© 1998 American Heart Association, Inc.
Original Contributions |
von Willebrand Factor Does Not Influence Atherogenesis in Arteries Subjected to Altered Shear Stress
From the Departments of Medicine (T.C.N., G.E., T. du L., T.J., T.R.G.) and of Pathology and Laboratory Medicine (T.C.N., D.A.B., R.L.R., J.L.S., G.E., T. du L., M.S.R., T.R.G.); The Center for Thrombosis and Hemostasis (T.C.N., D.A.B., T.R.G.); and the Division of Laboratory and Animal Medicine (D.A.B.), Biostatistics (G.G.K.), and Biomedical Engineering (T.J.), University of North Carolina at Chapel Hill.
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Abstract—The role of von Willebrand factor (vWF) in arterial neointimal formation that develops in arteries with altered shear stress was investigated using normal, heterozygous, and homozygous von Willebrand disease pigs (ie, vWD, or lacking vWF) that were fed normal pig chow. Shear stress was applied to carotid and femoral arteries with a Goldblatt clamp for 14 days, producing a
80% stenosis. Neointimal lesion size was
measured by computer-assisted morphometry. Expression of proliferative
cell nuclear antigen (PCNA) by neointimal and medial cells
was used as a relative index of proliferative activity. For
shear-stressed arteries, there was no significant difference in the
number of smooth muscle cell layers in the lesion, lesion size, and
percent of PCNA-positive neointimal or medial cells among
normal, heterozygous, and homozygous vWD pigs
(P.1, ANOVA). Lesions in pigs that expressed vWF
(normals and heterozygotes) contained large amounts of vWF in the
neointima, whereas lesions in vWD pigs had no detectable
vWF. Moreover, no foam cells were detected in the lesions. Thus, the
absence of vWF apparently does not alter the size of lesions in
shear-stressed arteries in vWD pigs or the number of
neointimal or medial cells expressing PCNA. Mechanism(s)
involved with shear-induced modulation of smooth muscle cell
proliferation, then, can operate independently of vWF in
normolipemic pigs.
Key Words: von Willebrand disease • von Willebrand factor • atherosclerosis • neointima • shear stress
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A role for vWF in atherogenesis has been suggested in some studies but not others (reviewed in References 1 through 31 2 3 ). Potential mechanism(s) have been suggested to be mediated by vWF-dependent delivery of platelet products to the vessel wall; however, such a mechanism remains unproven. These aforementioned studies have compared the burden of atherosclerosis in normal and vWD pigs by examining advanced atherosclerotic plaques. Such plaques contain foam cells, necrotic cores, SMCs, and inflammatory cells in a complicated extracellular matrix. Factors that control or regulate the contribution of each cell type or matrix constituent to plaque development are complex and multifactorial. It is possible that any role of vWF is obscured in the development of such a complex plaque. Thus, studies of an early or less complex lesion might help identify mechanism(s) whereby vWF contributes to atherosclerosis.
Positioning a collar or cuff around the carotid artery in normal rabbits induces neointimal lesions composed predominantly of SMCs in a matrix that contains abundant vWF.4 5 6 7 These vWF-rich neointimal lesions contain no foam cells unless the animal is fed an atherogenic diet.5 The cuffing procedure produces altered flow patterns and thus altered shear stress in the operated artery.8 Altered shear stress is a key force in vascular remodeling and gene expression and may be a critical determinant in restenosis following angioplasty or failure of saphenous vein and prosthetic vascular grafts (reviewed in Reference 99 ). Taken together, these findings raise the question of whether or not vWF influences the development of neointimal lesions in arteries with altered shear stress in vivo. The purpose of our study was to determine whether or not vWF supported the development of neointimal lesions in arteries with altered shear stress by using normolipemic pigs that express vWF and those that do not (ie, pigs with vWD). An abstract of this work has been published.10
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Normal, Heterozygous, and vWD Pigs
Male and female pigs (Sus scrofa) were used between 3 and 5 months of age. The animals were divided into three groups: pigs that express vWF (normal genotype, n=2, and heterozygous vWD, n=3) and pigs that do not express vWF (homozygous vWD pigs, n=3). All pigs were produced at the Francis Owen Blood Research Laboratory at the University of North Carolina, Chapel Hill. Porcine vWD is inherited as an autosomal recessive trait.11 12 The following baseline data were obtained for the animals before beginning the protocol: (1) bleeding time,13 (2) vWF activity by platelet agglutinating factor assays using formaldehyde-fixed and lyophilized human platelets,12 14 (3) vWF antigen by ELISA,15 16 (4) platelet counts, and (5) hematocrits. All animals were treated according to the standards set in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 85–23). All procedures were in accordance with institutional guidelines.
Application and Measurement of Shear Stress
The pigs were anesthetized with ketamine (10
mg/kg body weight intramuscularly administered), intubated, and
maintained with halothane (2% in air supplemented with 1 L/min
oxygen), and stainless steel 0.5-cm-long by 0.35-cm-wide Goldblatt
clamps were applied to one each of the carotid and femoral arteries as
described (see Figs 1
and 2 and References 15 and 1715 17 through 19).
The Goldblatt clamp was partially closed until reactive
hyperemia was blocked, which we and others have previously
shown produces a 80% stenosis.15
Measurements of blood flow velocity were performed in vivo by a
Doppler flowmeter using a custom-made Doppler system (Craig J.
Hartley, PhD, Baylor College of Medicine, Houston, Tex). The
contralateral artery was either surgically isolated in an identical
fashion but had no shear applied (ie, sham-operated control) or
maintained as a nonsurgical control. The pig was allowed to recover and
clamps were left in place for 14 days. The distribution of pigs
according to vWF genotype and arteries according to shear
status is summarized in Table 1.
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Time-averaged shear stress was calculated for the stenosed and
unstenosed arteries by using blood flow velocity and the dimensions of
the artery as measured by computer (see below). These two variables
were used to calculate volumetric flow rate, which was subsequently
used for determining shear stress. All equations assume laminar flow
conditions. Wall shear stress (
) at baseline was determined with the
Hagan-Poiseuille approximation:
 | (1) |
After the Goldblatt clamp was partially closed, the stenosed section of
the artery became rectangular due to the conformation of the clamp (Fig 2
). Shear stress in the stenosed arteries was calculated with the
following formula:
 | (2) |
Sacrifice of Pigs and Harvesting of Tissues
At the end of the protocol, blood flow velocity measurements
were repeated to confirm that reactive hyperemia remained
blocked. The pigs were then killed with an overdose of pentobarbital (6
grains/10 lbs i.v.). The arteries were removed and fixed with 4%
paraformaldehyde at pH
7.4.15 17 19 21 Cross sections of the arteries
were taken in the prestenotic, stenotic, and
poststenotic segments (Fig 1). Serial sections were cut in
order to use adjacent sections for morphometry and
immunohistochemistry. In preliminary analyses,
neointimal lesions were found to develop predominantly in
the proximal portion of the stenotic segment (ie, region 2,
serial section A of Fig 1). These sections were then subjected to
morphometric and immunohistochemical analyses.
Morphometry of Arterial Changes
The image of each vessel segment was digitized using a Nikon
Microphot-FXA connected to a Macintosh computer via an Optronics TEC
470 CCD video camera system (Optronics Engineering). Images of the
external elastic lamina, internal elastic lamina, and lumen were
measured using NIH image software. The extent of arterial
changes was evaluated by three indices calculated from the tracings:
(1) neointimal area in µm,2
(2) number of layers of neointimal cells in the thickest
region of the plaque, and (3) percent stenosis induced by the
Goldblatt clamp.22 The percent stenosis
induced by the clamp was 80% in all stenosed arteries.
PCNA Detection
Paraffin-embedded sections were cut at 5 µm, collected on
ProbeOn plus slides (Fisher Scientific Co), and then subjected to an
antigen retrieval process.23 The slides were then
placed in buffer for subsequent immunohistochemical staining with
standard techniques.24 PCNA was detected with a
commercially available antibody that has been used as a measure of cell
replication in porcine arteries (PC10, Dako Corp, at a dilution of
1:150).24 25 PCNA is a nuclear protein of
Mr of
36 000.26 27 It is an essential cofactor of DNA
polymerase-
, and thus necessary for the cell to proceed through the
S phase of the replication cycle.26 28 29 The
chromogen was 3,3'-diaminobenzidine, which was used as recommended by
the manufacturer (Vectastain ABC, Vector Laboratories). The slides were
lightly stained with hematoxylin. In a subset of pigs, serial sections
were stained with an anti-SMC actin antibody (A2547, Sigma). The
majority of neointimal cells expressing PCNA contained SMC
actin, as reported by Kockx et al.6 7 The number
of SMC nuclei in the media and neointima was determined by
point counting by using the imaging system described above. The vessel
was divided into four quadrants, and at least 100 cells were counted
per quadrant. The vessels were evaluated by two observers who had a 5%
interobserver variability. The percentage of cells positive for PCNA in
the neointima and media was then determined as a fraction
of the total number of cells counted in that cross section.
Endothelial cells were excluded from this
analysis. During each preparation of slides for PCNA detection,
a section of porcine small intestine was included for a positive
control. For negative controls, the primary antibody was omitted from
serial sections.
Immunohistochemical Detection of vWF Antigen Deposition in the
Vessel Wall
A purified polyclonal rabbit anti-human vWF (Dako Corp, code No.
A082) was used to detect vWF antigen in the tissue
sections.16 vWF antigen was then detected with
the chromogen diaminobenzidine (Vectastain ABC Kit, Vector
Laboratories). The sections were then examined by light microscopy
(Nikon Microphot-FXA). Positive and negative control sections were
included in each preparation. Negative controls were created by
omitting the primary antibody from selected sections or including
sections from a known vWD pig.
Statistical Analysis
The data for the intimal area, number of layers of
neointimal cells, and percentage of medial and
neointimal cells positive for PCNA are described as
means±SD. For these variables, multiple linear regression models
for three-way ANOVA were used for comparisons pertaining to normal,
heterozygous vWD, or homozygous vWD status of the pigs; shear-stressed,
sham-operated, or unoperated status of the arteries; and anatomic
location of the arteries (carotid or femoral). Also the results from
these comparisons had nonparametric confirmation with
Wilcoxon rank sum tests and their stratified extensions to
address any concern for the assumptions of the two-way ANOVA.
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In the sham-operated (ie, patent) arteries, the mean Doppler velocity shift was 5.2±1.5 kHz and the shear stress was calculated to be 40±20.1 dyne/cm2 (range, 26 to 69). In the shear-stressed arteries (ie, with the clamp partially closed), the mean Doppler velocity shift was 0.3±0.19 kHz and the shear stress was calculated to be 199.9±126.5 dyne/cm2 (range, 133 to 533), or a mean increase of 95.2±3.9% (range, 86% to 99.2%).
The primary difference in the neointima that developed in
arteries with altered shear stress among normal, heterozygous, and
homozygous vWD pigs was the presence of abundant vWF in the lesions of
pigs that express this protein (Fig 3).
However, the presence of vWF did not influence the size of the
shear-induced neointima, the number of cell layers in the
neointima, or the expression of PCNA in the
neointima and media at 14 days (Figs 3 and 4 and Tables 2 through 5, P0.1, ANOVA).
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The changes in the shear-stressed arteries compared with sham-operated
and unoperated arteries revealed a significant increase in the number
of neointimal cell layers, the size of the
neointima, and the degree of PCNA expression in the media
and neointima (P
.003, ANOVA). Also, there
was essentially no difference among normal, heterozygous vWD, and
homozygous vWD animals for these variables, regardless of whether
the artery had been shear stressed, sham operated, or unoperated, nor
was there any difference between femoral and carotid arteries or any
between sham-operated and unoperated arteries.
No foam cells or lipid incorporation was seen in the neointimal lesions. Normal and heterozygous vWD pigs had bleeding times <3 minutes and vWF antigen and activity levels that ranged from 24% to 112%. vWD pigs had bleeding times >15 minutes and vWF antigen and activity levels <1%. In this study, no pig required transfusion for control of bleeding.
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Our study addressed the question of whether or not expression of vWF influenced neointimal formation in arteries with altered shear stress. We found that the absence of vWF did not alter the size of the neointimal lesions, the number of layers of cells in the neointima, or the expression of PCNA in the neointima or media. The exact mechanism of neointimal formation in arteries with altered shear stress is unknown but is likely to be multifactorial. It has been known for some time that many forms of vascular manipulation will induce neointimal formation, and there appear to be important species-dependent responses (reviewed in References 30 through 3230 31 32 ). First, simple surgical isolation of an artery induces neointima in rabbits, and thus, either the operative procedure alone or the disturbed flow that likely occurs during healing contributes to neointimal hyperplasia.33 Second, vascular injury applied either externally to a rabbit artery or endoluminally in rats, rabbits, and pigs induces neointimal formation (reviewed in References 34 and 3534 35 ). Third, altered shear stress induces differential gene expression in vivo of key proteins involved in atherogenesis.36 In addition to shear stress, these injury mechanisms also could contribute to the development of neointima in our model. Fourth, perivascular cuffing with polyethylene collars has been shown by several laboratories to induce neointimal formation in rabbits, in part due to alterations in shear stress, release of factors that stimulate chemotaxis, mitogenesis from the cuff itself, or impairment of the transmural flow by the collar.4 5 6 7 37 Moreover, the disturbed flow patterns associated with shear stress gradients appear to contribute to the growth of the atherosclerotic plaque.38 We designed our study with these issues in mind: comparable operative procedures in all arteries, stainless steel Goldblatt clamps to standardize and minimize any potential reaction to the collar material, and application of the equivalent amount of stenosis to produce a uniform alteration of shear stress in all arteries. It is clear from our studies that under these conditions, vWF is not essential for neointimal formation.
The role of fluid shear stress in atherogenesis is critical in the initiation and progression of lesions. Obstructive atherosclerosis is less frequent in large arteries with undisturbed laminar flow conditions; on the other hand, obstructive lesions appear to develop in regions of altered fluid shear stress created by recirculation zones, such as are found in carotid, femoral, and coronary arteries; aortic bifurcations; and the abdominal aorta (reviewed in References 39 through 4139 40 41 ). These atherosclerosis-susceptible regions are found where there are sudden alterations (decreases, increases, or gradients) in shear stress. Our shear stress calculations yielded values within the range reported in rabbits carotid arteries36 and for arteries of similar size in humans.42 Positioning a clamp around the carotid and femoral arteries then essentially models this plaque development associated with altered shear stress.
It is interesting to note that application of high shear stress to SMCs in vitro in a parallel-plate system increases their production of cell-associated proteins and stimulates release of biologically active mitogens.43 44 These shear-stressed, cultured SMCs are actively replicating but at a reduced rate relative to control cells cultured under static conditions.43 44 45 Such a system essentially models SMCs abruptly and directly exposed to flowing blood (ie, with no overlying endothelium, subintima, or internal elastic lamina) after a period of growth under static conditions at atmospheric pressure (ie, without systemic blood pressure). Despite the differences between the in vivo and in vitro models, the proliferative neointimal lesions that develop with high shear stress may result from this local production of SMC mitogens. We focused our study on the region of the vessel that showed the greatest degree of neointimal formation. Because we were focusing on the role of vWF in neointimal formation, we used serial arterial cross sections for immunohistochemical and morphometric analyses. This design did not allow for measurement of the entire lesion. Thus, focal or local reduction of SMC proliferation by altered shear stress would not have been detected by our analyses.
Interestingly, we found that 13.9% of the SMCs in the media of control
carotid and femoral arteries exhibited detectable PCNA expression by
our methods. We have previously found that 
0.5% of SMCs in
uninjured, nonatherosclerotic coronary arteries of normal and
vWD pigs take up
[3H]thymidine.46 There
are several possible explanations for this apparent difference in SMC
proliferation in the carotid and femoral when compared with
coronary arteries. First, the two methods measure different
aspects of cell proliferation: [3H]thymidine
reflects DNA synthesis during the S phase of the cell cycle, and the
expression of PCNA reflects protein expression during the
G1 and G2 phases of the
cell cycle.47 48 It is possible then that the
relatively increased rate of proliferation suggested by the number of
PCNA-positive cells that we detected actually reflects a long half-life
of PCNA. This possibility is supported by Gordon et
al,49 who found PCNA and
[3H]thymidine labeling indices in
balloon-injured rat carotid arteries of 17.3% and 9.8%, respectively,
when measured concurrently.49 Second, studies
with mouse NIH3T3 cells using anti-human PCNA antibodies detected two
populations of PCNA: one that is present in serum-starved,
quiescent, cultured cells and can be extracted by Triton X and one that
is bound to nuclear structures and cannot be extracted by detergent or
high salt concentrations.50 Both forms were
detected when cells were fixed with paraformaldehyde,
but only the nonextractable form was detected when methanol fixation
and fluorescent secondary antibodies were used. However, this
antibody did not detect PCNA by immunoblotting in
nonproliferative tissues. In contrast, other studies have found that
paraformaldehyde fixation may reduce the amount of PCNA
detected by IgG or IgM anti-PCNA antibodies and peroxidase-conjugated
secondary antibodies.51 We fixed and processed
all our tissues in a standard fashion to minimize any variability due
to methodology by using techniques previously used on porcine
arteries.24 Third, our sampling method counts a
sample of cells in four quadrants of each cross section. This
semiquantitative method could bias results through an
inadvertent sampling error. However, we included scored
cells as "PCNA-positive" only if they had sharply defined nuclei
clearly distinct from "PCNA-negative" cells and if the positive
control tissue identified PCNA-positive cells processed
simultaneously. In addition, the cells were counted by two
observers who had <5% interobserver variability. Fourth, the pigs
were used at a young age when they are in an active growth phase. The
rate of baseline SMC growth in young growing animals is likely greater
than that found in mature adult pigs. Finally, all of our animals and
tissues were handled in an identical fashion. If our methods included a
systematic error, it would have affected all pigs equally. Given the
strengths and limitations of PCNA measurements, it is probably best to
interpret these data as an estimate of the relative rate of SMC
proliferation rather than an absolute rate.
Our study included normal, heterozygous vWD, and homozygous vWD pigs with normal, intermediate, and undetectable levels of vWF, respectively. This distribution provided an opportunity to perform a genetically determined "dose-response" curve on any effect vWF might have on atherogenesis in this model. However, after our analyses on the set of pigs in this study were completed, we found no differences by using multiple analyses. Still, our study is small, and it possible that with a very large number of pigs we could detect some difference among the three groups. Any difference would likely be small on the basis of our current findings and thus, would not justify the use of such a large number pigs.
The abundance of vWF in shear-induced neointima is
intriguing, especially if one considers the fact that vessel wall vWF
is essential in the development of occlusive coronary and
carotid arterial thrombosis in the Folts' stenosis
and injury model.15 The high local concentration
of vWF in the neointima may well contribute to plaque
thrombogenicity. Accumulation of vWF in the neointima is
likely a combined function of synthesis and release by
endothelial cells, uptake from plasma and/or
platelets, and the rate of removal of vWF from the extracellular
matrix. Elegant studies have characterized the complex intracellular
processing, storage, and secretion of vWF.52
Human umbilical vein endothelial cells release vWF
constitutively and in response to various stimuli that include high
shear stress: reactive oxygen intermediates, calcium ionophore A23187,
thrombin, phorbol ester, interleukin-1, fibrin, histamine, complement
C5a and C5b-9, vascular endothelial growth factor,
irradiation, and estrogens (reviewed in References 53 and 5453 54 ). Pigs and
humans have very little vWF in the unstimulated aortic or
coronary arterial endothelium or
subendothelium and normally none in the tunica media
(Fig 3 and References 55 and 5655 56 ). This finding likely reflects two
facts: (1) 95% of newly synthesized vWF is secreted via the
constitutive pathway and 5% is stored in Weibel-Palade
bodies57 and (2) deposition of plasma vWF into
the subendothelium across intact
endothelium is thought to be
minimal.58 59 It is possible, however, that there
is increased sequestration of vWF resulting from
transendothelial transport of plasma or platelet
vWF if the endothelial barrier is altered during
atherogenesis. Such a mechanism has been suggested to support increased
vWF deposition in the vessel wall after
angioplasty.60 We have previously found that the
operative procedure we used to apply shear stress in vivo injures or
disrupts 40% to 50% of the endothelium but leaves
the underlying internal elastic lamina nearly
intact.61 Determination of the relative
contribution of any of these mechanisms to the accumulation of vWF in
the neointima is beyond the scope of this study.
Identification of the key mechanisms that produce increased vWF
abundance in the neointima, however, would allow for the
development of strategies for reduction of vWF content. Such strategies
could be expected to reduce plaque
thrombogenicity.15 18
The role of vWF in neointimal lesions that develop in arteries with altered shear stress is unknown. Unlike previous studies of vWF and atherogenesis, this shear stress model produced lesions without foam cells. These less complex lesions, however, developed to the same size and had the same degree of proliferative activity independent of the presence of vWF. However, the abundance of vWF even in less complex lesions may contribute to the thrombogenicity of individual plaques. Taken together, these findings support the hypothesis that vWF is not essential for the development of shear-induced neointimal proliferation but likely plays a role in plaque thrombogenicity.
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Acknowledgments |
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This study was supported by the National Heart, Lung, and Blood Institute, Bethesda, Md (HL26309 to T.R.G., HL49818 to M.S.R.), and Naval Research Grant 14–89-J-1712 (to M.S.R.). G.E. and T. du L. were supported by a Training Grant in Experimental Cardiology (T32HL38885). We also thank Robin Raymer and Ivy McManus and the support staff at the Francis Owen Blood Research Laboratory, Chapel Hill, NC, for their excellent care and handling of the animals.
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Reprint requests to Timothy C. Nichols, MD, Department of Medicine, CB No. 7075, 349 Burnett-Womack Building, University of North Carolina, Chapel Hill, NC 27514-7075.
Received March 12, 1997; accepted November 17, 1997.
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