© 1997 American Heart Association, Inc.
Articles |
The Watanabe Heritable Hyperlipidemic Rabbit Is a Suitable Experimental Model to Study Differences in Tissue Response Between Intimal and Medial Injury After Balloon Angioplasty
From the First Department of Internal Medicine (M.T., K.T., T.T.) and the Department of Pathology (M.U), Osaka City University Medical School, Osaka, Japan; and the Department of Cardiovascular Pathology (A.E.B), Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands.
Correspondence to Dr Makiko Ueda, Department of Pathology, Osaka City University Medical School, 1-4-54, Asahi-machi, Abeno-ku, Osaka 545, Japan.
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Abstract |
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Abstract The study evaluates whether the Watanabe heritable hyperlipidemic (WHHL) rabbit is an adequate model to study mechanisms that underlie differences in tissue response after postangioplasty injury. Postangioplasty studies of human coronary arteries have revealed that healing and restenotic processes differ depending on whether the laceration is limited to the atherosclerotic plaque or whether injury extends into the media. Forty-five homozygous WHHL rabbits underwent percutaneous transluminal angioplasty of the left subclavian artery. The inflated arterial segment was studied histologically at 3, 7, 14, 28, and 56 days, using conventional and immunohistochemical techniques to identify macrophages, smooth muscle cell (SMC) phenotypes, and cell proliferation. Electron microscopy was done to study reendothelialization. There were marked differences in response between those segments with medial injury and those with injury limited to the atherosclerotic plaque tissues. The latter category shows a distinct retardation in redifferentiation of SMCs, confirming previous observations in human coronary arteries. In these segments, moreover, cell proliferation occurred mainly in macrophages and spindle cells. Medial injury showed a more florid fibrocellular response, as in human coronary arteries, with cell proliferation initially confined to areas with dedifferentiated SMCs in the preexistent media and, once neointimal tissue was formed, among spindle cells also. The similarities with the repair processes encountered in postangioplasty human coronary arteries suggest that the WHHL rabbit is an adequate model to study differences in the response related to different types of angioplasty injury.
Key Words: atherosclerosis • percutaneous transluminal coronary angioplasty • restenosis • animal models • vascular wall injury
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Introduction |
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Restenosis after an initial successful PTCA occurs in 30% to 40% of patients and thus remains a significant clinical problem.1 The mechanisms that underlie restenosis are still unclear. Initial reports have documented a proliferative cellular response as the main pathological feature.2 3 4 5 More recently, so-called late remodeling has been introduced as a mechanism, although the processes causing this phenomenon are unknown.6 7 8 Hence, a suitable experimental animal model to study postangioplasty phenomena is urgently needed. Thus far, several animal models have been tested, but the end-results of these experiments have not proven to be applicable to the situation in humans.9 The problem, in this respect, may relate to the fact that the majority of these experimental injury models do not reflect the condition of atherosclerotic disease in humans. The Watanabe heritable hyperlipidemic (WHHL) rabbit is an exception to this statement, because our previous immunohistochemical studies demonstrated that the cellular composition of advanced atherosclerotic lesions in WHHL rabbits appears similar to that in humans,10 11 and the cellular response after angioplasty medial injury is remarkably like that seen in humans.12
We have previously shown that in humans post-PTCA healing and restenotic processes are markedly different when related to the depth and the extent of the laceration, both with respect to the time sequence and to the make-up of the cellular components.5 13 14 15 16 17 This study was designed to evaluate whether the WHHL rabbit may serve as a suitable model to study the underlying mechanisms.
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Methods |
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Animal Preparation
Forty-five homozygous WHHL rabbits (17 to 24 months of age, 3.0 to 4.0 kg) of both sexes were used in this study. The rabbits were obtained from Dr M. Shiomi (Institute for Experimental Animals, Kobe University School of Medicine). It is known that all WHHL rabbits at these ages show severe atherosclerosis with distinct lesions in the aorta and the proximal segments of the head and neck arteries.18 19 All animal handling and experimental procedures, including putting to death, were conducted in a gentle manner to minimize the stress and discomfort to the rabbits. Each animal received human care in compliance with the Principles of Laboratory Animal Care of the National Society of Medical Research and Guide for the Care and Use of Laboratory Animals of the National Academy of Sciences.
PTA
Each WHHL rabbit was fully anesthetized with
sodium pentobarbital (25 mg/kg body weight). A 4F sheath introducer
(Medikit Co) was inserted into a femoral artery. An angioplasty balloon
catheter for use in PTCA in humans was advanced retrogradely into the
proximal portion of the left subclavian artery, using the over-the-wire
technique under fluoroscopy. The balloon was inflated to a pressure of
2 atm and sized at a length of 2 cm with a diameter of 3 mm (the
latter was 25% to 50% larger than the original diameter of the
artery). The balloon was inflated three times, 1 minute each, at
30-second intervals. The balloon catheter was then deflated and
withdrawn. After completion of angioplasty, the sheath introducer was
removed. The femoral arteries were ligated, and the incision was
closed. The rabbits were allowed to recover from anesthesia
and monitored.
Pathological Examination
Rabbits were killed, after being fully anesthetized at 3
(n=10), 7 (n=10), 14 (n=10), 28 (n=8), and 56 days (n=7). In each
animal the aortic arch and left subclavian artery were removed. The
proximal part of the left subclavian artery, which had been used for
angioplasty, was serially sliced at 1-mm intervals perpendicular to its
long axis. At 3 and 7 days postangioplasty, six slices of the dilated
segments were obtained from each rabbit; seven slices were obtained
from each rabbit at 14, 28, and 56 days, three of (every second,
fourth, and sixth slice) which were used for transmission electron
microscopy for the purpose of identifying endothelial
cell recovery at the site of injury (see below).
All six slices at 3 and 7 days and the remaining four slices at 14, 28,
and 56 days were fixed in methanol-Carnoy's fixative and processed
routinely, embedded in paraffin, and sectioned at 5-µm thickness. A
total of 220 slices was sectioned. The sections were then stained with
hematoxylin-eosin and Weigert's elastic van Gieson stain. After light
microscopic investigation of the sections obtained from each slice,
some were excluded because (1) there was no angioplasty-related injury,
(2) there were two different types of injury in the same section
(injury limited to an atherosclerotic plaque and injury extending into
the media [Fig 1
], see below), and (3)
the laceration extended outside the media into the adventitia. Thus,
137 arterial slices were excluded, and the remaining 83
were used for further study with immunohistochemical staining.
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Immunohistochemical Analysis
From each of the 83 arterial slices, 30 serial
sections were cut at a thickness of 5 µm. Every 9th and 10th
section was stained with hematoxylin-eosin and with Weigert's elastic
van Gieson stain, respectively; the other sections were used for
immunohistochemical staining. To identify SMCs and to evaluate
differences in the state of differentiation two antibodies were
used14 15 : the anti-muscle actin mAb HHF-35 (Enzo
Diagnostics Inc) and the anti-SMC mAb CGA-7 (Enzo
Diagnostics Inc). Fully differentiated SMCs are
vimentin+/HHF-35+/CGA-7+,
intermediately differentiated SMCs are
vimentin+/HHF-35+/CGA-7-,
and dedifferentiated SMCs or spindle-shaped cells are
vimentin+/HHF-35-/CGA-7-.14 15
The anti-rabbit macrophage mAb RAM-11 (Dako A/S) was used to
recognize macrophages.20 The media and
preexistent noninjured atherosclerotic intima were used as positive
control tissues for these antibodies, as previously
documented.10 11 An anti-vimentin mAb was used to
identify mesenchymal cells. The mAb to PCNA (Dako), which is expressed
in the late G1 (presynthetic), S (DNA synthetic),
and G2 (premitotic) phases of the cell cycle, was
used to detect proliferative cells.
The labeled streptavidin-biotin complex system with nickel chloride color modification was used in all instances. Sections were counterstained with methyl green.
Immunostained Area Quantification
The immunostained sections were used for surface
area quantification of HHF-35+ and
CGA-7+ SMCs as well as
RAM-11+ macrophages within the
neointimal lesion using a computerized morphometry system,
MacSCOPE Ver. 2.2 (Mitani Corporation). The
HHF-35+ SMC area, the
CGA-7+ SMC area, and the
RAM-11+ macrophage area were then
expressed as a percentage of the surface area occupied by cells
positive for vimentin. The results are expressed as mean±SE.
Intraobserver variability was determined from triplicate measurements.
The mean±SE differences among measurements was 3.2±0.29%.
Interobserver variability was determined from measurements by three
observers. The mean±SE differences among measurements were 4.9±0.2%.
Statistical comparisons between groups were performed by Student's
t test. A value of P<.05 was considered
significant.
Electron Microscopy
We performed electron microscopic studies because we have
no reliable immunocytochemical marker for endothelial
cells in rabbits. Tissue slices were fixed in 2.5%
glutaraldehyde and postfixed in 1% osmium tetroxide,
dehydrated in an ethanol series, and embedded in epoxy resin. After
light microscopic determination of the type of angioplasty-related
injury using sections from slices fixed in methanol-Carnoy's fixative,
one or two slices embedded in epoxy resin from each of the dilated
segments, which were immediately next to the methanol-Carnoy's-fixed
slices containing an angioplasty injury limited to the atherosclerotic
plaque or an injury extending into the media (see below), were selected
for further study. Semithin sections 1 to 2 µm thick were
stained with toluidine blue for a light microscopic survey. These
sections were screened for the presence of a neointimal
tissue response, and the appropriate areas were then selected.
Ultrathin sections were cut from the selected areas and stained with
uranyl acetate and lead citrate. The specimens were observed under a
JEOL JEM 1200EX2 electron microscope (JEOL Ltd).
Quantification of Endothelial Cell
Recovery
The degree of re-endothelialization of the
luminal surface of the neointima was quantified using 30
consecutive electron micrographs (magnification x6200) taken from the
neointima in specimens from 14 days onward. The number of
specimens with an injury limited to the atherosclerotic plaque was 3
slices (2 rabbits) at 14 days, 3 slices (3 rabbits) at 28 days, and 3
slices (2 rabbits) at 56 days; the number of specimens with an injury
extending into the media was 4 slices (3 rabbits) at 14 and 28 days and
3 slices (2 rabbits) at 56 days. The luminal lining by
endothelial cells was measured and expressed as a
percentage of the total length of the luminal surface. In this
investigation, endothelial cells were defined, based on
the criteria provided by Schwartz et al.21 On this basis,
for each case the mean±SE was calculated and provided as the
"regenerated endothelial cell score." Statistical
comparisons between groups were performed by the Student's
t test. A value of P<.05 was considered
significant.
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Results |
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Atherosclerotic Lesions of Noninjured Left Subclavian Artery
In the proximal part of the noninjured left subclavian artery in elderly WHHL rabbits, advanced atherosclerotic lesions were found, which were either concentric or eccentric. These lesions contained various degrees of extracellular deposits of lipids, foam cells with necrotic debris, and cholesterol crystals in the middle and deep parts of the plaque; SMCs were scattered in the superficial portion of the plaque. Collagen fibers were distributed throughout the plaque.
Immunohistochemical studies demonstrated that these advanced atherosclerotic plaques contained macrophages, SMCs, and actin-negative spindle-shaped cells (vimentin+/HHF-35-/CGA-7-).
Arterial Wall Injury Due to PTA
All 45 rabbits showed advanced
atherosclerosis in the proximal part of the left
subclavian artery. In all rabbits, the angioplasty procedure had
resulted in laceration of the arterial wall. Two different
situations had occurred. The first was characterized by angioplasty
injury limited to the atherosclerotic plaque (30 arterial
slices: 7 at 3 days, 8 at 7 days, 5 at 14 days, 6 at 28 days, and 4 at
56 days) and was encountered in the dilated arteries of 17 rabbits (4
at 3, 7, and 14 days; 3 at 28 days; and 2 at 56 days), which at the
most proximal segment contained a markedly thickened atherosclerotic
plaque with concentric morphology. These slices all showed laceration
of the plaque, but the internal elastic lamina and the media were
intact. In each of these arteries, the remaining slices from the more
distal segments were excluded from the analysis, because either
there were two different types of injury in the same section or the
laceration extended outside the media into the adventitia. The second
situation was characterized by angioplasty injury extending into the
media (53 arterial slices: 13 at 3 days, 14 at 7 days, 10
at 14 days, 9 at 28 days, and 7 at 56 days) and was encountered in the
dilated arteries of 28 rabbits (6 at 3, 7, and 14 days; and 5 at 28 and
56 days each). In the vast majority this type of injury was observed in
slices from the proximal segment, which contained an eccentric lesion;
it was found rarely in slices with a concentric lesion. The remaining
slices of these arteries showed an injury extending into the adventitia
or no injury and, therefore, were excluded from the
analysis.
These two types of injury, based on a per slice evaluation, were taken for further analysis of the reparative response.
Reparative Response to Injury Limited to the Atherosclerotic
Plaque
At each postangioplasty time interval, the reparative phenomena
observed were remarkably similar. This thus allowed a collective
description of the findings for each category.
At 3 days after angioplasty, the site of injury showed the presence of mononuclear round cells and a small number of spindle-shaped cells amid a fibrin-platelet thrombus. The round cells were identified as macrophages (vimentin+/RAM-11+). The spindle-shaped cells were positive only for vimentin, and none of these cells stained with the actin markers HHF-35 and CGA-7. PCNA positivity, indicative of cell replication, occurred in some of the round cells and in some of the spindle-shaped cells.
At 7 days after angioplasty, round cells and spindle-shaped cells were
more numerous at the site of injury. The immunohistochemical
characteristics were the same as those observed at 3 days
postangioplasty. Some round cells and some of the spindle-shaped cells
were positive for PCNA and, in addition, some spindle-shaped cells in
the adjacent preexistent plaque tissues also showed this positivity
(Fig 2).
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At 14 days after angioplasty, a distinct accumulation of macrophages was observed, partially covered by spindle-shaped (vimentin+/HHF-35-/CGA-7-) cells. Occasional macrophages showed a foamy appearance. PCNA+ cells were found in regions containing macrophages. Electron microscopic studies revealed that there were no endothelial cells at the luminal surface.
At 28 days after angioplasty a neointima had formed,
composed of macrophages covered by a thin layer of
spindle-shaped cells. A large number of the accumulated
macrophages revealed a foamy cytoplasm. Most spindle-shaped
cells were HHF-35+, but the majority were
CGA-7-. PCNA+ cells were
observed occasionally in macrophage-rich regions (Fig 3).
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At 56 days after angioplasty the neointima consisted of
macrophages, macrophage-derived foam cells, and a
distinct layer of fibrocellular tissue covering the
macrophages. The spindle-shaped cells within the fibrocellular
tissue were HHF-35+ and
CGA-7+ (Fig 4). A
few PCNA+ cells occurred at sites dominated by
macrophages. Electron microscopic studies revealed that the
surface was partially covered by endothelial cells (Fig 5).
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Reparative Response to Injury Extending Into the Media
As with injury limited to the atherosclerotic plaque, the
reparative phenomena in cases with medial injury at a given time
interval were remarkably similar and, thus, are collectively
described.
At 3 days the site of injury was characterized by a small number of
spindle-shaped
(vimentin+/HHF-35-/CGA-7-)
cells with infiltration of some macrophages
(vimentin+/RAM-11+) amid a
fibrin-platelet thrombus. PCNA+ cells were
found in the reactive tissue and in the preexistent media immediately
adjacent to the site of injury. Medial sites with PCNA positivity
showed a marked loss in staining reactivity of medial SMCs with both
actin markers HHF-35 and CGA-7 (Fig 6).
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At 7 days the site of medial injury contained spindle-shaped cells,
which formed a distinct neointimal layer. A few cells
within the neointima stained positive with HHF-35; there
was no staining with CGA-7. PCNA+ cells were
found in the neointimal tissue and in the preexistent media
adjacent to the laceration (Fig 7).
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At 14 days a prominent neointimal layer was present at
the site of medial injury. The spindle-shaped cells were
HHF-35+, but only some cells were
CGA-7+ (Fig 8).
Only few PCNA+ cells occurred in both
neointima and preexistent media.
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At 28 days the spindle-shaped cells within the neointima
stained positive with vimentin and actin markers HHF-35 and CGA-7 (Fig 9). Only a few
PCNA+ cells were found at the site of medial
injury. Ultrastructurally, the endothelial cell lining
was completed. At 56 days the neointima was as seen at 28
days; no PCNA+ cells were observed.
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Some Relevant Differences in Response Between Arteries With Injury
Limited to the Atherosclerotic Plaque and Those Extending Into the
Media
The differences observed affect primarily the cellular composition
of the repair tissue and the differentiation of SMCs, related to the
time interval between the angioplasty procedure and killing. The
salient features are summarized in the
Table.
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Immunostained Area Quantification
The HHF-35+ SMC area was significantly
larger (at 7 days: P<.05; at 14, 28, and 56 days:
P<.01) in arteries in which injury extended into the media
than in arteries with injury limited to an atherosclerotic plaque. At
28 and 56 days postangioplasty, moreover, the
CGA-7+ SMC area also was significantly larger
(P<.01) in specimens with injury extending into the media
than in those with injury limited to an atherosclerotic plaque. In
contrast, at all stages the RAM-11+
macrophage area was significantly larger (P<.01) in
arteries with injury limited to an atherosclerotic plaque than in those
with injury extending into the media (Fig 10).
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Quantification of Endothelial Cell
Recovery
At all stages from 14 days onward (Fig 11), the regenerated
endothelial cell score at the site of
neointima was significantly higher (P<.01) once
injury extended into the media than in a group with injury limited to
an atherosclerotic plaque.
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Discussion |
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Despite numerous intervention trials, the incidence of postangioplasty restenosis has not diminished.22 23 24 Recently, it has been suggested that restenosis is due to late remodeling of the vascular wall with little if any contribution of a fibrocellular tissue response at the site of injury.6 7 8 However, the fact remains that clinicopathological correlates in humans have indicated clearly that a local "response to injury" plays a role.2 3 4 5 25 Thus, it appears that there is controversy, and, hence, an adequate experimental animal model is urgently needed.
Several studies have revealed that the WHHL rabbit may serve as a good model for human atherosclerosis.10 11 19 20 26 Indeed, advanced atherosclerotic lesions of noninjured left subclavian arteries are dominated by atheroma and fibrosis, known to be common features in advanced atherosclerotic plaques in humans. Moreover, the atherosclerotic lesions in WHHL rabbits contain macrophages, SMCs, and actin-negative spindle-shaped cells (vimentin+/HHF-35-/CGA-7-), all of which are found generally in advanced atherosclerotic plaques of humans, including target lesions of PTCA.15 16 These observations support the concept that the major cellular components of advanced atherosclerotic lesions in the left subclavian artery of WHHL rabbits appear similar to those of advanced coronary atherosclerotic plaques in humans. Our previous study demonstrated that the cellular response at sites of angioplasty injury extending into the media is very similar to that observed in humans and is characterized by a time-related phenotypic shift of SMCs from synthetic to contractile.12 This study further highlights the point that the WHHL rabbit may serve as a model also to study differences in postangioplasty response between arteries in which the injury was limited to the atherosclerotic plaque and those in which the injury extended into the media.
Cellular Response at the Site of Injury Limited to the
Atherosclerotic Plaque
This study demonstrates that the response to medial injury
differs markedly from injury limited to the plaque (Table
and Fig 10),
thus endorsing previous observations in
humans.5 13 14 15 16 17 At the site of plaque injury the
early stages show a prominence of reactive macrophages amid
spindle cells. Because these macrophages accumulate
preferentially at the site of injury, often embedded in thrombus, one
may assume that these are responding cells rather than preexistent
cells. With time, moreover, the macrophages showed transition
into foam cells and, eventually, were covered by layers of spindle
cells. The latter presented different phenotypes at
different postangioplasty time intervals. Initially, spindle cells at
the site of injury were negative for both anti-actin markers. At 28
days, however, spindle cells were positive with HHF-35 and at 56 days
spindle cells were positive with both HHF-35 and CGA-7. It is likely,
therefore, that redifferentiation of SMCs occurs as a time-related
phenomenon, like that observed in postangioplasty repair processes in
the WHHL rabbit after medial injury12 and similar
to that described in human coronary
arteries.14 15 However, redifferentiation of SMCs
is retarded compared with the response after injury extending into the
media. In the latter situation, fully differentiated SMCs were
encountered at 28 days postangioplasty, but in arteries with injury
limited to the atherosclerotic plaque this was not the case.
Furthermore, electron microscopic studies revealed that the
endothelial cell lining was not completed in arteries
with injury limited to the plaque, even at 56 days. In contrast, in
arteries with medial injury, endothelial cells had
regenerated much earlier; this observation is supported by our previous
study in the same animal model.12 Indeed, the
regenerated endothelial cell score, introduced in this
study, clearly shows statistically significant differences between
arteries with injury extending into the media and those limited to
plaque tissue (Fig 11). In the electron microscopic studies, we defined
endothelial cells according to the criteria provided by
Schwartz et al,21 indicating that so-called type
B cells (cells of indefinite type) and type C cells (modified SMCs)
were not included in the evaluation of the regenerated
endothelial cell score.
These findings in the WHHL rabbit are of interest since our previous works on human coronary arteries demonstrated that the cellular response after angioplasty injury is different depending on whether the injury is limited to the atherosclerotic plaque or whether it extends into the media.5 13 14 15 16 17 In humans, angioplasty injury to an atheroma causes mural thrombosis, followed by a response that consists of an accumulation of macrophages intermingled with occasional spindle-shaped cells.13 27 28 In the repair process that follows angioplasty injury to an atherosclerotic plaque macrophages and dedifferentiated SMCs may play an important role, since these cells colocalize with platelet-derived growth factor and they express its receptor.16 17 Furthermore, restenosis lesions at sites of injury limited to an atheroma revealed macrophages and SMCs as the main cellular constituents, thus indicating that healing of an injured atherosclerotic plaque could be a mechanism underlying restenosis.5 These observations, therefore, strongly suggested that the type of response after angioplasty injury reflects the depth and the extent of the laceration and, hence, the nature of the tissue involved. In this context, it is of interest also that Moreno et al29 recently showed that the amount of macrophages in coronary atherosclerotic lesions can be used as a predictor for restenosis after coronary interventions.
The question arises why macrophages are preferentially retained at the site of plaque-limited injury, compared with medial injury. Previous in vitro studies have demonstrated that oxidized LDL has many properties that could contribute to the atherogenic process.30 31 32 33 Moreover, there is accumulating evidence that oxidized LDL is present in atherosclerotic plaques of the WHHL rabbit.34 35 36 Given the role of oxidized LDL, one may speculate that it plays a role in the recruitment of circulating monocytes, in the retention of macrophages at the site of plaque injury, and in the subsequent formation of macrophage-derived foam cells.30 31 32 33
Cellular Response at the Site of Injury Extending Into the
Media
The dominant cell type involved is the vascular SMC; only a
few macrophages occur within the repair tissue. Using two
different actin markers for SMCs, we have demonstrated that the spindle
cells encountered as part of the fibrocellular response show
differences in phenotypic differentiation, related to the
postangioplasty time interval. In the earliest stages of injury
extending into the media loss of staining with both markers of SMCs of
the preexistent media is seen, immediately adjacent to the site of the
tear. Spindle-shaped cells within the reactive tissue, moreover,
stained positive for vimentin, but failed to stain with the two actin
markers. With time, however, the spindle-shaped cells in the
neointima became positive first with HHF-35 and
subsequently also with CGA-7. These observations strongly support the
concept that dedifferentiation of medial SMCs had occurred at the site
of injury, followed by formation of neointima and gradual
redifferentiation to mature SMCs.15 37 Recently,
several experimental studies in rabbits have investigated the time
course and immunocytochemical characteristics of the
neointimal response after balloon
injury.38 39 40 41 In these studies, however, the
identification of SMCs was based on one anti-actin marker only, either
HHF-3540 41 or anti-
actin
antibody.38 39 Staged redifferentiation of
neointimal SMCs, therefore, was not seen by these authors.
Our present observations are relevant particularly because we
demonstrated in human coronary arteries after angioplasty,
using the same mAbs, almost identical shifts in the cytoskeletal
phenotype of neointimal
SMCs.14 15 The present observations in the
WHHL rabbit thus indicate that this model may be fit to study the
underlying biological processes.
Cell Proliferation at the Site of Injury
This study provides new data with regard to cell proliferation at
sites of injury. In the arteries with injury extending into the media,
the earliest stages show proliferation confined to the dedifferentiated
SMCs in the preexistent media. Once neointimal tissue was
formed, cell proliferation occurred also among spindle cells. At later
stages proliferation gradually diminished parallel to the
redifferentiation of neointimal SMCs. The validity of our
observations in the WHHL rabbit model is supported by kinetic studies
of SMC proliferation in rats42 43 and data on the
phenotypic changes of SMCs in a rat carotid balloon injury
model.37
In arteries with angioplasty injury limited to the plaque tissue, the early stages show PCNA positivity of macrophages and spindle cells. At later stages proliferation was found predominantly in macrophage-rich areas at sites where macrophages transformed into foam cells. Stadius et al41 reported recently that the neointima induced by balloon injury of iliac arteries in cholesterol-fed rabbits contained RAM-11+ macrophages which showed proliferative activity. Moreover, there is accumulating evidence that macrophage-derived foam cells are able to proliferate in situ, particularly in the early phase of atherosclerosis.44 45 To the best of our knowledge this is the first report to disclose that proliferation of macrophages and macrophage-derived foam cells occurs as part of the healing process after plaque-limited angioplasty injury.
Limitations of the Study
Although the WHHL rabbit model appears to be a suitable
animal model for the study of postangioplasty healing processes as they
occur in humans, some questions remain that should be addressed. First,
the model uses rabbit subclavian arteries as opposed to the epicardial
coronary arteries in humans and, hence, the question remains
whether the underlying basic biological processes can be compared.
Second, the role of hypercholesterolemia and
the hypercoagulable state in the development of the intimal response to
balloon injury is not characterized.46 However,
our purpose was to evaluate the suitability of this model in terms of
similarities of cellular response after balloon injury. Third, in this
study we have not measured a ratio of PCNA+
cells, defined as the number of PCNA positive staining cells divided by
the total cells counted, but instead have focused on the investigation
of immunohistochemical characteristics of PCNA+
cells. We selected this approach because of the fact that cell numbers
and cell types in the preexistent atherosclerotic plaque were not
uniform at each angioplasty site in our series using the WHHL
rabbit.
Despite these limitations it is our opinion that the similarities between conditions encountered in humans and those observed in this rabbit justify the use of the WHHL rabbit to study mechanisms involved in postangioplasty healing responses and restenosis in humans.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We thank Dr Masashi Shiomi for providing the facilities to prepare the studies, Dr Akiko Kojima for the tissue preparation, and Drs Ryoichi Matsuo and Kenji Nakamura for technical assistance during the angioplasty studies.
Received April 16, 1997; accepted September 5, 1997.
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