© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Complete Atherosclerosis Regression After Human ApoE Gene Transfer in ApoE-Deficient/Nude Mice
From the Laboratoire RTG, (C.D., J.-M.H.), Institut Pasteur, Paris; the Cardiovascular Department, Rhône-Poulenc Rorer/Gencell (J.-M.C., F.E., P.B., D.B., N.D.), Vitry/Seine; and the Laboratoire INSERM U325 (J.C.F., G.C.), Institut Pasteur de Lille, Lille, France.
Correspondence to Nicolas Duverger, PhD, Cardiovascular Department, Rhône-Poulenc Rorer/Gencell, 13, quai Jules Guesde, 94403 Vitry/Seine cedex, France. E-mail nicolas.duverger{at}aventis.com
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Abstract—The apolipoprotein E (apoE)-deficient mouse is a relevant animal model of human atherosclerosis. Although the prevention of atherosclerosis development has been documented after somatic gene transfer into animal models, regression of lesions remains to be demonstrated. Thus, we used this genetically defined mouse model nn the nude background to show atherosclerosis regression. ApoE-deficient nude mice were infected with 5x108 or 109 plaque-forming units of a first-generation adenovirus encoding human apoE cDNA. The secretion of human apoE resulted in a rapid decrease of total cholesterol, which normalized the hypercholesterolemic phenotype within 14 days (from 600±100 to <100 µg/mL). Transgene expression was observed during a period of >4 months, with a normalization of cholesterol and triglyceride levels during 5 months. At that time, we successfully reinjected the recombinant adenovirus and observed the appearance of the human protein as well as the correction of lipoprotein phenotype. In mice killed 6 months-after the first infection, we observed a dose-dependent regression of fatty streak lesions in the aorta. We showed sustained expression of a transgene with a first-generation adenoviral vector and a correction of dyslipoproteinemia phenotype leading to lesion regression. These data demonstrate that somatic gene transfer can induce plaque regression.
Key Words: atherosclerosis • regression • gene transfer • adenoviral vector • apolipoprotein
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Introduction |
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Atherosclerosis, the principal cause of morbidity in Western countries, is a process contributing to the pathogenesis of myocardial and cerebral infraction, gangrene, and peripheral vascular diseases. Although advances in prevention and treatment of cardiovascular diseases have been effective, there is still a need for effective new therapies. The concept of atherosclerosis regression remains a major challenge and has been approached in animal models and in humans during lipid-lowering drug trials. However, studies in both cholesterol fed-animals1 2 3 4 and humans5 6 7 8 lasted for several years and were difficult to manage owing to the variability of the results, and the regression, if it occurred, was marginal.
Mouse models of atherosclerosis have been developed for
10 years, and the most widely used model is the apolipoprotein E
(apoE)-deficient mouse (apoE–/–).9
ApoE is a surface constituent of lipoproteins and a ligand for
lipoprotein clearance. ApoE is synthesized by macrophages and
other cells of that lineage, but the apoE in plasma is derived largely
from the liver.10 ApoE–/– mice are
hypercholesterolemic and spontaneously develop
atherosclerosis on a chow diet.11 12
Lesions in this model are not only fatty streaks but also widespread
fibrous plaque lesions at vascular sites typically affected in human
atherosclerosis.13 14 Because the
evolution of atherosclerotic lesions involves complex mechanisms,
crossbreeding of apoE–/– mice with transgenic
or knockout mice has been an elegant approach to evaluate the impact of
specific genes in this polygenic process and to produce more improved
or appropriate mouse models.
In parallel, advances in somatic gene transfer techniques, notably with adenoviral vectors that allow a high level of transgene expression,15 have produced important information in gene function. However, adenovirus-mediated gene transfer is currently limited by the immune response directed against both the transgene product and the adenoviral proteins, which results in transient expression.16 17 18
In the current report, we took advantage of recent progress in the generation of animal models as well as in gene transfer techniques to gain insights into the atherosclerosis regression process. We generated apoE–/– mice on the nude background as a mouse model of atherosclerosis and transferred the human apoE cDNA into this animal with a first-generation adenoviral vector. Athymic nude (nu+/+) mice are deficient for cellular immunity (lack of mature T lymphocyte) but retain near-normal serum immunoglobulins.19 First, we showed that the T lymphocytes deficiency did not impair plaque formation in this mouse model. Then, owing to the immunodeficient background, the limitation of adenovirus-mediated gene transfer was overcome and expression of the human transgene was prolonged for several months, allowing us to demonstrate complete plaque regression by liver-derived human apoE overexpression in the apoE–/– nu+/+ mouse model.
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Methods |
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Animal Experiments
ApoE-deficient (apoE–/–) mice on a C57BL/6 background were obtained from Transgenic Alliance (Iffa Credo, France). These mice were homozygous for disruption of the apoE gene.9 ApoE-deficient/nude (apoE–/– nu–/–) mice were obtained by crossbreeding with nude (nu+/+) mice on a C57BL/6 background obtained from SPF Bomolgard (Bomice, Danemark). The F1 mice were heterozygous for the knockout mutation and for the nude character (apoE-/+ nu-/+). Then, these F1 progeny was crossed with apoE-deficient mice to obtain an apoE–/– background, and animals were selected for hypercholesterolemia (apoE–/– nu-/+ and apoE–/– nu+/+). The apoE–/– genotype was confirmed by polymerase chain reaction (PCR) analysis. These mice (F2) were crossed together. Animals sorted by the phenotypic character of no hair (nude mice) were considered apoE–/– nu+/+ and their parents as apoE–/– nu-/+. The colony was obtained by crossing male apoE–/– nu+/+ and female apoE–/– nu-/+ mice. The mice were fed a chow diet and allowed to acclimate for 2 weeks before vector administration.
Male mice, 17 weeks old, were treated by retro-orbital sinus injection of purified recombinant adenovirus stocks. Each group contained 5 or 6 mice. Blood was obtained from the retro-orbital plexus and collected in preheparinized pipettes. Plasma was separated by centrifugation at 2800g for 20 minutes at 4°C, and aliquots were stored at -20°C. At the end of the experiment, animals were killed for DNA/RNA and histological analyses. Liver, kidney, aorta, heart, spleen, and lung were harvested. Half of each tissue was directly frozen into a LN2 bath, and the rest was placed into a PBS–10% formalin solution before being embedded in paraffin blocks. The study protocol was approved by Animal Use Committee of Rhône-Poulenc Rorer.
Recombinant Adenoviral Vectors
The human apoE adenoviral vector
(AV1.0CMVapoE) was prepared by ligation of
SpeI-linearized plasmid vector pLAL.CMV/ApoE3 (containing
the left terminus of adenovirus type 5, the cytomegalovirus
promoter,20 and a 1-kb
EcoRI-BamHI fragment of human apoE3 cDNA followed
by the simian virus 40 polyadenylation signal) to
XbaI-restricted adenovirus type 5 DNA deleted for E1a and
most of the E1b region. Ligation products were transfected into 293
cells21 of low passage number and were used for cell
culture studies (American Type Culture Collection. Rockville, Md) by
using the lipofectamine reagent (GIBCO BRL). Recombinant
adenoviral vectors were propagated in 293 cells, and the resulting
plaques were screened for the presence of human apoE by using PCR with
specific human apoE primers (Sq 5531
5'-CAC-CAG-GCG-GCC-GCG-CAC-GTC-CTC-CAT-GTC-CGC-GCC-3' and Sq 4409
5'-TAT-GAA-GGT-GGA-GCA-AGC-GGT-GGA-GAC-AGA-GCC-GGA-GC-3'). The
recombinant viruses encoding thymidine kinase gene, deleted for the E1
region (AV1.0CMVTK) and deleted for both E1 and
E3 regions (AV3.0CMVTK), or the ß-galactosidase
gene, deleted for the E1 region (AV1.0CMVLacZ),
were prepared as previously described.22 Titers are given
as plaque-forming units (pfu) per milliliter. High-titer stocks,
1011 pfu/mL as determined by plaque assays on 293
cells, were produced in 293 cells and purified by CsCl gradients.
Apolipoprotein, Lipid, and Lipoprotein Analyses
Human apoE in mouse plasma was measured with a sandwich-type
ELISA (Apo-Tek ApoE, PerImmune, Inc). Cholesterol and
triglycerides were measured
colorimetrically with commercially available kits
(Boehringer Mannheim). Plasma lipoprotein distribution
was assayed by analytical gel filtration chromatography
with a Superose 6 HR 10/30 column (Pharmacia). The elution flow rate
was 0.4 mL/min in a running buffer consisting of 0.15 mol/L NaCl,
1 mmol/L EDTA, and 0.02% NaN3, pH 8.2.
Fractions of 0.5 mL were collected in which cholesterol
contents were determined.
Extraction of DNA and RNA From Organs for Slot Blotting
Analysis
Liver, spleen, kidney, and lung fragments previously
frozen in LN2 were minced and incubated overnight
in SDS–proteinase K buffer. DNA extraction was prepared by using the
QIAamp tissue kit protocol (Quiagen). For slot blot analyses,
10 µg of DNA was heated in 0.4 mol/L NaOH and 2.2 mmol/L EDTA at
95°C for 2 minutes and transferred to a Hybond N+ nylon membrane. DNA
was fixed by baking the membranes in a microwave oven for 2 minutes.
Hybridization of the membrane with a
32P-radiolabeled probe corresponding to the apoE
cDNA sequence was performed under classic conditions. RNA extraction
was prepared by using an RNeasy kit protocol (Quiagen). For slot blot
analyses, 10 µg of RNA was added to 30 µL of SSC x20 and
20 µL of 37% formaldehyde and then transferred to a Hybond N+ nylon
membrane. As previously described for the DNA slot blots, RNA was fixed
by baking the membrane in a microwave oven for 2 minutes. Hybridization
of the membrane with a 32P-radiolabeled probe
corresponding to the human apoE sequence or the murine erythropoietin
sequence was performed under classic conditions. Quantification was
performed with a Molecular Dynamics PhosphorImager (Image Quant
software).
Histological Analysis
Aortic sectioning, lipid staining, and lesion scoring were
performed in a blinded manner according to the methods described
previously.23 In brief, mouse hearts were fixed, stored in
4% formalin, and embedded in 25% gelatin. Then, 10-µm proximal
aortic sections, separated by 200 µm, were stained with oil red
O for neutral lipids. A lesion area value for each mouse was obtained
from the mean of 4 sections evaluated for lipid-stained areas. Livers
were harvested from mice, and formalin-fixed tissues were stained with
hematoxylin and eosin.
ApoE Distribution
ApoE distribution was determined in individual plasma samples by
nondenaturing 2-dimensional electrophoresis as previously
described.24 In brief, the first dimension was carried out
in 0.75% agarose gel in 50 mmol/L barbital buffer, pH 8.6, on Gel
bond (FMC Bioproducts) at 4°C and 200 V for 1 hour, 45 minutes.
The second dimension was carried out on 2% to 15% gradient
polyacrylamide gels and electrophoresis at 120 V for 16 hours.
Two pieces (corresponding to samples taken before and after injection)
were cut from the agarose gel and laid on the top of a
polyacrylamide gel. Each polyacrylamide gel contained 2
patterns, and after electrophoresis, the gel was divided in half
longitudinally. Transfer to the nitrocellulose sheet was carried out in
25 mmol/L Tris and 192 mmol/L glycine buffer, pH 8.3, for 1
hour, 45 minutes at 400 mA under semidry conditions
(Electrophoresis-Atta). Nitrocellulose sheets were blotted with
anti-human apoE goat polyclonal antibodies in a 50 mmol/L
Tris–0.5% NaCl buffer containing 2.5% powdered milk for 1 hour at
room temperature and the second time with anti-goat IgG rabbit
antibodies labeled with peroxidase. ApoE distribution was detected by
luminescence with Hyperfilm (ECL, Western blotting detection, Amersham
Life Science).
Statistical Analysis
All data are expressed as mean±SEM. Data were evaluated with
ANOVA.
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Characteristic of ApoE–/– nu+/+ Mouse Model
ApoE–/– nu+/+ mice were compared with immunocompetent littermates for lipoprotein metabolism and atherosclerosis development. Both types of mice had similar plasma cholesterol (524±37 versus 550±41 mg/dL for apoE–/– nu+/+ and apoE–/– nu–/–mice, respectively) and triglyceride (92±12 versus 80±21 mg/dL for apoE–/– nu+/+ and apoE–/– nu–/–mice, respectively) levels, cholesterol profile, and rate of progression of atherosclerosis over a period of 9 months, suggesting that the T lymphocyte deficiency did not impair atherosclerosis development in this mouse model. Characteristics of lesion development in apoE–/– nu+/+ mice correspond to the data shown below.
Human ApoE Expression in ApoE–/–
nu+/+ Mice
To determine the kinetics and plasma levels of human apoE
expression after adenovirus-mediated gene transfer into
apoE–/– nu+/+ mice,
17-week-old mice were injected in the retro-orbital sinus with either
purified, recombinant virus encoding human apoE
(5x108 pfu or 109 pfu) or,
as a control, with a ß-galactosidase–encoding vector
(109 pfu; Figure 1
). Human apoE was readily detectable in
sera at day 4 and peaked at day 21 (3.2±1.7 and 18.4±3.3 mg/dL with
5x108 pfu and 109 pfu of
AV1.0CMVapoE, respectively). No human apoE was
found in AV1.0CMVLacZ-infected control mice.
Persistence of human apoE expression lasted for at least 4 months after
the initial injection. Expression of human apoE was still detectable at
day 124 (0.15±0.10 mg/dL) and at day 145 (0.18±0.11 mg/dL) for mice
administrated 5x108 pfu and
109 pfu, respectively, of
AV1.0CMVapoE. When the same construct was
injected into immunocompetent mice, human apoE expression was
transient, lasting for 1 month.25 26 Thus, use of the
apoE–/– nu+/+ mouse
strain overcomes the limitation of transient, adenovirus-mediated
expression of human transgenes in mice.
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) of
AV1.0CMVapoE. In addition, mice that received the first
injection of 5x108 pfu of AV1.0CMVapoE were
injected with 109 pfu (
) of AV1.0CMVapoE on
day 138.A second injection of 109 pfu of AV1.0CMVapoE was performed at day 138 in a group of mice pretreated with 5x108 pfu AV1.0CMVapoE. Kinetics of human apoE expression in this group of mice followed exactly the same profile as the first injections, with a peak of human apoE at day 21 and reaching 54.4±11.6 mg/dL after the second injection by day 159. Interestingly, this second peak was 17-fold higher than the previous one measured in the same mouse and 3-fold higher than that in mice treated with a single dose of 109 pfu AV1.0CMVapoE.
To check whether the second elevation of the human transgene was due to any CMV promoter reactivation by liver inflammation as proposed in previous studies,27 we injected mice pretreated with 109 pfu AV1.0CMVapoE at day 177 with 2x1011 viral particles of a first- and a third-generation adenovirus encoding the TK gene (AV1.0CMVTK and AV3.0CMVTK). No elevation of human apoE was observed 21 days later (day 198, not shown), indicating that inflammation was not an efficient factor by itself to enhance the CMV promoter. Thus, the second peak of human apoE expression resulted from adenovirus-mediated human apoE gene transfer after reinjection of the AV1.0CMVapoE. Altogether, these data show that a high expression level, long duration, and reinjection can be achieved in injected apoE–/– nu+/+ mice with the use of an adenoviral vector. This model is therefore suitable for studying long-term effects of human transgenes on atherosclerosis.
Persistence of Human ApoE in ApoE–/–
nu+/+ Mice
To gain insight about prolonged transgene expression in
apoE–/– nu+/+ mice, the
presence of viral DNA and transgene mRNA was studied. By using slot
blot analyses of liver DNA extracted from
apoE–/– nu+/+ mice that
were administrated 109 pfu of
Ad1.0CMVapoE, apoE cDNA was detected in all mice
killed at days 15, 30, 45, 60, 75, and 199 after infection (Figure 2). Murine erythropoietin was used as a
positive control. No apoE signal was detected in
Ad1.0CMVLacZ-treated mice. About 0.3 to 3 copies
of apoE cDNA per genome were observed in
Ad1.0CMVapoE-treated mice (not shown). These data
indicate that infection with a first-generation adenovirus lead to
persistence of the viral genome for at least 6 months on a nude mouse
background.
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The presence of apoE mRNA was analyzed in the liver and spleen from mice injected at day 0 or at day 138 (second injection) and killed at day 198. Human apoE mRNA was detectable in the livers of mice treated at day 131 but was not detectable in the livers of mice injected at day 0. This result indicates a time-dependent decrease of transgene mRNA within the 6-month period. No apoE mRNA was detectable in the spleens of all treated animals, suggesting that macrophages did not express human apoE after the adenoviral injection.
Effect of Human ApoE Expression on Lipoprotein Metabolism
Total cholesterol and triglyceride levels
in the plasma of infected mice are presented in Figures 3 and 4,
respectively. Lipoprotein metabolism modifications were
observed from day 4 to day 198 in
AV1.0CMVapoE-treated mice but neither in
AV1.0CMVLacZ control mice nor after
AV1.0CMVTK and AV3.0CMVTK
injection.
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) of AV1.0CMVapoE on
day 138. Control mice were administrated 109 pfu 
of
AV1.0CMVLacZ.
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In mice administrated 109 pfu of AV1.0CMVapoE, the cholesterol levels dramatically decreased from 591±85 mg/dL (day 0) to 92±7 mg/dL (day 21), which is equivalent to normal cholesterol levels found in C57BL/6 mice.28 29 30 This corrected cholesterol level lasted 5 months, while a slow increase was observed after the sixth month. The normalization of cholesterol levels was significant in comparison with that of control mice infected with 109 pfu Ad1.0CMVLacZ for all time points. Mice administrated 5x108 pfu AV1.0CMVapoE showed the similar decrease as mice infected with the highest dose (513±40 mg/dL at day 0 and 84±6 mg/dL at day 21), but hypercholesterolemia reappeared earlier (198±55 mg/dL at day 77), indicating that the cholesterol level correction as well as the apoE secretion were dose dependent.
Mice infected with 108 pfu of AV1.0CMVapoE at day 0 and reinjected at day 138 with 109 pfu of AV1.0CMVapoE showed a second decrease in cholesterol level, from 265±61 mg/dL at the time of reinjection to 90±3 mg/dL, 21 days after the second injection. At the time the experiment was terminated (day 198), the total cholesterol measured in the plasma of those mice was still normal (71±20 mg/dL).
Triglyceride levels (Figure 4
) in mice injected with
109 pfu of AV1.0CMVapoE
decreased from 222±45 mg/dL at day 0 to 93±11 mg/dL at day 21, were
maintained for 5 months, and increased again during the last month
(157±17 mg/dL at day 199). For mice injected with
5x108 pfu of AV1.0CMVapoE,
triglyceride levels decreased from 145±15 mg/dL at day 0
to 110±12 mg/dL at day 21, but
hypertriglyceridemia reappeared at day 105
(143±27 mg/dL). This increase was not observed in mice receiving the
second injection of AV1.0CMVapoE.
Cholesterol Distribution in Lipoproteins
Cholesterol distribution among lipoproteins in
apoE–/– nu+/+ mice before
and 21 days after injection of 109 pfu of
AV1.0CMVapoE is presented in Figure 5. The lipoprotein elution profile showed
that the cholesterol was primarily transported in the
VLDL-LDL fractions in untreated mice, in agreement with previous
reports.11 12 After human apoE expression (day 21), the
lipoprotein distribution profile for cholesterol was
modified toward a marked reduction of the cholesterol in
VLDL-LDL fractions. Thus, when human apoE was expressed in
apoE–/– nu+/+, the major
cholesterol carrier was the HDL fraction, as observed in
normal mice. In addition, a slight increase in
HDL-cholesterol plasma levels was observed in mice
expressing human transgene in comparison with control mice. These data
indicate that expression of human apoE provoked a decrease in total
cholesterol levels and produced a modification in the
distribution of cholesterol in the different lipoprotein
fractions. No alteration of cholesterol distribution was
observed in mice treated with Ad1.0CMVLacZ (not
shown).
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ApoE Distribution
Human apoE distribution was studied with 2-dimensional gel
electrophoresis (data not shown). Human apoE was observed associated
with all HDL subtractions. In addition, a specific apoE-containing
lipoprotein named 
-LpE31 was detected. This lipoprotein
has been proposed to play a role in cellular cholesterol
efflux by acting, like pre-ß1-HDL, as an initial acceptor of
cell-derived cholesterol.
Histological Analysis in
ApoE–/– nu+/+ Mice
The type and development of atherosclerotic lesions are similar in
apoE–/– nu+/+ and
apoE–/– nu–/– mice. The
lesions developed not only in the aortic root but also throughout the
aorta and its principal branches. These lesions were characterized by
the adherence of mononuclear cells to the endothelial
surface. Foam cells lesions, or fatty streaks, are subsequently found
at the same sites.32 With increasing age, the lesions
progressed to intermediate, or fibrofatty, lesions containing multiple
layers of lipid-filled macrophages and smooth muscle cells and
ultimately, to fibrous plaques. As a consequence, at 17 weeks old, ie,
at the time when mice received the viruses, fatty streak lesions were
already well developed in apoE–/–
nu+/+ mice, with a mean size of 220±37
mm2 (Figure 6).
Lesions contained primarily macrophage foam cells under the
endothelial layer, which was sometimes interrupted
(Figure 7). They also contained some
cholesterol crystals. Lipid infiltration was weak in the
vessel wall and was accompanied by low smooth muscle cell
proliferation. A cellular inflammatory reaction was not observed within
the lesion, whereas adhesion of inflammatory cells onto the
endothelium was noticed.
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A 6-fold increase in lesion size was observed in control mice
administrated 109 pfu of
Ad1.0CMVLacZ during the 28-week observation
period. Lesion size was 1172±255 mm2 at day
199 in Ad1.0CMVLacZ-treated mice
(P<0.001, different from initial 17-week-old lesions).
Lesions were of a type similar to those described at day 0, but
oversized, with unambiguous lipid infiltration into the vessel wall and
endothelium lining and more cholesterol
crystal deposition (Figures 6 and 7).
In contrast, a dose-dependent regression of lesion size was observed in
mice administrated Ad1.0CMVapoE (Figures 6 and 7). Lesion size in mice treated with
5x108 and 109 pfu of
Ad1.0CMVapoE were 147±76 and 28±6
mm2 at day 199 (P<0.05 and
P<0.001, respectively, different from the initial
17-week-old lesions, and P<0.001 and P<0.001,
respectively, different from lesions in
Ad1.0CMVLacZ-treated mice at day 199). The size
of fatty streak lesions in mice treated with 109
pfu of Ad1.0CMVapoE represented 13%
of initial lesions (day 0) but only 2.2% of lesions in age-matched
mice, 28 weeks after injection. This regression was accompanied by a
complete remodeling of the arterial wall, with the
disappearance of macrophages and foam cells, the lack of
cholesterol crystals, and
reendothelialization of the arterial wall
(Figure 7). A strong inverse correlation (r=-0.807,
P=0.015) displayed in Figure 8 was found between the extent of lesion
sizes (µm2 ) and human apoE plasma level per day
(µg · mL-1 ·
d-1). No relationship was found between lesion
size and total cholesterol exposure (mg ·
dL-1 · day-1) in
apoE-treated mice.
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ApoE–/– nu+/+ mice were engineered by crossbreeding apoE–/– and nude mice on a C57BL/6 background. This homogeneous background corresponds to the only normal mouse strain that develops atherosclerosis when fed a cholesterol-rich diet.32 Nude C57BL/6 mice were previously studied with the aim to address the role of immune mechanisms in atherosclerosis development.33 34 These mice were fed the rich-cholesterol diet containing cholate to induce lesions. These lesions were very small fatty streaks with few T lymphocytes, limiting interpretation of the study. In contrast, apoE–/– mice spontaneously develop severe atherosclerosis, with T lymphocyte infiltration that mimics human plaque progression. Oxidation of LDL in the vessel wall induces the recruitment of T cells and macrophages, resulting in local secretion of cytokines in the plaque. T lymphocyte–derived cytokines modulate macrophage metabolism, such apoE secretion, lipid uptake, and oxidation, and induce smooth muscle cell proliferation, thereby affecting the atherosclerotic process. Our data do not support a major involvement of T cells in atherogenesis in the apoE–/– nu+/+ mouse model, in agreement with previous studies in Rag-1– and Rag-2–deficient mice on an apoE–/– background.35 36 37 38
Adenovirus-mediated human apoE gene transfer into the livers of apoE–/– nu+/+ mice resulted in long-term expression of the transgene. Previous studies25 26 with a first-generation adenovirus carrying the human apoE cDNA under control of the similar CMV promoter led to only very transient expression (<1 month), establishing our interest in using the nude background to avoid cytolytic T lymphocyte–mediated rejection of transduced hepatocytes expressing viral protein as well as a response against the human transgene. The use of a second-generation recombinant adenovirus increased gene expression duration to 3 months.39 In our study, expression of apoE in the sera of treated mice decreased and then disappeared after 4 months. ApoE cDNA was still detectable 6 months after infection, whereas apoE mRNAs were almost undetectable, indicating a decreased transcription level. The CMV promoter is known to shut off in the liver cells after few months.27 40 41 Thus, disappearance of apoE expression is probably due to extinction of the promoter. Nevertheless, repeated injections of this adenoviral vector were feasible in the apoE–/– nu+/+ mice without using immunosuppressive agents. The CMV promoter has also been reported to be sensitive to reactivation in response to local inflammation.27 However, reactivation of the CMV promoter by adenovirus-mediated liver inflammation was not effective after the systemic administration of an E1- or E1/E4-deleted adenovirus encoding TK.
Human apoE secretion was able to completely correct hyperlipidemia and to maintain normal levels for at least 5 months. Surprisingly, this normal lipid level was maintained even when the human protein was no longer detectable in the sera of treated mice. One possible explanation could be that a low expression of human apoE persisted in the liver, which thus led to the capture of VLDL. VLDL-containing human apoE taken up by the liver would prevent human apoE and VLDL to be transported to the circulation. After the second injection of AV1.0CMVapoE, a second peak of apoE secretion was observed. This peak was 3 times higher than that after the first injection at the same dose. Based on the hypothesis of local expression of the transgene that was undetectable in the circulation, all additional human apoE synthesis provided by the second injection of the virus may be available for entrance into the bloodstream, leading to higher plasma levels of the human transgene.
Liver overexpression of human apoE in this mouse model not only resulted in a decrease in plaque formation but also induced atherosclerotic plaque regression. The antiatherogenic effect of apoE has been widely explored, such as for its role in lipoprotein remnant clearance42 43 44 when secreted by the liver and in modulating cholesterol balance within the arterial wall when expressed by macrophages.45 46 47 48 In this study, human apoE was not secreted in the vessel wall, and the detected transgene in this tissue came only from apoE infiltration in the arteries (data not shown). Similar infiltrations of liver-secreted proteins with antiatherogenic properties in the vessel wall have been observed for mouse apoE49 and human tissue inhibitor of metalloproteinase-1.50
Atherosclerotic regression in our model resulted from synergistic
mechanisms. Whereas lipid lowering is a major component of this
process, it does not explain cholesterol removal from the
preexisting lesions, which is due to an active process. In addition, we
found no correlation between the reduction in cholesterol
level and lesion size, but we did observe a strong relationship between
plasma concentration of apoE and lesion size. Among candidates for
cholesterol removal in this study, we found an increase in
HDL particles and plasma apoE levels. Both apoA-I– and apoE-containing
lipoproteins have already been shown to be acceptors of excess
cholesterol that can be desorbed from human
monocyte–derived macrophages.51 52 Moreover, both
lipoproteins participate in reverse cholesterol transport
to the liver for cholesterol elimination.53 In
addition, both lipoproteins have a cooperative effect, with apoE acting
as a "bridging" molecule that attracts apoA-I–containing
lipoproteins in the atherosclerotic intima.54 When human
apoE is expressed in the mouse liver, an elevated concentration of
-LpE is observed. This peculiar association of apoE exists in the
physiological situation but usually
represents only 1% to 2% of the total concentration of apoE.
Krimbou et al24 have demonstrated that in vitro, this form
of apoE was able to induce efflux of cholesterol from the
arterial wall to transfer it to cholesterol
acceptors such as HDL particles.
Recently, reports suggest links between all of these mechanisms: (1) Cholesterol lowering reduces matrix metalloproteinase activity in the plaque.55 (2) Matrix metalloproteinase (L. Lindstedt, personal communication, 1998) and other proteases such chymase56 are able to trap and proteolyze apoA-I–containing lipoproteins. This may explain the high apoA-I content in atheromatous lesions.57 A similar mechanism could be envisaged for apoE.58 We propose that in our model, cholesterol lowering resulted in a reduction of matrix proteases, thus allowing both apoA-I– and apoE-containing lipoproteins coming from the circulation to act on cellular cholesterol removal in the arteries without being trapped. This active process induced a complete remodeling of the arterial wall, including the disappearance of macrophages and foam cells, and the reendothelialization of the arterial wall.
In conclusion, complete lesion regression was demonstrated in an apoE–/– nu+/+ mouse model by using a first-generation recombinant adenovirus. Besides the usefulness of this model for evaluating the role of candidate genes in atherosclerosis development, this mouse model will provide new insights into the plaque regression process.
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Acknowledgments |
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We thank the Groupement d’interêt écouomique d’aide à la formation par la recherche and the Association Française coutre les myopathies for their financial support. We also thank Isabelle Viry, James Clavier, Laurent Bassinet, Patrick Juvet, and Catherine De Geitère for their excellent technical assistance.
Received May 5, 1999; accepted September 1, 1999.
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