Induction of Rapid Atherogenesis by Perivascular Carotid
Collar Placement in Apolipoprotein E–Deficient and
Low-Density Lipoprotein Receptor–Deficient Mice
Jan H. von der Thüsen, MD; Theo J.C. van Berkel, PhD; Erik A.L. Biessen, PhD
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Background—Perivascular collar placement has been used as a means for localized atherosclerosis induction in a variety
of experimental animal species. In mice, however, atherosclerosis-like lesions have thus far not been obtained by this
method. The aim of this study was the development of a mouse model of rapid, site-controlled atherogenesis.
Methods and Results—Silastic collars were placed around the carotid arteries of apolipoprotein E– deficient (apoE⫺/⫺)
and LDL receptor– deficient (LDLr⫺/⫺) mice. The development of collar-induced lesions was found to occur
predominantly in the area proximal to the collar and to be dependent on a high-cholesterol diet. Lesions were evident
in apoE⫺/⫺ mice after 3 weeks and in LDLr⫺/⫺ mice after 6 weeks and were overtly atherosclerotic in appearance.
Lumen stenosis reached 85% in apoE⫺/⫺ mice and 61% in LDLr⫺/⫺ mice 6 weeks after collar insertion. Expression
levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were increased both proximal and
distal to the collar, whereas endothelial nitric oxide synthase expression was downregulated at the proximal site.
Conclusions—We propose that this model of collar-induced acceleration of carotid atherogenesis is of hemodynamic
cause. It may serve as a substrate for sequential mechanistic studies concerned with the underlying cause and
pathogenesis of atherosclerosis. The rapidity of lesion development will also aid the efficient screening of new
potentially antiatherogenic chemical entities and the evaluation of therapies with limited duration of effectiveness, such
as adenoviral gene therapy. (Circulation. 2001;103:1164-1170.)
Key Words: atherosclerosis 䡲 carotid arteries 䡲 cell adhesion molecules 䡲 hemodynamics
A
lesions in the rabbit carotid artery. The underlying mechanism,
however, appears to remain in essence fibroproliferative, and it
could, therefore, be valuable to study the effect of perivascular
collar placement in animals that are more susceptible to spontaneous and humanlike atherogenesis. The aforementioned transgenic mouse models are eminently suited to this purpose
because they are predisposed to developing severe atherosclerotic lesions, particularly when fed a high-fat diet.
The aim of this study was to evaluate the intimal response
of the carotid artery to Silastic collar placement in apoE⫺/⫺
and LDLr⫺/⫺ mice, which are known to spontaneously
develop extensive and complex atherosclerotic lesions. On an
atherogenic diet, LDLr⫺/⫺ mice have relatively fibroproliferative intimal lesions at sites of hemodynamic predilection,3
whereas lesions in apoE⫺/⫺ mice are more heterogeneous
and lipid-rich, even when a regular chow diet is given.4 We
hypothesized that the flow disturbance caused by the placement of a mildly constrictive perivascular collar could result
in site-controlled atherogenesis in these mouse strains; therefore, we applied such collars to the midsection of the common
carotid artery, an easily accessible site of low natural occur-
therosclerosis is a common and complex pathological
process characterized by intimal foam cell accumulation
and extracellular matrix deposition in medium and large-sized
arteries.1 In the pursuit of a representative, reproducible, and
practical model for this disease, several animal species and
atherogenic stimuli have been used. Systemic approaches have
included diet-induced hypercholesterolemia2 and genetically
modified mouse models, including LDL receptor knock-out
(LDLr⫺/⫺),3 apolipoprotein E knockout (apoE⫺/⫺),4 and apolipoprotein E3-Leiden transgenic5 strains. Local lesion induction
has been achieved by transluminal (eg, wire denudation)6 or
extravascular (eg, perivascular collar placement)7 arterial manipulation. Perivascular collar placement offers the advantage of
maintaining the structural integrity of the endothelium while
inducing rapid, site-controlled lesion formation. The neointima
in perivascular collar models tends to occur within the collar and
is generally fibroproliferative in appearance, with limited foam
cell formation and extracellular lipid deposition.8,9 Cholesterol
feeding10 or local (oxidized) LDL application11 in conjunction
with the placement of a perivascular Silastic collar have been
shown to promote the development of more atherosclerosis-like
Received April 18, 2001; revision received August 25, 2001; accepted September 11, 2000.
From the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Leiden University, Leiden, The
Netherlands (J.H.v.d.T., T.J.C.v.B., E.A.L.B.); and the Department of Cardiology, Leiden University Medical Centre, Leiden, The Netherlands
(J.H.v.d.T.).
Correspondence to Jan H. von der Thüsen, Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Leiden
University, Wassenaarseweg 72, PO Box 9503, 2300 RA Leiden, The Netherlands. E-mail [email protected]
© 2001 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
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von der Thüsen et al
Atherogenesis by Perivascular Carotid Collar
1165
Tissue Harvesting and Preparation
One to 18 weeks after collar placement, the animals were anesthetized
and exsanguinated by femoral artery transection. In situ perfusion
fixation through the left cardiac ventricle was performed by PBS
instillation for 15 minutes, followed by constant-pressure infusion (at 80
mm Hg) of 10% neutral buffered formalin (Shandon Inc) for 30
minutes. Subsequently, both carotid bifurcations and common carotid
arteries were removed. Formalin fixation was omitted for arteries that
were to be stained for vWF; these were immediately snap-frozen in
liquid nitrogen after having been embedded in OCT compound (TissueTek; Sakura Finetek), whereas the remaining arteries were left in 10%
formalin overnight before freezing. The specimens were stored at
⫺20°C until further use. Transverse 5-␮m cryosections were prepared
in a proximal direction from the carotid bifurcation and mounted in
order on a parallel series of slides.
Histology and Immunohistochemistry
Figure 1. Schematic representation of carotid collar positioning
and assessment sites.
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rence of atherosclerosis. The extent as well as the composition of the lesions thus obtained was assessed to identify any
potential differences in lesion development between the
strains, thereby validating their relative value as a substrate
for focal induction of atherogenesis. In addition, we used
immunohistochemistry to determine endothelial integrity
(von Willebrand factor [vWF]) and the expression levels of
endothelial cell nitric oxide synthase (eNOS) and two endothelial adhesion molecules that are known to be involved in
the development of atherosclerosis in humans (intercellular
adhesion molecule-1 and vascular cell adhesion molecule-1
[ICAM-1 and VCAM-1]) to further elucidate the underlying
mechanisms of lesion formation in this model.
Cryosections were routinely stained with hematoxylin (Sigma Diagnostics) and eosin (Merck Diagnostica) and with oil red O (Sigma
Diagnostics) for lipid visualization. Corresponding sections on separate
slides were stained immunohistochemically with antibodies against a
macrophage-specific antigen (MOMA-2, polyclonal rat IgG2b, diluted
1:10; Research Diagnostics Inc), ␣–smooth muscle cell actin (monoclonal mouse IgG2a (clone 1A4), dilution 1:500; Sigma), ICAM-1 (monoclonal rat IgG2a (clone BSA2), dilution 1:200; R&D Systems), VCAM-1
(monoclonal rat IgG2a (clone 429), dilution 1:100; Pharmingen), eNOS
(monoclonal rat IgG1 (clone 3), dilution 1:20; Transduction Laboratories), and vWF (peroxidase-conjugated polyclonal rabbit Ig, dilution
1:100; Dako). The slides were incubated with primary antibody for 2
hours at room temperature, except for anti–VCAM-1 (4°C overnight).
Goat anti-mouse IgG peroxidase conjugate (dilution 1:100 and 1:500;
Nordic) and goat anti-rat IgG alkaline phosphatase conjugate (dilution
1:100; Sigma Diagnostics) were used as secondary antibodies (1-hour
incubation at room temperature), with 3,3⬘-diamino-benzidine (Sigma
Diagnostics), nitro blue tetrazolium (Sigma Diagnostics), and 5-bromo4-chloro-3-indolyl phosphate (Sigma Diagnostics) as enzyme substrates.
Morphometry
Methods
Animals
All animal work was approved by the regulatory authority of Leiden
University and carried out in compliance with Dutch government
guidelines. Male LDLr⫺/⫺ and apoE⫺/⫺ mice were acquired from
TNO-PG and were 10 to 12 weeks old at the time of entry into the
study. Unless otherwise stated, the animals received a Western-type
diet (0.25% cholesterol and 15% cocoa butter, Hope Farms) 2 weeks
before surgery. Diet and water were provided ad libitum.
Carotid Collar Placement
Collars were prepared from Silastic tubing (Dow Corning) and stored in
70% ethanol until further use. An inside diameter of 0.5 mm was
nonconstrictive, whereas an inside diameter of 0.3 mm resulted in a
stenosis of ⬇30% because the average outside diameter of the common
carotid at 80 mm Hg perfusion pressure was 0.36 mm. Mice were
anesthetized by subcutaneous injection of ketamine (75 mg/kg, Eurovet), droperidol (1 mg/kg), fluanisone (0.75 mg/kg), and fentanyl
(0.04 mg/kg) (all from Janssen-Cilag). Access to the anterior cervical
triangles was gained through a sagittal anterior neck incision. Both
carotid sheaths were opened, and the common carotid arteries were
dissected free from the surrounding connective tissue, avoiding damage
to the vagus nerves and the carotid bodies. In the sham-operated
animals, the neck wound was closed in one layer with interrupted silk
sutures at this point (6-0, Braun). In the remaining animals, collars were
placed bilaterally around the common carotid arteries, and their axial
edges were approximated by placement of 3 circumferential silk ties
(Figure 1). Subsequently, the entry wound was closed and the animals
were returned to their cage for recovery from anesthesia.
Hematoxylin and eosin–stained sections were used for morphometric
analysis. Each vessel was assessed in cross section at 3 levels: 0.5 mm
proximal, in the mid-section, and 0.5 mm distal to the collar (Figure 1).
The images were analyzed with a Leica DM-RE microscope and
LeicaQwin software (Leica Imaging Systems). The intimal surface area
was calculated by subtracting the patent lumen area from the area
circumscribed by the internal elastic lamina, whereas the medial surface
area was defined as the area between the internal elastic lamina and the
external elastic lamina. The intima/media ratio and the intima/lumen
ratio were determined by dividing the intimal area by the medial area
and the total area confined by the internal elastic lamina, respectively.
Cholesterol Assay
Nonfasting serum samples were obtained by femoral artery transection.
Total serum cholesterol levels were quantified colorimetrically by
enzymatic procedures (Roche), with Precipath used as internal standard.
Statistical Analysis
All groups consisted of 3 animals. Values are expressed as mean⫾SD.
Data variance was analyzed by 1-way and 2-way ANOVA. A 2-tailed
Student’s t test was used in the comparison of individual groups, which
was used in a nonpaired form when comparing different animals and
paired when comparing contralateral values in the same animal. A level
of P⬍0.05 was considered significant.
Results
After recovery from anesthesia, the presence of perivascular
collars did not affect normal functioning of the animals. This
was corroborated by the absence of a difference in body
weight or lipid levels between collar-treated animals and
sham-operated control animals. There was no evidence of
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February 27, 2001
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Figure 3. Cross-sectional intimal surface area in apoE⫺/⫺ (A)
and LDLr⫺/⫺ (B) mice. Carotid arteries were analyzed at 6
weeks, after treatment with collar of 0.3 or 0.5 mm diameter, or
after sham surgery. Animals were maintained on chow or
Western-type diet. Error bars represent SD.
Figure 2. Cross-sectional intimal surface area (A and B), intimamedia ratio (C and D), and degree of lumen stenosis (E and F) in
collar-treated carotid arteries of apoE⫺/⫺ and LDLr⫺/⫺ mice.
Animals were fed Western-type diet; lesion formation was
assessed at 1, 3, and 6 weeks (n⫽3 at each time point). Error
bars represent SD. *Significant difference from sham-operated
animals (assessed at 6 weeks, t test, P⬍0.05).
105 ␮m2 in apoE⫺/⫺, 0.59⫾0.23⫻105 ␮m2 in LDLr⫺/⫺;
P⬍0.05 compared with sham-operated in both strains). The
increase with time in neointimal surface proximal to the collar
was found to be significant in both apoE⫺/⫺ and LDLr⫺/⫺
mice (P⬍0.01, 1-way ANOVA). The intimal area within and
distal to the collar remained virtually unaltered at all time points
examined; these values were confirmed to differ significantly
from the values obtained at the proximal site (P⬍0.0001, 2-way
ANOVA). The medial surface area did not rise significantly with
time at any site in either strain, and medial SMC loss was not
found to have occurred at the time points examined (data not
shown). No plaques were found in the corresponding sites of the
common carotid arteries of sham-operated animals of either
strain (Figure 3). The intima-media ratios of the proximal site are
in agreement with the absolute data, being highest in both strains
6 weeks after insertion (apoE⫺/⫺, 2.96⫾1.72; LDLr⫺/⫺,
1.42⫾0.88). The degree of proximal lumen stenosis (as ex-
collar-induced thrombosis in the analyzed common carotid
arteries at any time point of analysis (from 1 day to 18
weeks). The data discussed below were derived from the right
carotid arteries; these were not found to differ significantly
from the contralateral side.
Lesion Size
One week after bilateral insertion of a 0.3-mm diameter
collar, intimal thickening was not detectable in either strain
(Figure 2 and Table 1). By 3 weeks, however, a neointima
had appeared in the area proximal to the collar in apoE⫺/⫺
mice. Six weeks after collar insertion, a significant increase in
intimal surface area had occurred in both strains, although
this was more extensive in the apoE⫺/⫺ group (1.46⫾0.50⫻
TABLE 1. Cross-Sectional Intima Surface Area Proximal to Collar in ApoEⴚ/ⴚ,
LDLrⴚ/ⴚ, and ApoE3Leiden Transgenic Mice
Collar Size/Diet
0.3 mm,
Western Type
0.5 mm,
Western Type
0.3 mm,
Chow
Sham,
Western Type
0.07⫾0.04
ND
ND
ND
ApoE⫺/⫺
Week 1
Week 3
0.34⫾0.25
ND
ND
ND
Week 6
1.46⫾0.46
0.08⫾0.03
0.4⫾0.53
0.03⫾0.01
0.03⫾0.02
ND
ND
ND
LDLr⫺/⫺
Week 1
Week 3
0.10⫾0.09
ND
ND
ND
Week 6
0.59⫾0.23
0.02⫾0.005
0.01⫾0.003
0.006⫾0.004
0.005⫾0.0001
ND
ND
ND
ApoE3-Leiden
Week 26
ND indicates not done. Values are expressed as 10 ␮m ⫾SD.
5
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von der Thüsen et al
Atherogenesis by Perivascular Carotid Collar
1167
Figure 4. Hematoxylin-and-eosin–stained
cross sections of proximal carotid site 1
week (A and C) and 6 weeks (B and D)
after collar insertion in apoE⫺/⫺ (A and
B) and LDLr⫺/⫺ (C and D) mice.
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pressed by the intima-lumen ratio) was highest in the apoE⫺/⫺
mice, being 36⫾26% at 3 weeks and approaching occlusion
(85⫾5%) by 6 weeks. The corresponding values for LDLr⫺/⫺
mice were 9⫾7% and 61⫾23%. Distal and midsection sites did
not display a significant increase in intima-media or intimalumen ratio. A neointimal response was not observed in animals
of either strain 6 weeks after insertion of a nonconstrictive
0.5-mm diameter collar (Figure 3; 0.08⫾0.03⫻105 ␮m2 in
apoE⫺/⫺, 0.02⫾0.0005⫻105 ␮m2 in LDLr⫺/⫺; P⬍0.05).
LDLr⫺/⫺ mice on chow diet had not developed collar-induced
lesions by 6 weeks (P⬍0.05; serum total cholesterol [TC]
261.6⫾39.4 mg/dL as compared with 1281.2⫾298.4 mg/dL on
Western-type diet). Lesions in chow-fed apoE⫺/⫺ mice (serum
TC, 731.0⫾85.4 mg/dL) were found to be considerably but not
significantly smaller than in their Western-type diet–fed counterparts (serum TC, 1126.6⫾29.5 mg/dL), as depicted in Figure
3 (0.40⫾ 0.53⫻105 ␮m2 versus 1.46⫾0.50⫻105 ␮m2).
ApoE3-Leiden transgenic mice did not develop collarinduced plaques for up to 6 months after collar placement
despite having been on a Western-type diet (serum TC,
229.6⫾48.6 mg/dL; 0.005⫾0.0001⫻105 ␮m2).
Lesion Composition
The composition of the observed plaques was determined by
a variety of histological and immunohistochemical staining
techniques. Routine hematoxylin and eosin staining revealed
that collar-induced plaques in LDLr⫺/⫺ mice remained
homogeneous and relatively cellular with advancing maturity,
whereas plaques in apoE⫺/⫺ animals were seen to become
increasingly heterogeneous, consisting of an acellular necrotic core and a well-defined fibrous cap by 6 weeks (Figure
4). Significant extracellular matrix deposition was demonstrated in mature plaques by Weigert’s staining method (data
not shown). Oil red O staining revealed extensive intracellu-
lar and extracellular lipid deposits in the plaques of both
strains, which included abundant cholesterol crystal clefts.
Early plaques contained a particularly high number of foam
cells; these were more numerous and appeared to be more
lipid-rich in apoE⫺/⫺ mice than in LDLr⫺/⫺ mice (Figure
5). On immunohistochemical staining, the majority of these
cells were positive for MOMA-2 (Figure 5), thus confirming
their monocytic origin. Staining for smooth muscle actin was
restricted to the media in early lesions, but differentiated
smooth muscle cells (SMCs) appeared in the intima at 6
weeks, when positive cells were found to be primarily located
in the fibrous cap (Figure 5).
Endothelial Expression of Adhesion Molecules
and eNOS
Endothelial integrity was confirmed by staining for vWF in
all 3 sites examined (Figure 6). Endothelial ICAM-1 expression was seen circumferentially throughout the common
carotid artery in both collar-treated and sham-operated animals. Expression at the proximal and distal sites, however,
was strongly upregulated by placement of a 0.3-mm-diameter
collar, whereas expression within the collar was reduced
(Figure 6). ICAM-1 expression by endothelial cells overlying
collar-induced lesions decreased with increasing lesion maturity. Furthermore, its expression did not appear to be
dependent on the administration of a high-cholesterol diet
(data not shown). VCAM-1 expression was detected in a
limited number of endothelial cells in the proximal and distal
sites and found to be downregulated in the intracollar area
(Figure 7). eNOS expression was found to be limited to
several contiguous areas of the endothelium. Attenuation of
eNOS staining was observed proximal to the collar 1 week
after collar insertion (Figure 7).
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February 27, 2001
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Figure 5. High-power (⫻1000) view of proximal plaque at 6 weeks after collar insertion in apoE⫺/⫺ (A, B, and C) and LDLr⫺/⫺ (D, E,
and F) stained for lipid (with oil red O; A and D), MOMA-2 (B and E), and actin (C and F).
Discussion
Since the initial studies by Booth et al,7 the rabbit has been the
favored animal model for the investigation of collar-inflicted neointima formation. However, placement of a pericarotid or perifemoral collar has also been shown to induce intimal accumulation of
SMCs in rats9 and mice,12 respectively. In all of these models, the
neointima develops primarily in the collared section, with little
involvement of the adjacent proximal or distal uncuffed artery. A
range of causative mechanisms has been suggested for the source of
these lesions. Thus, direct mechanical or chemical injury of the
media by the collar may trigger the migration and proliferation of
SMCs within the intima. Alternatively, indirect activation of the
media might result from the disruption of its innervation and blood
supply caused by damage to the perivascular nerve plexus13 and/or
adventitial vasa vasorum.14 Furthermore, the presence of a collar
has been found to attenuate endothelial NO synthesis and to reduce
reactivity to NO.15,16 Finally, it may also elicit a localized inflammatory response, resulting in diapedesis and intimal accumulation
of leukocytes.17
In contrast, the collar-related plaques formed in our model are
located primarily in the area proximal to the collar. Minor effects
are observed in the distal area, whereas the area inside the collar
remains unaffected (Figure 2). These plaques are therefore
presumed to differ in pathogenesis from those seen in the
above-mentioned models. The location of the lesions, with
maximal stenosis occurring at some distance from the collar,
implies that induction of atherosclerosis by the collar through a
direct mechanical or chemical injury is unlikely. The absence of
Figure 6. ICAM staining (with alkaline phosphatase, using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as substrates) and vWF staining (with peroxidase ⫹ 3,3⬘-diamino-benzidine as substrate) of proximal (A), intracollar (B), and distal sites (C) 1
week after collar insertion in apoE⫺/⫺ mice. Note that vWF staining is present throughout artery; ICAM-1 staining is increased in sites
distal and particularly proximal to collar.
von der Thüsen et al
Atherogenesis by Perivascular Carotid Collar
1169
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Figure 7. Right carotid artery staining with alkaline phosphatase⫹BCIP⫹NBT for VCAM-1 (A, B) and peroxidase⫹DAB staining for
eNOS (C and D) 0.5 mm proximal to collar in apoE⫺/⫺ mice 1 week after collar insertion. VCAM-1 staining is augmented in proximal
site as compared with section inside collar, whereas reciprocal pattern is seen with eNOS staining.
lesions at the level of the collar also indicates that diminished
removal of waste products from the vessel wall or disruption of
the perivascular nerve plexus are unlikely to play a major
pathogenic role. Moreover, the limited thickness of the murine
carotid media, which consists of 3 SMC layers, obviates the need
for a transmural blood supply.18 The initial lesions seen in our
model contain mainly foam cells of monocytic origin, in addition to extracellular lipid deposits. With progressive maturation,
SMCs appear in the cap of the lesion and extracellular matrix is
deposited. This suggests that the pathogenesis of these lesions
depends on lipid accumulation as an initial stimulus rather than
migration and proliferation of SMCs. This is corroborated by the
dependency of lesion development on high-cholesterol feeding,
which was found to be relative in apoE⫺/⫺ mice and absolute
in LDLr⫺/⫺ mice. The lesional distribution pattern found in our
model mirrors the distribution of lipid deposition in aortic
coarctation models in cholesterol-fed rats19 and cynomolgus
monkeys,20 in which lipid deposition was found to be highest
proximal to the stenosis, with relative sparing20 and absence of
lesion development distal from the stenotic segment. The deposition of lipids in these models is known to be rheologically
determined in part,19 whereas it may also reflect a localized
increase in endothelial permeability and/or retention of LDL.21
These lesions were found to correspond to areas of low shear
stress and disturbed laminar flow. It is conceivable that equivalent hemorheological conditions exist in our model, in which a
collar of 0.3-mm inside diameter was found to cause a stenosis
of ⬇30% and attenuation of flow disturbance by placement of a
nonconstrictive collar resulted in considerably delayed atherogenesis. Hemodynamic factors also play an important indirect
role in human and experimental atherogenesis, through modulation of vascular adhesiveness for leukocytes. The endothelium
assumes a pivotal role in these processes because the expression
of various endothelial genes known to be upregulated in the
endothelium of atherosclerotic plaques and lesion-prone sites in
humans and experimental animals22 have been found to be
shear-stress responsive, including ICAM-1, eNOS, and VCAM1.23–25 Atherogenesis is known to occur predominantly at sites of
disturbed flow, typified by low and oscillating shear
stress.23,26 –28 The application of oscillatory shear stress to endothelial cells in culture has been found to greatly enhance
ICAM-1 expression and downregulate eNOS expression in
comparison with steady laminar flow as well as inducing a
transient rise in VCAM-1 levels.29 In our model, the expression
of ICAM-1 and VCAM-1 is in agreement with these findings,
being higher at sites of disturbed flow (proximal and distal to the
collar) than in the high laminar shear stress environment within
the collar (Table 2 and Figures 6 and 7). Conversely, downregulation of eNOS was observed at the proximal site (Table 2 and
Figure 7). It should be noted that besides shear stress, circumferential deformation has also been found to augment ICAM-1
and VCAM-1 expression, potentially through the generation of
reactive oxygen species.30 This type of deformation is more
likely to occur in the “free” proximal and distal sites than within
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February 27, 2001
TABLE 2. Endothelial Cell Expression Levels of Proatherogenic
Cell Adhesion Molecules, eNOS, and vWF in Collar-Treated and
Sham-Operated Carotid Arteries of ApoEⴚ/ⴚ Mice 1 Week
After Surgery
Proximal
Intracollar
Distal
Sham
Operated
ICAM-1
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
VCAM-1
⫹⫹
⫹
⫹
0
eNOS
0
⫹
⫹
⫹
vWF
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹ indicates strong, circumferential expression; ⫹⫹, circumferential
expression; ⫹, expression in cell clusters or individual cells; and 0, no evident
expression.
the collar, where the artery is prevented from full extension by
the “splinting” effect of the collar.
Summary
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Our model is characterized by site-controlled and accelerated
atherogenesis, which mirrors the differences seen between
apoE⫺/⫺ and LDLr⫺/⫺ mice in spontaneous atherosclerosis. The differences in lesion composition reflect the more
fibrocellular plaques normally seen in LDLr⫺/⫺ mice and
the more lipid-rich and heterogeneous plaques typical of
apoE⫺/⫺ mice. In addition, the temporal pattern of lesion
development is in agreement with the more delayed and
diet-dependent atherogenesis in LDLr⫺/⫺ mice in comparison with apoE⫺/⫺ mice. It may offer several advantages
over conventional animal models of mechanically induced
atherosclerosis. First, closer resemblance to human plaque
morphology, endothelial expression pattern, and plaque
pathogenesis should ensure its relevance as a model of human
atherosclerosis per se. This is of importance in the validation
of mechanistic studies and may improve the relevance of in
vivo assessment of potential therapeutic strategies. Second,
rapid atherogenesis will allow efficient screening of potentially antiatherogenic new chemical entities and the evaluation of therapies with a limited duration of effectiveness. The
latter category includes many adenoviral vectors, the expression of which often does not exceed a few weeks. Last, the
possibility of controlled lesion induction in easily accessible
sites is very much suited to further intraluminal instrumentation and application of therapeutic agents. This will also
prove valuable in the development of a more representative
murine model for restenosis because a double-injury restenosis model based on collar-inflicted atherosclerosis is likely to
reflect the complex pathogenesis seen in clinical practice.
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Induction of Rapid Atherogenesis by Perivascular Carotid Collar Placement in
Apolipoprotein E−Deficient and Low-Density Lipoprotein Receptor−Deficient Mice
Jan H. von der Thüsen, Theo J.C. van Berkel and Erik A.L. Biessen
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Circulation. 2001;103:1164-1170
doi: 10.1161/01.CIR.103.8.1164
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