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© 1997 American Heart Association, Inc.
Articles |
Fibrin(ogen) and von Willebrand Factor Deposition Are Associated With Intimal Thickening After Balloon Angioplasty of the Rabbit Carotid Artery
From the Departments of Cardiology (J.M.B., C.J.V.) and Pharmacology (H.B., G.R.Y. De M., A.G.H.), University of Antwerp, Wilrijk, and the Department of Pathology, AZ Middelheim, Antwerp (M.M.K.), Belgium.
Correspondence to Johan Bosmans, University Hospital Antwerp (UZA), Department of Cardiology, Wilrijkstraat 10, 2650 Edegem, Belgium.
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Abstract |
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Abstract The aim of the study was to assess the contribution of thrombus incorporation into neointimal thickening in the rabbit carotid artery after deep vascular injury induced by balloon angioplasty compared with superficial vascular injury induced by a perivascular collar. Besides CD 31 (PECAM I), vimentin,
-smooth
muscle actin, rabbit anti-macrophage monoclonal antibody and
proliferating cell nuclear antigen, fibrin(ogen) and von
Willebrand factor (vWF) deposition was assessed
immunohistochemically. Angioplasty was performed in 47 rabbits and
evaluated immediately (n=7), after 6 hours (n=4), and after 1 (n=7), 2
(n=9), or 3 (n=20) weeks. A collar was placed in 29 rabbits and
evaluated immediately (n=5), after 6 hours (n=5), and after 1 (n=7), 2
(n=10), or 3 (n=2) weeks. After dilatation, the arteries were
extensively denuded of endothelium, the internal
elastic membrane was ruptured and blood-filled clefts were present
in the media, pointing to deep vascular (type III) injury. Six hours
later, mural fibrin(ogen) thrombi were formed, specially at sites with
severe damage. This fibrin(ogen) matrix became infiltrated by
phagocytes and smooth muscle cells. A luminal cap covered by
regenerating endothelium was formed, demonstrating
increased immunoreactivity to vWF. vWF was deposited in the
extracellular neointimal spaces. Fibrin(ogen) thrombus
deposition and incorporation appeared to be protracted phenomena for at
least 2 weeks. After collar placement, minimal
endothelial denudation was documented, pointing to
focal superficial (type I) vascular injury. In subsequent weeks,
neointimal thickening was associated with vWF deposition
but not with fibrin(ogen) thrombus incorporation. In conclusion, mural
fibrin(ogen) thrombus formation and incorporation contribute to
neointima formation after deep vascular injury and seem to
occur for several weeks after the initial insult.
Key Words: angioplasty • collar • neointima • fibrin(ogen) • restenosis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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PTCA has found widespread application in the treatment of different subsets of ischemic heart disease since its introduction in 1979 by Grüentzig et al.1 Although the immediate success rate of both balloon dilatation and other newly introduced interventional techniques has increased to more than 95%, restenosis during early follow-up remains a major limitation of coronary angioplasty. Clinical and quantitative angiographic follow-up studies have demonstrated that within 4 to 6 months of successful balloon dilatation,
30% of patients develop significant
coronary artery restenosis.2 3 A wide
variety of pharmacological therapies has been tested and various new
technologies have been introduced, but no effective solution has yet
been found for this important clinical problem.4 5 6 Although the precise mechanism remains unclear, restenosis is thought to result from excessive myointimal proliferation.7 8 9 Medial SMCs respond to dilatation injury by migration, proliferation, and extracellular matrix synthesis. The myointimal growth process has been attributed to release of chemoattractants and growth factors released by activated platelets that adhere to the site of injury of the vessel wall10 11 12 13 14 and cytokines released by other cells.7 8 Myointimal proliferation leads to the formation of a space-occupying neointima that progressively obstructs the arterial lumen, thereby leading to restenosis. Inhibition of platelet aggregation by aspirin,15 16 dipyridamole,16 ticlopidine,17 or anti-glycoprotein IIb/IIIa antibodies18 has reduced neointima formation in animal models. Nevertheless, the role of platelets as the primary "trigger" of restenosis after balloon angioplasty remains controversial, since all platelet-inhibitor intervention studies have failed to prevent human restenosis.19 20
Concepts about the pathogenesis of restenosis after angioplasty were mainly derived from animal models, in which endothelial denudation was the major stimulus of platelet aggregation and neointimal proliferation.10 11 12 13 14 21 22 Endothelial denudation in most of those models was obtained by passing a Fogarty balloon through the artery, a technique that creates a superficial (type I) vascular wall injury.8 10 13 14 Balloon dilatation in the clinical setting, however, frequently induces a more severe and deeper (type III) lesion in the vascular wall23 that also may lead to important mural thrombus deposition.
Therefore, the aim of the study was to analyze the time course
and contribution of thrombus incorporation to intimal thickening
induced by balloon angioplasty, which induces deep vascular (type III)
injury, compared with the focal superficial injury induced by a
perivascular collar,24 25 26 which fulfills the
criteria8 of a type I injury. In view of this aim,
anticoagulant therapies were omitted. The rabbit carotid artery was
used as a model, and thrombi were evaluated by light microscopy and
specific immunohistochemistry for fibrin(ogen). Other vascular
components were identified by specific stains for -SM actin, vWF,
platelet-EC adhesion molecule 1 (CD 31, or PECAM I), a rabbit
anti-macrophage monoclonal antibody (RAM 11), vimentin, and
PCNA.
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Methods |
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Experimental Protocol
Male New Zealand White rabbits (2.5 to 3 kg, n=81) fed a normal laboratory diet without cholesterol supplementation were anesthetized with sodium pentobarbital (30 mg/kg body weight IV). The rabbits were posed in a supine position and the ventral neck region infiltrated with xylocaine for local anesthesia. Both carotid arteries were surgically exposed and dissected from the surrounding tissues.
Angioplasty
For angioplasty, direct arteriotomy was performed in the right
common carotid artery just proximal to the bifurcation. A standard
2-cm-long, 2.5-mm PTCA balloon dilatation catheter (Advanced
Cardiovascular Systems) was introduced over a 0.014-in.
floppy guide wire and advanced toward the aorta into the carotid artery
to 5 cm from the incision. The balloon was inflated three times with
an inflation pressure of 6 atm for a period of 2 minutes each. Between
subsequent inflations the deflated balloon was left in the artery for 1
minute. After removal of the catheter, the small distal incision was
surgically closed and arterial blood flow
restored.
Histological evaluation of the angioplastied region (2 cm) was performed immediately afterward without restoration of arterial blood flow (n=7) and after 6 hours (n=4) and 1 (n=7), 2 (n=9), or 3 (n=20) weeks. To this end and to prevent postmortem thrombus formation, the rabbits were anticoagulated with heparin (150 U/kg IV) and anesthetized with sodium pentobarbital (30 mg/kg body weight IV). The angioplastied region in the right carotid artery and the equivalent normal control region in the left carotid artery were surgically removed and immediately immersed in methacarn fixative (60% methanol; 30% 1,1,1-trichloroethane; and 10% glacial acetic acid). To preserve fresh mural thrombi, the arteries were not flushed with saline. Perfusion fixation was not used. Afterward the rabbits were killed with an IV overdose of pentobarbital.
In 5 rabbits a sham operation was performed. After induction of intravenous anesthesia and dissection of both carotid arteries, direct arteriotomy was performed in the right carotid artery. Eight minutes later the incision was closed again without inserting a balloon catheter. Histological evaluation was performed after 2 weeks. All surgical procedures conformed to the guidelines detailed in the Position of the American Heart Association on Research Animal Use and approved by the institutional animal use committee.
Collar
For focal, superficial injury induced–intimal thickening, a
nonocclusive, biologically inert, soft, flexible silicone collar was
placed around the middle part of the left common carotid artery and
closed with silicone glue. The right common carotid artery underwent
sham operation; ie, it was separated from the surrounding connective
tissue and vagus nerve and received a stretch similar to that applied
to the contralateral collared artery.24 25 26
Histological evaluation was also performed immediately
(n=5) and after 6 hours (n=5) and 1 (n=7), 2 (n=10), or 3 (n=2)
weeks.
Immunohistochemistry
For light microscopy, the arteries were fixed in methacarn.
Afterward the segments were cut to a length of 4 mm. At least 4
segments per artery were paraffin embedded. Transverse sections were
stained with Sirius red–hematoxylin and immunohistochemistry was
performed. The reactions were performed with the indirect peroxidase
antibody conjugate technique. The following monoclonal antibodies were
used: -SM actin (Sigma Chemical Co, 1/3000 dilution), PCNA (PC 10
cyclin, 1/10 dilution), vimentin (1/200 dilution), rabbit
anti-macrophage (RAM 11, 1/100 dilution), and CD 31 (1/10
dilution; all from Dako). The sections were preincubated with BSA to
omit specific binding of the primary antibody. The monoclonal
antibodies were diluted in PBS. After three washes in PBS, the sections
were incubated with rabbit anti-mouse peroxidase (The Jackson
Laboratory) for 45 minutes. For demonstration of the complex,
3-amino-9-ethylcarbazole was used as the chromogen. The specificity of
these antibodies was demonstrated in previous
studies.24 25 26
The following polyclonal antibodies (raised in sheep) were used: vWF (1/250, Binding Site) and rabbit fibrin(ogen) (1/10 000 dilution, Cappel). The specificity of the primary antibody against vWF was determined previously.25 The specificity of the primary antibody against fibrin(ogen) was tested by the staining pattern of prepared fibrin clots of rabbit plasma. The clots showed dense, threadlike, immunoreactive conglomerates that we considered to be fibrin. However, the antibody was not specific for fibrin because preabsorption of the antibody with fibrinogen had completely abolished immunoreactivity within the clots. This observation indicated that the antibody recognized both fibrin and fibrinogen. The term fibrin(ogen) is therefore used to indicate that the antibody does not distinguish fibrin from fibrinogen. The polyclonal antibodies were visualized by pig anti-sheep peroxidase as the secondary antibody (Binding Site), with 3-amino-9-ethylcarbazole as the chromogen or donkey anti-sheep fluorescein (The Jackson Laboratory) for double immunofluorescence. For double immunofluorescence both primary antibodies were used in parallel. For negative controls the primary antibody was omitted.
Quantification of Areas of vWF and Fibrin(ogen)
Accumulation
The presence of fibrin(ogen) and vWF in the
neointima or media was analyzed by a color image
analyzing system (PC-image Colour, Foster Findlay Associates).
Quantification was performed for at least 4 segments from each artery.
Only for those arteries harvested immediately after angioplasty or
collar placement was analysis not performed. The
arterial wall of each segment was divided into four
quadrants. In each quadrant the regions between the luminal margin and
the IEL (neointima) and between the IEL and the EEL (media)
were demarcated. The lengths of the IEL and EEL, the areas of
neointima and media, and the vWF- and
fibrin(ogen)-immunoreactive areas within both regions were measured.
Segmentation was performed by measuring the average brown color of 10
different points within the immunoreactive areas. Percentages of vWF-
and fibrin(ogen)-immunoreactive areas within the neointima
and media were calculated.
Quantification of Intimal and Medial Thickening
The -SM actin–stained sections were used to count the
maximum number of SMC layers in the media and neointima
(between the IEL and the luminal margin) in at least 4 segments from
each artery. The mean of the maxima of these 4 segments was
calculated.
Quadrant Injury Score
For each quadrant of arterial segment, a quadrant
injury score was calculated (minimum of 0, maximum of 3), as defined by
the sum of the presence (+1) or absence (0) of
endothelium, IEL ruptures, and clefts in the media.
Since all arteries were immersion fixed at ambient atmospheric
pressure, the IEL always has a regular undulating aspect in normal
rabbit carotid arteries. In contrast to those in human arteries, pores
are normally small in the IEL of rabbit carotid
arteries,27 28 29 and ruptures were defined as large
irregularities in the IEL, which sometimes appeared as calcified
discontinuities in regions of severe injury.
Presence of ECs
At least 4 segments from each artery were examined.
Lumen-lining, nucleated cells that were immunoreactive for vimentin,
demonstrated membrane-associated immunoreactivity for CD 31 and
granular cytoplasmic immunoreactivity for vWF, and were negative for
-SM actin were considered to be ECs. Their presence was classified
visually as absent, focal, or continuous.
Presence of Thrombus
A thrombus was identified as the luminal presence of amorphous
material consisting of fibrin(ogen), RBCs, and platelets. The
thrombus was regarded as organized if it had been infiltrated by
cellular elements, such as SMCs and macrophages (RAM
11–positive cells), or if it had been covered by ECs. Thrombi could be
occlusive or nonocclusive. Luminal threadlike, dense,
fibrin(ogen)-immunoreactive conglomerates were considered to be fibrin.
Platelets were defined as CD 31–positive, vWF-positive,
vimentin-negative, nonnucleated, nonendothelial
elements. The number of SMCs and PCNA-positive cells within each
thrombus was counted and the thrombus area measured in at least 4
segments from each artery. The mean of the 4 segments was calculated
and expressed as number per square millimeter.
Statistical Analysis
All data are expressed as mean±SEM; n refers to the number of
rabbits. The percentage of vWF- and fibrin(ogen)-immunoreactive area
within the neointimal or medial region of interest and the
extent of neointimal thickening of the collared and
dilatated arteries were compared by the nonparametric
Mann-Whitney U test. The SPSS for WINDOWS package (SPSS) was used for these purposes. A 5% level of
significance was selected.
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Results |
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Sham-Operated Arteries
Sham-operated arteries from both collared and dilatated animals were evaluated after 2 weeks. At this time an intact EC layer was present, resting directly on the intact IEL. The ECs demonstrated membrane-associated immunoreactivity for CD 31 and granular cytoplasmic immunoreactivity for vWF. Because fixation had been performed under ambient atmospheric pressure, the IEL always displayed a regular, continuous, undulating aspect in which pores were not visible. Mural fibrin(ogen) thrombi were never present. Subendothelial
-SM actin–positive cells were
absent. In the media, defects were not seen and fibrin(ogen) deposition
was absent. In every aspect these sham-operated segments were
indistinguishable from naive, nonmanipulated rabbit carotid arteries
(results not shown).
Angioplastied Arteries
Numerical results are summarized in Tables 1
and 2.
Immediately after angioplasty (Fig 1),
marked segmental loss of SMCs and deep, circular medial tears were
always observed (7/7). Although ruptures of the IEL were visible in
only 2 of 7 arteries (complete loss in one and ruptures in 3/4
quadrants in the other), focal, flattened distension of the IEL pointed
to injury in all segments (Fig 1). This distension, or focal loss of
the undulating aspect, was never present in the contralateral
control arteries. All arteries with remnants of an IEL(6/7) were
partially covered by scarce "islands" of remaining ECs (Fig 3b),
which were identified as such by the presence of nuclei and
immunoreactivity for vimentin, vWF, and CD 31 and the absence of
immunoreactivity for -SM actin.
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Arteries With Occlusive Thrombi After Angioplasty
In 30% (n=12/40) of dilatated arteries an occlusive thrombus was
observed. The 7 rabbits that were evaluated immediately after
angioplasty were not included in this series because in this group,
arterial blood flow had not been restored after dilatation.
The injury of the IEL in arteries with occlusive thrombi (ruptures in
2.2±0.5 quadrants) was significantly more severe (P<.001,
Mann-Whitney U test) than in arteries with nonocclusive
thrombi (ruptures in 0.2±0.1 quadrant).
Six hours after dilatation, 1 of 4 examined arteries was completely occluded by a very loose luminal thrombus consisting of fibrin(ogen) and RBCs on top of a severely damaged IEL and media, also containing fibrin(ogen) deposits.
After 1 week, 2 of 7 dilatated arteries were occluded by a loose
luminal thrombus, again consisting of fibrin(ogen) and RBCs. There was
a marked influx of RAM 11–immunoreactive macrophages and a few
-SM actin–positive SMCs in the thrombus in both cases.
Two weeks after dilatation, 3 of 9 arteries showed occlusive fibrin(ogen) thrombi with more pronounced infiltration by macrophages (RAM 11–positive cells) and SMCs. Severe damage to the media with marked circular tears and fibrin(ogen) deposits was still visible. The intima remained unchanged: neither intimal SMCs nor ECs were observed.
After 3 weeks (Fig 2), occlusive fibrin(ogen) thrombi
(6/20) were colonized further by numerous macrophages and many
SMCs (Fig 2a), and collagen deposition had started. Most of the RAM
11–positive macrophages at this time were loaded with iron or
hemosiderin (Fig 2b), probably due to phagocytosis of
RBCs. Clusters of foam cells and multinucleated giant cells (Fig 2c)
were sometimes seen. Neovascularization of the thrombus by several
capillaries was also observed (Fig 2b). Intimal thickening was not
present. Damage of the media with circular clefts and fibrin(ogen)
deposits was still always visible.
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Arteries With Nonocclusive Thrombi After Angioplasty
Seventy percent (n=28/40) of arteries remained patent after
balloon dilatation. Six hours after dilatation (Fig 3a
)
thin, mural thrombi consisting of dense, threadlike conglomerates of
fibrin(ogen) and RBCs were observed in all preparations (3/3), in close
contact with sites of severe, deep injury of the media. In these 3
arteries (12 segments, 48 quadrants), 5 mural thrombi were recognized.
The mean injury score of the quadrants covered by thrombi was
significantly higher than that of quadrants not covered by thrombi
(2.6±0.2 versus 1.3±0.3; P<.001, Mann-Whitney
U test). Fibrin(ogen) deposits (Fig 3a) and numerous RBCs
(Fig 1) were observed in deep medial clefts. Only scarce CD
31–positive, vWF-positive, vimentin-negative, nonnucleated
platelets were observed in 1 of 3 specimens. The ruptured and
extended internal elastic membrane was partially covered by islands of
remaining ECs (Fig 3b and Table 2).
One week after dilatation, in addition to a modest circular intimal
thickening (on average, two SMC layers; Table 1), mural thrombi were
present in all 5 arteries (Fig 3c) and again mainly consisted of
fibrin(ogen). This fibrin(ogen) matrix was infiltrated by several
-SM actin–positive cells (Fig 3c). Most of these SMCs had become
oriented longitudinally, although in more bulging thrombus areas the
organization pattern remained rather random. RAM 11–immunoreactive
macrophages were present in all thrombi and in areas of
medial injury. Fibrin(ogen) deposits could still be recognized in
medial regions of injury. At this time, CD 31– and vWF-positive ECs
were present in all specimens, predominantly in a focal way (4/5;
Table 2).
Two weeks after dilatation (Fig 4), a circular,
asymmetric intimal thickening with an average of five SMC layers (Table 2) was observed in all 6 arteries. This thickened layer consisted of
radially oriented SMCs above the IEL and longitudinally oriented cells
on the luminal side (Fig 4d). The ratio of intimal area to IEL length
was 28±6 µm. In bulging intimal regions clearly due to
incorporation of organizing mural fibrin(ogen) thrombi, the SMC
orientation pattern was rather random (Fig 4d). RAM 11–immunoreactive
macrophages were again detected predominantly in the organizing
thrombi and the remaining areas of medial injury. Throughout the entire
intima a striking fibrin(ogen) immunoreactivity was present,
sometimes most pronounced in the deeper parts in close contact with the
internal elastic membrane (Fig 3f). In all arteries, the intima had
become lined with ECs, which had already formed a continuous layer in
most areas (Figs 3e and 4e). A relatively high fraction of EC nuclei
stained positively for PCNA, suggestive of intense proliferative
activity (Fig 4b). The ECs showed dense, flocculent immunoreactivity
for vWF, and vWF-immunoreactive material was found beneath the
endothelium as well (Fig 3d). The latter material was
found to be extracellular, as demonstrated by double
immunofluorescence for -SM actin and vWF
(results not shown). The depth of subendothelial
accumulation varied. The media was nearly completely repaired. Some
scarce medial fibrin(ogen) deposits could still be detected (Fig 3f).
In most arteries (5/6), the mural thrombi, which had become
incorporated into the intima, demonstrated an unequal degree of
organization (Fig 4a). Some had been completely organized by SMCs and
macrophages and showed incipient neovascularization. Others
appeared to have formed only recently with a loose and amorphous
aspect, in which infiltration and organization had just started. PCNA
immunohistochemistry (Fig 4b) demonstrated that the cells in these
"younger"-looking thrombi showed a high cell replication rate,
whereas the media and older-looking thrombi in the same specimen did
not show PCNA-reactive nuclei, indicating a lower replication rate. At
this time the media appeared to be almost completely repaired, and the
number of SMC layers was clearly increased (on average, 13 SMC layers;
Table 2).
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Three weeks after dilatation a prominent, circular, frequently
eccentric intimal thickening had formed (14/14). In these eccentric
intimal areas, almost completely organized mural thrombi could be
recognized with significant collagen deposition, numerous SMCs with
rather random orientation pattern, some macrophages, and in
most cases (10/14) pronounced neovascularization with many small
capillaries. An unequal degree of thrombus organization was sometimes
still distinguishable (2/14). Again, striking fibrin(ogen)
immunoreactivity was observed throughout the whole intima, sometimes
still most pronounced in the deeper parts (Fig 3g). ECs were always
present, and the lining was often (10/14) continuous (Table 2). A
relatively high fraction of the EC nuclei still stained positive for
PCNA (results not shown). The ECs' dense, flocculent immunoreactivity
for vWF and extracellular vWF immunoreactive material was found beneath
the endothelium. The depth of
subendothelial accumulation varied. Some scarce medial
fibrin(ogen) deposits could still be detected (Fig 3g). The number of
SMC layers in the completely repaired media was still moderately
increased (on average, 10 SMC layers; Table 2).
Collared Arteries
Numerical results are summarized in Table 3.
Immediately after collar placement (n=5), only several small foci of EC
denudation were present.25 The IEL and the media were
without visible defect.24 25
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Six hours after collar placement (n=5), the media showed segmental
infiltration by polymorphonuclear cells and focal immunoreactivity
for fibrin(ogen) (Fig 5a). The fibrin(ogen) deposition
was most pronounced in the inner third of the media, especially in
regions with dense polymorphonuclear cell infiltration. vWF had not
been deposited in the media. In contrast to the dilatated arteries,
mural fibrin(ogen) thrombi were never observed and the intimal surface
was not covered by fibrin(ogen) deposits. However, the periadventitia
showed a slight concentric fibrinogen deposition, possibly related to
surgical manipulation.
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One week after collar placement (n=7), a small, luminal intimal thickening could be recognized consisting of a maximum of two to three layers of subendothelial SMCs. Mural fibrin(ogen) thrombi were absent, and fibrin(ogen) immunoreactivity could not be recognized in either intima or media. The EC layer was completely intact but demonstrated dense cytoplasmic immunoreactivity for vWF. Moreover, a variable diffuse immunoreactivity was found in the inner media.25
Two weeks after collar placement (n=10), a circular
subendothelial accumulation of two to five cell layers
of -SM actin–positive SMCs (Table 3) was present in all 10
rabbits. The continuous layer of CD 31–positive ECs (Fig 5d) showed a
dense flocculent immunoreactivity for vWF (Fig 5b). Also beneath the
endothelium extracellular vWF-immunoreactive material
was found. Double immunofluorescence for -SM
actin and vWF demonstrated that the subendothelial vWF
deposits lay in the extracellular space between the intimal SMCs
(results not shown). The depth of subendothelial
accumulation varied. In some regions focal immunoreactivity was found
in the inner media. Mural fibrin(ogen)-rich thrombi were absent (Fig 5c). Diffuse, intimal, fibrin(ogen)-positive material was seen in 1
specimen only, predominantly in subendothelial regions.
The media never contained fibrin(ogen). The internal elastic membrane
was intact. The ratio of intimal area to IEL length was 17±4
µm.
Three weeks after collar placement (n=2), a concentric neointima consisting of five to six cell layers was present. The medial SMCs appeared normal. Fibrin(ogen) deposition could not be demonstrated.
Quantification of the Areas of Fibrin(ogen) and vWF
Immunoreactivity
At all evaluated time points, the mean area of
fibrin(ogen)-immunoreactive material in the neointima and
media was significantly higher in dilatated arteries than in collared
arteries (Fig 6). Moreover, in dilatated arteries a
significant relation existed between quadrant injury score and the
quadrant area of fibrin(ogen)-immunoreactive material in the
neointima and media at all evaluated time points (Fig 7). However, a significant linear correlation between
quadrant area of fibrin(ogen)-immunoreactive material in the
neointima and total quadrant area of the
neointima after dilatation could not be demonstrated (1
week: r2=.078, NS; 2 weeks:
r2=.132, NS; 3 weeks:
r2=.019, NS; Fig 8).
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The mean vWF-immunoreactive area in the neointima of
collared compared with dilatated arteries was not statistically
different at all evaluated time points (Fig 9).25 No relation existed between quadrant
injury score and the quadrant area of vWF-immunoreactive material at
all evaluated time points after dilatation. A significant linear
correlation between quadrant area of vWF-immunoreactive material in the
neointima and total quadrant area of the
neointima after at different time points could not be
demonstrated (1 week: r2=.132, NS; 2 weeks:
r2=.174, NS; 3 weeks:
r2=.098, NS).
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Quantification of Mural Fibrin(ogen) Thrombus Organization
Over Time
During the first 2 weeks after angioplasty, significant
differences between the mean number of SMCs per unit area and the mean
number of PCNA-positive cells per unit area in different mural thrombi
from 1 artery could be demonstrated. From the third week on, no
quantitative regional differences could be documented (Table 4).
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Discussion |
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Choice of Models
To evaluate how the contribution of thrombus formation to intimal thickening could be influenced by the extent of injury, two completely different types of vascular injury were compared in the present study. The perivascular collar is one of the most gentle methods used to induce intimal thickening. In the early postoperative period, collared arteries demonstrate only small foci of endothelial denudation,24 25 which regenerate rapidly. However, there are some early changes in endothelial function, with impaired muscarinic receptor activity26 and increased vWF immunoreactivity.25 Moreover, experiments with radiolabeled fibrinogen point to an early increase in endothelial permeability.30 The latter finding was confirmed in the present study, with scarce subendothelial fibrin(ogen) deposition predominantly at sites of neutrophil infiltration. Because of the modest amount of morphological endothelial damage that it causes, the perivascular approach induces damage that can be classified as a focal type I vascular injury8 and is therefore related to the previously studied animal models.21 The lack of mural thrombus formation in the collar model is an additional similarity to the models of superficial vascular injury studied previously.21 On the contrary, inflation of a slightly oversized coronary angioplasty balloon catheter via a protocol that is similar to the one used during clinical coronary angioplasty created extensive vascular wall damage with intramural clefts. Since this model causes a deep type III injury, it appears to be more representative of clinical balloon angioplasty.23
Mural Fibrin(ogen) Thrombi and Intimal Thickening
After collar placement, neointimal thickening is not
associated with mural fibrin(ogen) thrombus incorporation despite the
presence of scarce early, transient medial fibrin(ogen) deposits. The
latter are possibly related to the increased permeability of the EC
layer, which is also indicated by the increased
transarterial fibrinogen flux in collared
arteries.30
Immediately after balloon dilatation the arteries were extensively denuded of endothelium, and deep intramural hemorrhages within the media, sometimes extending to the EEL, were present. Six hours later dense, threadlike conglomerates of fibrin(ogen)-immunoreactive material were present in both mural thrombi and the medial layer. The fibrin(ogen) matrix became incorporated into the neointimal thickening by extensive phagocytic invasion, infiltration by SMCs, and formation of a luminal cap covered by regenerating endothelium. This process was accompanied by neovascularization, which provides further proof for the important contribution of fibrin(ogen) thrombi.31 The neointimal fibrin(ogen) matrix could be detected as late as 3 weeks after angioplasty. A portion of this fibrin(ogen), in closer contact with the IEL, may be a remnant of the initial deposit. Another portion of this fibrin(ogen), however, may be due to increased permeability of the rapidly regenerating endothelium,30 with leakage of fibrinogen from the arterial lumen through the endothelium into the neointimal tissue and local secondary conversion to fibrin.32 33 34 35 36 37
Mural fibrin(ogen) thrombus deposition and incorporation seem to be
protracted phenomena that are not limited to the initial trauma. Mural
thrombi with different degrees of organization were frequently
demonstrated within 1 segment. Some were completely organized by SMCs
and macrophages and showed incipient neovascularization. Others
appeared to be only recently formed with a loose, amorphous aspect in
which infiltration and organization had just started. Quantitative PCNA
immunohistochemistry (Table 4) demonstrated that particularly in
younger-looking thrombi, the invading cells (SMCs and monocytes) were
actively replicating, while in the adjacent, older-looking thrombi the
cell replication rate was much lower. Although real age differences can
not be proved by our methodology, the striking differences between
adjacent thrombi within 1 segment clearly suggest differences in cell
proliferation rate and age. This continuous mural thrombosis is
possibly related to the areas with marked endothelial
denudation, which persist throughout the first 2 weeks. Afterward, when
the endothelium has almost completely regenerated, new
mural fibrin(ogen) thrombi are rarely formed. This element, which to
our knowledge is a new finding, has not been mentioned in other studies
and could have implications for the continuation of anticoagulant
therapy after clinical PTCA.
One third of these fibrin(ogen) thrombi became occlusive. As we have
demonstrated, mural fibrin(ogen) thrombi are mostly formed in quadrants
with a high injury score (Fig 7). Moreover, IEL injury in occluded
arteries was significantly more severe than that in nonoccluded
arteries. Therefore, the thrombotic process seems to be related to the
severity of the vascular insult. The occlusive thrombi can probably be
considered the experimental equivalent of the acute thrombotic
occlusions that sometimes occur after PTCA in humans. The higher
incidence of acute thrombosis in our model can be partially attributed
to the absence of any antiplatelet or anticoagulant therapy before,
during, or after balloon dilatation.
On the basis of findings for the perivascular collar and balloon
angioplasty models, we have demonstrated that intimal thickening can be
considered a general reaction pattern induced by vascular injury but is
produced by different mechanisms depending on the type or severity of
injury. Fibrin(ogen) accumulation is certainly not an essential element
for intimal induction, but it appears to contribute to
neointimal thickening after type III injury. However, no
clear linear mathematical correlation was found between intimal
thickening and fibrin(ogen) immunoreactivity; this lack is presumably
due to fibrinolysis. Indeed, the amount of fibrin(ogen)
immunoreactivity decreased in both the intima and media as time
progressed (Fig 6). To prove the contribution of fibrin(ogen)
deposition to restenosis more conclusively, further experiments
are necessary in which type III injury is combined with either the
blockade of fibrin and/or thrombin formation or the stimulation of
fibrinolysis. On the basis of results presented
herein, studies based on type I injury, like the frequently used
Fogarty balloon denudation model, can give misleading information with
respect to PTCA in the clinical situation.
Intimal Thickening and vWF
The increased flocculent immunoreactivity for vWF in regenerating
ECs after dilatation and the presence of vWF in the
subendothelial extracellular spaces parallel the
findings from the collar model.25 In normal arteries ECs
secrete vWF into the plasma and toward the
subendothelial space, where minimal deposition of this
glycoadhesive protein is present in the basal
lamina.38 Some authors attribute an important function in
EC adhesion to the basal lamina vWF.39 40 41 In cell culture
studies it has been demonstrated that ECs can be stimulated to release
additional large amounts of vWF under conditions of
stress.40 The effect of the increased
subendothelial vWF deposition on neointimal
formation remains speculative but might by itself contribute to SMC
migration and neointimal formation.25
Conclusions
In conclusion, in the rabbit carotid artery, balloon dilatation
created a type III injury of the vascular wall, resulting in the
formation of mural fibrin(ogen) thrombi that became progressively
incorporated into the intima. The thrombotic process was related to the
severity of the injury and appeared to proceed for weeks. On the
contrary, intimal thickening after collar placement was not associated
with fibrin(ogen) accumulation. Although vascular organization of
thrombi has been known for years, the present study suggests that
mural thrombus deposition and its incorporation into the intima play a
significant role in restenosis after coronary
angioplasty. Long-term prevention of continuing mural fibrin(ogen)
thrombus deposition may therefore offer an effective therapeutic
strategy for the prevention of restenosis.
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Selected Abbreviations and Acronyms |
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Received May 15, 1995; accepted May 23, 1996.
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References |
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