(Circulation. 1997;96:4349-4356.)
© 1997 American Heart Association, Inc.
Articles |
Effects of Adenovirus-Mediated Human Apo A-I Gene Transfer on Neointima Formation After Endothelial Denudation in Apo E–Deficient Mice
From the Center for Molecular and Vascular Biology, University of Leuven (Belgium).
Correspondence to D. Collen, MD, PhD, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium. E-mail desire.collen{at}med.kuleuven.ac.be
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background Inactivation of apolipoprotein (apo) E genes in mice markedly increases ß-VLDL levels and accelerates progression of complex atherosclerotic lesions. The present study investigated (1) the effect of apo E deficiency (apo E-/-) on neointima formation after endothelial denudation; and (2) the effect of increased HDL, induced by adenovirus-mediated transfer of a human apo A-I gene, on neointima formation.
Methods and Results Guidewire-induced abrasion of the
endothelium of the common carotid artery did not
produce neointima formation within 18 days after injury in
C57BL/6J mice (n=12) but was associated with an intima/media ratio of
0.82±0.25 in age-matched C57BL/6J apo E-/- mice
(n=12). Neointima consisted primarily of smooth muscle
-actin positive cells. Injection in C57BL/6J apo
E-/- mice of 2x109 (n=5) or
4x109 (n=7) plaque forming units (p.f.u.) of a recombinant
human apo A-I adenovirus 3 days before injury resulted in an increase
of HDL cholesterol from 36±5 to 75±3 mg/dL
(P<.05) and to 96±13 mg/dL (P<.05),
respectively, and of the HDL cholesterol/non–HDL
cholesterol ratio from 0.063±0.003 to 0.15±0.01
(P<.05) and to 0.16±0.015 (P<.05),
respectively. Intima/media ratio decreased to 0.28±0.06
(P=NS versus C57BL/6J apo E-/- mice)
with 2x109 p.f.u. of apo A-I virus and to 0.03±0.01 with
4x109 p.f.u. (P<.01 versus C57BL/6J apo
E-/- mice). Injection of 4x109 p.f.u. of
RR5 (n=7) or tissue plasminogen activator
(t-PA) control virus (n=6) did not result in a significant alteration
of HDL cholesterol (44±11 and 26±4 mg/dL, respectively)
nor in a reduction of intima/media ratio (0.81±0.35 and 0.86±0.23,
respectively).
Conclusions Apo E deficiency is associated with increased neointima formation after endothelial denudation. Gene transfer of apo A-I increases HDL cholesterol and significantly reduces neointima formation, which suggests a direct vascular protective effect of HDL.
Key Words: atherosclerosis • endothelial injury • lipoproteins • apolipoproteins
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Apolipoprotein A-I (apo A-I), a single-chain polypeptide of 243 amino acids, is the principal apolipoprotein of high-density lipoproteins (HDL). Epidemiological studies have demonstrated an inverse relationship between plasma levels of HDL and risk for ischemic cardiovascular disease.1–3 Studies in transgenic mice have demonstrated that apo A-I has an antiatherogenic effect4–6 and that the protein composition of HDL significantly affects its antiatherogenic potential.7,8 The antiatherogenic effect of HDL has been ascribed to reverse cholesterol transport, the process by which cholesterol is transported from peripheral tissues to the liver for excretion and to endocrine organs for steroid hormone synthesis.9,10
High HDL is associated with decreased risk for restenosis after percutaneous transluminal coronary angioplasty (PTCA) in humans.11,12 Infusion of apo A-I Milano, a natural apo A-I mutant with a cysteine for arginine mutation at position 173, reduces neointima formation in cholesterol fed New Zealand White rabbits.13 The protective effect of HDL on restenosis and on neointima formation may be independent of reverse cholesterol transport. Indeed, HDL inhibits the oxidation of low-density lipoproteins (LDL)14,15 and reverses the inhibitory effect of oxidized LDL on endothelium-dependent arterial relaxation in vivo.16
Mice with inactivation of both apo E alleles are characterized by very high levels of ß-VLDL and accelerated progression of complex atherosclerotic lesions.17–20 Because introduction of a human apo A-I transgene in C57BL/6J apo E-/- mice has been shown to be a potent suppressor of atherosclerosis,5,6 we speculated that adenovirus-mediated transfer of a recombinant human apo A-I transgene might suppress neointima formation in these mice. This study demonstrates that endothelial denudation caused neointima formation in C57BL/6J apo E-/- but not in C57BL/6J control mice. Gene transfer with a recombinant adenovirus that induced a transient production of human apo A-I resulted in a 2.7-fold increase in HDL cholesterol levels and in a significant suppression of neointima formation. The present study suggests a direct vascular protective effect of HDL.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Preparation of Recombinant Adenovirus and Viral Stocks
The entire human apo A-I cDNA, including presequences and prosequences,21 was cloned into the pACCMVpLpA vector.22 Five micrograms of this plasmid (pACCMVapo A-I) was cotransfected into 293 cells together with 2.5 µg of the pJM17 plasmid that contains the full-length adenovirus serotype 5 genome.23 Transfection efficiency was boosted after 6 hours by glycerol shock with 15% glycerol in Dulbecco's modified Eagle medium (DMEM, Gibco). Homologous recombination between pJM17 and pACCMVapo A-I leads to the formation of a recombinant adenovirus (AdCMVapo A-I) in which most of the early region 1 (E1) of the adenoviral genome is replaced by the expression cassette containing the apo A-I cDNA under control of the cytomegalovirus (CMV) promoter. The recombinant adenovirus is defective because of the absence of the E1 region, which is provided in trans by the 293 cells.24 The cells were overlaid with 0.65% noble agar in modified Eagle medium (MEM, Gibco) supplemented with 4% fetal bovine serum (Gibco) 24 hours after transfection. Viral plaques, appearing within 7 to 10 days, were picked and used to infect 293 cells. Recombinant adenovirus was identified by measuring human apo A-I concentration in the conditioned medium by ELISA.21 Large-scale production of recombinant adenovirus was performed by infecting confluent monolayers of 293 cells in 15-cm dishes. When 90% of the cells showed a cytopathic effect, Igepal CA-630 (Sigma) was added to a final concentration of 0.1% to lyse the cells. Cellular debris was removed by centrifugation at 12 000g for 10 minutes. After addition of 0.5 volume of 20% polyethylene glycol in 2.5 mol/L NaCl, virus-containing extracts were incubated on ice for 1 hour, and virus was precipitated by centrifugation at 12 000g for 20 minutes. The pellet was resuspended in 20 mmol/L Tris-HCl, pH 7.8, containing CsCl to obtain a relative density of 1.1. After CsCl step gradient, ultracentrifugation at 22 000 rpm for 2 hours, the virus was harvested from the 1.3 and 1.4 interface. The recombinant virus was desalted by chromatography on Sepharose CL4B in an isotonic saline buffer (10 mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2). The virus was stored at -80°C until use. Viral titers were determined by plaque assay, and doses were expressed in plaque forming units (p.f.u.). The experiments described in this report were performed with one batch of virus, although spot checks with other batches confirm the generality and reproducibility of the observations. The generation of the recombinant adenovirus AdRR5, which does not contain an insert after the CMV promoter, has been described earlier.25 The human tissue plasminogen activator (t-PA) virus was a kind gift of Dr R.D. Gerard (Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute of Biotechnology, Leuven). t-PA plasma levels were determined by ELISA as described previously.26
Animal Experiments
All experimental procedures in animals were performed in
accordance with protocols approved by the Institutional Animal Care and
Research Advisory Committee. Apo E-/-
mice,27 backcrossed for six generations into the
C57BL/6J background, were purchased from Jackson Laboratory (Bar
Harbor, Maine). These mice had 98.4% C57BL/6J background. Mice were
fed normal chow ad libitum. All mice used in this study were
approximately 4 months of age and weighed between 22 and 30 g.
Viral Injections
Mice were anesthestized by intraperitoneal
injection of 60 mg/kg pentobarbital (Abbott Laboratories). The jugular
vein was prepared by blunt-end dissection and a 2F gauge catheter was
introduced in the vein. Each viral dose was given in a final volume of
300 µL.
Guidewire Injury Model
Endothelial denudation of the common carotid
artery of mice was induced with a guidewire essentially as described by
Lindner et al.28 The right common carotid artery
and the right external carotid artery were exposed by blunt-end
dissection. A guidewire (epidural spinal anesthesia
guidewire; Portex; diameter of 320 µm) was introduced through
the external carotid artery and moved proximally into the common
carotid artery. The common carotid artery was abraded three times over
its entire length. Eighteen days after injury, mice were anesthestized
with 60 mg/kg Nembutal and a maximal blood volume was collected by
puncture of the inferior vena cava. Perfusion fixation
under physiological pressure was performed for 10
minutes with 4% formol in phosphate-buffered saline (PBS, pH 7.0)
after intracardiac puncture. The common carotid artery was dissected,
fixed in 4% formol in PBS for 5 hours and transferred to PBS
containing 20% sucrose. Arteries were embedded in OCT compound
(Tissue-Tek, Miles Inc), snap-frozen in precooled 2-methyl butane, and
stored at -80°C until further use. Seven-micron-thick cryosections
were made through the whole injured artery and stained with
hematoxylin-eosin.
Morphometric Analysis
Morphometric analysis was performed in a blinded manner
with the Leica 2 Quantimet system. The area within the external elastic
lamina, the area within the internal elastic lamina, and the lumen size
were determined in the injured segment of the artery at distances of
84 µm. The length of the injured segment was between 5 and
7 mm, and approximately 80 sections were analyzed per
artery. Media was defined as the area between the internal and external
elastic lamina. Intima was defined as the area within the internal
elastic lamina not occupied by vessel lumen, thrombus, and organized
thrombus. Intima/media ratio was defined as the ratio between the area
occupied by neointima and the area occupied by media.
Immunohistochemistry
Endothelial cells were immunostained
with rabbit anti–von Willebrand Factor antibodies (Dakopatts;
diluted 1:100), biotinylated goat anti-rabbit antibodies (Dakopatts),
and peroxidase-labeled avidin (Dakopatts; diluted 1:100). Peroxidase
reaction was performed in 0.05 mol/L Tris:HCl (pH 7.0) containing
0.06% 3,3'-diaminobenzidine and 0.01%
H2O2. Tissue sections were
counterstained with hematoxylin. Smooth muscle cells and inflammatory
cells were detected in an indirect staining procedure using
respectively a cross-reacting murine monoclonal biotinylated antibody
against human smooth muscle cell -actin (clone1A4; Sigma; diluted
1:500) or a rat biotinylated monoclonal antibody against murine common
leukocyte antigen/CD45 (clone 30F11.1; Pharmingen; diluted
1:100).
RNA Extraction and Northern Blotting
Total RNA was extracted from mouse liver by a single step
method (Ultraspec RNA isolation system, Biotecx) based on the
guanidinium isothiocyanate acid phenol method of Chomczynski and
Sacchi.29 Approximately 10 µg of RNA was
separated on a 1.25% agarose gel containing 0.21 mol/L formaldehyde
and 3.8 vol% formamide and was blotted overnight on a nylon membrane
(Hybond-N, Amersham Life Sciences). After UV cross-linking, the
membrane was prehybridized in Quikhyb (Stratagene) for 4 hours. A human
genomic 2.2 kb apo A-I fragment was labeled with a random primer
labeling kit (Prime-Random II Primer Labeling Kit, Stratagene) and 15
million cpm were added to the prehybridization solution. After
hybridization for 16 hours, the blot was washed for 20 minutes in a
solution containing 2xSSC (1xSSC is 0.15 mol/L NaCl, 0.015 mol/L
trisodiumcitrate 2H2O, pH 7.0) and 0.05% SDS and
then washed twice for 20 minutes in a buffer containing 0.1% SSC and
0.1% SDS. The membrane was exposed overnight at -80°C to a
Hyperfilm-MP (Amersham Life Sciences).
Plasma Lipid and Lipoprotein Analyses
Blood was obtained after an overnight fast by puncture of
the retro-orbital plexus or by puncture of the vena cava and
anticoagulated with 0.1 volume of 4% trisodiumcitrate. Plasma was
obtained by centrifugation and lipoprotein fractions
were separated by gel filtration of 200 µL of plasma on a Superdex
200HR column equilibrated with 20 mmol/L Tris:HCl buffer, pH 8.1,
containing 0.15 mol/L NaCl, 1 mmol/L EDTA, and 0.02 mg/mL sodium
azide in a FPLC system (Waters Associates). Phospholipid and
triglyceride levels were determined by standard enzymatic
assays (Biomérieux and Sigma, respectively).
Cholesterol of fractions obtained after gel filtration was
extracted with methanol/chloroform 2:1 (vol:vol). Esterified and
unesterified cholesterol was quantitated by
high-performance liquid chromatography on a
reversed-phase column (Zorbax ODS; Du Pont de Nemours) essentially as
descibed by Vercaemst et al.30 Samples were
eluted isocratically at 45°C with a mixture of
acetonitrile/isopropanol 50:50 (vol:vol).
Statistical Analysis
Intima/media ratio of control apo E–deficient mice, mice
treated with 4x109 p.f.u. of RR5 control virus
and 4x109 p.f.u. of t-PA adenovirus, were
compared by Kruskal-Wallis nonparametric ANOVA test on
logarithmically transformed values in the INSTAT V2.05a statistical
program (Graph Pad Software), which revealed the absence of a
statistically significant difference between the three control groups.
Therefore, these three control groups were pooled and compared with
mice treated with 2x109 p.f.u. and
4x109 human apo A-I adenovirus with a
Kruskal-Wallis nonparametric ANOVA test on logarithmically
transformed values followed by Dunn's multiple comparisons test.
Significance of differences in lipid values was assessed by a
two-tailed unpaired alternate Welch t
test.31 A probability value of <.05 was
considered statistically significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Expression of Human Apo A-I in C57BL/6J and C57BL/6J Apo E-/- Mice
Fig 1A
illustrates the time course
of apo A-I expression in C57BL/6J mice after injection of
10,9 2x10,9
4x109, and 1010 p.f.u. of
human apo A-I adenovirus. Injection of 2x109
p.f.u. resulted in 36-fold increased human apo A-I levels at day 3 and
6.5-fold higher human apo A-I levels at day 6 compared with injection
of 109 p.f.u. A dose of
4x109 p.f.u. did not produce a significant
further increase of the apo A-I levels. In C57BL/6J mice treated with
2x10,9 4x109, or
1010 p.f.u., human apo A-I levels decreased
3-fold between day 6 and day 9, whereas in mice treated with
109 p.f.u. maximal human apo A-I levels were
obtained at day 9. Fig 1B shows that the time course of human apo A-I
expression after injection of a dose of 2x109
p.f.u. of recombinant virus in C57BL/6J apo
E-/- mice was very similar to that in
C57BL/6J mice. Northern blot analysis of total liver RNA of
C57BL/6J apo E-/- mice at days 6, 9, 14, and
21 after virus injection demonstrated a strong signal of human apo A-I
RNA at days 6 and 9, which was markedly reduced at day 14 (data not
shown).
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), 2x109
(
), 4x109 (
) or of 1010 (
) p.f.u. of
human apo A-I adenovirus in C57BL/6J mice and (B) after injection of a
dose of 2x109 p.f.u. of apo A-I adenovirus in C57BL/6J apo
E-/- mice. Data represent mean±SEM of four
independent experiments.
Effect of Human Apo A-I Adenovirus on Lipoprotein Profiles and
Lipid Values in C57BL/6J and C57BL/6J Apo E-/-
Mice
The cholesterol and human apo A-I distribution
profiles in C57BL/6J mice at day 3 after gene transfer with
4x109 p.f.u. of human apo A-I adenovirus and the
cholesterol profile in C57BL/6J control mice, C57BL/6J mice
3 days after injection with 4x109 p.f.u. of RR5
or t-PA control virus, are illustrated in Fig 2A. The corresponding profiles in
C57BL/6J apo E-/- mice are shown in Fig 2B.
Ninety percent of the human apo A-I was associated with HDL fractions
both in C57BL/6J and C57BL/6J apo E-/- mice.
The Table summarizes cholesterol and
phospholipid levels of lipoprotein fractions after gel filtration and
triglyceride plasma levels in both C57BL/6J mice and
C57BL/6J apo E-/- control mice and in
C57BL/6J mice and C57BL/6J apo E-/- mice 3
days after injection with 4x109 p.f.u. of RR5
control virus, t-PA control virus, and human apo A-I adenovirus. HDL
cholesterol levels increased 2.8-fold in C57BL/6J mice
(P<.05) and 2.7-fold in C57BL/6J apo
E-/- mice (P<.05) 3 days after
treatment with 4x109 p.f.u. of human apo A-I
adenovirus (Table). Injection in C57BL/6J apo
E-/- mice of 2x109
p.f.u. of human apo A-I adenovirus resulted in a 2.1- fold increase of
HDL cholesterol (75±3 mg/dL; P<.05). Non–HDL
cholesterol levels after treatment with
4x109 p.f.u. of human apo A-I adenovirus
increased 3.1-fold in C57BL/6J mice (P<.05) and were not
significantly altered in C57BL/6J apo E-/-
mice treated with either 2x109 p.f.u. (490±29
mg/dL) or with 4x109 p.f.u. (Table). The HDL
cholesterol/non–HDL cholesterol ratio
increased 2.5-fold from 0.063±0.003 in untreated C57BL/6J apo
E-/- mice to 0.15±0.01 (P<.05)
and 0.16±0.015 (P<.05) in C57BL/6J apo
E-/- mice treated with
2x109 p.f.u. and 4x109
p.f.u. of human apo A-I adenovirus, respectively. No significant
alteration of HDL cholesterol was seen after treatment with
4x109 p.f.u. of RR5 or of t-PA control virus in
either C57BL/6J or C57BL/6J apo E-/- mice.
Injection of 4x109 human t-PA adenovirus
resulted in human t-PA plasma levels of 9.7±0.76 µg/mL in C57BL/6J
mice (n=4) and 8.8±1.0 µg/mL in C57BL/6J apo
E-/- mice (n=6), whereas baseline murine t-PA
levels are 2.5±0.65 ng/mL (mean±1.96 SEM).32
Non–HDL cholesterol levels in C57Bl/6J mice increased
1.8-fold after treatment with RR5 control virus (P<.05) and
3.1-fold after treatment with t-PA control virus (P<.01).
No significant alteration of non–HDL cholesterol occurred
in C57BL/6J apo E-/- mice after treatment
with of 4x109 p.f.u. of both control
viruses.
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), and human apolipoprotein (apo) A-I adenovirus (
) in C57BL/6J (A) and C57BL/6J apo
E-/- (B) mice was predominantly associated with HDL
fractions (fractions 19 to 25).
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Fig 3 illustrates phospholipid
distribution profiles in C57BL/6J (Fig 3A) and C57BL/6J apo
E-/- mice (Fig 3B) for control mice and for
mice 3 days after gene transfer with 4x109
p.f.u. of RR5 control virus, t-PA control virus, and human apo A-I
adenovirus. Phospholipid levels increased 2.9-fold in apo A-I
adenovirus–treated C57BL/6J mice (P<.05) and 5.6-fold in
apo A-I adenovirus–treated C57BL/6J apo E-/-
mice (P<.01); non–HDL phospholipids increased 4.2-fold in
(P<.05) and 3.0-fold (P<.05), respectively.
There was no statistically significant alteration of HDL phospholipids
in either C57BL/6J or C57BL/6J apo E-/- mice
after treatment with 4x109 p.f.u. of RR5 or t-PA
control virus. Non–HDL phospholipids increased 2.0-fold
(P=NS) and 1.3-fold (P=NS) in C57BL/6J and
C57BL/6J apo E-/- mice, respectively, after
treatment with 4x109 p.f.u. of RR5 control virus
and increased 3.6- fold (P<.01) and 1.8- fold
(P<.05) after treatment with 4x109
p.f.u. of human t-PA virus.
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Treatment with 4x109 p.f.u. of human apo A-I adenovirus induced a 2.1-fold (P<.05) and a 3.9-fold (P<.05) increase of triglycerides in C57BL/6J and C57BL/6J apo E-/- mice, respectively. No significant alteration of triglycerides was seen after treatment with 4x109 p.f.u. of RR5 or t-PA control virus in either C57BL/6J or C57BL/6J apo E-/- mice.
Endothelial Denudation and Neointima
Formation in C57BL/6J and C57BL/6J Apo E-/-
mice
Endothelial denudation of the common carotid
artery of mice was induced with a guidewire essentially as described by
Lindner et al.28 To confirm the reproducibility
of the model, neointima formation was measured in Swiss
Webster mice, which were previously used by Lindner et al. Mean
intima/media ratio was 0.44±0.29 (n=7) 18 days after injury (data not
shown). However, no detectable neointima formation was
obtained in 12 C57BL/6J mice notwithstanding the fact that
endothelial denudation was confirmed by the loss of
immunostaining for von Willebrand factor within
the internal elastic membrane 2 days after injury.
Representative photographs illustrating the extent of
neointima formation in injured arteries of nontreated and
human apo A-I adenovirus treated C57BL/6J apo
E-/- mice are shown in Fig 4. Recombinant adenoviruses were injected
3 days before injury and the extent of neointima formation
was assessed 18 days after injury. Thrombus was detected 18 days after
injury in 4 out of 16 C57BL/6J apo E-/-
control mice, in 2 out of 9 RR5-treated animals (P=NS), 1
out of 8 apo A-I adenovirus–treated mice (P=NS), and 1 out
of 7 t-PA–treated mice (P=NS). These arteries were not
included in the analysis. Mean intima/media ratio was
0.82±0.25 in C57BL/6J apo E-/- mice (n=12),
0.81±0.35 in C57BL/6J apo E-/- mice treated
with 4x109 p.f.u. of RR5 adenovirus (n=7),
0.86±0.23 in C57BL/6J apo E-/- mice treated
with 4x109 p.f.u. of human t-PA adenovirus
(n=6), 0.28±0.06 in C57BL/6J apo E-/- mice
treated with 2x109 p.f.u. of human apo A-I
adenovirus (n=5), and 0.03±0.01 in C57BL/6J apo
E-/- mice treated with
4x109 p.f.u. of human apo A-I adenovirus (n=7)
(Fig 5). Comparison of C57BL/6J apo
E-/- control mice, mice treated with
4x109 p.f.u. of RR5 control virus, and mice
treated with 4x109 t-PA adenovirus by
Kruskal-Wallis nonparametric ANOVA test on logarithmically
transformed values demonstrated the absence of a statistically
significant difference between these three control groups.
Subsequently, C57BL/6J apo E-/- control mice,
mice treated with 4x109 p.f.u. of RR5 control
virus, and mice treated with 4x109 t-PA
adenovirus were grouped and comparison with mice treated with
2x109 and 4x109 apo A-I
adenovirus by Kruskal-Wallis nonparametric ANOVA test on
logarithmically transformed values showed a probability value of .0029.
Comparison of all grouped control mice with mice treated with
4x109 apo A-I adenovirus was statistically
significant at P<.01.
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Endothelial denudation was confirmed at day 2 by the
loss of immunostaining for von Willebrand
factor within the internal elastica membrane (Fig 6A). At day 18 after
endothelial abrasion,
reendothelialization had occurred (Fig 6B).
Neointima was characterized by a dense population of smooth
muscle -actin immunoreactive cells (Fig 6C), whereas CD-45
immunoreactive cells were only occasionally observed (Fig 6D).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study demonstrates (1) that apo E deficiency was associated with increased neointima formation after endothelial denudation in the carotid arteries; and (2) that adenovirus-mediated transfer of human apo A-I, resulting in a significant increase of HDL in the absence of a decrease of VLDL, was associated with reduced neointima formation. These data suggest that HDL reduce neointima formation associated with endothelial injury, even in the presence of elevated levels of atherogenic ß-VLDL.
Previously it has been demonstrated that apo E-/- mice with elevated plasma levels of ß-VLDL due to delayed clearance of large atherogenic particles from the circulation showed accelerated progression of complex atherosclerotic lesions19,20 and that introduction of a human apo A-I transgene in apo E-/- mice significantly reduced the progression rate.5,6 Elevated HDL cholesterol levels in these mice accounted for 78% of the observed variance of mean lesion area.6 An approximately 50% increase in HDL cholesterol levels has previously been reported after gene transfer with 109 p.f.u. of a human apo A-I adenovirus.33
In the present study, adenovirus-mediated transfer of human apo A-I in C57BL/6J apo E-/- mice resulted in a 2.7-fold increase of HDL cholesterol that was similar to that observed after transfer of a human apo A-I transgene in apo E-/- mice.5,6 This increase was associated with a significant reduction of neointima formation after endothelial denudation. Human apo A-I gene transfer was, however, also associated with an increase of non–HDL phospholipids and triglycerides both in C57BL/6J and in C57BL/6J apo E-/- mice and an increase in non–HDL cholesterol in C57BL/6J mice. The increases of non–HDL cholesterol and phospholipids can be at least partially explained by an adenovirus-induced acute phase response, which is known to be associated with increases of phospholipids and ß- and pre–ß-lipoproteins.34,35 Although gene transfer with the RR5 control virus did not significantly alter non–HDL phospholipids, administration of t-PA control virus led to a significant increase in non–HDL phospholipids, both in C57BL/6J mice and C57BL/6J apo E-/- mice, probably due to the inflammatory response directed against viral gene products and the transgene. This response was more pronounced in mice treated with apo A-I or t-PA virus in comparison with the RR5 virus, which does not produce a transgene. The increase in non–HDL cholesterol seen after gene transfer with 4x109 p.f.u. of human apo A-I adenovirus was also observed after treatment with the same dose of RR5 and t-PA control virus but again was more pronounced after treatment with t-PA virus compared with the RR5 virus. A smaller but statistically significant increase in non–HDL cholesterol has also been observed in human apo A-I transgenic mice and rabbits.4,36
Apo E production by monocytes and macrophages in the vessel wall may reduce atherogenesis independent of circulating lipoproteins in the blood37,38 by redistributing excess cholesterol39 and enhancing reverse cholesterol transport.40 Overexpression of human apo E in the arterial wall of transgenic mice did not alter lipoprotein profiles but decreased lesion area with 70%,37 whereas macrophage-specific expression of human apo E significantly reduced atherosclerosis in the aortic sinus and proximal aorta compared with apo E-/- mice matched for plasma cholesterol.38 It is possible that local vascular production of apo E plays a protective role in neointima formation after endothelial denudation and that lack of apo E containing HDL in the vessel wall is associated with increased intimal hyperplasia. This has however to be tested in apo E-/- mice selectively expressing apo E in macrophages and backcrossed to the C57BL/6J background.
Alternatively, increased neointima formation may be due to a direct effect of atherogenic ß-VLDL on smooth muscle cells exposed after endothelial denudation. It has indeed been shown that lysophosphatidylcholine, a major phospholipid component of atherogenic lipoproteins such as ß-VLDL and oxidized LDL, that may be generated by the action of leukocyte-secreted phospholipase A2 at sites of inflammation, may induce growth factor gene expression that may contribute to the migration and proliferation of smooth muscle cells.41 The stimulation of smooth muscle cell proliferation by VLDL equalled that obtained by direct stimulation with platelet derived growth factor.42 Injury of the endothelium may have been associated with the release of radicals that induced oxidation of ß-VLDL and LDL that infiltrated the arterial wall.
There are different possible mechanisms by which HDL may inhibit neointima formation. Increase of HDL levels may result in a direct inhibition of smooth muscle cell proliferation by inhibition of growth factor synthesis43 or in an indirect inhibition by increasing the degradation of lysophosphatidylcholine by plasma lysolecithin acyltransferase44 or by neutralizing the effect of lysophosphatidylcholine.16
Residual thrombus 18 days after injury was seen in 4 of 16 C57BL/6J apo E-/- mice, 2 of 9 RR5 control virus–treated C57BL/6J apo E-/- mice, 1 of 7 t-PA control virus–treated mice, and 1 of 8 human apo A-I adenovirus–treated mice. The arteries with thrombus were discarded from the final analysis because in the presence of thrombus it is difficult to discern between clot colonization and neointima formation unrelated to presence of thrombus. Furthermore, neointima formation and clot colonization may represent distinct biological processes. The inclusion or exclusion of arteries with thrombus, however, did not affect the overall conclusions of this study.
The absence of detectable neointima formation in C57BL/6J mice was surprising and points to the importance of a fixed genetic background in these studies. Indeed, we confirmed that Swiss Webster mice, which were used in the original guidewire injury model described by Lindner et al,28 developed neointima after endothelial denudation. Our data obtained in C57BL/6J mice are also in agreement with the previous observation of Sullivan et al45 that 17ß estradiol replacement in ovariectomized C57BL/6J mice potently suppressed carotid response to injury.
In conclusion, the present study demonstrates that apo E deficiency induces substantial neointima formation after endothelial denudation in C57BL/6J mice that can be markedly reduced by a transient increase in HDL induced by adenovirus-mediated transfer of human apo A-I.
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Acknowledgments |
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Bart De Geest is a research assistant of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. This study was supported in part by the Interuniversitaire Attractiepolen (Program 4/34) and by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Program G 3063.94). The authors are grateful to H. Bernar, A. Bouché, E. Brouwers, E. Deridder, M. Landeloos, and M. Lox for technical assistance.
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Footnotes |
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Presented in part during the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 9 to 13, 1996.
Received January 22, 1997; revision received September 8, 1997; accepted September 11, 1997.
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