© 1995 American Heart Association, Inc.
Articles |
Stenosis Enhances Role of Platelets in Growth of Regional Thrombus and Intimal Wall Thickening in Rat Carotid Arteries
From The Third Tokushima Institute of New Drug Research (Pharmacology), Otsuka Pharmaceutical Co, Ltd, Tokushima, Japan.
Correspondence to Dr T. Igawa, Otsuka Pharmaceutical Co, Ltd, The Third Tokushima Institute of New Drug Research, 463-10 Kagasuno Kawauchi-cho, Tokushima 771-01, Japan.
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Abstract |
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Abstract The authors present the results of a study in which stenosis was induced, resulting in either thrombus or intimal wall thickening, in rat carotid arteries. At
75%
stenosis in mildly denuded arteries, an acute and occlusive
thrombus formation was induced, but the thrombus was significantly
reduced in thrombocytopenia. Thrombus formation near the site of
stenosis decreased with decreasing degree of stenosis,
whereas the percent formation in the distal region (percent total
thrombus) increased. Numerous mural platelet microthrombi were
noted at the distal region of the stenosed arteries. After chronic 50%
stenosis of the carotid artery for 2 weeks, significant intimal
thickening was observed, without any occlusive thrombus formation. The
combination with mild denudation was critical in eliciting the effect
of stenosis. The magnitude of intimal growth in the stenosed
artery was marked by day 6 and plateaued thereafter, whereas it was
slight in nonstenosed arteries. The 5-bromodeoxyuridine index of the
cells of the medial layer at day 3 was significantly increased by the
stenosis, and the effect was reversed in thrombocytopenia.
Complete reendothelialization of the intimal surface
was observed by 7 to 10 days after surgery in the stenosed arteries.
These findings suggest that the introduction of stenosis in
these arteries enhances the interaction of platelets with the
damaged arterial walls under abnormal fluid shear and that
this enhancement leads to acute and occlusive thrombus formation
associated with more marked stenosis as well as to sustained
increase of intimal wall thickness in less marked stenosis.
Key Words: rat carotid artery • intimal wall thickening • arterial thrombosis • mild denudation • abnormal fluid shear
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Introduction |
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Ischemic heart disease and stroke remain the most common causes of death in the industrialized nations. The relation between the development of thrombus and plaque formation is of prime importance in atherosclerotic arteries in such diseases.1 2 3 It has been recognized that the development of plaque formation is an adaptive or reparative process in the response to intimal injury4 and that platelets play an important role in regulating intimal SMC behavior in arteries by releasing growth factors.5 In spite of this recognition plaque and thrombus formation have so far been studied independently of each other. The balloon injury method, especially in rat carotid arteries, has been well investigated as a model for the study of intimal lesion formation in vivo. Fingerle et al6 suggested that platelets do not play a role in the initiation of SMC proliferation after injury by balloon catheter but may regulate their movement into the intima. They also demonstrated that the significantly higher proliferation seen in ballooned vessels might reflect a response of the medial cells to trauma that occurred during denudation.7 These observations suggest that the balloon model does not sufficiently reflect the clinical situation. On the other hand, various models of arterial thrombosis, such as electrical stimulation8 or chemical induction9 models, have also been postulated, but almost all of them have been established on the basis of artificial conditions; thus, it is also difficult to conclude that they reflect the clinical situation. It is generally recognized that clinical atherosclerotic plaque or thrombus formation is frequently located near the site of stenosis or bifurcation of the artery.10 11 It is likely that the abnormal fluid dynamics near the stenosis or bifurcation contributes to thrombus or intimal plaque formation by interaction of hyperactive platelets with the injured surface of the artery. On the basis of this background, we hypothesized that significant intimal thickening or thrombus formation could be induced by the introduction of stenosis in the rat carotid artery. We also evaluated the contribution of platelets to the development of thrombus and intimal thickening induced by stenosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgical Preparation
Six-week-old male Wistar rats weighing
200 to 250 g (Charles
River Japan, Shiga) were anesthetized with pentobarbital (25
mg/kg IP), and the iliac artery was exposed. A balloon catheter
(Fogarty arterial embolectomy catheter, maximally
inflatable to 200 µL, model 12-060-2F, CV-1035, Baxter Healthcare
Corp) was inserted into the iliac artery and introduced into the left
common carotid artery via the aorta. The inner lumen of the left
carotid artery, which for anatomic reasons was much easier to cannulate
than the right carotid artery, was mildly denuded; the balloon was
inflated once with 50 µL of saline, which was then slowly withdrawn.
Immediately after the balloon denudation, the external surface of the
common carotid artery at the corresponding position was stenosed with a
suture (Nescosuture, 7-0, RS849, Nihonshoji). A piece of stainless
steel wire was placed alongside an exposed segment of the carotid
artery, and a suture was firmly placed around both the wire and the
vessel. The wire was removed immediately, leaving a narrowed lumen. A
wire 0.75 mm in diameter (50% stenosis) was used to induce
intimal thickening, and wires with diameters of 0.60 mm (75%
stenosis), 0.45 mm (91% stenosis), or 0.35 mm (96%
stenosis) were used to induce thrombus formation. A suture was
placed on the artery 7 to 8 mm from the bifurcation of the common
carotid artery. All surgical procedures were carried out in a manner
designed to minimize trauma to the rat and under sterile conditions.
Some treated rats were kept at 37°C under anesthesia
until the thrombus weight was determined, and others were housed for
2 weeks under standard conditions until the intimal thickness was
determined.
Determination of Thrombus Formation
The rats (10 rats per group) were anesthetized with
pentobarbital, and their carotid arteries were fixed by the perfusion
of neutralized 4% formalin from the left ventricle at a pressure of
100 mm Hg. The treated left and nontreated right common carotid
arteries were isolated from the rat and fixed thoroughly by immersion
in the fixative for a few hours. The fixed arteries were trimmed, and
two segments, each 5 mm in length, proximal and distal to the
stenosis (total length, 10 mm) were obtained. For each pair of
carotid arteries, the wet weight of the segments was measured with an
electronic balance (Sartorius MC-1). A preliminary study revealed that
the mean wet weight of the untreated left common carotid arteries
(1.568±0.196 mg/cm artery) was not significantly different from that
of the contralateral untreated right arteries (1.628±0.087 mg/cm).
Thus, it was confirmed that the degree of thrombus formation could be
expressed as the difference between the wet weights of paired left
(treated) and right (nontreated) carotid arteries (L-R).
Determination of Intimal Thickness and Cell Proliferation of SMCs
in the Medial Layer
After the removal of the carotid segment (10 mm in length), the
DNA content was determined.12 The preliminary study
revealed no significant difference between the DNA content of the
uninjured left (3.02±0.17 µg/cm) and uninjured right (3.20±0.14
µg/cm) arteries. The change in intimal thickness was determined by
calculating the difference between the DNA content in the left
(treated) and right (nontreated) carotid arteries in each rat. In a
preliminary study, it was confirmed that the difference (L-R) in DNA
content was clearly correlated with the intimal thickness area (in
square millimeters) of balloon-treated carotid arteries (Fig 1
), which was determined planimetrically, indicating
that the difference reflected the changes in intimal thickness without
a marked structural alteration, at least for the first 4 to 14 days
after surgery. On the basis of these results, we expressed the intimal
thickness as the L-R DNA content per 10-mm length of carotid
artery.
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As a measure of the proliferative activity of the carotid artery, the incorporation of BrdU into the nuclei of SMCs in the medial layer was determined. On the third day after surgery, 30 minutes after the intravenous injection of BrdU (50 mg/kg, Wako) in rats, the carotid arteries were perfused and fixed with 4% formalin under ether anesthesia. A thin ring-shaped paraffin-embedded artery section was stained with antibody to BrdU, and the ratio of positively stained cells to total cells in the medial layer was determined for each rat. The value is expressed as the percent BrdU-labeled cells (BrdU labeling index) for at least five rats.
Preparation of APS and Induction of Thrombocytopenia
Pure platelet fraction was carefully isolated from the blood
of Wistar rats and inoculated into rabbits (New Zealand White, Kitayama
Labes, Nagano, Japan). The rabbit serum was collected after four
inoculations, and the cytotoxicity was evaluated by hematological cell
counts after two injections (at an interval of 2 days) of the rabbit
serum into rats (Fig 2). The rats were bled via the tail
vein (50 µL) into an EDTA microcontainer (No. 5961, Becton
Dickinson); the number of cells was measured with a hemocytometer
(Sysmex, Toa Medical Co, Ltd), and the white blood cell population was
determined by microscopy. The two consecutive injections of APS
resulted in a severe thrombocytopenia (30 000±4000/µL the first day
and 26 000±7000/µL the fourth day after injection) that persisted
for at least 4 days (Fig 2) without a marked change in white blood cell
count. Thus, the white blood cell count was not altered by the APS
treatment at least during the first 4 days. There was also no
significant difference between the differential cell counts in the
normal and the APS-treated rats; the respective values were as follows:
neutrophils, 13.3±2.3% and 15.5±2.4%; lymphocytes, 83.3±2.7% and
82.2±2.6%; monocytes, 2.8±0.8% and 2.3±0.3%; and eosinophils,
1.8±0.5% and 0.8±0.3%. Surgical operation was performed after the
confirmation of severe thrombocytopenia. The plasma level of
thromboxane B2 (EIA Kit, TiterZyme-TXB2,
PerSpective Diagnosis) was below the detection limit (<10 ng/mL) in
the APS-treated rats at day 2. The results confirmed that highly
selective platelet depletion was induced by this method.
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Morphological Observations and Measurement of Intimal Surface
Reendothelialization
The morphology of the luminal surface of the carotid artery and
of the platelets was observed, after balloon denudation, by
scanning electron microscopy (Hitachi, S-450). The rats were
anesthetized and treated with balloon denudation once with or
without stenosis induction, and immediately after the
treatment, the arteries were perfused and fixed with 2.5%
glutaraldehyde solution at 100 mm Hg for a few minutes
and processed in the usual manner. The percent intimal surface
reendothelialization was evaluated in arteries treated
with ballooning to induce mild denudation with and without
stenosis. Evans blue dye in PBS (80 mg/kg) was infused
intravenously 30 minutes before fixation to aid in
distinguishing denuded (blue) regions from
reendothelialized (white) regions. The percentage of
white region to the total intimal surface area was measured and
calculated by computer-assisted morphometry (LA-500, PIAS).
Measurement of Blood Flow and Luminal Diameter of Stenosed
Arteries
The blood flow of the rat carotid artery was measured with a
pulsed-Doppler flowmeter. The rats were anesthetized under
pentobarbital, and velocity recordings were obtained with a
20-MHz pulse-Doppler device (model VF-1, flow probe with 1.0-mm
lumen diameter, Crystal Biotech). Doppler-shifted signals were
recorded on an instrument recorder (RECTI-HORIZ-8K, Sanei). The
frequency shifts were converted to volume flow data by using the
Doppler equation. Luminal stenosis was obtained in the same
manner as described above. A suture was placed on the dorsal surface of
the vessel downstream from the flow probe. The degree of
stenosis was regulated in accordance with the diameter of the
steel wire (range, 0.75 to 0.30 mm). The luminal diameter of the
stenosed portion of the carotid artery was determined by measuring the
thickness of a plastic cast of the artery. The rats were
anesthetized, and the treated arteries were perfused with resin
(Mercox CL-2B-5, Dai-nihon Ink Co, Ltd) at a pressure of 100 mm Hg,
and the fixed resin was isolated from the surrounding tissues by
immersion in 10N NaOH. The diameter of the normal and constricted
points was measured under the microscope with a micrometer,
and the luminal stenosed area was calculated. The following relation
was observed between the wire diameter and the degree of
stenosis: a diameter of 0.75 mm corresponded to 50%
stenosis; 0.60 mm, to 75% stenosis; 0.45 mm, to 91%
stenosis; and 0.35 mm, to 96% stenosis.
Data Analysis
All data are expressed as mean±SEM. Each statistical
analysis sequence was conducted by using the SAS package. The
group means of blood flow and thrombus formation in arteries with
various degrees of stenosis were assessed by one- or two-way
ANOVA, and multiple comparisons were performed by Dunnett's test. The
correlation coefficients for the linear regressions of the data
acquired in the preliminary study were analyzed, and the slopes
were compared. All other data were analyzed by Student's
t test when the groups showed equal variances (F test) or by
Welch's test when they showed unequal variances (F test). A
significant difference was accepted at a value of
P<.05.
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Results |
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Relation Between Blood Flow and Thrombus Formation According to Degree of Stenosis
The mean blood flow volume decreased with increasing percent stenosis of the carotid arteries (Fig 3
). It was
significantly reduced at 96% stenosis but was not markedly
altered at <75% stenosis. The thrombus wet weight (L-R) 1
hour after surgery increased with increasing percent stenosis,
and it was markedly increased at >75% stenosis. The wet
weight of thrombi that developed downstream from the stenosis
(distal region) reached its maximum value at 91% to 96%
stenosis, and the percentage of distal thrombus weight relative
to the total weight was increased with decreasing percent
stenosis. At 96% stenosis, the combination with mild
denudation resulted in greater thrombus formation compared with severe
denudation (Table) and also compared with the nondenuded
arteries (data not shown). Although the thrombus weight in the carotid
artery increased rapidly during the first hour after denudation and
then continued to increase for 6 hours, the minimum period for
production of stable and occlusive thrombus was 1 hour.
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Effect of Thrombocytopenia on Thrombus Formation With and
Without Stenosis
In the presence of stenosis, the thrombus wet weight at 1
hour was significantly reduced in the thrombocytopenic rats (Fig 4). In the absence of stenosis, no difference
between the thrombus weights in arteries with and without APS
pretreatment was seen.
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Effect of Stenosis on the Morphology of Platelets and
Thrombi on the Intimal Surface
In the stenosed arteries, numerous mural microthrombi were
scattered on the subendothelial surface of the artery
in the region distal to the stenosis (Fig 5a).
These microthrombi sometimes grew into giant mural thrombi, including
red blood cells or a fibrin network around the platelet aggregate
core (Fig 5b). The intimal surface of nonstenosed carotid arteries was
completely covered by a platelet monolayer immediately after the
mild denudation (Fig 5c). Platelets adhered to and spread over the
subendothelium of the denuded surface (Fig 5d).
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Course of Change of Intimal Thickness in Stenosed
Artery
The course of change of intimal thickness (DNA content) in the
arteries with mild denudation (Fig 6a) was markedly
different from that in the arteries with severe denudation (Fig 6b). At
2 weeks, in the absence of stenosis, only a slight increase in
intimal thickness was observed in the arteries with mild denudation,
whereas a marked increase was observed in arteries with severe
denudation. In the presence of 50% stenosis, the increase of
intimal thickness was significantly enhanced in arteries with both mild
and severe denudation, particularly in the early stage (by 6 days). The
magnitude of growth was rapidly curtailed and almost plateaued after 6
days in the mildly denuded arteries, whereas it continued to increase
until day 14 in the severely denuded arteries. A slight increase in DNA
content was found in nondenuded arteries treated with stenosis
alone (data not shown).
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Effect of Thrombocytopenia on Proliferation of Medial SMCs in
Stenosed Arteries
The BrdU labeling index of SMCs of the medial layer was
significantly higher than that of mildly denuded nonstenosed arteries 3
days after surgery of mildly denuded arteries with stenosis
(Fig 7). This increased labeling index was significantly
reduced in the thrombocytopenic rats. The BrdU labeling index of
nonstenosed arteries with severe denudation was higher than that of
nonstenosed arteries with mild denudation. Thrombocytopenia did not
affect the BrdU labeling index in the nonstenosed arteries with severe
denudation.
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Reendothelialization of Denuded Arteries With
Stenosis
Complete reendothelialization was observed in
stenosed arteries by 7 to 10 days, whereas the percent
reendothelialization in nonstenosed arteries plateaued
at 60% (Fig 8). It was significantly different from
that of stenosed arteries at day 14. The slope of the change in the
percentage of the intimal surface showing
reendothelialization was not significantly altered by
the stenosis in mildly denuded arteries.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most important finding of the present study is that both acute occlusive thrombus and chronic intimal thickening could be induced in the corresponding position in the rat carotid arteries by only a slight change in the degree of stenosis. What is common to both phenomena is the disturbance of the bloodstream by the stenosis. How the stenosis affects the bloodstream is discussed below with an analysis of acute thrombus formation, together with a discussion of why intimal wall thickening is observed in the abnormal blood flow in this model.
Occlusive Thrombus Formation and Hemodynamic
Alteration by Stenosis
We estimated the accumulated thrombus formation 1 hour after
surgery by measuring the occlusive thrombus weight. Since it is
recognized that an arterial thrombus formation is produced
as the result of dynamic imbalance of various factors, it is essential
to understand the course of events during the process of thrombus
formation. Folts13 demonstrated that the stenosis
was closely related to the induction of dynamic thrombus formation by
detecting cyclic flow variation in canine coronary arteries.
The essential finding of the present study corresponds to the
finding by Folts, since the conditions applied are the same, except for
the kind of artery and the animal species. We suggest that the process
of thrombus formation is related to the amount of accumulated thrombus,
because we observed a good correlation between thrombus weight and
degree of stenosis. The reliability of the analysis of
the process of thrombus formation in our model is under investigation.
The acquisition of findings for a reproducible parameter
(thrombus wet weight) is a first step in a reliable analysis.
To clarify how thrombus formation occurs near the stenosis in
our model, we determined the thrombus weight separately in the regions
distal and proximal to the stenosis. We demonstrated that the
thrombus weight in the distal region, as a percentage of the total
thrombus weight, increased with decreasing stenosis. This
finding suggests that thrombus formation is initiated in the distal
region and that the formation of occlusive thrombi near the
stenosis (including proximal thrombus) is a secondary phase in
this process. Thus, it can be recognized that there are conditions
triggering thrombus formation in the distal region in the mild
stenosis range, even though there is little occlusive thrombus
formation. The electron microscopic finding of numerous microthrombi
near the surface of the stenosed artery also provides support for this
concept.
We found the maximum thrombus formation in the distal region at 91% to 96% stenosis. Because this is the threshold condition that affects the total blood flow volume, the shear rate at the apex of the stenosis site is at its maximum under this stenosis condition. Thus, it is suggested that blood cells are exposed to the maximum shear during their passage through the stenosis site under this condition,14 15 and the platelets are among the major candidates for activation by the shear.16 This is supported by the complete reduction of thrombus formation observed in thrombocytopenia. Although the rheological features downstream from the stenosis site are not simple, flow disturbance consistent with recirculation has recently been shown to exist downstream from the stenosis.17 Several studies18 19 have demonstrated in vitro that the reattachment point of the bloodstream with the distal recirculation zone is the peak site at which platelet adhesion is seen.
We conclude that the stenosis of the artery produced an alteration of shear stress between the circulating platelets and the denuded surface of the artery, followed by the activation of the platelets themselves and their adhesion to the surface. This rat carotid artery model could be proposed as an arterial thrombosis model, because a reproducible and occlusive thrombus formation in a short period of time was observed, especially in the setting of more severe stenosis.
Intimal Thickening by Stenosis
Significant intimal thickening was observed in the mildly denuded
arteries when the mild stenosis condition was maintained for 2
weeks. As was suggested above, the introduction of stenosis in
the arteries enhances the relation of platelets to the injured
arteries under the influence of the hemodynamic
alterations, and we will attempt to apply this concept in relation to
findings for intimal thickening in the stenosed arteries as well.
Difference Between Profiles of Arteries With Mild and Severe
Denudation
In balloon-injured arteries without stenosis, which have
been extensively studied by many investigators, it has been recognized
that the intimal thickening is the result of migration of SMCs from the
medial layer by the mitogenic stimulation of platelet
factors.6 On the other hand, there is another view of this
higher proliferation seen in ballooned vessels, in which the mechanical
traumatization by ballooning of medial cells, not only the platelet
factors, is a causative factor of intimal proliferation.7
This suggestion has important implications for the present study,
since it is a possible explanation for our findings of different
profiles in intimal thickening for arteries with mild and with severe
ballooning. We developed a technique by which mild denudation was
obtained by complete removal of endothelial cells from
the luminal surface of the rat carotid artery with single inflation of
the balloon catheter with a small volume (<75 µL). Since the luminal
diameter of the carotid artery in 6-week-old rats is 
1 mm, we could
obtain complete denudation without overinflation of the balloon
catheter. Complete denudation was confirmed on day 0. The intimal
lesion formation in the arteries treated with mild denudation was
markedly different from that in arteries with severe denudation (with
denudation repeated five times). The former arteries also showed a
significantly lower BrdU index (4%) compared with the latter (10%).
Fingerle et al6 reported that the thymidine index of
arteries treated with a loop of monofilament suture never exceeded 2%.
This evidence suggests that our mild denudation technique is clearly
distinguishable from that of severe repeat ballooning in terms of the
minimal medial damage, and this idea is also supported by the
observations of Richardson et al.20
Relation of Platelets to Intimal Thickness in Stenosed
Arteries
The introduction of stenosis in mildly denuded arteries
significantly enhanced the intimal thickening. The enhancement of the
BrdU index in the stenosed artery was significantly reduced in the
thrombocytopenic rats. These findings suggest the role of platelets
as a potent stimulus in the medial layer of the artery under the
stenosed condition. On the other hand, the BrdU index of arteries with
severe denudation was not affected by thrombocytopenia, and this
finding corresponds to that of Fingerle et al.6 It is
suggested that the severely denuded medial cells are left with no
further capacity to respond to the platelet stimuli because they
are already maximally stimulated by the mechanical traumatization of
the repeated balloon denudations. In this view, mild denudation is an
essential condition in eliciting the effect of the stenosis on
intimal growth. Holycross et al21 reported that PDGF-BB
may play a generalized role in the modulation of the SMC
phenotype to a less differentiated state. It is suggested that
the medial SMCs in the stenosed artery are capable of proliferation or
migration to the intima when they are exposed to the platelet
stimuli. The stenosis is one of the factors that can elicit an
extremely active role of platelets in the denuded arteries. The
scanning electron microscopic findings are indicative that there are
numerous mural microthrombi on the surface downstream from the stenosed
artery.
Relation of Reendothelialization to Intimal Wall
Thickness in Stenosed Artery
Intimal growth in mildly denuded arteries was rapidly stimulated
by 6 days after surgery, and it plateaued thereafter. This finding
correlates well with the finding that endothelial
regeneration was completed by 7 to 10 days in stenosed arteries, and
many reports refer to this phenomenon.22 23 24 Our findings
clearly demonstrate that the reendothelialization plays
a key role in the regulation of intimal thickening. The period of
exposure of the subendothelium to blood platelets
is an important factor in regulation of the development of intimal
lesions. The marked platelet adherence (accumulation) in the
stenosed artery suggests that platelets play a role in the increase
of the intimal wall thickness by the effective exposure of the
arterial wall to the mitogenic factors. It has
recently come to be accepted that intimal thickness formation results
from the migration of SMCs induced by PDGF.25 The
successive phases of SMC modulation in the stenosed artery, such as
differentiation, proliferation, and migration, suggest that the
platelet stimuli have multiple forms of action.
In conclusion, it was demonstrated that mildly denuded rat carotid arteries with stenosis show a markedly different proliferative and thrombogenic response profile compared with arteries with severe denudation. This finding suggests that arteriosclerotic plaque formation, together with secondary plaque (intimal thickness) formation under the influence of hemodynamic alterations, is a critical event leading to thrombus formation and that platelets strongly contribute to the induction of this vicious cycle. It is also suggested that regeneration of the endothelium could play an important role in regulating neointimal thickness formation in stenosed arteries. The contribution of hemodynamic factors in such a situation as that advocated in the "response to injury hypothesis"4 requires reconsideration.
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Selected Abbreviations and Acronyms |
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Received August 10, 1994; accepted April 12, 1995.
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