Apoptosis After Stent Implantation Compared With Balloon Angioplasty in Rabbits
Role of Macrophages
Abstract Both cell proliferation and apoptosis (programmed cell death) are supposed to play a role in restenosis after angioplasty. We studied these processes in smooth muscle cells (SMCs) and macrophages 1, 4, and 12 weeks after balloon angioplasty or Palmaz-Schatz stent implantation in rabbit iliac arteries. Proliferating cells were visualized by immunostaining with antibodies directed against proliferating cell nuclear antigen. Apoptotic cells were detected using the TUNEL (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) technique, propidium iodide staining, and transmission electron microscopy. At all time points, the neointimal cross-sectional area of the arteries was twofold to fourfold greater after stent implantation than after balloon angioplasty. The total number of neointimal cells was similar 1 and 12 weeks after both interventions. The neointimal cell density, however, decreased by 58% between the 1st and the 12th week after stent implantation compared with a 20% decrease after balloon angioplasty (P<.01). Stent implantation induced more cell proliferation but also more apoptosis in the media than balloon angioplasty after 1 and 4 weeks. In addition, stent implantation caused more macrophage accumulation and apoptosis in the neointima, but cell proliferation rates did not differ significantly in comparison with balloon angioplasty. The higher rate of apoptosis in the neointima 1 week after stent implantation compared with balloon angioplasty is due to an increased rate of SMC and macrophage death. Macrophage accumulation and apoptosis in the early phase after stent implantation appear to play a role in extracellular matrix secretion, which increases neointima formation after 4 and 12 weeks compared with balloon angioplasty in this model.
- Received February 10, 1997.
- Accepted June 30, 1997.
Numerous studies have shown that both balloon angioplasty and stent implantation induce neointimal hyperplasia, eg, by migration and proliferation of SMCs.1 2 3 4 5 These cells produce and also modulate the extracellular matrix in the newly formed intima.6 Moreover, monocytes/macrophages have been associated with neointima formation after stent implantation in rabbits.7
Apoptosis is a distinct form of programmed cell death involved in the development and homeostasis of tissues.8 9 10 11 This process has also been suggested to play a role in the regulation of intimal thickening in vessel wall lesions.12 13 14 In recent investigations, programmed cell death of SMCs in the neointima has been detected by the TUNEL method, PI, and TEM after arterial balloon injury in rats.12 14 The TUNEL method allows the identification of apoptotic cells by detecting DNA strand breaks.15 16 PI, which is generally used in cytology, strongly binds to nucleic acid and is excluded by an intact plasma membrane.17 18 19 With TEM, apoptotic cells are defined by their nuclear and cytoplasmic alterations.9 12 13 14 20 21 22 Apoptosis can be discriminated from necrosis by a cascade of morphologically characteristic steps including nuclear and cytoplasmic condensation, membrane budding, DNA fragmentation, and appearance of apoptotic bodies and their phagocytosis by neighbor cells or macrophages.9 12 13 14 20 21 22
The purpose of this study was to determine the influence of proliferation and apoptosis of SMCs and macrophages on neointimal lesion formation after stent implantation versus balloon angioplasty in iliac arteries of rabbits. The TUNEL method, staining with PI, and TEM were applied to detect apoptotic cells, while PCNA immunoreactivity was used to detect proliferating cells. These cells were further characterized as SMCs and macrophages by double immunostaining with α-actin or RAM 11 antibodies, respectively.
Animal Care and Surgical Procedure
All experiments were performed in accordance with the guidelines for animal research established by the American Heart Association and were approved by the state committee for animal research. New Zealand White rabbits of either sex weighing 2.5 to 3.0 kg housed individually were used for the study. Anesthesia was performed with ketamine (35 mg/kg) and xylazine (5 mg/kg). The protocol for angioplasty and stent implantation was described previously.24 In brief, both femoral arteries were exposed and ligated distally. Arteriotomies were performed, and a 4F pediatric sheath was inserted into each femoral artery. Heparin (500 U) and acetylsalicylic acid (60 mg) were given intra-arterially. An articulated Palmaz-Schatz stent was cut in two pieces at the bridging strut, and a single piece (7-mm length) was mounted on an angioplasty catheter. One iliac artery of the rabbits was balloon dilated to a diameter of 3 mm and the contralateral iliac artery was stented. The rabbits received a 60- mg dose of acetylsalicylic acid IM every third day for 4 weeks and were killed after 1 (n=5), 4 (n=5), and 12 weeks (n=5).
Tissue Collection and Fixation
The rabbits were killed by a lethal dose of sodium pentobarbital (120 mg/kg). The abdominal aorta was cannulated and the animals were exsanguinated by flushing with physiological saline solution at 100 mm Hg pressure. One half of the stented region and one half of the balloon-injured segments were cut off of each artery and immersed in 1.5% PFA and 1.5% glutaraldehyde overnight. The specimens were dehydrated with graded alcohols and embedded in epoxy-araldite resin (Serva). The second half of both stented region and balloon-injured arteries was cryopreserved. Before fixation, the stent wires were removed as described previously.25 The specimens were cut at −20°C into 6-μm cross-sections.
Cryosections were postfixed in 4% PFA (20 minutes, RT) and were treated with a permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate, 10 minutes, 4°C). Endogene peroxidase was blocked with 0.3% H2O2 solution (30 minutes, RT). The TUNEL reaction was performed by using an in situ cell death detection kit (Boehringer Mannheim). Antibody binding was visualized with diaminobenzidine (Pierce), yielding a brown color. In each experimental set up, a positive and negative control was carried along. After fixation and permeabilization, positive controls were treated with DNAse (1 mg/mL, 10 minutes, RT) to induce DNA strand breaks; negative controls were stained with labeling solution (without terminal transferase) instead of the TUNEL reaction mixture.
Specimens of the iliac arteries were incubated in a solution of PI (10 μg/mL, 5 minutes, RT) and cryopreserved for fluorescence microscopy. To obtain positive controls, pieces of a bland artery were preincubated with Triton (30 minutes, RT) and then incubated in PI solution (10 μg/mL, 5 minutes, RT). The nuclei of the apoptotic cells were identified by the presence of a strong red fluorescence.
To further characterize apoptotic cells, double staining of TUNEL-positive cells was performed using a mouse anti-rabbit SMC α-actin monoclonal antibody (Boehringer Mannheim, 1:800). A mouse anti-rabbit macrophage antibody (RAM 11, DAKO Corp, 1:500) was used to identify macrophages. The detection of proliferating cells was assessed by staining sections with a mouse antibody against PCNA (clone PC10, DAKO, 1:100). The tissue was incubated with primary antibodies for 1 hour in a humidified chamber at 37°C. Antibody coupling was achieved using the indirect biotin-streptavidin horseradish peroxidase (Amersham) or alkaline phosphatase (Sigma) methods. Antibody binding was visualized with diaminobenzidine (Pierce) for peroxidase yielding a brown color, and with fast red (Boehringer Mannheim) for alkaline phosphatase yielding a red color. Nuclei of the sections were counterstained with hematoxylin. Uninjured arteries, lung with alveolar macrophages, and ileum were used as positive controls. Omission of the first antibody abolished the immunohistochemical reaction completely and was used as negative control.
Histomorphometry and Cell Counting
Segments of stented and balloon-dilated arteries were sectioned into 70-μm slices with a rotating diamond-coated saw (Leica). After staining with toluidine blue, vessel perimeter and neointimal area were measured computer-assisted using a light microscope (Olympus) connected to a video camera (Sony) and a computer-based high-resolution digitizing image analyzer.24 Total cell number of neointima and media in three to six cross-sections was counted in each arterial specimen. Cell density was measured at ×150 light magnification in a 0.1-mm2 neointimal or medial area, respectively. The number of TUNEL-positive nuclei of SMCs and macrophages was expressed as percentage of the total number of SMCs and macrophages.
After fixation with 1.5% PFA and 1.5% glutaraldehyde solution, the arterial segments were postfixed in 1% osmium tetroxide and 0.1 mol/L cacodylic acid for 1 to 2 hours, dehydrated in graded ethanol baths, and embedded in epoxy-araldite. Specimens were sectioned into 500-μm slices and stent struts were removed. The segments were then re-embedded in epoxy-araldite, and four segments of each study group of the stented arteries were cut into 40- to 80-nm sections. The sections were mounted on copper nets and 2% uranyl acetate and lead citrate added for contrast. Then the specimens were examined on a Zeiss microscope (EM 10C/CR) at 80 kV accelerating voltage.
Analysis of Apoptosis by TEM
Cell counting and morphological analysis was performed from electron microscopic images at a magnification of ×4050. The morphological analysis of the cells was specified by detail pictures (×13 290). Apoptosis was defined according to criteria devised by Desmoulière et al.8
Data are presented as mean±SD. The two-tailed paired Student’s t test was used to compare group means. Comparisons of more then two means were performed with the unpaired Student’s t test and additional Bonferroni correction. A probability value of P<.05 was considered significant.
Morphometric measurement of cross-sections of the balloon-dilated or stented iliac arteries revealed differences in neointimal hyperplasia after 1, 4, and 12 weeks. The neointimal area increased continuously up to 12 weeks after stent implantation, whereas a peak of neointimal growth was found 4 weeks after balloon angioplasty. At all time points, the neointimal area was twofold to fourfold greater after stent implantation than after balloon angioplasty (Fig 1⇓).
Neointimal and Medial Cell Number
The number of neointimal cells per cross-section revealed a peak 4 weeks after both interventions (Table⇓). Numerous macrophages were located in the neointima after stent implantation, whereas no macrophages were observed after balloon angioplasty (Table⇓). The relation between SMCs and macrophages 1, 4, and 12 weeks after stent implantation is shown in Fig 2⇓.
The number of medial cells was twofold to threefold lower after stent implantation than after balloon angioplasty. At 1 week, 147±27 SMCs were found in the media after stent implantation, at 4 weeks 109±23, and at 12 weeks 199±47. After balloon angioplasty, the number of medial SMCs was 298±2 at 1 week, 323±21 at 4 weeks, and 309±47 at 12 weeks.
Neointimal Cell Density
Both interventions caused a similar cell density in the neointima after 1 week. Thereafter, the cell density of the neointima decreased continuously up to the 12th week after stent implantation, whereas a maximum was reached 4 weeks after balloon angioplasty (Fig 3⇓). Twelve weeks after stent implantation, the cell density in the neointima was significantly smaller than after balloon angioplasty (P<.01, Fig 3⇓).
Proliferation of Neointimal and Medial Cells
The rates of proliferating SMCs were similar in the neointima after both interventions. After stent implantation, the rate of proliferating SMCs decreased from 30±2% at 1 week to 8±3% at 4 weeks and to 1±0.4% at 12 weeks, respectively (P<.01, 1 week versus 4 and 12 weeks). After balloon angioplasty, it decreased from 28±2% at 1 week to 3±2% at 4 weeks and to 1±0.5% at 12 weeks (*P<.01, 1 week versus 4 and 12 weeks).
More proliferating SMCs were found in the media of stented arteries than of balloon-dilated arteries. After 1 and 4 weeks, the rate of proliferating SMCs was increased after stent implantation in comparison with balloon angioplasty (stent: 28±3% and 9±3% versus balloon: 14±3% and 1.0±0.7%, P<.01 stent versus balloon). After 12 weeks, 2±0.5% SMCs were proliferating in the media after stent implantation, whereas 1±0.5% SMCs were proliferating in the media after balloon angioplasty.
Apoptosis of Neointimal and Medial Cells
Neointima. One week after both interventions, TUNEL-positive cells were scattered within the rather small neointima (Fig 4A⇓ and 4C⇓). After 4 weeks, TUNEL-positive cells were more numerous in the center portion of the neointima (Fig 4⇓ B and 4D). After 12 weeks, TUNEL-positive cells were predominantly located in the subendothelium. The cells were identified as being SMCs or macrophages by double immunostaining (Fig 5⇓). After stent implantation, more TUNEL-positive cells were identified as macrophages compared with balloon angioplasty, and these cells were located around the stent struts and near the internal elastic lamina.
Morphometric measurement revealed that 1 and 4 weeks after stent implantation, more neointimal cells were undergoing apoptosis than after balloon angioplasty. The data of this analysis is shown in Fig 6⇓ and the Table⇑. The differentiation of the apoptotic cells revealed that more macrophages were apoptotic at 1 week than at 4 weeks after stent implantation, whereas no apoptotic macrophages were found after balloon angioplasty (Table⇑).
Media. In the media of stented arteries, 36±1% of the cells were TUNEL positive at 1 week, whereas only 4±1% were apoptotic after balloon angioplasty (P<.01). At 4 weeks, TUNEL-positive cells decreased to 19±0.1% after stent implantation and to 1±0.3% after balloon angioplasty (P<.01). At 12 weeks, 2±1% apoptotic cells were present in the media of stented arteries, whereas 0.2±0.1% apoptotic cells were found after balloon angioplasty.
We measured 93±22 PI-positive cells in the neointima 1 week after stent implantation. The number of PI-labeled nuclei increased to 123±24 PI-positive cells after 4 weeks. After balloon angioplasty, the number of PI-labeled nuclei increased from 30±11 at 1 week to 75±32 PI-positive cells in the neointima at 4 weeks.
Apoptosis of macrophages and SMCs was identified by a number of structural criteria including nuclear and cytoplasmic condensation, membrane budding, and cell fragmentation (Fig 7⇓). The mean number of evaluated cells per study group was 284±91. The rate of apoptotic cells was markedly higher after stent implantation than after balloon angioplasty. The rate of apoptosis assessed by TEM was 10% to 20% lower compared with the TUNEL technique.
In this study, stent implantation increased the rate of macrophage accumulation and apoptosis in the neointima of rabbit arteries in comparison with balloon angioplasty. This difference was associated not only with a reduction in neointimal cell density but also an increase in the amount of neointima formation. We conclude that a possible relation exists for macrophages and the accumulation of extracellular matrix in neointimal lesion formation after stent implantation.
Several authors suggested that SMC proliferation is one of the major reasons for neointimal hyperplasia after balloon angioplasty, as well as stent implantation.4 26 27 28 Our results corroborate results showing that SMC proliferation occurs after both procedures. A peak of proliferation of neointimal cells has been shown by Zeymer et al28 to occur 1 week after balloon denudation of the rat aorta. Using the same PCNA detection kit, we found comparing the proliferation rates at 1 week with those at later observation periods that most of the neointimal SMCs proliferate during the first week in rabbits.
It was previously indicated that balloon angioplasty can induce apoptosis in arteries.12 14 Our results further demonstrate that stent implantation causes more apoptosis in the media and neointima than balloon angioplasty. This result was reproduced using three different methods to detect cell death; namely the TUNEL technique, PI staining, and TEM. The TUNEL technique has been criticized for not being specific in the detection of apoptosis.29 30 31 However, Sanders and Wride16 showed that only 10% to 15% of TUNEL-positive cells had ultrastructural criteria not related to apoptosis when using a modified TUNEL method for electron microscopy. We found similar rates of cell death comparing the TUNEL technique with PI staining, but detected a 10% to 20% lower apoptotic cell rate by analyzing the TEM images. The number of apoptotic cells after balloon angioplasty detected with the TUNEL technique in our study was very similar to the results obtained by Han et al.14 Bochaton-Piallat et al12 reported lower apoptotic rates after balloon injury of the rat aorta. Kockx et al32 suggested that the TUNEL technique may underestimate the extent of apoptotic cell death in the atherosclerotic plaque. Therefore, the rate of vascular apoptosis detected by TUNEL may vary depending not only on the severity of the injury but also on the structure and type of the vessel, ie, whether it is elastic or muscular, and on arteriosclerotic disease. In concordance with previous results obtained by Han and colleagues,14 we found that the maximum rate of apoptosis occurs in the first week. It decreases to less than 3% within 12 weeks after arterial injury in the rabbit model. The present study shows that balloon angioplasty or the implantation of a stent directly initiates apoptosis and that the vascular repair mechanisms include the reduction of the cell death stimuli over time. Isner et al13 reported that a substantial amount of apoptosis persists in human restenotic lesions several months after angioplasty. Kockx et al32 showed that apoptotic cells can still be found in human saphenous vein grafts even 9 years after coronary artery bypass surgery. Thus, the time course and extent of vascular apoptosis in animal models of restenosis may be different from that in human restenotic lesions.
Stent Implantation and Balloon Angioplasty
Stent implantation induced more cell proliferation but also more apoptosis than balloon angioplasty in the media of the arteries. However, stent implantation caused markedly more apoptosis than balloon angioplasty in the neointima, whereas the proliferation rates of the neointimal cells were similar. Therefore, we could have expected a reduction in neointimal hyperplasia after stent implantation but found the contrary. Schwartz and coworkers33 observed that stent implantation increases the amount of neointima formation in comparison with balloon angioplasty. This result was corroborated in our previous experiments and in this study.24
Substantially more macrophages were located in the neointima after stent implantation compared with balloon angioplasty, which was also shown by Rogers and Edelman.7 This finding suggests that inflammatory mechanisms play a pivotal role in neointima formation after stent implantation. In addition, our results suggest that the apoptosis found in the neointima after stent implantation is largely due to macrophage death, whereas balloon angioplasty primarily induces SMC death in this model. The diminished neointimal cell density after stent implantation was associated with an increase in neointima formation, which suggests that a reduced secretion of matrix-degrading enzymes had occurred and more extracellular matrix accumulated. Chung et al34 recently proposed that extracellular matrix synthesis may be an important component of restenosis after stent deployment. We conclude that apoptosis of macrophages may play a pivotal role in neointima formation and may influence the production of extracellular matrix after stent implantation.
Selected Abbreviations and Acronyms
|PCNA||=||proliferating cell nuclear antigen|
|SMC||=||smooth muscle cell|
|TEM||=||transmission electron microscopy|
|TUNEL||=||terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling|
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (He 1603/2-1) and a grant from the Medical Faculty, University of Heidelberg, Germany (36/95). We would like to thank Silke Vorwald for her expert technical assistance.
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