© 1999 American Heart Association, Inc.
Thrombosis |
Intimal Deposition of Functional von Willebrand Factor in Atherogenesis
From the Division of Pharmacology (G.R.Y. De M., M.M.K., A.G.H., H.B.), University of Antwerp (UIA); the Department of Pathology (M.M.K.), General Hospital Middelheim, Antwerp; and the Center for Molecular and Vascular Biology (M.F.H, H.Y.), University of Leuven, Leuven, Belgium.
Correspondence to Guido R.Y. De Meyer, Division of Pharmacology, University of Antwerp (UIA), Universiteitsplein 1, B-2610 Wilrijk, Belgium. Email gdemeyer{at}uia.ua.ac.be
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Abstract—During the formation of intimal thickening in normocholesterolemic rabbits, von Willebrand factor (vWF) is increased in the endothelial cells (ECs) and deposited in the intima. We investigated whether this also occurs during cholesterol-induced plaque formation, whether the synthesis of vWF increases, and whether this influences platelet adhesion. Rabbits were fed a cholesterol-rich (0.3%) diet for 26 weeks. Thereafter, half of the animals received a normal diet for another 26 weeks (cholesterol withdrawal). To induce intimal thickening in normocholesterolemic rabbits, collars were positioned around the carotid artery. Arterial segments were studied using immunohistochemistry, reverse transcription–polymerase chain reaction, electron microscopy, and platelet adhesion tests. Cholesterol treatment induced plaque formation in the aorta. The ECs had a cuboidal aspect, showed a dense immunoreactivity for vWF, a pronounced rough endoplasmic reticulum, and numerous Weibel-Palade bodies. There were subendothelial vWF deposits in the plaques and vWF mRNA was significantly increased as compared with controls. Similar changes were seen after collar-induced intimal thickening. After cholesterol withdrawal, both vWF mRNA and the ultrastructural morphology of the ECs normalized, and the vWF deposits disappeared from the plaque. Perfusion studies with anticoagulated rabbit blood over cross-sections of atherosclerotic aortas revealed increased vWF-mediated platelet adhesion in the subendothelial plaque region. Whereas rabbit platelets perfused through the lumen adhered to the same extent to de-endothelialized aortas of normocholesterolemic and atherosclerotic rabbits, vWF mediated platelet adhesion to endothelium was observed in atherosclerotic but not in normal aortas. Our results show an increased synthesis and (sub)endothelial presence of vWF after vascular injury, with functional consequences for platelet deposition on the vessel wall.
Key Words: atherosclerosis • intima • reverse transcription–polymerase chain reaction • hyperlipidemia • von Willebrand factor
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Introduction |
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Intimal thickening is a site of predilection for the development of atherosclerotic plaques. By positioning a flexible silicone collar around the rabbit carotid artery, a localized intimal thickening is induced within 14 days,1 2 3 which is probably the consequence of both media damage due to collar placement and the retention of cytokines and/or growth factors by the collar.1 The intimal thickening develops via migration of smooth muscle cells (SMCs) from the media to the intima and further SMC proliferation, similar to other models of intimal thickening and to the human situation.
We previously showed that during intimal thickening, the
immunoreactivity for von Willebrand factor (vWF) increases in
the endothelial cells (ECs) and that vWF is deposited
in quantities in the extracellular space of the intimal
thickening.2 The glycoprotein vWF is
synthesized by only 2 cell types: ECs and megakaryocytes. One can
discriminate the following 3 pools of vWF in the body: soluble plasma
vWF, basement membrane (extracellular matrix) vWF, and cellular vWF
found in storage granules of ECs and platelets (Weibel-Palade
bodies and 
-granules).4 The 3 pools contribute to
adhesion of platelets and formation of a platelet plug during
blood vessel injury. The basement membrane vWF may also promote EC
adhesion in the intact vessel.4 Plasma vWF has an
additional role in hemostasis because it carries factor VIII and
protects it against proteolysis. Secretion of vWF from ECs can occur
through 1 of the following 2 pathways: a constitutive pathway directly
linked to synthesis (major pathway in cultured ECs) and a regulated
pathway involving storage in the Weibel-Palade bodies and release after
stimulation by secretagogues.5 In megakaryocytes and
platelets, only the regulated pathway may be effectively
operative.6 Accumulation of vWF in intimal thickening may
be the result of increased synthesis and release by ECs, an influx from
the plasma and/or platelets, or a decreased rate of removal of vWF
from the extracellular matrix.7
The aim of the present study was to investigate whether the intimal deposition of vWF is due to an increased synthesis of the protein and whether subendothelial deposition of vWF also occurs after cholesterol-induced plaque formation and whether this has functional consequences for platelet adhesion.
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Methods |
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Collar-Induced Intimal Thickening
Male New Zealand white rabbits (2.5 to 3 kg) (Rijksstation voor Kleinveeteelt, Merelbeke, Belgium) were anesthetized with sodium pentobarbital (30 mg/kg IV). Both carotid arteries were surgically exposed and dissected from the surrounding tissues. A soft silicone collar was placed around the left carotid artery.1 The right carotid artery was sham operated; ie, it was isolated from the surrounding connective tissue and the vagus nerve and was exposed to a similar stretch as the contralateral collared artery. All the rabbits of the collar experiment were fed a normal diet. Animals were killed by an overdose of pentobarbital at 7 days (transmission electron microscopy experiment) or 14 days. A segment of the sham-operated and collared carotid artery was fixed in 4% formalin. The remaining part was frozen as fast as possible in liquid nitrogen for RNA isolation.
Cholesterol-Induced Atherosclerotic Plaques
Male New-Zealand white rabbits (2.8 to 3.5 kg) were fed a diet
supplemented with a low-dose cholesterol (0.3%) for 26
weeks (n=20). Ten randomly selected animals were killed after this
period (chol 26 wk group); the other animals were fed a normal diet for
another 26 weeks, the cholesterol withdrawal (chol wd)
group. After this period, these animals were also killed for study. A
control group was included. Serum was stored at -20°C and total
cholesterol, LDL and VLDL cholesterol, and
triglycerides were determined. For
histological examination, a segment of the thoracic
aorta was fixed in 4% formalin. The remaining part of the thoracic
aorta of 4 rabbits of the control group, 4 rabbits of the
cholesterol group, and 4 rabbits of the
cholesterol withdrawal group was frozen as fast as possible
in liquid nitrogen for RNA isolation. In a second experiment,
platelet adhesion studies were performed (cf. infra).
In addition, 8 rabbits were given an even lower cholesterol diet (0.125%) for 9 weeks, and the evolution of platelet numbers was followed with time.
RNA Extraction and Reverse Transcription-Polymerase Chain
Reaction Analysis
Total RNA was isolated using the guanidinium
isothiocyanate and phenol/chloroform extraction
method.8 Reverse transcription-polymerase chain reaction
(RT-PCR) was performed with an automatic thermal cycler (MultiCycler
PTC-200, MJ Research Inc) using a 1-step RT-PCR system (Titan,
Boehringer Mannheim). The avian myoblastiosis virus–reverse
transcriptase was applied for first strand synthesis and an enzyme
blend, which consisted of Taq DNA polymerase and Pwo DNA polymerase for
the PCR part. The following specific primers were used: rabbit vWF mRNA
sense (5' CAG CAA ACG GAG CCT GTC CT 3' [GenBank accession No. S64544,
positions 102 to 121]), rabbit vWF mRNA antisense (5' ACG CAT GTG TTG
CAG TCC AT 3' [GenBank accession No. S64544, positions 352 to 371]).
Rabbit ß-actin mRNA sense (5' GCG GCA CCA GGG CGT 3' [GenBank
accession No. X60733, positions 189 to 203]) and rabbit ß-actin mRNA
antisense (5' ATG GCC GGC GTG TTG AAC 3' [GenBank accession No.
X60733, positions 453 to 470]) primers were used as
controls.9 The final concentrations in the RT-PCR mixture
(50 µL) were sense and antisense primer, 0.4 µmol/L each;
dNTP, 0.2 mmol/L each; dithiothreitol, 5 mmol/L;
MgCl2, 1.5 mmol/L; RNase
inhibitor, 8 U; total RNA, 1 µg; and enzyme mix, 1
µL. Reverse transcription was performed at 50°C for 30
minutes. The thermocycling parameters were denaturation of
the template at 94°C for 2 minutes; 10 cycles consisting of
incubations at 94°C for 30 seconds, 55°C for 30 seconds, and 68°C
for 45 seconds; 25 cycles consisting of incubations at 94°C for 30
seconds, 55°C for 30 seconds, and 68°C for 45 seconds plus cycle
elongation of 5 seconds for each cycle; followed by a prolonged
elongation time of 7 minutes at 68°C. Products were
analyzed by agarose gel electrophoresis (2.5%, Gibco BRL Life
Technologies) and visualized by Sybr Green I nucleic acid gel stain
(1:10 000 dilution of the commercially available stock solution, FMC
BioProducts) under ultraviolet light. Quantification of the bands
(optical densityxmm2) was done using the
Diversity One 1.1 software (pdi).
Immunohistochemistry
The immunohistochemical reactions were carried out by an
indirect peroxidase antibody conjugate method using a polyclonal
antibody raised in sheep against human factor VIII–related antigen
(vWF), dilution 1:250 (Binding Site). To enhance antigenicity the
sections were pretreated with 1 mg trypsin/mL
tris(hydroxymethyl)aminomethane buffer, pH 7.8, at 37°C
for 10 minutes (Fluka). The specificity of the primary antibody against
vWF has been reported previously.3 The polyclonal sheep
anti-vWF antibody was visualized by a pig anti-sheep peroxidase as a
secondary antibody (Binding Site,) by using aminoethylcarbazol as a
chromogen. For negative controls, primary antibody was
omitted.
The vWF deposits were quantified by using a color image analysis system (PC_image) as described elsewhere.2 For this purpose, a rectangle with a length of 100 µm along the luminal margin was drawn. The parallel segment of the internal elastic lamina formed the opposite outer border of this rectangle. Within each intimal rectangle, the area of vWF immunoreactivity (brown color) was measured in a standardized way. The cutoff value for the vWF immunoreactivy was defined by the RGB (red-green-blue) levels of the vWF staining in the cytoplasm of the ECs.2 Measurements were repeated within several intimal rectangles until the vWF deposits were quantified along the whole circumference of the artery. For each artery, summation of the data of all intimal rectangles was expressed as immunoreactive area (µm2 per 100 µm ECs).
Double immunostains were performed for vWF and -SMC
actin and for vWF and macrophages (RAM-11 antibody). The vWF
antibody was visualized using a peroxidase technique and
aminoethylcarbazol as a chromogen. The -SMC actin or RAM-11 antibody
was visualized using an alkaline phosphatase technique and Fast Blue BB
salt (Sigma) as a chromogen.
The presence of adhering platelets in aorta segments from
atherosclerotic rabbits was verified immunohistochemically using a
monoclonal anti-GPIb antibody (G28E5) raised in our laboratory
against human GPIb-chain but cross-reactive with rabbit GPIb.
Bound G28E5 was detected via secondary peroxidase conjugated goat
anti–mouse-Ig antibodies (Dako) and staining essentially as reported
by van Zanten et al10 using diaminobenzidine (0.5 mg/mL)
supplemented with CoCl2 (0.25 mg/mL) and
NiNH4SO4 (0.2 mg/mL) in
50 mmol/L Tris-HCl buffer, pH 7.6, to increase the sensitivity of
detection.
Transmission Electron Microscopy
Segments of the sham-operated and collared carotid artery
(collar experiment) and the thoracic aorta (cholesterol and
cholesterol withdrawal group) were cut in small
arterial rings. The rings were rinsed in a cacodylate
buffer and were postfixed in glutaraldehyde 0.5%
(vol/vol) in 0.1 mol/L sodium cacodylate buffer (pH 7.4) for 24 hours.
After fixation, the specimens were washed with sucrose phosphate buffer
and postfixed for 30 minutes in 1% (vol/vol) osmium tetroxide in 0.1
mol/L sodium cacodylate buffer (pH 7.4). After dehydration in ethanol
and propylene oxide, they were embedded in Epon. Selection of the zones
most characteristic for the lesions was done on semithin sections
stained with toluidine blue. Thin sections were cut with an LKB
ultratome, stained with 2% uranyl acetate, and examined with a Jeol
1200 EX electron microscope at 80 kV.
In Vitro Platelet-Vessel Wall Interactions Under Flow
Conditions
Rabbit aorta cross-sections (7 µm) were mounted on Denhardt
coated coverslips as described elsewhere10 11 and were
perfused in a parallel-plate flow chamber at 1300
s-1 with normal citrate anticoagulated rabbit
blood for 5 minutes. After perfusion, the sections were fixed with
0.5% glutaraldehyde and bound platelets were
detected with the anti-GPIb antibody G28E5, as described above.
In addition, the thoracic aorta from
normocholesterolemic and atherosclerotic rabbits was
harvested, divided in segments, and opened longitudinally. After
careful de-endothelialization of some segments with
cotton-wool sticks, both nondamaged and damaged rabbit aorta segments
were mounted in a circular perfusion chamber. The aorta segment (0.5
cm2 exposed to the perfusate) was
perfused for 5 minutes at 1100 s-1 with normal
anticoagulated rabbit blood supplemented with autologous
111In-labeled rabbit platelets (1 to
2x106 cpm/10 mL blood). As a consequence
of the labeled platelet addition, total platelet numbers
increased by 15% to 20%. After perfusion, segments were carefully
rinsed, and the number of bound platelets (radioactivity
measurement) was calculated as a function of the total platelet
number in the perfusate. The role of vWF in platelet
deposition was investigated by performing perfusions in the presence of
increasing concentrations of the antihuman vWF antibody AJvW-2
(0 to 10 µg/mL) (gift of Ajinomoto Co, Inc, Yokohama, Japan, code
AJvW-2),12 cross-reactive with the A1 domain of rabbit vWF
and neutralizing vWF binding to its receptor on the platelet, the
GPIb/V/IX complex. The specificity of platelet binding was further
investigated in the presence of inhibitors of platelet
activation such as iloprost (1 µg/mL, Ilomédine,
Schering-Plough), the NO-donor sodium nitroprusside (100 µmol/L,
Sigma), and prostaglandin E1 (2
µmol/L, Sigma). In addition, perfusions were performed with the NO
synthase inhibitor N
-nitro-L-arginine methyl ester
(400 µmol/L, Sigma).
Statistical Analysis
In the collar experiment, the sham-operated and collared
arteries were compared using the 2-tailed paired Student's
t test. In the cholesterol experiment control
group, cholesterol group and cholesterol
withdrawal groups were compared using ANOVA followed by the
Student-Newman-Keuls test. The serum lipid values at the start of the
experiment and after 26 weeks of cholesterol
supplementation or 26 weeks cholesterol followed by 26
weeks cholesterol withdrawal were compared using the paired
Student's t test. Comparison of the degree of platelet
adhesion with rabbit (sub)-endothelium was done via the
Kruskal-Wallis test. The number of circulating platelets was
compared before and after the cholesterol-rich diet (week
9) using the Wilcoxon test. The SPSS package for Windows (SPSS
Inc) was applied for these purposes. Differences were considered
significant at P<0.05.
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Results |
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Serum Lipid Values
After feeding rabbits a cholesterol-rich diet (0.3%) for 26 weeks the total serum cholesterol levels increased from 43±6 to 1245±148 mg/dL (P<0.001), the LDL cholesterol levels from 7±1 to 396±140 mg/dL (P<0.05), and the VLDL cholesterol levels from 20±8 to 412±46 mg/dL (P<0.001). The triglyceride levels did not significantly change (from 91±12 to 105±45 mg/dL). After 26 weeks of cholesterol withdrawal, the total cholesterol normalized to 28±2 mg/dL, the LDL cholesterol to 6±1 mg/dL, and VLDL to 8±2 mg/dL. The level of the triglycerides amounted to 65±9 mg/dL.
vWF mRNA
Intimal Thickening
vWF mRNA was detected both in sham-operated and collared arteries
as the expected 270-bp amplified product. After collaring the
RT-PCR signal for vWF mRNA was significantly increased
(P<0.05, n=5). Omitting either the RNA or the RT step did
not result in a band on the gel. The signal for ß-actin mRNA, which
was used as internal control, did not significantly differ between the
sham-operated and collared arteries (Figures 1A
and 2A).
The ratio of vWF mRNA to ß-actin mRNA was significantly increased
(0.81±0.07 versus 0.48±0.05 in sham, P<0.05, n=5).
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Cholesterol-Induced Atherosclerotic Plaques
vWF mRNA was demonstrated in thoracic aortas of both control and
cholesterol-fed rabbits. The RT-PCR signal for vWF mRNA was
significantly increased after cholesterol-induced plaque
formation and was normalized after 26 weeks of cholesterol
withdrawal (P<0.05, n=4). The signal for ß-actin mRNA did
not significantly differ between the 3 groups (Figures 1B
and 2B). The ratio of vWF mRNA to ß-actin mRNA was significantly
increased after the cholesterol treatment (0.87±0.05
versus 0.52±0.12 in controls, P<0.05, n=4) and normalized
after cholesterol withdrawal (0.57±0.07).
vWF Deposition
Intimal Thickening
The sham-operated carotid arteries were lined by ECs showing a
granular staining for vWF. Deposition of vWF in the
subendothelium or the media was not found (Figure 3A). After 14 days of collar placement, a
thickening of the intima composed of -SMC actin-positive SMC had
formed. The ECs showed a dense flocculent immunoreactivity for vWF. vWF
was deposited in the matrix of the intimal thickening (Figure 3B). Macrophages were not present in the intimal
thickening. The depth of the intimal vWF accumulation varied. The
immunoreactive area per 100 µm ECs amounted to 289±75 versus
109±18 in sham, P<0.05, n=9 (Figure 4A). In some regions, some focal
immunoreactivity was present in the inner media.
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Cholesterol-Induced Atherosclerotic Plaques
The thoracic aortas from the rabbits fed a normal diet were lined
by ECs showing a granular staining for vWF. Atherosclerotic plaques
were not present, and deposition of vWF in the
subendothelium or the media was absent (Figure 3C). After 26 weeks of cholesterol treatment,
plaques had formed with a mean thickness of 170±64 µm (n=9). In
the ECs, vWF could clearly be demonstrated. In the plaques, vWF
deposits were present (immunoreactive area per 100 µm ECs;
1006±556 (n=9) versus 79±18 in controls (n=3), Figure 4B). The
vWF area correlated with the thickness of the plaque
(R=0.85, P=0.004, n=9). Three types of vWF
deposits could be distinguished. In a first type, the deposits were
diffusely present in the plaque, showing a great similarity to the
vWF deposition in the collar-induced intimal thickening (Figure 3D). In a second type, vWF deposition was prominently
present in the luminal part of the plaque, just beneath the ECs
(Figure 3E). In a third type, vWF was present in the deeper
regions of the plaque (Figure 3F). Omitting the primary antibody
resulted in complete disappearance of the immunoreactivity, which
excludes aspecific binding of the secondary antibody. Furthermore, the
specificity of the primary antibody against vWF has been documented
previously.2 The media showed no immunoreactivity for
vWF. After cholesterol withdrawal the vWF deposits
disappeared from the plaque (Figures 3G and 3H). The
immunoreactive area per 100 µm ECs amounted to 114±19 (n=10), a
value that was similar to the control arteries (Figure 4B).
The localization of the vWF deposits was further documented using
double immunostaining for vWF and -SMC actin and for
vWF and macrophages (RAM-11 stain). In the thoracic aorta of
rabbits fed a diet supplemented with 0.3% cholesterol for
26 weeks, vWF-immunoreactive granules were present in the ECs, and
vWF was deposited in the spaces between the SMCs of the plaque (Figure 3I). There was an accumulation of macrophages in the
plaque, but vWF was not present within these macrophages
(Figure 3J). After 26 weeks of cholesterol
withdrawal, the vWF deposits had disappeared from the plaque (Figures 3K and 3L). Rare macrophages remained in the plaque
(Figure 3L), and vWF immunoreactivity was present in the
ECs.
Transmission Electron Microscopy
Intimal Thickening
Transmission electron microscopy showed that after collaring, the
ECs had lost their normal flattened shape, became cuboidal, and
contained a pronounced rough endoplasmic reticulum. Weibel-Palade
bodies, which are single-membrane-bound cylindrical rod-like bodies
that contain a number of microtubules set in an electron-dense
matrix,13 were more abundant in comparison with
sham-operated carotid arteries (Figures 5A and 5B).
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Cholesterol-Induced Atherosclerotic Plaques
After 26 weeks of cholesterol feeding, the plaques
were covered by an uninterrupted layer of ECs14 with a
cuboidal aspect and a pronounced rough endoplasmic reticulum.
Weibel-Palade bodies were abundantly present (Figure 6A). However, after 26 weeks of
cholesterol withdrawal, the plaques were covered by an
uninterrupted layer of ECs, which had become flattened, similar to
control arteries, and their ultrastructural morphology had normalized.
Weibel-Palade bodies were scarcely present (Figure 6B).
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Platelet-Vessel Wall Interactions
Mild cholesterol feeding (0.125%) was associated with
a progressive reduction in circulating platelet numbers from
669 000±45 000 platelets/µL platelet-rich plasma at day 0
to 551 000±76 000 platelets/µL at the end of the 9th week
(P=0.0026). The immunohistochemical detection of vessel wall
associated platelets via the platelet membrane protein GPIb
confirmed the presence of essentially single adhering platelets to
the aorta endothelium of cholesterol-fed
rabbits but not to normocholesterolemic rabbits.
Therefore, perfusion experiments were performed with normal rabbit
blood over cross-sections prepared from the aorta and the carotid
artery of hypercholesterolemic and
normocholesterolemic rabbits.
Platelet Adhesion to Aorta Cross-Sections
In agreement with similar perfusions performed over human
lesions,10 platelet deposition on normal aorta
occurred primarily in the adventitia (Figures 7A and 7C). However, the weak adhesion
observed in the media area was concentration-dependently inhibited by
AJvW-2, confirming that platelet adhesion occurred in a
vWF-dependent fashion to vessel wall collagens. Higher AJvW-2
concentrations were required to prevent adventitial platelet
adhesion than medial adhesion (Figure 7E). Perfusion with normal
rabbit blood over atherosclerotic cross-sections revealed a higher
platelet deposition both in the medial and the intimal area
(Figures 7B and 7D). Except for the adventitial platelet
deposition, aggregate formation was concentration-dependently inhibited
by AJvW-2 (Figure 7F). These experiments suggested that the
higher platelet reactivity with the atherosclerotic rabbit vessel
wall was related to vWF but did not enable distinction between
platelet adhesion via vascular vWF and plasma vWF-mediated
platelet binding to cross-section exposed collagens. Therefore,
additional perfusions were performed over nondenuded rabbit
endothelium of hypercholesterolemic and
normocholesterolemic rabbits and over
de-endothelialized subendothelium.
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Platelet Adhesion to De-Endothelialized
Subendothelium From
Normocholesterolemic and Hypercholesterolemic
Rabbits
During the perfusion in a cylindrical flow chamber of cotton-wool
damaged aorta of normal or atherosclerotic rabbits with
111In-labeled platelets added to normal
anticoagulated rabbit blood, very similar platelet adherence was
observed around 0.08% of total platelets (Figure 8A). Platelet adhesion to cotton-wool
damaged subendothelium was inhibited by AJvW-2 with an
IC50 around 2.5 µg/mL (Figure 8B).
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Platelet Adhesion to Endothelium From
Normocholesterolemic and Hypercholesterolemic
Rabbits
Perfusions over normal endothelium resulted in
some apparent adhesion (0.026±0.0032%; Figure 8A). However,
this signal resulted from nonspecific accumulation of
platelet-released radioactivity in the vessel wall, as evident from
radioactivity measurements in postperfusion plasma (not shown) and from
control experiments in the presence of PGE1,
iloprost, and sodium nitroprusside, which substances had no effect on
this background signal (not shown). Similarly, neither AJvW-2 nor
N-nitro-L-arginine methyl ester had any effect on this low value
(not shown).
In contrast to nondenuded controls, perfusions over nondenuded
atherosclerotic aorta revealed specific platelet adhesion
(P=0.001; Figure 8A). This specific platelet
adhesion (ie, corrected for background signal) equaled 0.015±0.005%
of circulating platelets, corresponding to an average specific
deposition around 600 000 platelets/cm2
endothelium. This adhesion was
concentration-dependently inhibited by AJvW-2 with an
IC50 around 2.5 µg/mL (Figure 8B).
Perfusions at 300 s-1, revealed no specific
platelet adhesion (Figure 8A), in agreement with the absence
of vWF interactions with its receptor on platelets at these low
shear rates.12
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Discussion |
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In normal arteries, the ECs secrete vWF both in the plasma and toward the subendothelial space, where a minimal deposition of this glycoadhesive protein is present in the basal lamina and plays a role in the adhesion of the ECs.4 However, ECs can also be stimulated to release additional amounts of vWF basolaterally under conditions of "stress" (eg, hemodynamic changes). In cultured ECs, approximately 5% of the newly synthesized vWF is directed to the Weibel-Palade bodies. The majority of the protein is secreted constitutively.15
The present study showed that cholesterol-induced
plaque formation was associated with an increased ratio of vWF mRNA to
ß-actin mRNA and deposition of vWF in the plaque. The ECs that
covered the plaques formed an uninterrupted layer14 but
showed cytoplasmic vWF accumulation, a pronounced rough endoplasmic
reticulum, and abundant Weibel-Palade bodies, similar to advanced human
atherosclerotic plaques.16 Therefore, our results point to
an increased synthesis of vWF by the ECs after a
cholesterol-rich diet. The reason for the vWF deposition is
unclear. Hypercholesterolemia may be a factor
that can regulate vWF gene transcription, as suggested by the
present study. This could be explained by the presence of
inflammatory mediators in atherosclerotic plaques17 or by
a direct action of cholesterol on vWF synthesis. It has
been shown that activated transcription factor nuclear
factor-
B (NF-B) is present in the ECs covering an
atherosclerotic lesion.18 Activation of NF-B by
(oxidized-)LDL may lead to transcription and enhanced synthesis of
adhesion molecules19 20 21 but possibly also of vWF. In
addition, in cultured ECs, LDL enhances the concentration of
cell-related vWF.22 In rabbits on a 0.3%
cholesterol diet the increase of serum
cholesterol levels is only partly due to the increase of
LDL cholesterol levels; VLDL cholesterol levels
are also increased. LDL cholesterol and ßVLDL
cholesterol may have different effects on
endothelium. However, a dense immunoreactivity for vWF
was only present in ECs covering an atherosclerotic plaque or
intimal thickening. In those parts of the aorta or carotid artery that
were lesion-free, the ECs showed a normal vWF expression despite that
they had been in contact with the same amount of blood lipids.
Qualitative immunohistochemical and quantitative platelet adhesion studies showed that in atherosclerotic aortas there was an increased adhesion of platelets to the ECs, which was mediated by vWF, possibly explaining the drop of platelet numbers throughout atherogenesis. After mechanical removal of the ECs, the degree of platelet adhesion to the subendothelium was similar in atherosclerotic and control aortas. Perfusion studies over cross-sections of atherosclerotic aortas revealed increased vWF-mediated adhesion in the plaque area in comparison with normal sections. This shows that an increased platelet adhesion results both from a direct effect mediated via (sub)endothelial vWF and an indirect effect exerted via reactive plaque collagens, this binding, however, being mediated via plasma vWF. It is conceivable that the increased production of vWF by ECs not only resulted in subendothelial vWF deposition but probably also in an increase in plasma vWF. High vWF levels may reflect endothelial dysfunction and may be an indicator of poor prognosis of cardiovascular events.23 24 25 26 27 However, we were unable to measure vWF levels in rabbit plasma.
Finally, we demonstrated that cholesterol withdrawal normalized vWF mRNA levels, the EC morphology, and their number of Weibel-Palade bodies, and it led to the disappearance of the vWF deposits from the plaques. Possibly, vWF is taken up by macrophages, which subsequently disappear from the plaques after cessation of cholesterol feeding.14 However, we did not find a colocalization of vWF and RAM-11.
Also in a collar-induced intimal thickening that almost completely consisted of smooth muscle cells, an increase in vWF mRNA, deposition of vWF in the intima, and changes in the ultrastructural morphology of the ECs were seen, although the rabbits were normocholesterolemic. In this case, the deposition of vWF could be related to shear stress1 28 or an increased biosynthesis and/or release of vWF mediated by inflammatory stimuli.29 30 31 32 33 34
The increased synthesis and intimal deposition of vWF in atherosclerotic plaques and intimal thickenings may have important implications for human vascular disease. The possible importance of vWF deposition is underscored by the fact that it is not limited to atherosclerotic plaques and intimal thickenings but that it occurs after balloon angioplasty,35 36 37 38 in the human ductus arteriosus,39 and in human stenosed vein grafts as well (unpublished data, 1998). Possibly, deposition of vWF in the matrix of intimal thickenings is merely a marker of endothelial activation.25 On the other hand, increased synthesis and deposition of vWF by itself may stimulate intimal thickening and plaque formation. Indeed, balloon angioplasty of the porcine carotid artery was associated with deposition and medial absorption of vWF over a time period that preceded and overlapped vascular smooth muscle proliferation and endothelial recoverage.36 Moreover, it has been demonstrated that inhibition of vWF function by aurintricarboxylic acid for only 24 hours after deep arterial injury attenuated neointimal hyperplasia at 2 to 3 weeks.40 In addition, both the thickness of cholesterol-induced plaques and collar-induced intimal thickening2 correlated with the deposition of vWF. Furthermore, homozygote vWF deficient pigs show a resistance to the initiation and progression of spontaneous and diet-induced atherosclerosis,41 but vWF appeared not essential for shear-induced smooth muscle cell accumulation in the intima.7 Therefore, the vWF deposits seem not to be important for this early step in the development of atherosclerosis. However, deposition of vWF could play a role in a later step of atherogenesis. The finding that vWF is not universally distributed in the vascular wall of normal pigs and is absent in the coronaries in which spontaneous atherosclerosis occurs with low frequency provides support for a role of vascular vWF in atherogenesis.42 Furthermore, it is well known that vWF is important in platelet-vessel wall interactions and that it has prohemostatic properties.6 The present study demonstrated that the increased amounts of vWF on the ECs of atherosclerotic aortas resulted in an increased adhesion of platelets to the vessel wall, as compared with normal aortas. Also, the subendothelial vWF deposits were functional with regard to platelet adhesion in the plaque, as shown by both types of perfusion studies. This may have important clinical consequences, eg, during plaque rupture when vWF deposits in the plaque become exposed to blood. It has been shown that vWF is essential for the development of occlusive thrombosis and that it appears to support the progression of a platelet-fibrin microthrombus to an occlusive thrombus both after cholesterol-induced atherosclerosis43 and after stenosis and pinch-injury in the pig.44 Although severe von Willebrand disease did not protect completely against the development of atherosclerosis, autopsy findings in 3 patients with von Willebrand disease types IIB and III revealed the presence of atherosclerotic lesions without occlusive arterial thrombi.45 The organization and incorporation of mural thrombi appears to be an important component of atherosclerotic plaque growth.42
In conclusion, our results show an enhanced synthesis and (sub)endothelial deposition of vWF during atherogenesis in rabbits. The raised vWF production facilitated platelet adhesion to activated endothelium and the intraplaque vWF deposits remained functional and may promote platelet adhesion on plaque rupture. These findings may be relevant for our understanding of endothelial cell activation during atherogenesis.
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Acknowledgments |
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Dr De Meyer is a research associate of the Fund for Scientific Research—Flanders (Belgium). Dr Kockx is a holder of a fund for fundamental clinical research of the Fund for Scientific Research—Flanders (Belgium). The authors acknowledge the assistance of Martine De Bie, François Jordaens, Michiel Knaapen, Frank Lakiere, Johannes Muhring, Rita Van Den Bossche, and Ludo Zonnekeyn. The authors owe much gratitude to Prof Dr N. Buyssens for his help and advice during the experiments. Prof Dr W. Jacob is thanked for providing facilities for electron microscopy. The neutralizing anti-vWF antibody AJvW-2 was a kind gift of Ajinomoto Co, Inc. (Yokohama, Japan). The financial support of the FWO Levenslijn projects 7.0022.98 and 7.0014.98 is greatly appreciated.
Received August 28, 1998; accepted April 24, 1999.
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