic profiles,
nondenaturing gel electrophoresis, or lipoprotein clearance rates that
were due to the low levels of apoE when they expressed apoE in
macrophages via a bone marrow transplantation protocol. They
observed a reduction in the fraction of total lipoproteins of <30-nm
diameter via negative-stain electron microscopy of total plasma from
mice with low-level apoE expression. We observed no differences in
particle diameters between low-level apoE–expressing mice and
apoE-/- mice by dynamic light scattering with particles in the
LDL+VLDL size range (Figure 4C). Similarly, in a single
analysis of the <1.063-g/mL density fraction via
negative-stain electron microscopy (measuring diameters of 800 to 900
particles for each population), we did not detect significant
differences in particle diameters due to low- level apoE expression
(R.H. Hamilton, F.E. Thorngate, D.L. Williams, unpublished data, 1999).
Thus, in transgenic mice and their nontransgenic littermates with
equivalent hypercholesterolemia, low-level apoE
expression provided protection against atherosclerosis
without altering the total level, size distribution, or mean particle
size of remnant lipoproteins in the LDL+VLDL fraction.
The response to retention hypothesis states that a key event in the initiation of atherogenesis is the subendothelial retention of atherogenic particles within the artery wall,43 thereby initiating a cascade of events that lead to macrophage recruitment and intimal foam cell formation. In a recent report, freeze-etch electron microscopy showed an accumulation of remnant lipoproteins associated with the intimal extracellular matrix of aortas in apoE-/- mice as early as 3 weeks of age.44 This event precedes the appearance of adherent monocytes and intimal foam cells and may be the initial event in atherosclerosis. One hypothesis derived from the present results is that low levels of systemic apoE suppress this early remnant lipoprotein retention. Other possibilities are that apoE acts at downstream steps to suppress the upregulation of endothelial vascular cell adhesion molecule-145 or to suppress the action of chemokines that promote monocyte migration and macrophage foam cell formation.46 Each of these is an early event that precedes significant lipid accumulation in the artery wall. It is interesting to note that apoE has a variety of hormone- or cytokine-like effects in steroidogenic cells,47 48 platelets,49 and lymphocytes.50 Thus, there is significant precedent to speculate that cytokine-like effects of apoE in the artery wall may underlie its atheroprotective actions.
Another potential antiatherogenic effect of low levels of apoE is to promote cholesterol efflux from macrophages in the arterial wall, as suggested by Bellosta et al16 on the basis of their results with transgenic mice expressing apoE in macrophages. If this mechanism is operative, the present results with adrenal-specific apoE expression indicate that low levels of systemic apoE can produce this effect without the need to have apoE expressed by the macrophage itself. The lines of transgenic mice described in the present study can be used to test each of the above hypotheses.
These studies demonstrated that only 2 µg/mL of plasma apoE is
sufficient to maintain normal TPC on an apoE-knockout background.
Between 2 µg/mL and 0 µg/mL of plasma apoE, an inverse linear
correlation between apoE concentration and cholesterol
(r=-0.819, P<0.0001) was seen. This result
suggests a simple dose-response relationship between plasma apoE
concentration and the clearance of remnant lipoproteins. A similar
result was seen by Hasty et al42 when apoE was
expressed by macrophages derived from bone marrow transplants.
Our data as well as those of Hasty et al yield the surprising result
that only 3% of wild-type plasma apoE was sufficient to correct the
hypercholesterolemia of apoE-/- mice. On the
basis of the results with our transgenic lines 621 and 630, in which
liver apoE expression was not detected, it appears that this low level
of plasma apoE is sufficient to mediate remnant lipoprotein clearance
even when the apoE is made in a peripheral tissue and not
in either the liver or macrophages. An interesting question
arising from these results is why apoE is normally expressed in mice at
levels greater (
30-fold) than are necessary to prevent
atherosclerosis and to maintain normal remnant particle
clearance. One possibility is that there are other actions of apoE that
are less sensitive to apoE concentration and that have dose-response
relationships shifted to higher plasma apoE concentrations. Such
possibilities could include the effects of apoE on ovarian or
adrenal steroid production,47 48 platelet
aggregation,49 lymphocyte activation,50 or
activation of hepatic lipase.51 Additional studies will be
required to test these possibilities.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-32868 (D.L.W.), HL-49373 (L.L.R.), and HL-6084401 (R.L.W.). F.E.T. was partially supported by Institutional National Research Award IT32-DK-07521. The authors thank P.A. Strockbine, J.K. Sawyer, and Dr M.D. Wilson for technical assistance. S. Busby made the apoE–glutathione-S-transferase fusion protein construct. We appreciate the expertise and advice of Dr T. Rosenquist of the University Transgenic Mouse Facility and D. Colflesh of the University Microscopy and Imaging Center. We thank Drs K.L. Parker, N. Maeda, and W.H. Strätling for plasmids and Dr P. Roheim for antiserum.
Received February 21, 2000; accepted March 20, 2000.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Breslow JL. Cardiovascular disease burden increases, NIH funding decreases. Nat Med. 1997;3:600–601.[Medline] [Order article via Infotrieve]
2.
Ross R. Atherosclerosis: an
inflammatory disease. N Engl J Med. 1999;340:115–126.
3.
Mahley RW. Apolipoprotein E: cholesterol
transport protein with expanding role in cell biology.
Science. 1988;240:622–630.
4. Schaefer EJ, Gregg RE, Ghiselli G, Forte TM, Ordovas JM, Zech LA, Brewer HB Jr. Familial apolipoprotein E deficiency. J Clin Invest. 1986;78:1206–1219.
5. Mahley RW, Rall SC Jr. Type III hyperlipoproteinemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. New York, NY: McGraw-Hill; 1989:1195–1213.
6.
Reddick RL, Zhang SH, Maeda N.
Atherosclerosis in mice lacking apo E: evaluation of
lesional development and progression. Arterioscler Thromb. 1994;14:141–147.
7.
Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R.
ApoE-deficient mice develop lesions of all phases of
atherosclerosis throughout the arterial
tree. Arterioscler Thromb. 1994;14:133–140.
8.
Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D,
Witztum JL. ApoE-deficient mice are a model of lipoprotein oxidation in
atherogenesis: demonstration of oxidation-specific epitopes in lesions
and high titers of autoantibodies to malondialdehyde-lysine in serum.
Arterioscler Thromb. 1994;14:605–616.
9.
Seo HS, Lombardi DM, Polinsky P, Powell-Braxton L,
Bunting S, Schwartz SM, Rosenfeld ME. Peripheral vascular
stenosis in apolipoprotein E-deficient mice: potential roles of
lipid deposition, medial atrophy, and adventitial inflammation.
Arterioscler Thromb Vasc Biol. 1997;17:3593–3601.
10. Yamada N, Inoue I, Kawamura M, Harada K, Watanabe Y, Shimano H, Gotoda T, Shimada M, Kohzaki K, Tsukada T, et al. Apolipoprotein E prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits. J Clin Invest. 1992;89:706–711.
11. Shimano H, Ohsuga J, Shimada M, Namba Y, Gotoda T, Harada K, Katsuki M, Yazaki Y, Yamada N. Inhibition of diet-induced atheroma formation in transgenic mice expressing apolipoprotein E in the arterial wall. J Clin Invest. 1995;95:469–476.
12.
Smith JD, Wong E, Ginsberg M. Cytochrome P450 1A1
promoter as a genetic switch for the regulatable and
physiological expression of a plasma protein in
transgenic mice. Proc Natl Acad Sci U S A. 1995;92:11926–11930.
13. Boisvert WA, Spangenberg J, Curtiss LK. Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation. J Clin Invest. 1995;96:1118–1124.
14.
Linton MF, Atkinson JB, Fazio S. Prevention of
atherosclerosis in apolipoprotein E-deficient mice by
bone marrow transplantation. Science. 1995;267:1034–1037.
15.
Van Eck M, Herijgers N, Yates J, Pearce NJ,
Hoogerbrugge PM, Groot PH, Van Berkel TJ. Bone marrow
transplantation in apolipoprotein E–deficient mice: effect of apoE
gene dosage on serum lipid concentrations, (beta)VLDL catabolism, and
atherosclerosis. Arterioscler Thromb Vasc
Biol. 1997;17:3117–3126.
16. Bellosta S, Mahley RW, Sanan DA, Murata J, Newland DL, Taylor JM, Pitas RE. Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice. J Clin Invest. 1995;96:2170–2179.
17.
Zhu Y, Bellosta S, Langer C, Bernini F, Pitas RE,
Mahley RW, Assmann G, von Eckardstein A. Low-dose expression of a human
apolipoprotein E transgene in macrophages restores
cholesterol efflux capacity of apolipoprotein E-deficient
mouse plasma. Proc Natl Acad Sci U S A. 1998;95:7585–7590.
18.
Blue M-L, Williams DL, Zucker S, Khan SA, Blum CB.
Apolipoprotein E synthesis in human kidney, adrenal gland, and liver.
Proc Natl Acad Sci U S A. 1983;80:283–287.
19.
Williams DL, Dawson PA, Newman TC, Rudel LL.
Apolipoprotein E synthesis in peripheral tissues of
nonhuman primates. J Biol Chem. 1985;260:2444–2451.
20. Dawson PA, Lukaszewski LM, Ells PE, Malbon CC, Williams DL. Quantification and regulation of apolipoprotein E expression in rat Kupffer cells. J Lipid Res. 1989;30:403–413.[Abstract]
21.
Morely SD, Viard I, Chung BC, Ikeda Y, Parker KL,
Mullins JJ. Variegated expression of a mouse steroid
21-hydroxlase/b-galactosidase transgene suggests centripetal migration
of adrenocortical cells. Mol Endocrinol. 1996;10:585–598.
22. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA. Current Protocols in Molecular Biology. New York, NY: John Wiley & Sons, Inc; 1995.
23.
von Kries JP, Phi-Van L, Diekmann S, Stratling WH. A
non-curved chicken lysozyme 5' matrix attachment site is 3' followed by
a strongly curved DNA sequence. Nucleic Acids Res. 1990;18:3881–3885.
24.
McKnight RA, Shamay A, Sankaran L, Wall RJ,
Hennighausen L. Matrix-attachment regions can impart
position-independent regulation of a tissue-specific gene in transgenic
mice. Proc Natl Acad Sci U S A. 1992;89:6943–6947.
25.
Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda
N. Generation of mice carrying a mutant apolipoprotein E gene
inactivated by gene targeting in embryonic stem cells.
Proc Natl Acad Sci U S A. 1992;89:4471–4475.
26.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–275.
27.
DeMattos RB, Thorngate FE, Williams DL. A test of the
cytosolic apolipoprotein E hypothesis fails to detect the escape of
apolipoprotein E from the endocytic pathway into the cytosol and shows
that direct expression of apolipoprotein E in the cytosol is cytotoxic.
J Neurosci. 1999;19:2464–2473.
28. Meltzer MS. Peritoneal mononuclear phagocytes from small animals. In: Methods for Studying Mononuclear Phagocytes. New York, NY: Academic Press; 1981:63–67.
29.
Sensel MG, Binder R, Lazier CB, Williams DL.
Reactivation of apolipoprotein II gene transcription by cycloheximide
reveals two steps in the deactivation of estrogen receptor-mediated
transcription. Mol Cell Biol. 1994;14:1733–1742.
30.
Nicosia M, Prack MM, Williams DL. Differential
regulation of apolipoprotein-E messenger RNA in zona fasciculata cells
of rat adrenal gland determined by in situ hybridization. Mol
Endocrinol. 1992;6:288–298.
31.
Rexin M, Feussner G. A competitive reverse
transcription-PCR to study apolipoprotein epsilon gene expression.
Clin Chem. 1998;44:773–778.
32. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345–1353.
33. Walzem RL, Davis PA, Hansen RJ. Overfeeding increases very low density lipoprotein diameter and causes the appearance of a unique lipoprotein particle in association with failed yolk deposition. J Lipid Res. 1994;35:1354–1366.[Abstract]
34.
DeMattos RB, Curtiss LK, Williams DL. A
minimally-lipidated form of cell-derived apolipoprotein E exhibits
isoform-specific stimulation of neurite outgrowth in the absence of
exogenous lipids or lipoproteins. J Biol Chem. 1998;273:4206–4212.
35.
Rudel LL, Kelley K, Sawyer JK, Shah R, Wilson MD.
Dietary monounsaturated fatty acids promote aortic
atherosclerosis in LDL receptor-null, human
apoB100-overexpressing transgenic mice. Arterioscler Thromb Vasc
Biol. 1998;18:1818–1827.
36. Stavenhagen JB, Robbins DM. An ancient provirus has imposed androgen regulation on the adjacent mouse sex-limited protein gene. Cell. 1988;55:247–254.[Medline] [Order article via Infotrieve]
37. Prack MM, Nicosia M, Williams DL, Gwynne J. Relationship between apolipoprotein E mRNA expression and tissue cholesterol content in rat adrenal gland. J Lipid Res. 1991;32:1611–1618.[Abstract]
38. Daugherty A, Pure E, Delfel-Butteiger D, Chen S, Leferovich J, Roselaar SE, Rader DJ. The effects of total lymphocyte deficiency on the extent of atherosclerosis in apolipoprotein E-/- mice. J Clin Invest. 1997;100:1575–1580.[Medline] [Order article via Infotrieve]
39.
Fazio S, Babaev VR, Murray AB, Hasty AH, Carter KJ,
Gleaves LA, Atkinson JB, Linton MF. Increased
atherosclerosis in mice reconstituted with
apolipoprotein E null macrophages. Proc Natl Acad Sci
U S A. 1997;94:4647–4652.
40.
Aiello RJ, Bourassa PK, Lindsey S, Weng W, Natoli E,
Rollins BJ. Monocyte chemoattractant protein-1 accelerates
atherosclerosis in apolipoprotein E-deficient Mice.
Arterioscler Thromb Vasc Biol. 1999;19:1518–1525.
41. Veniant MM, Zlot CH, Walzem RL, Pierotti V, Driscoll R, Dichek D, Herz J, Young SG. Lipoprotein clearance mechanisms in LDL receptor-deficient ‘apo-B48-only’ and ‘apo-B100-only’ mice. J Clin Invest. 1998;102:1559–1568.[Medline] [Order article via Infotrieve]
42.
Hasty AH, Linton MF, Swift LL, Fazio S. Determination
of the lower threshold of apolipoprotein E resulting in remnant
lipoprotein clearance. J Lipid Res. 1999;40:1529–1538.
43.
Williams KJ, Tabas I. The response-to-retention
hypothesis of early atherogenesis. Arterioscler Thromb Vasc
Biol. 1995;15:551–561.
44.
Tamminen M, Mottino G, Qiao JH, Breslow JL, Frank JS.
Ultrastructure of early lipid accumulation in apoE-deficient mice.
Arterioscler Thromb Vasc Biol. 1999;19:847–853.
45.
Nakashima Y, Raines EW, Plump AS, Breslow JL, Ross R.
Upregulation of VCAM-1 and ICAM-1 at
atherosclerosis-prone sites on the
endothelium in the apoE-deficient mouse.
Arterioscler Thromb Vasc Biol. 1998;18:842–851.
46. Terkeltaub R, Boisvert WA, Curtiss LK. Chemokines and atherosclerosis. Curr Opin Lipidol. 1998;9:397–405.[Medline] [Order article via Infotrieve]
47.
Reyland ME, Williams DL. Suppression of cAMP-mediated
signal transduction in mouse adrenocortical cells which express
apolipoprotein E. J Biol Chem. 1991;266:21099–21194.
48.
Dyer CA, Curtiss LK. Apoprotein E-rich high density
lipoproteins inhibit ovarian androgen synthesis. J
Biol Chem. 1988;263:10965–10973.
49.
Riddell DR, Graham A, Owen JS. Apolipoprotein E
inhibits platelet aggregation through the L-arginine:nitric oxide
pathway: implications for vascular disease. J Biol
Chem. 1997;272:89–95.
50. Pepe MG, Curtiss LK. Apolipoprotein E is a biologically active constituent of the normal immunoregulatory lipoprotein, LDL-In. J Immunol. 1986;136:3716–3723.[Abstract]
51. Thuren T, Weisgraber KH, Sisson P, Waite M. Role of apolipoprotein E in hepatic lipase catalyzed hydrolysis of phospholipid in high-density lipoproteins. Biochemistry. 1992;31:2332–2338.[Medline] [Order article via Infotrieve]
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