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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Localized reduction of atherosclerosis in von Willebrand factor–deficient mice
Nassia Methia, Patrick André, Cécile V. Denis, Maria Economopoulos, and Denisa D. Wagner
To examine the role of the platelet adhesion molecule von Willebrand factor (vWf)
in atherogenesis, vWf-deficient mice
(vWfⴚ/ⴚ) were bred with mice lacking
the low-density lipoprotein receptor
(LDLRⴚ/ⴚ) on a C57BL/6J background.
LDLRⴚ/ⴚvWfⴙ/ⴙ and LDLRⴚ/ⴚvWfⴚ/ⴚ
mice were placed on a diet rich in saturated fat and cholesterol for different
lengths of time. The atherogenic diet
stimulated leukocyte rolling in the mesenteric venules in both genotypes, indicating an increase in P-selectin–mediated
adhesion to the endothelium. After 8
weeks on the atherogenic diet, the fatty
streaks formed in the aortic sinus of
LDLRⴚ/ⴚvWfⴚ/ⴚ mice of either sex were
40% smaller and contained fewer monocytes than those in LDLRⴚ/ⴚvWfⴙ/ⴙ
mice. After 22 weeks on the atherogenic
diet (early fibrous plaque stage), the difference in lesion size in the aortic sinus
persisted. Interestingly, the lesion distribution in the aortas of LDLRⴚ/ⴚvWfⴚ/ⴚ
animals was different from that of LDLRⴚ/ⴚ
vWfⴙ/ⴙ animals. In vWf-positive mice,
half of all lesions were located at the
branch points of the renal and mesenteric
arteries, whereas lesions in this area were
not as prominent in the vWf-negative
mice. These results indicate that the absence of vWf primarily affects the regions
of the aorta with disturbed flow that are
prone to atherosclerosis. Thus, vWf may
recruit platelets/leukocytes to the lesion
in a flow-dependent manner or may be
part of the mechano-transduction pathway regulating endothelial response to
shear stress. (Blood. 2001;98:1424-1428)
© 2001 by The American Society of Hematology
Introduction
von Willebrand factor (vWf) is a multimeric glycoprotein essential
for thrombus formation at high shear stress.1 It is found in plasma,
platelet ␣-granules, Weibel-Palade bodies of endothelial cells, and
the subendothelium.2 Even though the involvement of vWf in
thrombus formation at the site of atherosclerotic plaque rupture is
well established,3 the role of vWf in atherosclerotic lesion development is less clear. Several indications suggest that vWf may
directly participate in plaque formation. First, Weibel-Palade
bodies are found in great number at the sites of atherosclerotic
lesions.4 Second, oxidized low-density lipoprotein (LDL) and fluid
mechanics, 2 factors involved in atherosclerotic lesion development, can induce Weibel-Palade body exocytosis.5,6 Third, vWf
expression is particularly prominent near branch points and bifurcations, areas prone to atherosclerotic lesion development.7 Fourth,
platelets may contribute to the atherosclerotic process by releasing
growth factors after their vWf-dependent interaction with a damaged endothelial surface.8,9 Experimental studies performed in pigs
with von Willebrand disease (vWd), though supportive of a role for
vWf in atherosclerosis,3 have not been conclusive because of the
genetic heterogeneity existing among the animals.10
The recent generation of vWf-deficient mice has provided an
opportunity to directly test the importance of vWf in atherosclerosis. Because mice are resistant to atherosclerosis, the vWf-deficient
mice were first bred with an atherosclerosis-susceptible strain, the
LDL receptor (LDLR)-deficient mice (LDLR⫺/⫺).11 When fed an
atherogenic diet, LDLR⫺/⫺ mice acquire widespread arterial
lesions that progress from simple fatty streaks to complex fibrous
plaques over an extended time frame.12 We thus developed a vWd
model highly susceptible to atherosclerosis, and our results substantiate an important and complex role for vWf in atherosclerotic
lesion development.
Materials and methods
Animals and diets
vWf⫺/⫺mice13 were backcrossed 4 times to C57BL/6J and then crossed
with LDLR⫺/⫺mice on a C57BL/6J background (The Jackson Laboratory,
Bar Harbor, ME). Thus, the mice were 5 times backcrossed to the C57BL/6J
strain and shared more than 98% of its genetic background. Littermates
from the F2 generation of this intercross were genotyped by polymerase
chain reaction for LDLR deficiency14 and by Southern blot analysis for vWf
deficiency13 and were used to establish LDLR⫺/⫺vWf⫹/⫹ and LDLR⫺/⫺
vWf⫺/⫺ matings. The progeny of these mice were used in our study.
Age-matched animals were fed a standard low-fat mouse chow diet
containing 5% fat (wt/wt) (Prolab 3000; PMI Feeds, St Louis, MO). When
they were 8 weeks old, mice were placed on an atherogenic diet (ICN
Biomedicals; Aurora, OH) containing 1.25% (wt/wt) cholesterol, 17.84%
(wt/wt) butter, 0.98% (wt/wt) corn oil, and 0.48% (wt/wt) sodium cholate.
Experimental procedures were approved by the Animal Care and Use
Committee of the Center for Blood Research.
Cholesterol determination
Mice were fasted overnight, and blood was collected through the retroorbital venous plexus. Cholesterol concentrations in plasma were determined using an enzymatic assay kit (Cholesterol kit 352-50; Sigma
Chemical, St Louis, MO).
From the Center for Blood Research and the Department of Pathology, Harvard
Medical School, Boston, MA.
School, 800 Huntington Ave, Boston, MA 02115; e-mail: [email protected].
harvard.edu.
Submitted January 31, 2001; accepted April 9, 2001.
Supported by grant R37 HL41002 from the National Heart, Lung and Blood
Institute of the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Denisa D. Wagner, The Center for Blood Research, Harvard Medical
© 2001 by The American Society of Hematology
1424
BLOOD, 1 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 5
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BLOOD, 1 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 5
Intravital microscopy
Mice were injected with 6 g rhodamine (0.2 mg/kg body weight), and a
midline abdominal incision was made to expose the mesentery.15 Venules
100 to 200 ␮m in diameter were recorded for 3 minutes. The number of
rolling leukocytes was determined by counting the number of cells passing
through a perpendicular plane in 1 minute. Four to 5 vessels for each mouse
were recorded, then averaged to determine the number of rolling leukocytes
per minute.
Quantification of lesions
Lesions in the aortic sinuses and the aortas, collected between the
subclavian and iliac branches, were measured as described.12 To quantify
the lesions at the branch points of the renal and mesenteric arteries, arteries
were identified and marked on the slide to which the aorta was mounted.
Aortas were compared to determine the relative distance of renal and
mesenteric arteries region from the top of the aorta (approximately 11 mm).
For each measurement, a ruler was placed next to the aorta, and the top was
designated as 0 mm. Aortas were stained with Sudan IV, and the surface
area between 11 and 15 mm, corresponding to the region marked, was
measured using a Leica (Deerfield, IL) Q500MC image analysis program.
ROLE OF VON WILLEBRAND FACTOR IN ATHEROSCLEROSIS
1425
Table 1. Body weight and total plasma cholesterol in LDLR-deficient mice
on atherogenic diet
Diet length (wks)
Genotype
Cholesterol (mg/dL)
n
8
vWf⫹/⫹
21 ⫾ 1.47
2317 ⫾ 118
10
vWf⫺/⫺
21.9 ⫾ 0.93
2449 ⫾ 171
12
22
vWf⫹/⫹
24.3 ⫾ 1.57
2045 ⫾ 245
12
vWf⫺/⫺
25.5 ⫾ 1.70
2033 ⫾ 128
14
vWf⫹/⫹
31.7 ⫾ 0.96
1707 ⫾ 148
12
vWf⫺/⫺
32.5 ⫾ 1.43
1768 ⫾ 181
12
37
Body weight (g)
Values represent the mean ⫾ SEM. n ⫽ number of animals evaluated (similar
numbers of males and females were present). There were no significant differences
between the 2 genotypes in body weight and total plasma cholesterol.
Statistical analysis
P-selectin is stored in the same storage granules as vWf and plays
an important role in atherosclerosis,12,19 we were concerned that its
expression on endothelium may be absent in vWf⫺/⫺ mice. Our
laboratory has shown previously that leukocyte rolling in the
mesenteric venules of LDLR⫺/⫺ mice on an atherogenic diet is
dependent on P-selectin.12 Similarly, leukocyte rolling on a segment of carotid artery was recently shown to be P-selectin
dependent.20 To investigate whether P-selectin expression was
induced by the atherogenic diet in our model, we compared
leukocyte rolling in LDLR⫺/⫺ mice with or without vWf. They
were fed either mouse chow or an atherogenic diet for 4 weeks and
then subjected to intravital microscopy of the mesenteric venules.
The atherogenic diet significantly increased the number of rolling
leukocytes in both LDLR⫺/⫺vWf⫹/⫹ and LDLR⫺/⫺vWf⫺/⫺
mice in comparison with the mice maintained on mouse chow
(Figure 1A). The number of rolling leukocytes was comparable in
the 2 genotypes on the atherogenic diet, but on chow diet fewer
leukocytes rolled in the LDLR⫺/⫺vWf⫺/⫺ mice, as we have also
observed in mice deficient in vWf only.21 It is likely that the
atherogenic diet, containing cholic acid, increased P-selectin
synthesis and direct plasma membrane deposition in both genotypes, making it less dependent on regulated secretion from
Weibel-Palade bodies.21 Immunohistochemical staining of the
aortic sinus of these mice also showed the presence of P-selectin in
both genotypes (Figure 1B), though there was less P-selectin in the
LDLR⫺/⫺vWf⫺/⫺ mice. Thus, the absence of vWf reduces, but
does not eliminate, P-selectin expression on endothelium.
Data are presented as mean ⫾ SEM using the Student t test analysis.
Effect of vWf deficiency on lesion size in the aortic sinus
Immunohistochemical and histologic analyses
Macrophages and smooth muscle cells in aortic sinus lesions were assessed
as described,12 and calcium deposits were determined.16 To quantify cell
density in the 8-week lesions, 4 sections for each mouse were stained with
hematoxylin, and all nuclei in the lesion areas were counted by light
microscopy and divided by the area of the lesion evaluated. We have
estimated the number of leukocytes recruited per section by multiplying the
density obtained by mean lesion size in that animal. To evaluate P-selectin
expression in the lesions, frozen sections of the aortic sinus from mice on
atherogenic diet for 4 weeks were fixed in cold acetone for 5 minutes and
incubated with 3% hydrogen peroxide to block the endogenous peroxidase
activity. Slides were then incubated with a goat serum blocking solution
(Histostain-SP kit; Zymed Laboratories, San Francisco, CA) for 15 minutes,
followed by incubation for 1 hour with a rabbit anti–human P-selectin
antibody (1:50) that recognizes mouse P-selectin (kindly provided by Dr
M. C. Berndt, Baker Medical Research Institute, Melbourne, Australia). A
biotinylated second antibody, a horseradish peroxidase-streptavidin conjugate, and a substrate-chromogen mixture (Histostain-SP kit; Zymed Laboratories) were added sequentially to visualize the P-selectin.
Results
Generation of mice with combined vWf and LDLR deficiencies
vWf-deficient mice were crossed with LDLR-deficient mice to
generate LDLR⫺/⫺vWf⫹/⫹ and LDLR⫺/⫺vWf⫺/⫺ mice. Because the mice had been backcrossed 5 times to C57BL/6J and all
study mice descended from the same grandparental breeding pair,
the colony had minimal genetic variation apart from the vWf
genotype. LDLR⫺/⫺vWf⫹/⫹ and LDLR⫺/⫺vWf⫺/⫺ mice responded similarly to the atherogenic diet, as demonstrated by
similar body weights and comparable levels of cholesterol in the
blood at all 3 observation times (Table 1). Gender was not found to
be a significant variable.
Diet-induced leukocyte rolling
In rabbits, it has been shown that a cholesterol-rich diet induces
increased endothelial synthesis not only of vWf17 but also of other
adhesion molecules such as P-selectin, intercellular adhesion
molecule-1, and vascular cell adhesion molecule-1.18 Because
The aortic sinus has been a major focus for qualitative and
quantitative studies of atherosclerosis in the mouse because this
region is particularly prone to intimal lesion development, and the
cusps of the valves provide a useful positional cue in comparative
studies of sectioned tissues.22 To quantitatively examine the impact
of vWf deficiency on the development of atherosclerotic lesions in
the aortic sinus, mean lesion areas were measured in oil red-O–
stained tissue. All measurements presented in this study were
performed blindly to the genotype of the animals. After 8 weeks on
the diet, the fatty streak lesions in LDLR⫺/⫺vWf⫺/⫺ mice were
40% smaller than in the LDLR⫺/⫺vWf⫹/⫹ mice (0.12 ⫾ 0.01
mm2 vs 0.20 ⫾ 0.01 mm2; n ⫽ 16; P ⫽ .002; Figure 2). When
mean lesion areas of either male or female animals were evaluated,
the difference between the LDLR⫺/⫺vWf⫹/⫹ and LDLR⫺/⫺
vWf⫺/⫺ mice was significant, indicating no gender differences in
the role of vWf. In mice killed after 22 weeks on the diet, the
difference in the early fibrous plaque lesions persisted between the
2 genotypes (0.81 ⫾ 0.05 mm2 for vWf⫹/⫹ mice vs 0.53 ⫾ 0.04
mm2 for vWf⫺/⫺ mice; n ⫽ 10-14; P ⫽ .0004; Figure 2). However, at 37 weeks—the complex fibrous plaque lesion stage—the
absence of vWf no longer affected lesion size (0.84 ⫾ 0.03 mm2 vs
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1426
METHIA et al
BLOOD, 1 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 5
plaque lesions in most of the LDLR⫺/⫺vWf⫹/⫹ mice contained
calcification (6 of 10 mice), whereas calcifications were rare in the
lesions from LDLR⫺/⫺vWf⫺/⫺ mice (2 of 15 mice). Therefore,
the absence of vWf in LDLR⫺/⫺ mice delayed calcification of the
lesions. This difference in calcification persisted at 37 weeks with
80% of LDLR⫺/⫺vWf⫹/⫹ lesions calcified compared to 47% of
LDLR⫺/⫺vWf⫺/⫺ lesions (P ⬍ .002).
Lesion distribution in the entire aorta
Figure 1. Effect of vWf deficiency on P-selectin expression. (A) Leukocyte rolling
in LDLR-deficient mice on an atherogenic diet. Four-week-old LDLR⫺/⫺vWf⫹/⫹ and
LDLR⫺/⫺vWf⫺/⫺ mice were fed either normal mouse chow (white bars) or
atherogenic diet (black bars) for 4 weeks. Animals were injected with 6 g rhodamine,
and the mesenteric venules were exposed. The number of leukocytes was quantified
by counting the number of cells passing through a perpendicular plane per minute.
The atherogenic diet significantly increased rolling in both LDLR⫺/⫺vWf⫹/⫹ (P ⫽ .03)
and LDLR⫺/⫺vWf⫺/⫺ (P ⫽ .0004) mice (n ⫽ 6-9). The number of rolling leukocytes
was comparable in the 2 genotypes of mice on atherogenic diet (P ⫽ .1). (B)
Immunohistochemical staining of the aortic sinus with an anti–P-selectin antibody.
Sections were obtained from mice that were on an atherogenic diet for 4 weeks and
were stained with a polyclonal antibody against P-selectin. Staining for P-selectin
was weaker in mice lacking vWf. Immunohistochemical staining with a PECAMspecific antibody confirmed the integrity of the endothelial monolayer (not shown).
Bar ⫽ 100 ␮m.
0.85 ⫾ 0.02 mm2, respectively; P ⫽ 0.9; n ⫽ 10), indicating that
vWf does not play an important role in smooth muscle cell
recruitment/proliferation in the advanced atherosclerotic lesion.
The extent of atherosclerosis was also determined in a large
segment of the aorta, from the subclavian branch to the iliac
bifurcation. To quantify the lesions, aortas from LDLR⫺/⫺
vWf⫹/⫹ and LDLR⫺/⫺vWf⫺/⫺ mice were stained with Sudan
IV to visualize lipid in the vessel. Unlike the protective effect of
vWf deficiency on lesion development in the aortic sinus at 22
weeks (Figure 2), the percentage surface area occupied by the
lesions in the rest of the aorta was comparable in the 2 genotypes
(Figure 3). This was surprising because the extent of lesion
formation in the entire aorta usually correlates with lesion size in
the aortic sinus,22 an observation also confirmed in all of our
previous studies on the role of adhesion molecules in atherosclerosis.12,16,19 On closer inspection, we noticed a difference in the lesion
distribution in the aortas between vWf-positive and -negative
animals. There was a clear “hot spot” of lesion formation at the
branch points of the renal and mesenteric arteries in LDLR⫺/⫺
vWf⫹/⫹ mice. This hot spot was not as prominent relative to the
rest of the aorta in the LDLR⫺/⫺vWf⫺/⫺ animals (Figure 3). To
evaluate this more objectively, an investigator blinded to the
genotype of the animals determined the percentage of lesion
located in this portion of the aorta. It was found that approximately
half of the lesion areas in LDLR⫺/⫺vWf⫹/⫹ mice were located in
this hot spot region, whereas only one third of total lesion area was
detected in the same region in LDLR⫺/⫺vWf⫺/⫺ mice. The
decreased level of atherosclerosis in the hot spot in the LDLR⫺/⫺
vWf⫺/⫺ mice was consistent between the animals—14 of 17
showed a lower percentage of lesions in this region than the mean
48.4% observed in LDLR ⫺/⫺vWf ⫹/⫹ mice. This difference in
lesion localization was significant (Figure 3). Lesion distribution in
the LDLR⫺/⫺vWf⫺/⫺ mice appeared more diffuse and had a
tendency toward an increase in lesion formation outside the hot
spot area, but this did not reach statistical significance (P ⫽ .09).
Comparison of aortic sinus lesion composition in the
LDLR-deficient mice with and without vWf
As expected for a fatty streak,8,12 the 8-week lesions in both
genotypes were composed of CD11b⫹ macrophages engorged with
lipids (not shown). We counted the nuclei in hematoxylin-stained
lesions at this stage and found that there was no difference in cell
density between the 2 genotypes (2320 ⫾ 152/mm2 in LDLR⫺/⫺
vWf⫹/⫹ mice vs 2350 ⫾ 192/mm2 in LDLR⫺/⫺vWf⫺/⫺ mice;
P ⫽ .7; n ⫽ 12-14). However, because the lesions were smaller in
the LDLR⫺/⫺vWf⫺/⫺ mice, the total number of macrophages
recruited into the lesions was lower than that in LDLR⫺/⫺
vWf⫹/⫹ mice (288 ⫾ 24 per average lesion section in LDLR⫺/⫺
vWf⫺/⫺ mice vs 430 ⫾ 33 in LDLR⫺/⫺vWf⫹/⫹ mice; P ⫽ .002;
n ⫽ 12-14). We found that there were no differences in percentage
lesion area positive for ␣-actin staining in both genotypes after 22
weeks on diet (31% ⫾ 4.9% for LDLR⫺/⫺vWf⫹/⫹ mice vs
29% ⫾ 2.9% for LDLR⫺/⫺vWf⫺/⫺ mice; P ⫽ 0.8; n ⫽ 8-10).
The proportion of smooth muscle cells increased only slightly in
the 37-week lesions, remaining similar in the 2 genotypes (P ⫽ .6).
Thus, the proportion of smooth muscle cells in the lesion was not
influenced by vWf. However, after 22 weeks on the diet, fibrous
Figure 2. Size comparison of atherosclerotic lesions in the aortic sinus of
LDLR-deficient mice with and without vWf. Eight-week-old mice were put on an
atherogenic diet for 8 or 22 weeks. Hearts were collected and processed, and oil
red-O–positive lesions were measured. Mean values of 5 sections per mouse were
compared. Bars represent standard error of the mean. Differences between LDLR⫺/⫺
vWf⫹/⫹ and LDLR⫺/⫺vWf⫺/⫺ mice were significant at both time points.
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BLOOD, 1 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 5
ROLE OF VON WILLEBRAND FACTOR IN ATHEROSCLEROSIS
1427
At 37 weeks, the lesions became prominent in both genotypes.
Differences in their distribution could not be discerned because
they were distributed uniformly throughout the aortas (50.6% in
LDLR⫺/⫺vWf⫹/⫹ mice and 44.3% in LDLR⫺/⫺vWf⫺/⫺
mice; P ⫽ .34; n ⫽ 11-12). As in the aortic sinus region at this
advanced stage, there were no significant differences between
the 2 genotypes.
Discussion
A role for vWf in atherosclerosis has long been suspected but was
difficult to prove because studies performed with pigs deficient in
vWf yielded more questions than answers. The first experimental
data suggested that the absence of vWf provided an atheroprotective effect in pigs.23,24 However, it was later shown that the
atherogenic diet leads to a variable degree of hypercholesterolemia
caused by a polymorphism in apolipoprotein B100,10,25 making it
difficult to draw a definitive conclusion about the involvement of
vWf in atherosclerosis. In contrast, mice deficient in vWf represented a model with a defined genetic background and allowed us
to show in the present study that the absence of vWf significantly
delays the formation of atherosclerotic lesions in mice on the
LDLR-deficient background. The absence of vWf seems to provide
a protective effect mainly in regions of disturbed flow, which are
highly prone to atherosclerotic lesion development.26,27 The regional importance of vWf for lesion growth distinguishes it from
P-selectin, which impacts evenly on lesion development throughout the aorta.12 The slight defect in P-selectin expression in the
LDLR⫺/⫺vWf⫺/⫺ mice on atherogenic diet (Figure 1) could not
account for the observed reduction in atherosclerosis. In fact, the
protective effect provided by the absence of vWf was much
stronger than that of P-selectin because the reduction in lesion size
in P-selectin–deficient mice was gender dependent and was only
prominent after 8 weeks on the atherogenic diet.12 Although the
defect in regulated secretion of P-selectin observed in vWf⫺/⫺
mice21 likely contributes to reduced atherosclerosis observed in the
LDLR⫺/⫺vWf⫺/⫺ mice, vWf clearly fulfills additional function(s) in lesion growth. These will be discussed in more detail below.
In the LDLR⫺/⫺vWf⫺/⫺ mice, the fatty streaks in the aortic
sinus were smaller than in the LDLR⫺/⫺vWf⫹/⫹ mice because
fewer monocytes were recruited. vWf could participate in the
recruitment of monocytes directly or indirectly. Monocytes can
adhere to the endothelium by various integrins, such as ␣4␤1, on
their surfaces. This integrin has been recently shown to play an
important role in the initiation of the atherosclerotic lesion.28
Interestingly, the vWf propolypeptide is a ligand for ␣4␤1.29 The
vWf propolypeptide, which is also absent in the vWf-deficient
mice, is processed from a large precursor of vWf and stored in the
same granules as mature vWf.2 Thus, vWf may participate in the
recruitment of monocytes directly through the vWf propolypeptide.
vWf may also act indirectly by recruiting platelets9 that, in turn,
may facilitate the recruitment of leukocytes.30,31 The possibility
also remains that the absence of vWf affects the shear stress
responses of endothelium. Indeed, shear stress is a modulator of the
endothelial cell phenotype.27,32,33 Shear stress regulates endothelial
cell gene expression and is thought to be mediated by mechanotransducing receptors.34 vWf is part of the basement membrane to
which endothelial cells adhere through their integrin receptors.
Integrins can transduce mechanical stimuli to biochemical signals,35 and a blocking antibody to ␤3 integrin has been shown to
affect the shear stress-induced vasodilatation of coronary arteries.36
Figure 3. Distribution of atherosclerotic lesions in the aorta of LDLRⴚ/ⴚ
vWfⴙ/ⴙ and LDLRⴚ/ⴚvWfⴚ/ⴚ mice. Eight-week-old mice were fed an atherogenic
diet for 22 weeks. Aortas were collected between the subclavian and iliac branches
and stained with Sudan IV. The area between and around the superior mesenteric
artery and renal artery, which is highly prone to lesion formation (see “Materials and
methods”) is referred to as the hot spot. The percentage of coverage for LDLR⫺/⫺
vWf⫺/⫺ is 16 ⫾ 2.6 and 15.2 ⫾ 1.9, respectively; P ⫽ .79. The percentage of the
lesion in the hot spot is 48.4 ⫾ 5.4 and 33.7 ⫾ 3.1, respectively; P ⫽ .02. Representative specimens from both genotypes are shown, and mean values from 16 to 17
animals of each genotype are given.
Therefore, the absence of a major ␤3 ligand, in this case vWf,
might affect the endothelial cell response to shear stress. Our
laboratory is addressing the effect of ␤3 integrin deficiency on
atherosclerotic lesion development and lesion distribution.
If vWf is not directly involved in the regulation of endothelial
responses to flow, the observed protection seen in vWf⫺/⫺ mice in
early stages of atherosclerosis in regions of disturbed flow could be
explained by the unusually flow-sensitive biology of vWf. First, at
the molecular level, shear stress induces a conformational transition from globular to extended chain in vWf, as demonstrated by
atomic force microscopy.37 This conformational change is likely to
expose or mask binding sites for cellular adhesion receptors
altering vWf adhesion properties. Second, at the endothelial
expression level, laminar shear was shown to induce the secretion
of vWf from Weibel-Palade bodies.6 Although this has not yet been
examined, changes in shear stress, such as would occur under
disturbed flow, may also have such an effect. Mechanosensitive
stretch-activated Ca⫹⫹ channels have been described,38 and, interestingly, Ca⫹⫹ influx causes the release of Weibel-Palade bodies.39
Third, the cell-cell adhesion processes mediated by vWf are shear
dependent. For example, we have shown recently that vWf released
from activated endothelial cells in vivo recruited resting platelets to
the endothelium in large numbers, preferentially under low shear
stress (80-100 seconds⫺1),9 conditions likely occurring in areas of
disturbed flow. This process may deliver platelets and with them
leukocytes, specifically to these regions of the aorta. All these
possibilities for a shear stress-dependent role of vWf in atherosclerosis are, of course, not mutually exclusive.
Thus, the role of vWf in atherosclerosis appears important but
complex. vWf promotes monocyte, and likely platelet, recruitment
at the most prominent sites of lesion development. Moreover, the
role of vWf in atherosclerosis is not limited to lesion growth. In
life-threatening moments, when plaque ruptures, it is only vWf that
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1428
BLOOD, 1 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 5
METHIA et al
can form bonds strong enough to lead to a complete occlusion of
the vessel,40,41 resulting in myocardial infarction.
Note added in proof. We have now finished analyzing the role
of vWf in atherosclerosis in a different mouse model, ie, mice
deficient in apoE and fed normal chow. We observed remarkably
similar results to those presented above with significant reduction
in fibrofatty lesion size in the aortic sinus at 4 months of age
(apoE⫺/⫺vWf⫹/⫹, 0.32 mm2; apoE⫺/⫺vWf⫺/⫺, 0.15 mm2;
n ⫽ 10; P ⬍ .0001) but no reduction at the later time point of 15
months. Interestingly, at 15 months when lesions spread throughout
the aorta, although the surface covered by the lesions was similar in
the 2 genotypes (apoE⫺/⫺vWf⫹/⫹, 28.4 mm2; apoE⫺/⫺vWf⫺/⫺,
28.3 mm2), the percent lesion found in the “hot spot” was again
significantly reduced by the absence of vWf (apoE⫺/⫺vWf⫹/⫹,
31.2 ⫾ 6.1%; apoE⫺/⫺vWf⫺/⫺, 16.6 ⫾ 1.6%, n ⫽ 11-13, P ⬍
.05). Lesions were slightly increased outside the hot spot area, P ⬍
.05. Thus, we are persuaded that vWf indeed plays a role in
atherosclerotic lesion distribution. We thank Kennard Thomas for
help with the analysis.
Acknowledgments
We thank Dr Michael A. Gimbrone for critical reading of the
manuscript, Lesley Cowan for assistance with its preparation,
and Dr Michael C. Berndt for generously providing the rabbit
anti–human P-selectin antibody. We also thank Dr Zhao Ming
Dong and Allison A. Brown for their help with histologic and
morphometric analyses.
References
1. Ruggeri ZM. Structure and function of von Willebrand factor. Thromb Haemost. 1999;82:576-584.
2. Wagner DD. Cell biology of von Willebrand factor.
Annu Rev Cell Biol. 1990;6:217-246.
3. Badimon L, Badimon JJ, Chesebro JH, Fuster V.
von Willebrand factor and cardiovascular disease. Thromb Haemost. 1993;70:111-118.
4. Trillo AA, Prichard RW. Early endothelial changes
in experimental primate atherosclerosis. Lab Invest. 1979;41:294-302.
5. Vora DK, Fang ZT, Liva SM, et al. Induction of
P-selectin by oxidized lipoproteins: separate effects on synthesis and surface expression. Circ
Res. 1997;80:810-818.
6. Galbusera M, Zoja C, Donadelli R, et al. Fluid
shear stress modulates von Willebrand factor release from human vascular endothelium. Blood.
1997;90:1558-1564.
7. Senis YA, Richardson M, Tinlin S, Maurice DH,
Giles AR. Changes in the pattern of distribution of
von Willebrand factor in rat aortic endothelial cells
following thrombin generation in vivo. Br J
Haematol. 1996;93:195-203.
8. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med. 1999;340:115-126.
9. Andre P, Denis CV, Ware J, et al. Platelets adhere
to and translocate on von Willebrand factor presented by endothelium in stimulated veins. Blood.
2000;96:3322-3328.
10. Nichols TC, Bellinger DA, Davis KE, et al. Porcine
von Willebrand disease and atherosclerosis: influence of polymorphism in apolipoprotein B100
genotype. Am J Pathol. 1992;140:403-415.
11. Ishibashi S, Goldstein JL, Brown MS, Herz J,
Burns DK. Massive xanthomatosis and atherosclerosis in cholesterol-fed low density lipoprotein
receptor-negative mice. J Clin Invest. 1994;93:
1885-1893.
12. Johnson RC, Chapman SM, Dong ZM, et al. Absence of P-selectin delays fatty streak formation
in mice. J Clin Invest. 1997;99:1037-1043.
13. Denis C, Methia N, Frenette PS, et al. A mouse
model of severe von Willebrand disease: defects
in hemostasis and thrombosis. Proc Natl Acad
Sci U S A. 1998;95:9524-9529.
14. Shimada M, Ishibashi S, Inaba T, et al. Suppression of diet-induced atherosclerosis in low-density lipoprotein receptor knockout mice overexpressing lipoprotein lipase. Proc Natl Acad Sci
U S A. 1996;93:7242-7246.
and E-selectins in atherosclerosis. J Clin Invest.
1998;102:145-152.
17. De Meyer GR, Hoylaerts MF, Kockx MM,
Yamamoto H, Herman AG, Bult H. Intimal deposition of functional von Willebrand factor in atherogenesis. Arterioscler Thromb Vasc Biol. 1999;19:
2524-2534.
18. Scalia R, Appel JZ III, Lefer AM. Leukocyte-endothelium interaction during the early stages of hypercholesterolemia in the rabbit: role of P-selectin, ICAM-1, and VCAM-1. Arterioscler Thromb
Vasc Biol. 1998;18:1093-1100.
19. Dong ZM, Brown AA, Wagner DD. Prominent role
of P-selectin in the development of advanced atherosclerosis in ApoE-deficient mice. Circulation.
2000;101:2290-2295.
20. Ramos CL, Huo Y, Jung U, et al. Direct demonstration of P-selectin– and VCAM-1–dependent
mononuclear cell rolling in early atherosclerotic
lesions of apolipoprotein E-deficient mice. Circ
Res. 1999;84:1237-1244.
21. Denis CV, Andre P, Saffaripour S, Wagner DD.
Defect in regulated secretion of P-selectin affects
leukocyte recruitment in von Willebrand factordeficient mice. Proc Natl Acad Sci U S A. 2001;
98:4072-4077.
22. Tangirala RK, Rubin EM, Palinski W. Quantitation
of atherosclerosis in murine models: correlation
between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions
between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J Lipid Res. 1995;36:
2320-2328.
23. Fuster V, Bowie EJ, Lewis JC, Fass DN, Owen
CA Jr, Brown AL. Resistance to arteriosclerosis in
pigs with von Willebrand’s disease: spontaneous
and high cholesterol diet-induced arteriosclerosis.
J Clin Invest. 1978;61:722-730.
24. Fuster V, Lie JT, Badimon L, Rosemark JA, Badimon JJ, Bowie EJ. Spontaneous and dietinduced coronary atherosclerosis in normal swine
and swine with von Willebrand disease. Arteriosclerosis. 1985;5:67-73.
25. Griggs TR, Reddick RL, Sultzer D, Brinkhous KM.
Susceptibility to atherosclerosis in aortas and coronary arteries of swine with von Willebrand’s disease. Am J Pathol. 1981;102:137-145.
26. Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an
atheroprotective force. Arterioscler Thromb Vasc
Biol. 1998;18:677-685.
15. Baatz H, Steinbauer M, Harris AG, Krombach F.
Kinetics of white blood cell staining by intravascular administration of rhodamine 6G. Int J Microcirc
Clin Exp. 1995;15:85-91.
27. Topper JN, Gimbrone MA Jr. Blood flow and vascular gene expression: fluid shear stress as a
modulator of endothelial phenotype. Mol Med Today. 1999;5:40-46.
16. Dong ZM, Chapman SM, Brown AA, Frenette PS,
Hynes RO, Wagner DD. The combined role of P-
28. Shih PT, Brennan ML, Vora DK, et al. Blocking
very late antigen-4 integrin decreases leukocyte
entry and fatty streak formation in mice fed an
atherogenic diet. Circ Res. 1999;84:345-351.
29. Isobe T, Hisaoka T, Shimizu A, et al. Propolypeptide of von Willebrand factor is a novel ligand for
very late antigen-4 integrin. J Biol Chem. 1997;
272:8447-8453.
30. Theilmeier G, Lenaerts T, Remacle C, Collen D,
Vermylen J, Hoylaerts MF. Circulating activated
platelets assist THP-1 monocytoid/endothelial
cell interaction under shear stress. Blood. 1999;
94:2725-2734.
31. Diacovo TG, Puri KD, Warnock RA, Springer TA,
von Andrian UH. Platelet-mediated lymphocyte
delivery to high endothelial venules. Science.
1996;273:252-255.
32. Gimbrone MA Jr, Nagel T, Topper JN. Biomechanical activation: an emerging paradigm in endothelial adhesion biology. J Clin Invest. 1997;99:
1809-1813.
33. Topper JN, Cai J, Falb D, Gimbrone MA Jr. Identification of vascular endothelial genes differentially
responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase,
and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear
stress. Proc Natl Acad Sci U S A. 1996;93:1041710422.
34. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519-560.
35. Ingber D. Integrins as mechanochemical transducers. Curr Opin Cell Biol. 1991;3:841-848.
36. Muller JM, Chilian WM, Davis MJ. Integrin signaling transduces shear stress-dependent vasodilation of coronary arterioles. Circ Res. 1997;80:
320-326.
37. Siedlecki CA, Lestini BJ, Kottke-Marchant KK,
Eppell SJ, Wilson DL, Marchant RE. Sheardependent changes in the three-dimensional
structure of human von Willebrand factor. Blood.
1996;88:2939-2950.
38. Lansman JB, Hallam TJ, Rink TJ. Single stretchactivated ion channels in vascular endothelial
cells as mechanotransducers? Nature. 1987;325:
811-813.
39. Birch KA, Pober JS, Zavoico GB, Means AR,
Ewenstein BM. Calcium/calmodulin transduces
thrombin-stimulated secretion: studies in intact
and minimally permeabilized human umbilical
vein endothelial cells. J Cell Biol. 1992;118:15011510.
40. Ruggeri ZM. Old concepts and new developments in the study of platelet aggregation. J Clin
Invest. 2000;105:699-701.
41. Ni H, Denis CV, Subbarao S, et al. Persistence of
platelet thrombus formation in arterioles of mice
lacking both von Willebrand factor and fibrinogen.
J Clin Invest. 2000;106:385-392.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2001 98: 1424-1428
doi:10.1182/blood.V98.5.1424
Localized reduction of atherosclerosis in von Willebrand factor−deficient mice
Nassia Methia, Patrick André, Cécile V. Denis, Maria Economopoulos and Denisa D. Wagner
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