(Circulation. 1995;92:2995-3005.)
© 1995 American Heart Association, Inc.
Articles |
Vascular Injury, Repair, and Restenosis After Percutaneous Transluminal Angioplasty in the Atherosclerotic Rabbit
Presented in part at the 64th Scientific Sessions of the American Heart Association, Dallas, Tex, November 11-14, 1991, and the 41st Scientific Sessions of the American College of Cardiology, Dallas, Tex., March 1992.
From the Krannert Institute of Cardiology, Department of Medicine, Roudebush VA Medical Center, Indiana University School of Medicine and Eli Lilly and Co. (G.S.), Indianapolis, Ind.
Correspondence to Robert L. Wilensky, MD, Krannert Institute of Cardiology, 1111 W 10th St, Indianapolis, IN 46202-4800.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Several nonatherosclerotic animal models of restenosis exist and are used for the evaluation of the vascular response to angioplasty-induced injury. However, few studies have evaluated the response of an atherosclerotic vessel to angioplasty. The present study examined the radiographic, histological, immunohistochemical, and morphometric responses over time of atherosclerotic rabbit femoral arteries after percutaneous transluminal angioplasty (PTA).
Methods and Results Rabbits (n=94) underwent arterial desiccation and were fed a hypercholesterolemic diet for 3 weeks, and then PTA was performed. Arteries were obtained before PTA and 1, 3, 5, 7, 14, and 28 days after PTA. PTA caused radial stretching of the artery, medial compression, intramural hemorrhage, injury to normal arterial segments, and dissection within the intima and media. Thrombus filled and cellular accumulation repaired the dissection. Peak smooth muscle cell and macrophage DNA synthesis was noted at 3 to 5 days after angioplasty, generally at the dissection but also in normal sections of the artery. Adventitial injury and subsequent adventitial cellular proliferation and collagen production were observed. A rapid decrease in the radiographic minimal luminal diameter was noted at 3 days, resulting from vascular recoil or thrombus filling the dissection. At 7 to 14 days, only 24% to 33% of the luminal loss was accounted for by an increase in the intimal area, and 22% to 28% of the intima was neointima.
Conclusions Restenosis in an atherosclerotic artery results from a variable combination of intimal proliferation, vascular remodeling/wound contraction, and recoil of the normal section of the artery. The variability of an atherosclerotic artery to PTA injury results from variable dissection, thrombus formation, and cellular response to injury as well as variable scar contraction and elastic recoil.
Key Words: atherosclerosis • stenosis • remodeling • angioplasty
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Although new advances in percutaneous transluminal coronary angioplasty catheter design and operator expertise have reduced the procedural complication rate, the angiographic restenosis rate remains at 35% to 40%.1 2 No currently devised pharmacological approach has been shown to reduce the rate of restenosis, and an incomplete understanding of the pathophysiology of restenosis limits all approaches to therapy. At the present time, there are at least seven animal models for the investigation of restenosis,3 and these can be divided into single- or double-injury models, depending on the number of steps performed to obtain an atherosclerotic or restenotic lesion. Balloon endothelial denudation and medial layer damage with or without a subsequent high-cholesterol diet constitutes the single-injury model in which there is de novo intimal growth in a previously normal artery. Interventions designed to evaluate the capacity for a device or medication to reduce the resulting intimal proliferation are performed at initial injury. The single-step approach has been evaluated in rat carotid and aortic arteries, rabbit iliac and ear4 arteries, and porcine coronary arteries (see Reference 3 for review). An advantage of such single-injury models is the homogeneity of the resulting lesion, which consists largely of smooth muscle cells (SMCs). Disadvantages include the lack of a preexisting atherosclerotic lesion and the relatively small degree of intimal proliferation in small animal models. The absence of lesion complexity may explain in part why single-injury models of restenosis respond to various antiproliferative regimens, whereas similar strategies in humans have been without effect.
The double-injury model involves initial injury of the endothelial and/or medial layer by a balloon,5 electrical current,6 or air desiccation.7 The animal is generally placed on a high cholesterol diet until angioplasty is performed. At the time of the angioplasty, medications are administered and the subsequent growth of neointima is evaluated as the measure of restenosis. This approach has been evaluated in the rabbit aortic, iliac, and femoral arteries and porcine coronary and iliac arteries.3 A disadvantage of the double-injury model is the heterogeneous nature of the second lesion and thus the difficulty in differentiating the original from the restenotic tissue. Nonetheless, the two-step approach creates greater lesion complexity and thereby more closely mimics human conditions. The temporal response to injury in atherosclerotic vessels has not been evaluated. Accordingly, in this study we have characterized the response to vessel injury after angioplasty to elucidate the resulting variability of injury and repair mechanism. The studies were performed in atherosclerotic rabbit femoral arteries obtained before and up to 4 weeks after percutaneous transluminal angioplasty.
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Methods |
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All procedures followed were in accordance with Indiana University Animal Resource Committee guidelines. New Zealand White rabbits weighing between 8 and 10 pounds each were used for this study. Endothelial denudation and medial damage of a 1-cm section of both deep femoral arteries was achieved by air desiccation in a procedure described previously.7 A metal clip was placed in the periadventitia near the center of the desiccated segment to aid in subsequent balloon placement as well as histological and morphometric evaluation. After desiccation, the rabbits were placed on a 150 g/d rabbit chow diet containing 2% cholesterol and 6% peanut oil (Dyets Inc) for 3 weeks until arteriography and angioplasty.
Angioplasty
The technique of angiography and angioplasty used
was a
modification of a previously published technique.8 After
placement of a 5F sheath into the aorta via the right carotid artery,
heparin 150 U/kg body wt was administered and a multipurpose catheter
was placed at the level of the aortic bifurcation. After administration
of 2 mL 1% lidocaine to reduce arterial spasm, angiography
of the femoral arteries was performed with a straight, multipurpose
catheter (Medi-Tech). Angioplasty, performed in the atherosclerotic
segment of arteries with 
20% but <100% stenosis, was
achieved by use of a standard angioplasty balloon (Advanced
Cardiovascular Systems), 
0.5 mm larger than the
proximal, normal arterial segment, which was inflated for 1
minute at 10 atm pressure. The balloon was deflated for 1 minute, and
the cycle was repeated for a total of three inflations. If the lesion
showed >50% improvement at subsequent angiography, the procedure was
considered successful. Thereafter, the contralateral artery underwent
angioplasty. The sheath was removed and the carotid artery and skin
sutured. The rabbit was administered cefazolin 2 g IM and placed on a
150 g/d normal rabbit chow diet until euthanasia.
Two groups of rabbits underwent only angiography, and these served as controls. One group, the original lesion group, was euthanatized 3 weeks after the original air desiccation injury. The other, a sham angioplasty group, underwent original injury and subsequent angiography but did not undergo angioplasty. This group was placed on a normal diet for 14 days until euthanasia 5 weeks after the original air desiccation injury.
Euthanasia
The rabbits were euthanatized at six time periods
after
angioplasty: 1, 3, 5, 7, 14, and 28 days. Most rabbits were given the
thymidine analogue bromodeoxyuridine (BrdU).6 Briefly,
BrdU 100 mg/kg and deoxycytidine 75 mg/kg were administered in a
subcutaneous depot 18 hours before euthanasia in addition to
administration of 30 mg/kg IM BrdU and 25 mg/kg IM deoxycytidine. A
second intramuscular injection of BrdU and deoxycytidine was
administered 12 hours before euthanasia to maintain constant serum
concentrations. In principle, all cells entering the S phase during the
last 18 hours would stain positive with antibodies for BrdU.
Deoxycytidine was given because an administration of BrdU in the
absence of deoxycytidine causes an inhibition in the internal
production of thymidine and cytidine, resulting in a decrease
in DNA synthesis (M. Oberhoff, personal communication, 1995). At the
time of euthanasia, the rabbit was anesthetized with
ketamine and xylazine and a 16-gauge angiocath was placed in
the abdominal aorta via a transverse abdominal incision. Two
milliliters of 1% lidocaine was injected into the aorta, and a final
angiogram was obtained. The venous effluent was collected via the
inferior vena cava. The aorta was infused with zinc
formalin solution (Anatech) at a pressure of 100 mm Hg, and the rabbit
was euthanatized with an overdose of pentothal.
The arteries were obtained en bloc and placed in a zinc formalin bath. The presence of zinc reduces formalin cross-linking, thereby enhancing antibody staining. The overlying skeletal muscle was dissected away from the artery, and the arterial segment was isolated and cut into pieces 3 to 4 mm in length (5 to 8 segments per artery). The segment nearest the metal clip was identified and noted. The distal end of each segment was marked with a short stripe of india ink, numbered, and placed in an individual canister for embedding and sectioning.
Histological and Immunocytochemical
Staining
The arterial sections, embedded in paraffin and
sectioned at a thickness of 5 to 6 µm, were stained with hematoxylin
and eosin and a combined Gomori's aldehyde fuchsin with a trichrome
counterstain. Other unstained sections were deparaffinized and used for
immunohistochemical analysis by the
avidin-biotin-peroxidase complex method.9 Primary
antisera were used at the following dilutions: factor VIII (Dako Corp)
1:100 and fibrinogen/fibrin10 (Cappel Labs)
1:1200. HHF-3511 (Enzo Laboratories), a monoclonal
antibody specific for vascular 
-actin at a dilution of 1:400,
was used to evaluate for the presence of vascular SMCs. RAM-11 (gift
from Dr Allen Gown, University of Washington, Seattle, Wash), a
monoclonal antibody specific for rabbit macrophages, was
diluted to 1:6500.12 The presence of
endothelial cells was determined by antibody staining
to von Willebrand's factor (factor VIII) at a dilution of
1:200.13 Staining for BrdU incorporated into DNA used a
monoclonal antibromodeoxyuridine antibody (Amersham) at a dilution of
1:100, counterstained with Gill's hematoxylin.
Selected arterial segments (n=24) underwent resectioning and double-antibody staining to evaluate the relative proliferative indexes of cells staining positive for the presence of RAM-11 or HHF-35. Sections (5-µm) were mounted, deparaffinized, and stained for the presence of either RAM-11 and BrdU or HHF-35 and BrdU according to protocols included in the Dako LSAB 2-AP and PAP kits.
Histological Analysis
The arterial cross sections were
evaluated by a
pathologist blinded to the arterial group. The segments
were evaluated for the following: (1) internal elastic lamina (IEL)
fracture; (2) intimal fracture; (3) inflammation, defined as increased
neutrophil infiltration (compared with nonangioplastied arteries); (4)
medial compression, defined as a thinned medial layer in relation to
adjacent areas or segments; (5) medial dissection; and (6) medial
necrosis defined as a reduction of SMCs and the presence of pycnotic
nuclei. The presence of a characteristic was noted, and if present
in any segment, the characteristic was considered present in that
artery. The pathologist was then unblinded, and the
arterial segments were characterized further to determine
the effect of time after angioplasty on the
histological characteristics.
To determine the cellular proliferation index, arterial segments were photographed at x40 to x100 magnification and all nuclei were counted in the intimal and medial layers. A proliferation index was obtained by dividing the number of BrdU-positive nuclei by the total nuclei in that cell layer. The presence of BrdU staining and the location of positive cells and their focality within the arterial layer were noted. Because it was difficult to determine the external border of the adventitia, adventitial BrdU uptake was reported as either focal or generalized. Cellular proliferation indexes were also determined for populations staining positive for BrdU and either HHF-35 or RAM-11 within the intimal and medial layers.
Data Analysis
The luminal, (neo)intimal, and medial areas
were evaluated by
morphometric analysis of arterial cross sections
obtained 1, 2, and 4 weeks after angioplasty and the cross sections of
the sham angioplasty and original groups. Morphometric analysis
was done on arterial cross sections imaged on a Macintosh
computer with an NIH image software package. All arterial
segments exhibiting an intimal layer cut in an axis perpendicular to
the long axis of the artery and exhibiting no fixation or
sectioning-related artifacts were evaluated. The endoluminal
border, the circumference bounded by the IEL, and the external elastic
lamina (EEL) were traced by hand, and the luminal, intimal, and medial
areas were calculated. Areas of mural thrombus were excluded from
measurement. To qualitatively evaluate the longitudinal variability of
these parameters within given arteries, four arteries were
sectioned at 200-µm distances and each section was evaluated for the
aforementioned areas.
Radiograms from all time points were evaluated by measuring the minimal luminal diameter (MLD) of the greatest stenosis. If arterial spasm or aneurysm formation was noted, the radiogram was excluded from further evaluation.
The segment exhibiting the greatest degree of intimal area, regardless of the luminal area, was considered the representative sample for that artery and was used for future comparisons between groups. For determination of the proliferation index, the results in all segments within a single artery were averaged and the artery was treated as an independent observation. Analysis of the morphometrically derived luminal, intimal, and medial areas from the different groups was compared by use of a one-way ANOVA and a protected least-significant-difference test for multiple comparisons. In addition, the EEL circumference was compared between groups by ANOVA and a protected least-significant-difference test. The protected least significant difference identifies differences in groups (as determined by ANOVA) while protecting for the performance of multiple comparisons, with the resultant increased chance of finding a nonmeaningful difference. Data are expressed as mean±SEM. A value of P<.05 was considered significant.
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Results |
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In total, 94 arteries were evaluated. The average cholesterol level after 3 weeks of diet was 1535±76 mg/dL. Despite discontinuation of the diet at the time of percutaneous transluminal angioplasty, serum cholesterol levels remained elevated over the duration of the study, with mean levels between 1091 and 1945 mg/dL. For example, in the 28-day group, the mean cholesterol level was 1400±214 mg/dL before angioplasty and was 1235±398 mg/dL at euthanasia.
The intima and media of the arterial segments obtained
before angioplasty were composed of SMCs, foam cells, collagen, and
elastin (Fig 1
). The relative number of foam cells
within the intimal and medial layers was heterogeneous
within arteries of the original lesion group and between arteries from
the same rabbit. The intima was intact, and the media exhibited no
compression, necrosis, or fibrosis. IEL fracture was seen in
approximately half (7 of 13) of the arteries. No inflammation was
observed, but increased fibrinogen/fibrin staining within the intima,
indicating arterial leakiness, was noted. Sham angioplasty
arteries were similar in composition to original lesion arteries. The
histology is summarized in Table 1.
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IEL fracture was noted in 70 of 75 (93%) of arteries obtained between 1 and 28 days after angioplasty. In addition, neointimal fracture and medial damage, as manifested by compression, necrosis, and eventual fibrosis, were observed. The arterial response to angioplasty-induced injury could be differentiated into three phases: stage 1, the acute phase of mural thrombus formation and inflammation; stage 2, the subacute phase, consisting of cellular accumulation presumably as a result of proliferation and migration; and stage 3, the chronic phase of connective tissue production.
Stage 1: Mural Thrombus/Inflammatory Phase (1 to 5
Days)
Arteries obtained early after angioplasty (1 to 5 days) showed
angioplasty-induced dissection within the intima and often deep
dissections extending into the medial and adventitial layers (Fig
2). The dissection planes generally contained thrombus,
which stained strongly for fibrinogen/fibrin. Within the mural
thrombus, platelets, neutrophils, lymphocytes, and monocytes were
observed.
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Inflammation was noted in all three arterial layers the day
after angioplasty (Table 1
) and decreased thereafter.
Intramural
hemorrhage was apparent. Focal but occasionally general medial
compression and necrosis without fibrosis were noted in most arteries.
Focal injury of the "normal" nonatherosclerotic areas of the
artery consisting of medial compression and necrosis (Fig 3)
was observed. Endothelial denudation
was observed in the majority of arterial segments between
days 1 and 5, although slight regeneration was seen on days 3 and 5
(Table 2).
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Stage 2: Cellular Accumulation Phase (1 to 14 Days)
Arteries
obtained 5 to 14 days after angioplasty exhibited less
neointimal fracture and little inflammation (Table 1). The
dissection was repaired by 7 days after angioplasty, and it was
difficult to differentiate between the intima and original
neointima. In areas in which the angioplasty dissection
appeared to have occurred, cells staining for HHF-35 were present,
filling in the original dissection planes, and in some arteries, areas
of organized thrombus with interdigitated SMCs were noted. Foam cells
generally were seen in the media, but foam cells observed in the intima
were located near the internal elastic lamina.
The intima was rich in
SMCs that stained for HHF-35. Collagen and
elastin staining of the intima was relatively weak. Four of 12 arteries
exhibited medial fibrosis at 7 days after angioplasty in areas of
previous compression and necrosis. Endothelial coverage
of the repaired intima as determined by the presence of continuous
layer of factor VIII–positive cells was observed in a majority of
arterial segments at 7 days after angioplasty (Table
2).
Stage 3: Connective Tissue Production Phase (>14
Days)
Two weeks after angioplasty, no neointimal fractures
were evident, although evidence of previous IEL fracture was observed.
Medial fibrosis was seen in 28 of 30 (93%) arteries obtained 2 to 4
weeks after angioplasty. Increased adventitial fibrosis, observed as a
thick band around the EEL, was also seen. An increased density of SMCs
was observed on the luminal surface of the neointima,
whereas increased extracellular matrix formation was noted throughout
the rest of the intima. Few foam cells were observed in the
neointima. Endothelial regeneration was
completed by 28 days after angioplasty. Collagen and elastin increased
over the weeks after angioplasty. Neovascularization was first observed
at 28 days. In most cases, it was impossible to differentiate the
restenotic neointima from the original
atherosclerotic intima (Fig 4).
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Cellular Proliferation
The total number of visible nuclei in
the intimal and medial
layers (Table 3) per cross section obtained from those
arteries that did not undergo angioplasty was 1561±414 and
1288±206,
respectively, and in the sham angioplasty group, the intimal nuclei
totaled 1182±219 and the medial, 1103±273. By 3 days after
angioplasty, the nuclei had decreased to 533±208 in the intimal layer
(P<.001) and 763±251 in the medial layer
(P<.001). Total cell number increased thereafter to a
maximum of 2664±400 and 1749±214 in the intimal and medial
layers,
respectively, at day 14 after angioplasty (Table 3).
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Three weeks after initial air desiccation injury, the original
lesion showed BrdU uptake in 
10% of the cells in the intima and
media (Table 3). The sham angioplasty arteries obtained 2 weeks
later
exhibited a proliferation index of 3.0% to 3.7% in both
arterial layers. In the experimental group, increased BrdU
uptake could be observed in all three arterial layers as
early as 1 day after angioplasty, and approximately one third of all
intimal and medial cells stained positive at 3 days after surgery.
Areas of dissection exhibited increased DNA synthesis (Fig 5),
although increased focal DNA synthesis was also seen
in nonatherosclerotic but injured areas of the artery. Arteries
obtained 1 to 5 days after angioplasty exhibited statistically
significant increases in BrdU uptake both in the intima and the media
(P<.001). BrdU labeling showed a heterogeneous
uptake of BrdU after angioplasty in that some arteries showed areas of
a focal intimal and medial BrdU staining, whereas other arteries showed
a more generalized BrdU uptake.
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Double-antibody staining for the
presence of BrdU and either RAM-11
or HHF-35 revealed that DNA synthesis was present in cells staining
positive for RAM-11 as well as those positive for HHF-35 (Fig
6). Comparison of the proliferation indexes of the two
cell types indicates that cells staining positive for the presence of
RAM-11 exhibited earlier DNA synthesis in the intimal layer than did
the cells staining positive for HHF-35. In the media, however, earlier
DNA synthesis activity was noted in the HHF-35 cells (34.2% versus
9.3% at day 3 after angioplasty, Table 3). Activated
macrophages demonstrated more sustained DNA synthesis over the
initial 2 weeks after angioplasty in both the intimal and medial
layers. At all time points, cells were noted that were BrdU-positive
but stained for neither the presence of RAM-11 nor HHF-35 (Fig
7). In the intima, these cells primarily were near the
luminal surface. In the medial layer, the proliferating cells were
interspersed within the media or represented discrete
intramedial areas. These cells appeared histologically
similar to cells staining positive for HHF-35.
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Adventitial Changes
An adventitial response was observed
after angioplasty. By
24 hours after angioplasty, increased BrdU activity and cellularity
were observed in the adventitia. These cells did not stain for HHF-35
or RAM-11 and did not have histological characteristics
of lymphocytes or neutrophils. The cells showing increased BrdU
positivity were focally localized in 26 of 40 segments obtained 1 to 7
days after angioplasty, with the remaining 14 segments exhibiting
diffuse uptake of BrdU positive cellularity. By 14 days, little BrdU
uptake was observed in the adventitia. Increased collagen was noted in
the adventitia starting at 5 days after surgery and increasing
thereafter. In some segments, the EEL was disrupted and it was
impossible to differentiate the cellular composition of the damaged
media from the adventitia. In those arteries with disrupted EEL and
IEL, the adventitia extended into the neointima (Fig 4).
Comparison of the Original and Restenotic
Lesions
Although the initial lesion frequently was eccentric, the
restenotic lesion frequently was concentric. The
neointima of restenotic arteries observed >14
days after angioplasty varied from the intima of the original,
atherosclerotic arteries before angioplasty in that the composition of
the intimal plaque stained more strongly for HHF-35 and fewer RAM-11
positive cells were seen. The foam cells that were observed were
located near the IEL. The media of the initial lesion was a combination
of SMCs and foam cells, whereas after angioplasty, media exhibited
areas of acellularity associated with fibrosis in addition to the
mixture of predominant SMCs and foam cells. Increased vasa vasorum
within the media was observed 4 weeks after angioplasty. The
arterial lesions from the sham angioplasty group were
similar in composition to the original lesions, although fewer foam
cells were present.
Radiographic Changes After Angioplasty
The initial mean
radiographic MLD was 0.64±0.05 mm
(Table 4). Angioplasty increased the MLD to 1.07±0.05
mm for an acute gain averaging 0.43 mm, whereas the mean MLD was
1.06±0.43 mm 1 day after angioplasty. Between days 1 and 3, a rapid
decrease in MLD was noted, followed by a slower decrease in MLD between
days 3 and 14. The MLD at 28 days was increased over the 14-day MLD and
approximated the postangioplasty MLD. The period between days 14 and 28
coincided with the period noted in histological
analysis of decreased SMC proliferation and increased
extracellular matrix formation. The radiographic MLD
significantly correlated with the lumen area determined by morphometry
(r=.44, P=.001) with a slope of
0.63±0.18
(P=.001) and an intercept of 0.35±0.11.
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Morphometric Analysis
Morphometric analysis of arteries
obtained before
angioplasty and 7 days after angioplasty revealed a significant
increase of luminal area after angioplasty (P<.001) but
showed no statistically significant difference in the mean intimal and
medial areas (Table 5). The summed intima-media area
increased after angioplasty, although the change was not statistically
significant. The EEL circumference at 7 days after angioplasty was
significantly increased (P<.001) but did not change
thereafter. To determine whether a sampling bias that minimizes the
morphometrically derived intimal area was present, four arteries
obtained 14 days after PTA underwent serial sectioning at 200-µm
distances. The results of a representative artery are
plotted in Fig 8, which indicates the varied intimal and
medial areas within the artery as well as ratios of intima to
media.
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Given the substantial variability within this model and the resulting absence of statistical difference between the average intimal areas found at 1, 2, and 4 weeks after angioplasty, computation of the sample sizes required in this model to detect given changes in area was performed with two data sets. If only the segment of each artery exhibiting the greatest intima area was used, the variability of lesion area resulted in a sample size of 34 arteries necessary to detect a 50% increase in intimal formation, at a significance level of P=.05 (two-tailed test) and a power of 0.8. For a power of 0.9, the sample size is 44 arteries. If the averaged intimal area for all segments of the artery was used, then 64 arteries were necessary to show a statistically significant 50% increase in area with a power of 0.80 and a significance of P=.05, whereas 86 arteries per group were needed for a power of 0.90. This higher number reflects the increase in variability of data when all diseased arterial segments are used, reflecting an overall plaque mass. Conversely, a similar number of arteries per group would be required to show a 50% decrease in intimal formation (ie, 50% reduction in lesion restenosis) resulting from a pharmacological treatment to reduce restenosis.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Angioplasty in this model caused radial stretching of the artery, compression injury of the media, intramural hemorrhage, and dissection of the intima and/or media. The subsequent vascular response to this injury exhibited characteristics common to the acute inflammatory response to injury. Restenosis resulted from a combination of mural thrombus filling the dissection planes and forming a framework for subsequent cellular ingrowth as well as deposition of collagen and elastin in the intima, media, and adventitia.14 The extracellular matrix presumably was produced by fibroblasts in the adventitia and by SMCs in the media and may have been a component of intramural wound contraction. Collagen and elastin may have exerted arterial constriction from the adventitia. Proliferating SMCs and macrophages were noted in the intima and media of regions possessing a preexisting lesion as well as in regions of the media of nonatherosclerotic sections damaged by angioplasty. The vascular reaction to injury may thus be divided into three overlapping stages of vascular repair and restenosis: stage 1, the acute response to injury, consisting of mural thrombus formation, inflammation, intramural hemorrhage, and vessel recoil, which resulted immediately from angioplasty and was noted for 5 days; stage 2, the subacute phase of cellular accumulation in the area of thrombus formation or arterial injury (days 1 through 14); and stage 3, the subacute and chronic phases of extracellular matrix and adventitial connective tissue formation (days 7 through 28).
We noted substantial variability in the intimal area between and within arteries as well as variability in the histological appearance of the restenotic lesion. The variability is postulated to have resulted from (1) inconsistent formation of dissection, (2) variable thrombus formation within dissection planes, (3) variable cellular proliferation rates, and (4) an unpredictable degree of elastic recoil and scar contraction. In addition, the arterial response to injury was superimposed on variable degrees of preexisting atherosclerosis, rendering the determination of the subsequent neointimal accumulation within individual arteries difficult to assess. In some arteries, the extent of neointimal accumulation may have been small in relation to the preexistent atherosclerotic area.
A rapid decrease in the radiographic MLD was noted in
the initial 3 days after angioplasty, which may represent
contributions from elastic recoil of the stretched normal segment as
well as the formation of thrombus within fracture planes leading to
decreased luminal diameter and wound contraction. A significant
decrease in the total number of cells in the intima and media was noted
on the third and fifth days, suggesting cell loss due to necrosis or
apoptosis. Concurrently, a threefold increase in BrdU labeling
of intimal and medial SMCs and macrophages was noted at 3 to 7
days. Since the cell cycle for SMCs is 24 to 30 hours,15
entry into S phase by some cells occurred early after injury. Although
focal areas of angioplasty-induced injury and DNA synthesis of
nonatherosclerotic media were observed and may represent a
source of accumulating SMCs, the greatest degree of BrdU uptake was
noted in SMCs and macrophages near a dissection (Fig 5) and may
herald the subsequent growth of the cells into the thrombotic
framework, as some dissections exhibited both thrombus as well as
cellular elements. A population of cells exhibited DNA synthesis but
did not stain with either RAM-11 or HHF-35 and may represent
endothelial cells, fibroblasts, or SMCs in the
synthetic phenotype possessing depolymerized actin (Fig 7).
Proliferation of a minimum of three cell types was accordingly
observed, which may contribute to variability in cellular
accumulation.
By 7 days after angioplasty, little evidence of thrombus was
observed, the artery was repaired without signs of the original
dissection, and the composition of the newly formed
neointima was primarily cellular, with little connective
tissue noted. By 14 days after angioplasty, the cellular density of the
intima decreased (Table 2) and an increased connective tissue
matrix
consisting of collagen and elastin fibers was observed. SMCs express
increased mRNA for collagen when density-arrested,16
and increased collagen synthesis has been observed when proliferating
SMCs reach confluence.17 18 By 14 days after
angioplasty,
the artery showed a decrease in radiographic lumen;
however, over the subsequent 14 days, the lumen either remained
constant or increased in size, mirroring observations in the rat model
of balloon injury.19 Insofar as markedly elevated
cholesterol levels were noted during the entire
postangioplasty period, it is not anticipated that the
histological or radiographic increase at 28
days resulted from lesion regression secondary to a decline in serum
cholesterol levels, although this possibility cannot be
excluded. The decrease in intimal area may have resulted from a
decrease in cellular elements within the lesion and an increase in
contracture resulting from the increased connective tissue
accumulation.
There was a significant relation between the radiographic
MLD and the morphometrically derived luminal area, although the
correlation coefficient was low, at r=.44. One possible
explanation for the low correlation coefficient includes the inherent
limitation of extrapolating a two-dimensional area from a single
radiographic image. Thus, an eccentric lesion of a given
morphometric MLD may be reflected by either a smaller
radiographic MLD if the x-ray beam is parallel to the
long axis of the lumen or a larger MLD if the x-ray beam is
perpendicular. Second, despite attempts to match
radiographic MLD with morphometric luminal area by use of
anatomic landmarks such as side branches as well as placement of the
surgical clip near the site of induced atherosclerosis,
sampling error was possible. Additional sampling error may have been
present, because the morphometric luminal area as shown in Table
5
represents the mean area of the sections with the greatest
amount of intimal mass. However, intimal mass may not necessarily
correlate with luminal area, owing to compensatory arterial
enlargement.20 A third possibility is related to the
presence of thrombus. Thrombus within dissection planes (Fig 2)
or
intraluminal thrombus would reduce the radiographic MLD; on
morphometric analysis, however, thrombus was excluded from
measurement, thereby yielding a larger morphometric MLD. Fourth,
fixation of the artery results in tissue shrinkage and may therefore
result in a smaller morphometric lumen than observed in vivo.
The finding that the radiographic loss of MLD was not
accompanied by a statistically significant increase in intimal area was
unexpected but confirms data published by other
investigators.20 21 By other indirect measures of
cellular
accumulation, including increased BrdU labeling, increased SMC
composition, increased absolute cell number of the
restenotic lesion, and the radiographic
appearance, an increase in neointimal area would be
expected. By comparing the radiographic luminal loss with
the morphometric area at euthanasia, a relative contribution of
neointimal formation to luminal loss can be grossly
calculated. This comparison is simplified, and it does not take into
account the arterial contraction at fixation or the
vasoreactivity at the time of euthanasia and assumes a concentric
lesion. Thus, it is intended only for illustrative purposes. The
radiographic MLD immediately after angioplasty was
1.01±0.08 mm in the 7-day group, with a MLD of 0.81±0.11 mm at
day 7.
The corresponding minimal luminal areas are, therefore, 0.95 and 0.52
mm2, respectively (Table 4), and the late luminal
area loss is 0.43 mm2. The increase in intimal area was
0.142 mm2 (Table 5). Hence, the intimal increase
represented only 33% of the loss of luminal area. The
neointima (the increase in measured intima after
angioplasty) contributed only 28% (0.142 of 0.500
mm2, Table 5) to the 7-day total intimal area of
0.500 mm2. Thus, with this simplified calculation, the
relative contribution of neointima at the point in time
when it is most cell-rich is approximately one third of the total
luminal loss and about one fourth of the resulting total intimal area.
For 14 days after angioplasty, the percentage of luminal loss caused by
intimal gain is similarly estimated at 24% and the contribution of
neointima to the resulting intima is 22%. The remaining
loss of MLD must have resulted from scar contraction and vascular
remodeling (Table 5). Given the multifactorial cause of
radiographic MLD reduction, it may be anticipated that a
treatment strategy designed to affect only SMC proliferation may not be
effective in reducing restenosis.
The lack of statistical significance in the morphometric determination
of the increase in intimal area reflects the large standard deviation
of the intimal area. By post hoc statistical analysis of the
variability, we determined that a sample size of 34 arteries would be
needed to show a statistically significant 50% increase in
neointimal area, rendering the sample size in the
present study insufficient to show a statistical difference. This
is consistent with the observations of Strauss et
al,14 in which a two-injury rabbit iliac model showed
a trend toward increasing intimal area 1 to 4 weeks after the
angioplasty, although statistical significance was not obtained until
12 weeks after angioplasty. The variability in the intimal area as well
as the ratio of intima to media within an artery may lead to false low
estimates of intimal area due to sampling bias (Fig 8) and
indicates
that a large number of sections should be evaluated for each artery,
with the section exhibiting the greatest amount of intima chosen to be
the representative section. The statistical
determination also indicates that large sample sizes (ie, >34 arteries
per group) are necessary to evaluate the effect of an agent to reduce
restenosis in this model.
The presence of adventitial inflammation and increased DNA synthesis
shortly after angioplasty suggests that adventitial fibroblasts also
may play a role in the restenotic process. An increased
density of circumferential adventitial collagen and intimal-medial
fibrosis were observed and may have played a role in subacute
vascular constriction. In specimens exhibiting severe injury, the EEL
was disrupted and connective tissue extended from the adventitia into
the intima and media and so contributed to the restenotic
intimal cell mass (Fig 4). Thus, it is possible that the
restenotic lesion derives not only from medial SMCs, but in
cases of extreme injury with adventitial dissection, from adventitial
fibroblasts that migrate into the media and/or neointima as
myofibroblasts. These cells may then undergo apoptosis when the
wound is healed, revert to a quiescent fibroblast form, or become SMCs
expressing -actin.22
Human restenosis is the vascular response of an atherosclerotic artery to angioplasty-induced injury. In the rabbit model characterized here, the initial fibrofatty atherosclerotic lesion is only an approximation of human atherosclerosis, which tends to be more fibrocalcific, contains lipid pools, and has areas of necrosis. The purpose of the present study was to evaluate the response of an atherosclerotic artery to balloon injury, and although the initial lesion may not represent human atherosclerosis in all its complexity, the arterial response to injury exhibits similarities to the human response noted at necropsy23 24 25 26 and in vivo intracoronary ultrasonography,27 28 29 in which plaque fracture, intimal compression, and vessel stretching have been shown after angioplasty.28 29 Tears or dissections involving the intima, media, and/or adventitia have been observed at necropsy in postangioplasty patients.23 24 25 26 Mural thrombus formation has been observed early,24 26 whereas neointimal growth has been observed at 11 days after angioplasty, with the incidence of neointimal proliferation subsequently increasing to 83%.24 The proliferating intima was seen predominantly around the circumference of the lumen and into the angioplasty-induced dissection, similar to the observations in the present study. These investigators24 also observed increased intimal collagen 6 months after angioplasty. Although it would appear that the sequence of vascular changes occurring in response to angioplasty injury in this model parallel the human process as observed in vivo and at necropsy, it is impossible to know with certainty in humans that these events occur in the months after angioplasty. An extension of the present study into a larger animal in which angioplasty of an atherosclerotic artery is used may aid in the determination of the universality of the proposed vascular response to angioplasty-induced injury and restenosis.
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Acknowledgments |
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This work was supported by a Grant-in-Aid from the American Heart Association, Indiana Affiliate (R.L.W.), and an educational grant from the Krannert Institute of Cardiology and Eli Lilly and Co. (K.L.M., R.L.W., I.G.-P., D.R.H.). Gratitude is hereby expressed to David Mendel and R. Gerald Dreesen, RRT, for technical assistance, Linda Smith in processing the histological samples, and Linda Rohyans for secretarial assistance.
Received February 9, 1994; revision received April 10, 1995; accepted April 17, 1995.
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