From the Department of Medicine and ¶ Center for
Experimental Therapeutics, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104, the ** University of
Delaware, Newark, Delaware 19711, and the
Wistar
Institute, Philadelphia, Pennsylvania 19104
Received for publication, April 18, 2000, and in revised form, September 20, 2000
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ABSTRACT |
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Apolipoprotein E is a multifunctional protein
synthesized by hepatocytes and macrophages. Plasma apoE is largely
liver-derived and known to regulate lipoprotein metabolism.
Macrophage-derived apoE has been shown to reduce the progression of
atherosclerosis in mice. We tested the hypothesis that liver-derived
apoE could directly induce regression of pre-existing advanced
atherosclerotic lesions without reducing plasma cholesterol levels.
Aged low density lipoprotein (LDL) receptor-deficient
(LDLR Apolipoprotein E (apoE) is a multifunctional protein produced
largely by the liver and mononuclear phagocytes (1). Although its
physiologic role is uncertain, macrophage-derived apoE can inhibit
progression of atherogenesis. Expression of macrophage apoE in
apoE-deficient mice using bone marrow transplantation reduced plasma
cholesterol levels and slowed progression of atherosclerosis (2, 3).
Tissue-specific transgenic expression reduced atherosclerosis independent of plasma cholesterol levels (4). Transplantation of
apoE-deficient bone marrow in wild-type mice increased atherosclerosis (5), consistent with a role of macrophage-derived apoE in inhibiting progression of atherosclerosis.
Liver-derived apoE is the major source of plasma apoE and has an
important physiologic role in regulating lipoprotein metabolism (1).
However, the potential role of liver-derived apoE in atherogenesis independent of its role in lipoprotein metabolism is uncertain. Expression of apoE in the liver of apoE-deficient mice using an adenoviral vector slowed the progression of atherosclerosis (6). We
demonstrated that adenoviral-mediated hepatic expression of apoE in
apoE-deficient mice resulted in marked regression of both early and
advanced atherosclerotic lesions (7), an observation confirmed
subsequently by another group (8). However, in all of these
experiments, hepatic expression of apoE in apoE-deficient mice markedly
reduced plasma cholesterol levels, making it impossible to assess the
direct effect of liver-derived apoE on atherosclerosis.
A previous study showed that repeated injection of purified apoE to
Watanabe Heritable Hyperlipidemic rabbits lacking functional LDL
receptors resulted in reduced progression of atherosclerosis without
lowering plasma cholesterol levels (9). We subsequently demonstrated
that hepatic expression of apoE in
LDL1 receptor-deficient mice
fed a western-type diet did not reduce plasma cholesterol levels but
nevertheless slowed the progression of early fatty streak
atherosclerotic lesions (10). Expression of apoE by the adrenals at
plasma levels below the threshold for reducing plasma cholesterol
levels reduced atherosclerosis in apoE-deficient mice (11). However,
regression of atherosclerosis by apoE derived from any source in the
absence of cholesterol reduction has never been demonstrated. In the
current study, we extended previous observations by testing the
hypothesis that apoE expression would induce regression of pre-existing
advanced atherosclerotic lesions in the absence of significant
reduction in plasma cholesterol.
The mechanisms by which apoE directly inhibits atherosclerosis are
poorly understood. One of the key factors that plays a causative role
in development and progression of atherosclerosis is oxidative stress
(12). Oxidative stress in the artery wall contributes to the
inflammatory nature of atherosclerotic lesions (12, 13) and could
potentially contribute to plaque rupture (14). Products of lipoprotein
oxidation are present in vessel wall and have proinflammatory effects
that could promote atherogenesis (15). ApoE-deficient mice exhibit
evidence of markedly increased lipoprotein oxidation as assessed by
autoantibodies to oxidized lipoproteins (15). ApoE has been shown
in vitro to have antioxidant (16) and anti-inflammatory (17)
properties. In this study, we investigated effects of apoE expression
on in vivo oxidative stress by quantitating isoprostanes in
multiple tissues. Isoprostanes are stable prostaglandin isomers
generated during free radical-mediated lipid peroxidation and have been
shown to be reliable markers of lipid peroxidation and oxidative stress
generation in vivo (18). We previously reported that urine,
plasma, and arterial iPF2 Animal Experimental Protocol--
Recombinant second generation
adenovirus encoding the human apoE3 and the control empty adenovirus
(Adnull) were constructed as described previously (7, 20). A total of
27 LDLR Lipid and Lipoprotein Analyses--
Plasma cholesterol and
triglyceride levels were measured enzymatically on a Cobas Fara II
(Roche Diagnostic Systems Inc.) using Sigma reagents. Plasma
apoE levels were measured using an immunoturbidometric assay (Sigma).
Plasma samples (200 µl pooled from nine mice each of Adnulll and
AdhapoE3 group) were analyzed by FPLC gel filtration (Amersham
Pharmacia Biotech, Uppsala, Sweden) on two Superose 6 columns as
described previously (20). The cholesterol concentrations in the FPLC
fractions were determined using an enzymatic assay (Wako Pure Chemical
Industries Ltd., Osaka, Japan).
Lipoproteins were isolated from plasma samples by sequential
ultracentrifugation. The plasma samples obtained at various time points
during the experiment were pooled from nine mice each (250 µl) of
Adnull and AdhapoE3 group. The samples were subjected to ultracentrifugation in 1-ml polycarbonate tubes at 90,000 rpm in a
Beckman TLA 100.2 rotor for 3 h at 10 °C (TL-100 centrifuge, Beckman Instruments Inc.). The VLDL (d<1.006), LDL (1.006 < d < 1.063) and HDL (1.063 < density < 1.21) were isolated by tube slicing in a volume of 250 µl. The
lipoprotein fractions were analyzed for the lipid and protein content.
Western blotting analysis for the detection of human apoE in aortas was
performed using extracts prepared from the lower abdominal abdominal
portion of the aortas from base-line, Adnull, and AdhapoE3 mice. The
aortas were minced on ice and homogenized in 250 µl of PBS containing
a mixture of protease inhibitors (complete miniTM, Roche Molecular
Biochemicals). Aliquots of the aortic extracts (10 and 15 µg
of protein) were resuspended in 40 µl of Laemli buffer (Bio-Rad) and
heated at 95 °C for 5 min. The samples were subjected to 10-20%
linear gradient SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose membrane (HybondTM ECL, Amersham Pharmacia
Biotech). The presence of human apoE was detected using anti-human apoE3 monoclonal antibody as primary and horseradish peroxidase-labeled donkey anti-mouse IgG as the secondary antibody.
Analysis of Atherosclerosis--
The extent of atherosclerosis
in the aorta and in aortic cross-sections was analyzed as described
previously (7, 19, 21, 22). Mice were killed by carbon dioxide
inhalation, and the aorta was immediately perfused in situ
with ice-cold PBS for 10 min via the left ventricle. The heart and
aorta were removed en bloc, and the heart was severed from
the aorta just above the aortic root. The apical half of the heart was
cut off in a plane perpendicular to the aortic root. The upper half of
the heart containing the aortic root was immediately embedded in Tissue Tek O.C.T. compound and frozen at
Atherosclerosis was also quantified in the aortic root cross-sections
from the fresh-frozen OCT-embedded hearts (7, 22). Serial
8-µm sections of the aortic root were cut and mounted on slides, then fixed in acetone, rehydrated in PBS containing 0.02% NaN3, and blocked with 1% bovine serum albumin in
PBS/NaN3. For detection of macrophages, sections were
immunostained with monoclonal rat anti-murine MAC-1 antibody,
monoclonal hamster anti-CD18, and monoclonal hamster anti-CD11c. For
detection of an extracellular matrix protein, monoclonal hamster
laminin antibody was used as a primary antibody, followed by incubation
with mouse anti-rat or goat anti-hamster IgG in the presence of 200 µg/ml normal mouse IgG. Antibody reactivity was detected using
horseradish peroxidase-conjugated biotin-streptavidin complexes and
developed with diaminobenzidine tetrahydrochloride as substrate.
Images of immunostained aortic root sections, captured digitally with a
video camera connected to a Leica microscope, were analyzed using
computerized image analysis (Image Pro Plus, Media Cybernetics, Silver
Spring, MD). Total lesion area in aortic root sections was measured by
manually tracing entire lesions in eight equally spaced aortic root
sections per mouse. Both macrophage and fibrous areas were quantified
in sections by determination of area that stained positively for macrophage markers and laminin, respectively. The acquisition of images
and analysis of lesions were performed in a blinded fashion.
Isoprostane Analysis--
For isoprostane measurement total
lipids from urine, LDL, and aortic homogenate samples were extracted
with ice-cold Folch solution, chloroform/methanol (2:1, v/v) as
described previously (19). After removing aliquots for phospholipid
measurement, the organic phase was dried under nitrogen. The samples
were hydrolyzed by the addition of aqueous KOH (15%), and total
iPF2 Effects of Hepatic ApoE Expression on Plasma Lipids and
Lipoproteins--
Mice that received Adnull had no significant change
in plasma apoE levels during the course of the experiment, whereas mice that received AdhapoE3 had significantly increased plasma apoE levels,
with the peak expression at day 7 (Fig.
1A). The plasma apoE levels in
AdhapoE3-injected mice remained about 2-fold higher than in Adnull mice
6 weeks after administration at the termination of the experiment.
Despite the increase in plasma apoE levels, no significant changes in
the plasma cholesterol levels from base line were observed (Fig.
1B). On day 7 after injection, the mean plasma cholesterol
level in mice injected with AdhapoE3 (573 ± 48 mg/dl) were
modestly lower than mice injected with Adnull (696 ± 61 mg/dl, p = not significant), but there
were no significant differences in cholesterol levels during the 6 weeks of the study. The mean post-injection cholesterol levels were not
different between the two groups (AdhapoE3, 594 ± 18 versus Adnull, 663 ± 23 mg/dl,
p = NS). Thus, hepatic expression of apoE had no
significant effect on plasma cholesterol levels in
LDLR
To evaluate the effect of hepatic apoE expression on plasma
lipoproteins, pooled plasma samples drawn on day 7 were analyzed by
FPLC gel filtration. This analysis showed no significant differences in
the plasma lipoprotein profiles between Adnull control and AdhapoE3
mice (Fig. 1C). These results demonstrate that gene transfer and hepatic expression of apoE3 in LDLR Effects of Hepatic ApoE Expression on Quantitative
Atherosclerosis--
The extent of atherosclerosis was assessed by two
independent methods: 1) en face quantitation of
atherosclerosis in the aorta from just above the root to the renal
arteries and 2) cross-sectional analysis of atherosclerotic lesions in
the aortic root. The Sudan IV stained en face
preparations of the LDLR
Atherosclerosis was also quantified in aortic root cross-sections by
manually tracing the entire lesions in five equally spaced sections
from each mouse. Consistent with the en face data of the
aorta, Adnull-injected mice showed progression of aortic root atherosclerotic lesions, whereas AdhapoE3-injected mice demonstrated significant 34% regression of lesion compared with base-line mice (Fig. 2C). Thus, by two independent assays, hepatic
expression of apoE induced significant regression of advanced
atherosclerosis over a 6-week period.
Effects of Hepatic ApoE Expression on Morphology of Atherosclerotic
Lesions--
To evaluate the effects of liver-derived apoE on the
morphology of lesions, we used immunocytochemistry to characterize
their composition. Aortic root cross-sections from base-line, Adnull, and AdhapoE3 mice were immunostained for macrophages with antibodies to
Analysis of Human ApoE in Artery Wall--
In previous studies, we
have shown that liver-derived apoE after gene transfer gained access to
the artery wall and was specifically retained in areas of
atherosclerotic lesions in apoE Effects of Hepatic ApoE Expression on Isoprostanes--
To examine
whether hepatic apoE expression affected in vivo oxidant
stress, we measured iPF2 In this study, we demonstrated that hepatic overexpression of
human apoE3 for 6 weeks induced regression of pre-existing advanced atherosclerosis in LDLR-deficient mice without reducing plasma cholesterol levels. This substantially extends our previous finding that apoE expression reduced the progression of early fatty streak lesions in LDLR-deficient mice (10) and indicates that liver-derived apoE has the potential to induce loss of foam cell mass from
pre-existing advanced atherosclerotic lesions. Furthermore, we
demonstrated that apoE overexpression in aged LDLR-deficient mice
markedly reduced levels of isoprostanes in urine, plasma, and aorta
compared with base-line and control treated mice. These results
strongly suggest that hepatic apoE expression resulted in anti-oxidant effects in vivo. Whether these effects were directly
responsible for the induction of atherosclerosis regression remains to
be determined.
It is not surprising that hepatic overexpression of human apoE3 on the
background of endogenous mouse apoE and in the absence of the LDLR did
not significantly reduce plasma cholesterol levels. The LDLR is the
major physiologic receptor for apoE in the liver (23), and although
there are backup mechanisms for clearance of lipoprotein remnants, such
as LDL receptor-related protein and heparan sulfate
proteoglycans, they are much less efficient. Furthermore, the majority
of plasma cholesterol in western diet-fed LDLR mouse is in LDL, which
are generally not cleared from the plasma through an apoE-mediated
mechanism. In fact, apoE-containing remnant lipoproteins can compete
with LDL for uptake, resulting in delayed catabolism of LDL (24).
Furthermore, apoE has been shown to inhibit lipolysis of
triglyceride-rich lipoproteins (25). Finally, hepatic apoE expression
enhances hepatic VLDL triglyceride (26-28) and apoB
production (29). Therefore any modest effect of apoE in promoting
clearance of VLDL in the absence of the LDLR may be offset by its
effect in competing with LDL for clearance, inhibiting VLDL lipolysis,
and promoting VLDL production. Our data demonstrate that apoE
overexpression had a very modest effect in reducing VLDL cholesterol on
day 7, but that this effect was no longer seen at subsequent time
points. These data are consistent with studies that expressed apoE
using bone marrow transplant (30) or by liver-directed gene transfer
(25) in the absence of both apoE and the LDLR and found little effect
on plasma cholesterol levels. Transgenic overexpression of apoE in the
absence of the LDL receptor in mice demonstrated reduction in VLDL
cholesterol and increased VLDL turnover, but no reduction in LDL
cholesterol or change in turnover (31), and transgenic overexpression
of apoE in rabbits resulted in a 70% increase in plasma cholesterol due largely to increased LDL cholesterol (32). Therefore, apoE expression has relatively little ability to reduce plasma cholesterol levels in the absence of the LDLR, making it a good animal model for
assessing the direct effects of apoE expression on atherosclerosis and
other markers without the confounding effects of a reduction in plasma cholesterol.
Macrophage-specific transgenic expression of apoE in apoE-deficient
mice reduced progression of atherosclerosis even after controlling for
changes in plasma cholesterol levels (4). Furthermore, when
apoE-deficient bone marrow was transplanted into wild-type mice,
atherosclerotic lesion formation was increased despite a lack of effect
on plasma cholesterol levels (5). Thus, macrophage-derived apoE appears
sufficient to inhibit the progression of atherosclerotic lesions
independent of its effects on plasma lipoproteins. In a previous study,
we demonstrated that liver-derived apoE gained access to and
accumulated within atherosclerotic lesions (7). Therefore, it is likely
that in these studies liver-derived apoE gained access to the vessel
wall and had direct effects that promoted regression of lesions. Plasma
apoE may gain access to the vessel wall in the context of small
lipoprotein particles such as There are a number of potential mechanisms by which hepatic apoE may
have induced regression of atherosclerosis. ApoE promotes cellular
cholesterol efflux (38-42) and is a contributor to the ability of
plasma to induce cholesterol efflux from cells (43, 44). However, apoE
has been demonstrated in vitro to have other cellular
effects such as inhibition of T-lymphocyte proliferation (17),
inhibition of vascular smooth muscle cell proliferation (45), and
anti-oxidant effects (16). However, the in vivo relevance of
these observations has been uncertain. In these studies, we used
quantitation of iPF2 The findings in our studies advance the concept of apoE as an
anti-atherogenic molecule in several important ways. First, we
expressed apoE in the liver instead of macrophages, demonstrating that
apoE is anti-atherogenic regardless of whether it is presented to the
vessel wall via macrophages or via the plasma compartment. This
suggests that interventions to raise hepatic production and plasma
levels of apoE could be anti-atherogenic, even if macrophage production
of apoE is not increased. Second, we demonstrated that apoE induced
regression of pre-existing advanced atherosclerotic lesions,
which has not been previously demonstrated for apoE derived from any
source. Inhibition of progression and induction of regression involve
very different cellular and molecular processes (47), and the
difference between these two processes is of potential clinical
significance. Third, we demonstrated that apoE changed the morphology
of advanced lesions, reduced foam cells and increased matrix, in a way
that could be interpreted as consistent with "stabilization" of
lesions (48). Finally, we showed that hepatic apoE expression markedly
reduced isoprostanes, an accepted index of in vivo oxidant
stress, providing important evidence that the in vitro
anti-oxidant properties of apoE that were described previously are also
functional in vivo and suggesting another mechanism by which
apoE may be anti-atherogenic.
In summary, we demonstrate that liver-directed gene transfer and
hepatic expression of apoE in LDLR/
) mice were fed a western-type
diet for 14 weeks to induce advanced atherosclerotic lesions. One group
of mice was sacrificed for evaluation of atherosclerosis at base line,
and two other groups were injected with a second generation
adenoviruses encoding human apoE3 or a control empty virus. Hepatic
apoE gene transfer increased plasma apoE levels by 4-fold at 1 week,
and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol
levels or lipoprotein composition induced by expression of apoE. The
liver-derived human apoE gained access to and was retained in arterial
wall. Compared with base-line mice, the control group
demonstrated progression of atherosclerosis; in contrast, hepatic apoE
expression induced highly significant regression of advanced
atherosclerotic lesions. Regression of lesions was accompanied by the
loss of macrophage-derived foam cells and a trend toward increase in
extracellular matrix of lesions. As an index of in vivo
oxidant stress, we quantitated the isoprostane iPF2
-VI
and found that expression of apoE markedly reduced urinary,
LDL-associated, and arterial wall iPF2
-VI levels. In
summary, these results demonstrate that liver-derived apoE directly
induced regression of advanced atherosclerosis and has anti-oxidant
properties in vivo that may contribute to its anti-atherogenic effects.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-VI levels are markedly
increased in apoE-deficient mice and are suppressed by the
administration of vitamin E accompanied by reduction in atherosclerosis
(19). Therefore, we also tested the hypothesis that hepatic expression
of apoE would reduce oxidant stress in LDLR-deficient mice as assessed
by quantitation of isoprostanes in urine, plasma, and aorta.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (back-crossed 10 times to C57BL/6 mice)
obtained from Jackson Laboratory were fed a western-type diet (normal
chow supplemented with 0.15% cholesterol and 20% butter fat) for a
total of 14 weeks prior to base-line analysis or administration of
vector. Blood was drawn 1 week prior to vector administration, plasma
cholesterol was quantified, and mice were divided into three groups
with mean cholesterol levels that were not significantly different. One day prior to vector administration, one group of mice
(n = 9) were killed for analysis of atherosclerosis
(base-line group). Remaining mice were injected intravenously with
either AdhapoE3 (n = 9) or control Adnull virus
(n = 9) at a dose of 1.0 × 1011
particles (~6.0 × 109 plaque-forming uints/g of
body weight). The western-type diet was continued after vector
administration. Blood samples from mice were obtained by retro-orbital
plexus prior to injection and 7, 14, 21, 28, and 42 days after
injection. Blood samples were collected into tubes containing 2 nM EDTA, 0.2% NaN3, 0.77% gentamycin and were
centrifuged to obtained plasma, which was stored at
20 °C for
lipid analyses and at 4 °C for FPLC. Urine samples were collected by
placing mice in metabolic cages (Nalgene, Rochester, NY) for a 24-h
urine collection at various time points. The samples were stored at
80 °C. Mice were killed 6 weeks after vector administration for
analysis of atherosclerosis.
80 °C. The remainder of
the aorta was cleaned free of adventitial fat and tissue, opened
longitudinally, stained with Sudan IV, and pinned out as
described (7, 19, 21, 22). The extent of atherosclerosis in aortas was
quantified by capturing images of aorta with a Dage-MTI 3CCD three-chip
color camera (Dage-MTI Inc., Michigan City, IN) connected to a Leica MZ
12 dissection microscope. The captured 24-bit digitized color images
were analyzed, and the lesion areas covering aortas were determined
using Image Pro Plus image analysis software (Media Cybernetics, Silver
Spring, MD). The acquisition of aortic images and the analysis of
lesion areas were both performed in a blinded fashion.
-VI was measured as described previously
(19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice fed a western-type diet. Plasma triglyceride
levels in mice injected with AdhapoE3 were transiently elevated at day
7 compared with control mice (data not shown) and did not differ at the
remaining time points.
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Fig. 1.
Plasma apoE, total cholesterol, and
lipoprotein profile after hepatic gene transfer in
LDLR /
mice. A, plasma apoE levels in
mice injected with second generation adenovirus expressing human apoE3
(AdhapoE, n = 9) (squares) and control virus
(Adnull, n = 9) (triangles). Plasma samples
obtained from mice at indicated time points after adenovirus injection
were analyzed for human apoE levels using an immunoturbidometric assay.
B, plasma total cholesterol levels after gene transfer in
mice injected with AdhapoE (n = 9) (squares)
and control virus (n = 9) (triangles). The
total cholesterol levels of plasma samples obtained at each time point
were measured by enzymatic assay. Data are mean ± S.E.
C, lipoprotein cholesterol distribution. Pooled day
7 plasma samples from nine LDLR
/
mice each from Adnull
(triangles) and AdhapoE3 (circles) group were
subjected to FPLC gel filtration using two Superose 6 columns.
Cholesterol levels of the fractions obtained were determined by an
enzymatic assay.
/
mice did not
significantly alter the distribution of cholesterol among lipoprotein
fractions. The distribution of human apoE3 was determined in the
lipoprotein fractions, and apoE was found to be primarily associated
with VLDL and large HDL particles, with a smaller amount of apoE
present on intermediate density lipoprotein and LDL fractions
(data not shown). To determine whether lipoprotein composition was
influenced, we isolated VLDL, LDL, and HDL by sequential
ultracentrifugation from pooled plasma samples. Analysis of the
lipoprotein cholesterol (free and esterified), phospholipid and protein
composition revealed no significant differences in lipoprotein
composition between Adnull and AdhapoE3 groups at days 7, 14, 28, or 42.
/
mouse aortas showed extensive
atherosclerotic lesions throughout the entire aorta at base line. The
aortas from Adnull-injected control mice demonstrated further
progression of atherosclerosis. In contrast, the lesions in AdhapoE3
mice were markedly reduced. Quantitation of atherosclerosis using
morphometric image analysis of the aortas demonstrated a marked 61%
regression of atherosclerosis in mice injected with AdhapoE3 compared
with base-line mice (Fig. 2A).
In LDLR
/
mice fed a western-type diet, atherosclerotic
lesions develop initially in the proximal portions of the aorta, the
aortic root and arch, and with progression of atherosclerosis, extend
distally into thoracic and abdominal portions of the aorta. To assess
the pattern of apoE-induced regression in different portions of the aorta, the lesion areas in the arch, thoracic, and abdominal portions of the aorta were analyzed. The results show that hepatic apoE expression not only induced marked regression of advanced lesions in
the thoracic and abdominal portions of the aorta but also effectively regressed relatively more abundant lesions in the aortic arch (Fig.
2B).
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Fig. 2.
Extent of atherosclerosis in
LDLR /
mice fed high fat diet for 14 weeks
(Baseline) and an additional 6 weeks after injection
with a control (Adnull) and human apoE3 expressing
virus (AdhapoE). A, the
atherosclerotic lesion area covering the aortic surface was quantified
in en face aortic preparations and expressed as percentage
of total aortic area. B, evaluation of apoE-induced
regression in aortic arch and thoracic + supra-abdominal
(S.Abd) segments of the aorta. The lesions areas in each of
these segments are expressed as µm2. The reduction in
lesion areas in AdhapoE group (black bars) relative to
base-line group (hatched bars) in arch and throracic + supra-abdominal portions of aorta were 48 and 81%, respectively
(open bars represent Adnull group). C, aortic
root lesion area measured in serial cross-sections through the aortic
origin. The mean lesion areas per each group (n = 9)
were shown. All data are mean ± S.E. with n = 9 in each group. Asterisks indicate statistically significant
difference from base-line and Adnull groups (p < 0.001).
2-integrin (Mac-1, CD18, and CD11c) and for extracellular matrix-rich regions with an antibody to laminin. Lesions contained a
large amount of non-macrophage foam cell components and laminin, consistent with advanced atherosclerotic lesions. Collagen staining was
similar to laminin staining (data not shown). Quantitative computer-assisted image analysis of immunostained aortic root serial
cross-sections revealed significant reduction of macrophage-derived foam cells in atheroscleroic lesions of AdhapoE3 mice compared with
base-line and Adnull mice (Fig.
3A). In contrast, the
distribution of laminin-rich areas showed no change (data not shown),
and therefore the ratio of macrophage foam cells to laminin was
significantly reduced in mice injected with AdhapoE3 (Fig.
3B). These results show that liver-derived apoE induced
regression of pre-existing advanced lesions primarily by reducing the
macrophage-derived components of lesions but not the extracellular
matrix components.
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Fig. 3.
Composition of aortic root atherosclerotic
lesions in base-line, Adnull, and AdhapoE mice. A,
macrophage foam cell area; B, ratio of laminin-rich fibrous
area and macrophage foam cell area. All data are mean ± S.E. with
n = 9 in each group. Asterisks indicate
statistically significant difference from base-line and Adnull groups
(p < 0.001).
/
mice (7). To
demonstrate the retention of liver-derived human apoE in the artery
wall in this study, aortic extracts prepared from base-line mice, as
well as Adnull and AdhapoE3 mice at the end of the experiment, were
analyzed by Western blotting using monoclonal antibody specific for
human apoE. The presence of liver-derived human apoE in the artery wall
was detected in AdhapoE3-injected mice but not in Adnull and base-line
mice (Fig. 4). No human apoE mRNA was
detected (data not shown), indicating that the human apoE present in
the aorta was not synthesized within the vessel wall. Thus,
liver-derived apoE gained access to the artery wall from the plasma
compartment and was retained in substantial amounts within the artery
wall several weeks after vector injection.
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Fig. 4.
Western blotting analysis for detection of
human apoE in artery wall. Aortic extracts from base-line mice and
Adnull and AdhapoE mice after 6 weeks of viral injection were subjected
to 10-20% gradient SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membrane. The presence of human apoE was
detected using anti-human apoE monoclonal antibody as primary and
horseradish peroxidase-labeled donkey anti-mouse IgG as secondary
antibody. Lane 1, positive control, human hyperlipidemic
plasma (1:100 dilution). Aortic extracts from base-line (lanes
2 and 3), Adnull (lanes 4 and 5),
and AdhapoE (lanes 6 and 7) mice were loaded on
the gel at two concentrations (10 µg of aortic protein, lanes
2, 4, and 6, and 15 µg of aortic protein,
lanes 3, 5, and 7). The 34-kDa bands
corresponding to human apoE are detectable in the aorta from AdhapoE
mice 6 weeks after viral injection. This band is absent in aortas from
base-line and Adnull mice.
-VI levels in urine and plasma LDL before and at several time points after vector administration and
in aorta at the termination of the experiment. Injection of Adnull had
no effect on urinary iPF2
-VI levels; in contrast, hepatic expression of apoE significantly reduced urinary
iPF2
-VI levels by 7 days, and they remained at 60% of
base-line values at 6 weeks (Fig.
5A). Analysis of the
LDL-associated iPF2
-VI levels showed no change in
control mice, but a rapid reduction in the AdhapoE3-injected mice, with
levels by 14 days only 6.3% of base-line values and remaining in that
range through the 6 weeks of the experiment (Fig. 5B).
Finally, aortic iPF2
-VI levels in AdhapoE mice were
significantly reduced compared with both base-line and Adnull mice
(Fig. 5C). These results suggest that hepatic apoE
expression reduced oxidant stress in vivo. The apoE-mediated
inhibition of lipid peroxidation and oxidative stress may contribute to
its anti-atherogenic mechanisms in the regression of advanced
atherosclerotic lesions.
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Fig. 5.
Effect of liver-derived apoE gene transfer on
isoprostane F2 -IV levels.
A, urinary isoprostane levels AdhapoE (black
bars) and control Adnull mice (white bars) during the 6 weeks following apoE gene transfer. B, LDL-associated
isoprostane levels in AdhapoE (black bars) and Adnull mice
(white bars). LDL samples from 9 mice in each group were
analyzed for isoprostane levels. C, aortic isoprostane
levels in base line, Adnull, and AdhapoE. Data are mean ± S.E. *,
significantly different from base line; **, significantly different
from both base line and Adnull.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-LpE (33, 34) and pre
-LpE
(34). Once within the intima, apoE could associate with components of
the extracellular matrix, such as heparan sulfate proteoglycans (35,
36) and laminin (37), for which it has affinity.
-VI, a major isoprostane that is not
influenced by cyclo-oxygenase activity and therefore felt to be a
specific marker for lipid peroxidation and oxidant stress (18, 46), to
test the hypothesis that expression of apoE reduced oxidant stress
in vivo. We found a dramatic effect of hepatic apoE
expression in reducing urine and LDL-associated and aortic levels of iPF2
-VI. This is the first in vivo
evidence that apoE has direct anti-oxidant effects. Combined with our
previous study in which vitamin E administration in apoE-deficient mice reduced isoprostane generation accompanied by reduction in
atherosclerosis (19), this finding further supports the concept that
isoprostanes may be a surrogate marker for anti-oxidant effects of
interventions, which are likely to beneficially influence atherosclerosis.
/
mice induced
significant regression of advanced atherosclerotic lesions without
substantial alteration in plasma cholesterol and lipoprotein levels. In
addition, apoE expression markedly reduced isoprostane levels in urine,
plasma LDL, and aortic tissue. These results demonstrate that
liver-derived apoE has direct anti-atherogenic and antioxidant
properties in vivo that are independent of cholesterol and
lipoprotein modulation.
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ACKNOWLEDGEMENTS |
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We are indebted to Dawn Marchadier, Robert Hughes, Anna Lillethun, and Linda Morrell for excellent technical assistance and Dr. Jane Glick for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by Grants HL-57811 and HL-59407 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an initial investigatorship from the American Heart Association Pennsylvania-Delaware Affiliate.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: University of Pennsylvania Medical Center, Rm. 614 BRB II/III, 421 Curie Blvd., Philadelphia, PA
19104. Tel.: 215-662-9097; Fax: 215-573-6725; E-mail:
rader@mail.med.upenn.edu.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M003324200
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ABBREVIATIONS |
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The abbreviations used are: LDL, low density lipoprotein; LDLR, LDL receptor; FPLC, fast protein liquid chromatography; VLDL, very low density lipoprotein; PBS, phosphate-buffered saline; HDL, high density lipoprotein.
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