© 1998 American Heart Association, Inc.
Original Contributions |
Apoptosis in Restenosis Versus Stable-Angina Atherosclerosis
Implications for the Pathogenesis of Restenosis
From the Department of Internal Medicine/Cardiology, University of Bonn, Bonn (G.B., R.H., B.L.); the Institute of Anatomy, University of Munich, Munich (S.S., U.W.); the Department of Molecular Pathology, Institute for Pathology, University of Tübingen, Tübingen (R.K.), Germany; and Metabolic and Cardiovascular Disease Research, Pharmaceutical Division, Novartis Corp, Summit, NJ (M.F.P.).
Correspondence to Gerhard Bauriedel, MD, FACC, Department of Internal Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str 25, D-53105 Bonn, Germany.
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Abstract |
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Abstract—Decreases in programmed cell death (apoptosis) may contribute to restenotic hyperplasia by prolonging the life span of intimal cells. Apoptotic events were compared in restenotic versus primary lesions, by using atherectomy samples from 16 restenotic and 30 primary human peripheral and coronary lesions from patients presenting with stable angina. We used transmission electron microscopy to identify apoptosis, quantify its frequency, distinguish apoptosis from necrosis, and relate these events to cellular composition. Smooth muscle cell (SMC) density was higher in restenotic versus primary lesions (P<0.0001), whereas the number of macrophages was significantly reduced (P<0.01) and the number of lymphocytes was lower, but not significantly (P=0.06). As the main finding, restenotic lesions contained fewer apoptotic cells compared with primary lesions (3% versus 13%, P=0.002), whereas no differences were found for cellular necrosis. With regard to cell type, the lower frequency of apoptotic cells observed in restenotic tissue was attributable to both SMCs and macrophages. The key finding of less apoptosis in restenotic versus primary lesions was in agreement with terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) analysis (2% versus 9%, P<0.001). For all lesions analyzed, significant inverse correlations were observed between the density of SMCs and the frequency of apoptotic cell death (r=-0.60, P<0.001) as well as the density of SMCs and that of macrophages (r=-0.74, P<0.001). No relationship was seen between the frequency of apoptosis and the density of macrophages. In conclusion, the data of the present study indicate that a low level of apoptosis may be an important mechanism leading to restenotic intimal lesion development after interventional procedures.
Key Words: apoptosis • atherosclerosis • necrosis • restenosis
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Introduction |
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Apoptosis, or programmed cell death, is thought to play a key role in the regulation of tissue mass.1 2 Previously, apoptosis has been extensively studied in the fields of cancer and immunity.2 3 4 Recent reports suggest that apoptosis may be a mechanism for the loss of myocardial cells in end-stage cardiomyopathy and arrhythmogenic right ventricular dysplasia.5 6 Apoptosis also appears to play a role in the regulation of neointimal cell number after balloon injury in the rat.7 8 Although the proliferative activity of human smooth muscle cells (SMCs) after angioplasty remains controversial,9 10 O'Brien et al10 have proposed that a mitigated cell death rate may accelerate the growth of restenotic tissue. Previous studies have demonstrated apoptosis in both atherosclerotic7 11 12 13 and restenotic13 human lesions. However, the extent that programmed cell death may play in lesion development is still unclear. Importantly, it has recently been shown that the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) technique, designated to identify apoptotic cells, can also label nonnuclear structures in atherosclerotic plaques and thus may signal false high levels of intimal apoptosis.14
The present study was designed to obtain more detailed information on the frequency of apoptotic events by comparing atherectomy tissue samples from primary stable atherosclerotic lesions and restenotic lesions. Transmission electron microscopy (TEM) analysis allows for careful documentation of various apoptotic events. With the use of TEM, the current study demonstrates that the frequency of apoptosis is lower in restenotic versus primary lesions. TUNEL labeling according to a modified technique recently described by Kockx et al14 was in agreement with these results. Our data indicate that a low intimal level of apoptosis is involved in human restenosis.
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Methods |
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Arterial Specimens
Percutaneous directional atherectomy was performed in patients presenting with stable angina attributed to the presence of stenotic primary atherosclerotic lesions or restenotic lesions after previous balloon angioplasty or atherectomy (2.2 to 30 months after the initial interventional procedure). Tissue samples were obtained from 18 coronary and 28 peripheral arteries, including 16 restenotic and 30 primary lesions (angiographic stenosis degree >75%). Atherectomy samples were obtained from 12 left anterior descending, 5 right coronary, 1 circumflex coronary, 25 superficial femoral, 1 iliac, and 2 popliteal arteries. Samples from 2 patients were studied from a primary as well as a subsequent restenotic lesion (the Table
: lesions 12 and 42 and
13 and 45). One lesion exhibited repeated restenoses and
therefore was treated twice with atherectomy (the Table, lesions 44 and
46). Restenosis was defined according to previously reported
clinical and angiographic criteria.13 15 Informed
consent for the analysis of tissue samples was obtained from
all patients prior to revascularization.
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Fixation and TEM
Immediately after percutaneous atherectomy, all
specimens were fixed in phosphate-buffered 3.5%
glutaraldehyde (pH 7.4). After 2 hours, the tissue was
placed in 1% glutaraldehyde. Additional fixation was
performed for 2 hours in 1% OsO4 in phosphate
buffer, followed by a thorough rinse in buffer. Samples were then
dehydrated twice through graded concentrations of alcohol and propylene
oxide. After additional incubation in a mixture of propylene oxide and
Araldite (1:1), the blocks were embedded in a mixture of Araldite,
(2-dodecen-1-yl)succinic anhydride, and accelerator. Tissue sections
(1.0 µm) were cut from the Araldite blocks and stained with
standard hematoxylin for light microscopy. Ultrathin sections were
contrasted with uranyl acetate dihydrate and 1% lead nitrate and were
viewed at 80 kV in a Philips CM 10 electron microscope.
Ultrastructural Analysis by TEM
TEM photomicrographs were evaluated quantitatively and
qualitatively. A primary magnification of x3600 was used.
Nonoverlapping images of randomly selected intimal regions were
photographically enlarged an additional 2.3x to a final magnification
of x8300. For each lesion studied, 8 photographs (17x21 cm each) were
taken, and 15 to 32 plaque cells were classified as SMCs,
macrophages, or lymphocytes. Altogether, recognition of >900
plaque cells was performed according to ultrastructural features, as
previously reported.16 17 18 19 The identification of
apoptotic cells and cellular necrosis was based on the specific
morphological criteria of apoptosis as defined in several
reports.1 20 21 22 The frequency of
apoptotic cells was calculated as the number of
apoptotic cells divided by the total number of cells for each
lesion. Morphometric evaluation was performed by 2 independent
investigators.
Assessment of Cell Density
Hematoxylin-stained histological sections
allowed the detection of cellular nuclei from intimal regions; adjacent
medial areas of the vessels were not analyzed. Assessment of
cell density was performed by a computer-assisted morphometry system
(VFG-1 graphic card) to count stained cell nuclei per area (0.04
mm2 ) and to calculate the final cell
density.23 The image from the microscope
(Optiphot-2, Nikon Inc) was relayed by a miniaturized video
camera/downstream monitor (KP-C-553-CCD, Hitachi Inc). Ten randomly
selected intimal areas, each encompassing 0.04
mm2, were assessed per tissue sample, and cell
counts were performed. Morphometric examination of intimal cellularity
and that of TUNEL labeling (see below) were performed by 2 independent
investigators.
TUNEL Assay
A limited number of atherectomy specimens were of sufficient
size to allow processing for both TEM and TUNEL evaluation. TUNEL
analysis was thus used on 15 primary and 7 restenotic
tissue samples to confirm the extent of apoptosis determined by
TEM evaluation. TEM evaluation was given preference, since it allows
evaluation of different forms of apoptosis, is extremely
helpful in distinguishing necrosis from apoptosis, and even
detects apoptotic cell death without nuclear condensation or
DNA fragmentation, as recently reported.24 25
Specimens used for TUNEL analysis were fixed in 4.5% formalin
and processed for paraffin embedding. In brief, after deparaffinization
and rehydration, tissue sections were incubated with 3% citric acid
for 1 hour according to the method described by Kockx et
al.14 22 The terminal
deoxynucleotidyl transferase (Tdt)–mediated TUNEL
test (In Situ cell death detection kit, AP, Boehringer
Mannheim) was performed according to the kit directions of the
manufacturer and was similar to previously published
protocols.12 13 For permeabilization, 20 µg/mL
of proteinase K in 10 mmol/L Tris HCl, pH 7.4, was applied for 10
minutes at 37°C. In the kit, Tdt, which catalyzes polymerization of
nucleotides to free 3'-hydroxyl DNA ends in a
template-independent manner, was used to label DNA strand breaks.
Incorporated fluorescein was detected by
anti-fluorescein antibody sheep Fab fragments conjugated
with alkaline phosphatase. After the substrate (fast red, Sigma
Chemical Co) reaction, stained cells exhibited distinct dark red
signals. Endogenous alkaline phosphatase activity was
blocked by 3 mmol/L levamisole included in the fast red solution.
Negative controls included omission of the enzyme Tdt as well as tissue
samples taken from nonimplanted saphenous vein grafts and normal
mammary arteries. Tissue from a tonsil served as a positive control.
The percentage of TUNEL-positive cells was determined as the number of
TUNEL-positive cells per total number of cells for each lesion, as
recently described.14 22
Statistical Analysis
For data analysis the SPSS/PC+ software
package for Windows 5.02 (Microsoft Inc) was used. Group differences in
densities of plaque cells, apoptotic cells, cellular necrosis,
and apoptotic bodies were evaluated by the Mann-Whitney
rank-sum test. Correlation coefficients were determined by Pearson's
test. All probability values were two tailed and corrected for ties.
Values of P<0.05 were considered significant. Group data
are given as mean±SD or mean±SEM, as indicated.
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Results |
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Atherectomy samples from primary coronary and peripheral lesions from patients presenting with stable angina were compared with those from restenotic lesions in regard to cell density, cell type, and frequency of cell death, specifically apoptosis and necrosis. In the Table
, age, sex,
and lesion characteristics of patients with primary and
restenotic lesions are outlined. Cell density was observed to
be higher in restenotic compared with primary lesions (623±62
versus 221±32 cells/mm2, P<0.0001;
Figure 1a). When lesion composition was
analyzed by TEM to identify SMCs, macrophages, and
lymphocytes, the cellular composition was found to differ between
primary and restenotic lesions. Although SMCs comprised the
largest cell population in both primary and restenotic lesions,
restenotic lesions were found to contain significantly more
SMCs than primary lesions (94% versus 81%, P<0.01; Figure 1b). In contrast, the number of macrophages and lymphocytes was
higher in primary lesions compared with restenotic lesions
(P<0.01 and P=0.06; Figure 1b).
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Apoptotic SMCs and macrophages, as identified by TEM
demonstration of cell shrinkage, chromatin condensation/margination, or
membrane budding, were observed in all primary and restenotic
lesions. Representative TEM photomicrographs are
illustrated in Figure 2. Importantly,
however, the frequency of apoptotic cells was found to be
significantly lower in restenotic versus primary lesions (3.2%
versus 12.8%, P=0.002; Figure 3a). With regard to cell type, this was
observed for apoptotic cells of both smooth muscle (2.8%
versus 10.8%, P=0.007) and macrophage (0.4% versus
2.0%, P=0.009) origin. Cellular necrosis, as defined by TEM
as a loss of cytoplasmic organelles and disrupted membrane integrity,
was also observed in all primary and restenotic lesions (Figure 4). In contrast to the frequency of cells
undergoing apoptosis, the frequency of necrotic cells appeared
to be similar in primary and restenotic lesions (10% versus
12%, NS; Figure 3b). TUNEL analysis of a subset of atherectomy
specimens from 22 patients confirmed the lower frequency of
apoptosis in restenotic (n=7) versus primary (n=15)
lesions (2.1±2.1% versus 9.2±3.8%, P<0.001).
Representative photomicrographs of TUNEL
immunohistochemistry illustrating fewer TUNEL-positive cells in
restenotic versus primary lesions are depicted in Figure 5. Sections from nonimplanted saphenous
vein grafts and normal mammary arteries did not show labeled nuclei (in
parallel with no detection of apoptosis by TEM).
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Statistical analysis of all quantitative data revealed a strong inverse correlation between the density of SMCs and the frequency of apoptotic cell death (r=-0.60, P<0.001) as well as the density of SMCs and that of macrophages (r=-0.74, P<0.001). In contrast, no relationship was seen between the frequency of apoptosis and the density of macrophages (r=0.03, P=0.83). In conclusion, the data from the present study indicate that a low level of apoptosis may be an important mechanism leading to hyperplastic restenosis after arterial interventions.
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Discussion |
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The present study compared apoptotic events in atherectomy-derived restenotic versus primary lesions from both the coronary and the peripheral vascular bed. Coronary lesions were assessed exclusively from patients who presented with stable angina. Patients with unstable angina were specifically excluded, since inflammatory cells are frequently found in atherectomy samples from this patient population compared with patients with stable angina,26 and cytokines have been demonstrated to induce apoptosis.12 26 27 As shown in Figure 1b
, SMCs were the predominant cell type in all primary and
restenotic lesions examined. SMC predominance and the
relatively low numbers of macrophages and lymphocytes for both
lesion types (Figure 1b) were also reported in a recent
immunohistochemical study of atherectomy
specimens.26
As the central finding in this study, we demonstrated that programmed
cell death occurs less frequently in restenotic compared with
primary lesions. Importantly, this conclusion was based on both TEM
determination of typical ultrastructural features of apoptosis
(Figures 2 and 3a) and immunohistochemical TUNEL labeling (Figure 5).
In contrast, the frequency of cell necrosis was evenly distributed in
primary and restenotic lesions (Figures 3b and 4). These data
reveal that apoptosis may be an important regulatory mechanism
in determining intimal cellularity, whereas necrosis does not appear to
play a regulatory role. As for primary atheromas, our
finding that the proportion of 9% apoptotic events determined
by TUNEL versus 13% apoptotic events determined by TEM is in
close agreement with a recent report showing 12% (in situ nick
translation–labeled elements in human carotid plaques rich in matrix
vesicles).14 Other reported percentages of
TUNEL-positive cells from human plaque tissue show remarkable
differences. Isner et al13 reported a low
apoptotic index of 0% to 6.6% in primary atherosclerotic
lesions. In contrast, Geng and Libby12 found an
apoptotic index of 34±5%. Han et al7
reported an apoptotic index ranging between 10% and 46%,
depending on plaque composition, with maximal levels for
macrophage-rich areas and the lowest levels in myxomatous
regions. However, several studies7 12 13 did not
employ TUNEL or in situ nick translation techniques, which includes
pretreatment with calcium-chelating agents or citric acid to exclude
nonspecific labeling of calcium-containing nonnuclear
structures.14 This pretreatment was
systematically employed in the current study.
Support for the propensity of atheromatous plaque cells
to undergo apoptosis also comes from a recent study on
(sub)occluded aortocoronary saphenous vein
grafts.22 Kockx et al22
reported a close spatial relationship between pronounced intimal SMC
loss, apoptotic cell death, and foam cell accumulation.
Interestingly, the ultrastructural features of smooth muscle
apoptosis, as outlined in our current article (Figure 2),
closely resemble the apoptotic SMCs found in diseased vein
grafts.22 However, whereas our data only
demonstrate a strong inverse relationship between SMC density and
apoptotic cell death, macrophage infiltration and
apoptosis did not appear to be correlated. Bennett et
al11 found that cultured plaque SMCs had a far
higher rate of spontaneous cell death in serum-deficient medium than
did SMCs from normal, nonlesioned arteries (17% versus 3%). Moreover,
several groups have reported that cultivation of primary plaque SMCs is
difficult, even with high serum and growth factor
concentrations.28 29 30 Taken together, these data
suggest a propensity of primary plaque tissue to undergo
apoptosis. The present labeling of 9% and 13%
apoptotic cells (by TUNEL and TEM, respectively) may be lower
than other indices reported recently7 12 13 14 but
may still be considered relatively high. An incomplete course of
apoptotic events and an impaired phagocytotic capacity for
apoptotic remnants in human plaque tissue have been suggested
to explain the phenomenon.12 22
The lower rate of apoptosis that we observed in restenotic lesions (2% by TUNEL and 3% by TEM) could be due to higher local concentrations of survival factors. Recently, the expression of platelet-derived growth factor-AA was reported to be far higher in restenotic lesions compared with primary lesions from patients with stable angina.26 31 The growth factors insulin-like growth factor-1, platelet-derived growth factor-AA, and platelet-derived growth factor-BB have been found to markedly suppress apoptosis of SMCs derived from both plaques and normal arteries.11 We18 and others19 have previously reported on other differences of cells obtained from primary and restenotic lesions. Ultrastructurally, restenotic SMCs were characterized by phenotypic alteration; ie, they revealed more synthetic organelles than did SMCs of primary lesion origin, suggestive of a larger susceptibility to growth-promoting stimuli.18 19 Also, SMCs cultured from restenotic lesions displayed both an increased migratory velocity29 and a higher rate of proliferation30 than did SMCs from primary lesions.
The relationship between the rates of cell proliferation and
those of apoptosis is not yet clear and may vary between cell
types or disease states. In several forms of cancer, the rate of
proliferation has been shown to be positively correlated with the rate
of apoptosis.2 32 Indeed, a high rate of
apoptosis was found to be related to disease progression and to
low survival probability.32 33 In contrast, in
chronic atherosclerosis the percentage of cells
undergoing proliferation appears to be low10
while the rate of apoptosis is quite
high.7 12 14 In restenosis, however, both
the degree of proliferation and that of apoptosis are
controversial. O'Brien et al10 reported 
1% of
proliferating cell nuclear antigen–positive cells in
restenotic lesions, whereas Pickering et
al9 reported 15% to 20% proliferating cell
nuclear antigen–positive cells. Likewise, in the present study we
report an average of 2% and 3% of cells (by TUNEL and TEM,
respectively) undergoing apoptosis in restenotic
lesions, whereas Isner et al13 reported an
incidence rate of up to 18%. Our present data are more
consistent with the paradigm that lower rates of
apoptosis result in hyperplasia, as originally postulated by
O'Brien et al.10 Also, our findings
indicate that the programmed form of cell death is diminished in the
late phase after angioplasty. This concept is supported by the
observation of high cell density found in restenotic tissue up
to 30 months after the initial intervention (the Table). There was only
a trend toward decreasing cellularity over time observed in plaques
studied 609 days after angioplasty, as recently
reported.34 Since both we18
and others9 34 35 36 have observed high cellularity
as a key finding in late human restenosis, a low level of
apoptosis is a potential mechanism to explain this lack of
decreased cellularity.
Another important argument originates from the apparent similarities observed between vascular healing after angioplasty/atherectomy and wound healing. Desmoulière et al37 recently demonstrated that apoptosis plays a role in the decrease in cellularity that occurs as granulation tissue evolves into a scar. Their data revealed that apoptosis of granulation tissue cells takes place essentially after wound closure and affects target cells consecutively rather than producing a single "wave" of cell disappearance. Indeed, the authors speculated that a lack of apoptosis could result in the establishment of a hypertrophic scar or keloid, both characterized by a high degree of cellularity.37
If our concept of low programmed cell death in the remodeling restenotic intima is valid, the development of antirestenotic approaches to enhance apoptosis may be possible. Since numerous agents have been reported to promote the occurrence of apoptosis, including NO38 and cytostatic agents such as protein kinase C inhibitors,39 local delivery of these agents may have a positive influence on remodeling. In addition, local somatic gene therapy using tumor suppressor genes, such as p53, or blockade of proto-oncogenes, such as bcl-x and bcl-2, may have beneficial effects.38 39 40 Most recently, the use of catheter-based radiotherapy has been propagated to inhibit restenosis in patients.41 42 Based on knowledge of radiation-induced apoptosis43 and on recent work from animal models in which neointimal formation and cellularity were markedly suppressed by a ß-particle–emitting stent,23 beneficial therapeutic effects of intracoronary radiation could be explained by an additional pro-apoptotic action. However, induction of apoptosis may also increase the higher basal levels of apoptosis in adjacent primary lesions. This would lead to a pronounced SMC loss in vulnerable regions of the atherosclerotic lesion and might result in plaque rupture and thrombosis.44
In summary, our finding that apoptosis, but not cell necrosis, was markedly lower in restenotic compared with primary lesions indicates that decreases in apoptosis may play an important role in restenotic lesion formation.
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Acknowledgments |
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This work was supported in part by the Deutsche Forschungsgemeinschaft grant Ba 1076/2–1 (to G.B.), the German Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie project 01 KV 9559 (to G.B.), and project 01 KV 9526 (to R.K.) and by the Bonner Forschungsförderung BONFOR 107/21 and 107/22 (to G.B.). We thank Susanne Dreher and Sabine Herzmann for their excellent assistance and the performance of the TEM analysis.
Received October 17, 1997; accepted February 3, 1998.
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