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(Circulation. 1995;91:2703-2711.)
© 1995 American Heart Association, Inc.

Articles

Apoptosis in Human Atherosclerosis and Restenosis

Jeffrey M. Isner, MD; Marianne Kearney, BS; Scott Bortman, MD; Jonathan Passeri, BS

From the Departments of Medicine (Cardiology) and Biomedical Research, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass.

Correspondence to Jeffrey M. Isner, MD, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail jisner@ opal.tufts.edu.

	Abstract

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Background Apoptosis has been recognized in normal, including rapidly proliferating, cell populations and is inferred to be potentially responsible for the maintenance of stable cell numbers in tissues with various degrees of proliferative activity. Previous studies performed in rats indicated that despite the persistence of a relatively high level of injury-induced proliferative activity, total arterial smooth muscle content at 12 weeks remained unchanged from that measured at 2 weeks, suggesting that accrual of vascular smooth muscle cells is mitigated by cell death. The extent to which apoptosis may be observed in human atherosclerosis and/or restenosis, however, has not been previously established.

Methods and Results We performed immunohistochemical studies on 56 specimens retrieved from patients undergoing directional atherectomy for primary atherosclerotic lesions or recurrent arterial narrowing after percutaneous revascularization (restenosis). Immunohistochemical staining disclosed evidence of apoptosis in 35 (63%) of the 56 specimens studied. When present, immunohistochemical evidence of apoptosis was typically limited to <2% of cells in the specimen. The finding of apoptosis, however, was not distributed equally among four groups of specimens studied. Specimens retrieved from patients with restenosis were more frequently observed to contain foci of apoptosis than specimens retrieved from patients with primary atherosclerotic lesions. Among 14 peripheral arterial specimens from patients with restenosis, 13 (93%) contained foci of apoptosis; in contrast, apoptosis was observed in only 6 (43%) of 14 peripheral specimens from patients with primary lesions (P=.0046). Among coronary arterial specimens, apoptosis was observed in 12 (86%) of 14 specimens from patients with restenosis versus 6 (29%) of 14 specimens from patients with primary obstructions (P<.0075).

Conclusions Apoptosis is a feature of human vascular pathology, including restenotic lesions and, to a lesser extent, primary atherosclerotic lesions. The findings of the present study suggest that apoptosis may modulate the cellularity of lesions that produce human vascular obstruction, particularly those with evidence of more extensive proliferative activity.

Key Words: apoptosis • programmed cell death • proliferation • BCL-2

	Introduction

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Apoptosis, a genetically encoded cell death program, was recognized nearly 30 years ago by Kerr¹ as a distinctive morphology that he called "shrinkage necrosis." Subsequent studies performed with Wyllie and Currie² disclosed evidence of such morphology in a wide range of pathological and normal tissues. Examinations performed on a variety of neoplasms led to the suggestion that apoptosis might account for much of the spontaneous cell loss previously documented in kinetic studies of many tumors.³ The recognition of apoptosis in normal, including rapidly proliferating, cell populations suggested a potential role in the maintenance of stable cell numbers in tissues with various degrees of proliferative activity.

Previous studies by Clowes et al⁴ in the balloon-injured rat carotid artery indicated that proliferative activity of vascular smooth muscle cells (SMCs) persisted at relatively high levels (3.8%) for up to 12 weeks after injury. In the absence of cell death, Clowes et al calculated that this level of ongoing proliferation should have led to a 36% increase in cell number. Total arterial SMC content at 12 weeks, however, was unchanged from that measured at 2 weeks. Because ". . . this increase was not observed," the authors concluded that "cell death must account for our finding." Subsequently, in vitro studies⁵ established evidence that rat vascular SMCs may indeed exhibit evidence of apoptosis. The extent to which apoptosis may be observed in human atherosclerosis and restenosis, however, has not yet been established.

Apoptosis was best characterized biochemically by the cleavage of genomic DNA into nucleosomal fragments of 180 base pairs (bp) or multiples thereof that are readily detected as a DNA ladder by gel electrophoresis of lysate from cells grown in culture.⁶ This approach is less applicable for examination of whole tissue specimens. Identification of apoptosis in tissue sections has been greatly facilitated by specific immunolabeling of nuclear DNA fragmentation with terminal deoxynucleotidyl transferase (TdT).⁷ This method, TdT/dUTP–biotin nick-end labeling (TUNEL) relies on incorporation of biotinylated deoxyuridine at sites of DNA breaks.

Accordingly, we performed immunohistochemical studies on 56 specimens retrieved from patients undergoing directional atherectomy for primary atherosclerotic lesions or recurrent arterial narrowing after percutaneous revascularization (restenosis). Apoptosis was in fact documented in both types of vascular lesions. While typically present at low frequency among cells that make up these excised tissues, apoptosis was observed more consistently in specimens retrieved from restenotic lesions. These findings suggest that apoptosis may modulate the cellularity of lesions that produce human vascular obstruction, particularly those with evidence of more extensive proliferative activity.

	Methods

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Tissue Specimens
A total of 56 tissue specimens were retrieved from 56 patients by directional atherectomy according to previously described techniques.⁸ An equal number of specimens retrieved from peripheral and coronary arterial sites were studied. Coronary specimens had been accessioned previously but not examined; specimens were thus included without knowledge of any light microscopic features. The only criterion for inclusion into this study of coronary lesions was that one half of the specimens represent primary lesions and one half represent restenotic lesions. Peripheral specimens were obtained prospectively. Criteria for inclusion were otherwise identical. A total of 28 lesions removed by directional atherectomy were obtained from patients undergoing percutaneous revascularization for the first time; these lesions were designated primary lesions. Another 28 lesions were identified at the site of a previous percutaneous intervention and were therefore designated restenotic lesions. In no case was light microscopic analysis of any feature, including apoptosis, performed with knowledge of either the clinical history (primary versus restenosis) or associated light microscopic findings. The Table summarizes clinical data for the 56 patients.

View this table: [in this window] [in a new window]   Table 1. Clinical Features and Pathological Findings in 56 Patients From Whom Vascular Specimens Retrieved by Directional Atherectomy Were Studied for Apoptosis

All coronary and peripheral specimens used in this study were placed in fresh 4% (wt/vol) paraformaldehyde immediately after retrieval of the specimen. After 2 hours, the tissue was transferred to a 30% sucrose–PBS solution before it was embedded in paraffin. In some cases of peripheral atherectomy, a portion of the typically larger tissue specimen was fixed in 100% methanol (rather than paraformaldehyde) before it was embedded in paraffin, and these tissues were used for immunohistochemistry to detect the proliferating cell nuclear antigen (PCNA).

For each tissue specimen, several 4-µm sections were cut. One section from each specimen was stained with hematoxylin and eosin and one with Richardson's combination elastic-trichrome stain for conventional light microscopic analysis. Additional sections were cut and left unstained for use in the immunohistochemical analyses described below.

DNA Nick-End Labeling of Tissue Sections
To detect apoptosis in situ, fragmented DNA was nick-end labeled with biotinylated dUTP introduced by TdT and then stained with avidin-conjugated peroxidase as previously described by Gavrieli et al.⁷ Briefly, tissues were deparaffinized and rehydrated by transferring the slides through the following solutions: xylene three times for 5 minutes, 100% ethanol two times for 2 minutes, 95% ethanol two times for 2 minutes, and finally PBS for 5 minutes. Nuclei were stripped of proteins by incubation with 20 µg/mL proteinase K (Sigma Chemical Co) for 10 minutes. Slides were then washed in dH₂0 twice for 5 minutes. Endogenous peroxide was blocked by covering the sections with 3% hydrogen peroxide for 5 minutes. After washing, slides were immersed in TdT buffer (30 mmol/L Trizma base, pH 7.2; 140 mmol/L sodium cacodylate; 1 mmol/L cobalt chloride). TdT (0.3 eu/uL) and biotinylated dUTP in TdT buffer were then added to cover the tissues sections and incubated at 37°C for 1 hour. One negative control slide per tissue was incubated in the absence of the TdT enzyme. The slides were washed in TB buffer (300 mmol/L sodium chloride, 30 mmol/L sodium citrate) for 15 minutes, followed by two washes in dH₂O for 5 minutes. Sections were then covered with 2% aqueous BSA for 10 minutes, rinsed in dH₂O, and immersed in PBS for 5 minutes. Next, the sections were incubated with an avidin-biotin complex (Vector Labs) for 30 minutes, immersed in dH₂O, and stained with 3,3'-diaminobenzidine (Biogenex Labs) for 10 minutes. One section of each tissue was processed as a positive control by pretreatment with DNase. Sections were deparaffinized and processed through proteinase K and 3% hydrogen peroxide as described above. Sections were pretreated with DN buffer (30 mmol/L Trizma base, pH 7.2; 140 mmol/L sodium cacodylate; 4 mmol/L magnesium chloride) for 5 minutes. DNase I, 1 µg/mL (Sigma) dissolved in DN buffer, was added to cover the sections. After 10 minutes, the slides were washed in dH₂O twice for 5 minutes and nick-end labeled as described above.

The age of the paraffin blocks used for TdT immunostaining ranged from 1 week to 15 months (mean, 5 months). Primary specimens were preserved in paraffin for a mean time of 6.9 months; for restenosis specimens, the mean interval was 3.7 months. Pilot studies performed on primary coronary tissues retrieved fresh and tissues embedded for >2 years showed apoptosis limited to <2% of cells in the specimen in all cases.

Immunohistochemistry
After DNA nick-end labeling, tissue sections in which apoptosis could be identified were further incubated with the mouse monoclonal antibody HHF-35 (Enzo Diagnostic) to identify SMC actin or HAM-35 (Enzo) to identify macrophages. Bound primary antibody was detected with labeled streptavidin biotin (Super Sensitive Immunodetection System, Biogenex).

The BCL2 antigen was unmasked by incubating sections in 10 mmol/L citric acid, pH 6.0, for 25 minutes at 98°C, followed by incubation for 15 minutes at room temperature. Sections were next incubated for 1 hour at 37°C with a mouse monoclonal antibody for the human BCL2 (Dako Corp) protein diluted 1:40 in 1% BSA:PBS. Negative controls were incubated with MOPC-21, a purified, nonspecific mouse monoclonal antibody (Sigma). Human tonsil was used as a positive control tissue. Bound primary antibody was detected with labeled streptavadin biotin (LSAB 2 Kit Alkaline Phosphatase, DAKO).

The p53 antigen was unmasked by incubating sections in 10 mmol/L citric acid, pH 6.0, for 25 minutes at 98°C, followed by incubation for 15 minutes at room temperature. Sections were then incubated for 1 hour at 37°C with a mouse monoclonal antibody for the human p53 (BioGenex) protein. A negative control slide for each tissue was incubated with MOPC-21. Operatively excised specimens from human colon cancer were used as a positive control. Bound primary antibody was detected with labeled streptavidin biotin.

Proliferative activity was evaluated by immunohistochemical analysis for PCNA (clone PC10, Signet) as previously described.⁹

Histological Analysis
Histological analysis was performed without knowledge of the clinical data. For those specimens in which apoptosis was identified, the total number of intimal cells for that section was manually counted. This procedure was facilitated by projection of the microscopic image (Bausch and Lomb Inc). If portions of the arterial media or adventitia were present on the tissue specimen, cells within these areas were excluded from manual counting. The number of intimal cells ranged from (1797±354, mean±SEM) for coronary specimens to 2833±866 for peripheral specimens. At least two observers reviewed all sections.

Transmission Electron Microscopy
Portions of tissue from 12 patients were fixed in 2.5% glutaraldehyde (pH 7.3) buffered with 0.1 mol/L sodium cacodylate overnight at 4°C and then washed with 0.1 mol/L sodium cacodylate buffer for 15 minutes before postfixation with 1% osmium tetroxide buffered with 0.1 mol/L sodium cacodylate for 1 hour on ice. After another wash with 0.1 mol/L sodium cacodylate buffer for 15 minutes, tissues were dehydrated with increasing concentrations of alcohol (30%, 50%, 70%, 80%, 90%, and 100%, three times at each concentration) for 10 minutes each. Next, each tissue was infiltrated with propylene oxide for 15 minutes, followed by 1:1 propylene oxide:epon for 1 hour, 1:2 propylene oxide:epon for 2 hours, and finally 100% epon for 2 hours. Tissues were embedded with fresh epon into molds and placed in a 60°C oven for 24 hours. Semithin sections were stained with toluidine blue, and three ultrathin sections of the areas of interest stained with uranyl acetate and lead citrate were examined per tissue with a Philips 300 electron microscope.

Statistical Analysis
Data are expressed as mean±SEM. Statistical significance was established by use of contingency table analysis. Statistical significance was inferred when the probability value was <.05.

	Results

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Apoptosis
Histochemical staining disclosed evidence of apoptosis in 35 (63%) of the total 56 specimens studied (Figs 1 through 3). The finding of apoptosis, however, was not distributed equally among the four principal groups of specimens studied. Specimens retrieved from patients with restenosis were more frequently observed to contain foci of apoptosis than were specimens retrieved from patients with primary atherosclerotic lesions. This was true whether the specimens were retrieved from peripheral or coronary arteries (Fig 1).

View larger version (0K): [in this window] [in a new window]   Figure 1. Photomicrographs showing apoptosis in human vascular lesions retrieved by directional atherectomy. This composite photograph illustrates the use of terminal deoxynucleotidyl transferase (TdT)–mediated dUTP-biotin nick-end labeling to immunostain sections from specimens representative of the four groups of lesions studied: peripheral restenosis (A through D), peripheral primary (E through H), coronary restenosis (I through L), and coronary primary (M through P). Positive staining appears as a yellow-brown reaction product. Occasional blue artifact (see K) serves as a consistent landmark in tissue section. Columns 1 and 2 were photographed at magnifications of x100 and x200, respectively. Column 3 shows a negative control (incubated in the absence of the TdT enzyme) for each specimen. Column 4 shows one section of each tissue processed as a positive control by pretreatment with DNase.

View larger version (0K): [in this window] [in a new window]   Figure 2. Photomicrographs showing representative results of double immunostaining. A, Terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling (TUNEL) and HHF-35 identify apoptotic nucleus (yellow-brown reaction product) of vascular smooth muscle cells (cytoplasmic red reaction product). B, TUNEL and HAM-56 identify the apoptotic nucleus of a macrophage by use of a similar color scheme. Analysis of a peripheral restenosis specimen for apoptosis and proliferative activity is shown. A specimen retrieved by atherectomy was divided in half; one portion was preserved in 4% paraformaldehyde and the other in 100% methanol. C, TUNEL immunostaining identifies multiple apoptotic cells. D, Methanol-preserved portion of a specimen immunostained with mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA). The persistence of proliferative activity is indicated by multiple positively stained cells.

View larger version (91K): [in this window] [in a new window]   Figure 3. Transmission electron photomicrographs showing sections taken from two restenotic peripheral lesions. A, The cell in the lower right corner illustrates early ultrastructural changes of apoptosis, including compaction of nuclear chromatin and cytoplasmic condensation. For comparison, no such changes are evident in the adjacent cell in the upper left corner. B, Similar ultrastructural changes of apoptosis in another cell. No apoptotic bodies were observed in either specimen. Note that the other cellular organelles such as the endoplasmic reticulum were preserved intact. Original magnification x6600 (A) and x8.900 (B).

Among 14 peripheral arterial specimens from patients with restenosis, 13 (93%) contained foci of apoptosis; in contrast, apoptosis was observed in only 6 (43%) of 14 peripheral specimens from patients with primary lesions (P=.0046).

Among coronary arterial specimens, apoptosis was observed in 12 (86%) of 14 specimens from patients with restenosis versus 6 (29%) of 14 specimens from patients with primary obstructions (P<.0075).

Among those specimens in which apoptosis was observed, histochemical evidence of nick-end labeling was typically limited to <2% of cells in the specimen. Among the 13 peripheral restenosis specimens in which apoptosis was recognized, for example, the frequency of apoptotic cells ranged from 0.04% to 12% (2.81±1.20); 10 (77%) of the specimens, however, contained <2% positively stained cells. Among 6 peripheral primary lesions with evidence of apoptosis, the frequency of positively stained cells was <2% in all cases.

Similarly, of 12 restenotic coronary specimens with foci of apoptosis, the finding of positively stained cells ranged from 0.10% to 18% (4.30±1.87). In 6 (50%) of these cases, the frequency of apoptotic cells was <2%. For primary coronary specimens with apoptotic foci, the frequency of positive cells was 6.6% in one case but <2% in the remaining 3 cases.

Immunohistochemical staining of adjacent sections and/or double immunostaining with HHF-35 or HAM-56 was performed to identify the nature of the apoptotic cell. For example, Fig 2A shows a double-immunostained section in which nick-end labeling identifies apoptosis of a vascular SMC, the cytoplasm of which was positively stained with HHF-35. Macrophages also contributed to apoptosis in some specimens, as illustrated by the double-immunostained section in Fig 2B, in which the nuclear yellow-brown reaction product indicative of apoptosis is surrounded by a rim of red reaction product, which in this case is the result of staining with HAM-56. Double immunostaining, however, failed to establish the identity of approximately 50% of apoptotic cells in a given tissue section; similar experience was reported with nonvascular tissues,¹⁰ apparently reflecting the impact of advanced cellular degeneration on cell-specific immunohistochemistry.

No positive immunostaining was observed in sections of operatively excised normal internal mammary artery or unused portions of saphenous vein.

Transmission electron microscopy was performed at three different levels of 12 atherectomy specimens to identify cells with ultrastructural changes of apoptosis. Fig 3A is a photomicrograph of a specimen retrieved from a restenotic peripheral lesion; the cell in the lower right portion illustrates early ultrastructural changes of apoptosis, including compaction of nuclear chromatin and cytoplasmic condensation. For comparison, no such changes are evident in the adjacent cell in the upper left portion. When such features were observed, other cellular organelles, as previously reported³ and illustrated in Fig 3B, often were preserved intact.

Relation of Apoptosis to Other Light Microscopic Findings
Apoptosis was most often observed in specimens with evidence of intimal hyperplasia, ie, foci of hypercellularity in which cells having phenotypic characteristics of proliferative vascular SMCs are associated with a loose extracellular matrix having tinctorial properties distinct from the matrix of primary atherosclerotic plaque.^{9 11 12 13} Intimal hyperplasia was characteristically observed in both coronary (12 of 14, 86%) and peripheral (9 of 14, 65%) restenosis specimens in the current study. Among the 12 coronary restenosis specimens with apoptosis, 11 contained foci of intimal hyperplasia; among the 13 peripheral restenosis specimens in which apoptosis was observed, intimal hyperplasia was present in 9.

Relation of Apoptosis to Evidence of Ongoing Cellular Proliferation
Immunostaining for PCNA was used to inspect selected tissue specimens for evidence of ongoing cellular proliferation. Only tissue preserved in methanol was evaluated in this fashion because our experience^{9 13 14} and that of others¹⁵ has been that alternative fixatives typically attenuate the antigenicity of PCNA. Consequently, this analysis was limited to certain peripheral specimens collected since the study was initiated, specifically those large enough to be divided into two portions, one to be preserved in paraformaldehyde and one in methanol. If one portion of a specimen disclosed evidence of apoptosis, the remaining portion frequently contained foci of PCNA-positive cells (Fig 2C and 2D).

Immunostaining for BCL2
The BCL2 gene has been regarded as the prototype of a family of genes that inhibit apoptosis¹⁶ but has not been reported previously to be present in vascular tissue. Immunohistochemical evidence of the BCL2 protein was identified in control sections of normal vascular tissues excised intraoperatively, including internal mammary artery and saphenous vein. Among vascular lesions retrieved by directional atherectomy, however, BCL2 protein was identified only in those that contained a portion of the media of the arterial wall and was always limited to the media component of the specimen. This was true regardless of the site (peripheral or coronary) or nature (restenosis or primary) of the excised lesion.

Immunostaining for p53
There is increasing evidence that the protein encoded by the tumor-suppressor gene p53 may induce apoptosis.^{17 18} In the present series of specimens, immunopositivity for p53 was in fact recognized in a subset of restenotic lesions, including those with apoptosis. These findings are consistent with the recent observation that immunopositivity for p53 may be identified in atherectomy specimens retrieved from restenotic but not primary lesions.¹⁹

	Discussion

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Apoptosis in Cardiovascular Tissues
Documented evidence of apoptosis in cardiovascular tissues has been limited. James²⁰ recently reviewed the extent to which apoptosis may be a factor in various cardiac disorders, including possible examples of apoptosis in SMCs of the media of a sinus node arteriole.²¹ In vitro studies established the principle that apoptosis could in fact be identified in both cardiomyocytes and vascular SMCs. Tanaka et al,²² for example, documented the finding of apoptosis by both immunostaining and gel electrophoresis in neonatal rat cardiomyocytes subjected to hypoxia. Gottlieb et al²³ used identical techniques, ie, immunohistochemical nick-end labeling and identification of nucleosomal ladders of DNA, to establish that reperfusion injury but not ischemic injury resulted in apoptosis of cardiac myocytes of New Zealand White rabbits.

Bennett et al⁵ established that apoptosis could be observed in vascular SMCs in vitro; apoptosis was recognized not only in cell populations with high proliferative activity (transfected cells constitutively expressing the proto-oncogene c-myc) but also in normal vascular SMCs deprived of serum. These findings led Bennett et al to suggest that apoptosis was likely to modulate the number of normal and neointimal vascular SMCs in the arterial wall. While Bennett et al observed cleavage of DNA into nucleosomal fragments in these in vitro studies, others^{24 25} demonstrated that DNA fragmentation is not necessary for occurrence of apoptosis.

Apoptosis in Excised Human Vascular Lesions
The present study establishes that apoptosis is indeed a feature of human vascular pathology. Apoptosis was identified by TUNEL of fragmented nuclear DNA.⁷ This technique has been used successfully to identify apoptosis in slowly proliferating cell populations such as liver epithelium, prostate, and adrenal cortex and in rapidly proliferating tissues such as intestinal crypt epithelium and spermatogonia.³ Only occasionally has the extent of apoptosis in these tissues or neoplasms been quantified.²⁶ However, even in cases in which the frequency of apoptotic cells is apparently low, eg, including many of the specimens described in the current report, previous reports emphasized that a small proportion of apoptotic cells visualized in tissue section can represent a considerable magnitude of cell loss.²⁶ This results primarily because the light microscopic appearance of apoptotic cells is limited to only a few minutes; even ultrastructural identification of apoptotic bodies may be seen for only a few hours before they are phagocytosed.⁷ Thus, relative to observations made in cell culture, the lower rates of cell turnover typical of even proliferative tissues compromise visualization of apoptosis in histological sections obtained from live organisms.

Apoptosis in Primary Atherosclerotic Lesions
Apoptosis was observed less consistently in primary atherosclerotic plaque than in restenotic specimens. The limited extent of apoptosis in primary atherosclerotic plaque is consistent with the results of previous pathological studies of primary atherosclerotic lesions obtained at necropsy^{27 28 29 30 31 32 33 34 35 36 37} or by directional atherectomy.^{11 19 38 39} Primary lesions are characterized by a paucity of cellular elements, consisting predominantly of well-organized collagen and ground substance. Among tissue specimens retrieved from 425 patients treated by directional atherectomy of de novo (primary) lesions as part of the Coronary Angioplasty Versus Excisional Atherectomy Trial (CAVEAT) study, 394 (93%) were judged to be hypocellular by light microscopy.⁴⁰ Similar findings were reported by Johnson et al.¹¹

Studies of cellular proliferation in primary lesions likewise indicated a low rate of proliferative activity. For example, Gordon and coworkers,⁴¹ used PCNA immunostaining to quantify proliferative activity in the coronary arteries of hearts explanted from patients undergoing cardiac transplantation; few of these patients had angina, and most had progressed to advanced heart failure. They observed that some atherosclerotic lesions (3 of 14) displayed no evidence of proliferation and that overall the proportion of PCNA-positive cells was low (mean, 0.85%). Subsequently, Pickering et al⁹ used PCNA immunostaining to study the proliferative activity in primary lesions retrieved by directional atherectomy from coronary or peripheral arteries of patients with active symptoms of ischemia; even in this actively symptomatic population, a fraction of PCNA-positive cells achieved a mean value of only 3.6%. Thus, given the low probability of visualizing apoptotic cells in tissues where the turnover rate of the cell population is very slow,⁷ the low frequency of apoptosis in primary atherosclerotic lesions is not surprising.

Apoptosis in Restenosic Lesions
The more frequent association between apoptosis and restenosis established in the present study is consistent with the concept that restenotic lesions are characteristically more proliferative than primary lesions. This concept is supported by necropsy reports of patients who died after multiple angioplasty procedures¹³ and of larger studies of restenotic lesions retrieved by directional atherectomy.^{9 11 19 38 39} Foci of hypercellularity within these specimens were interpreted as an indirect reflection of antecedent proliferative activity, a notion that was reinforced by experimental observations recorded in a variety of animal models.^{42 43 44 45 46} Indeed, in the present study, apoptosis was observed most frequently in the hypercellular tissue specimens typical of restenosis.

PCNA immunostaining has been used to assess the extent of proliferative activity that is ongoing when a patient is treated for restenosis with atherectomy.^{9 47} Because other preservatives attenuate the antigenicity of PCNA immunostaining,^{13 15} only tissues fixed in methanol can be used for this purpose. The TUNEL method used in the present study to label apoptotic cells has been optimized for tissues preserved in paraformaldehyde rather than in methanol. Whereas the typically larger peripheral atherectomy specimens can routinely be divided in two for differential fixation, this strategy is not appropriate for the smaller coronary specimens, particularly for a generally low-frequency event such as apoptosis. Therefore, in the present study, direct assessment of ongoing proliferative activity was restricted to analysis of peripheral specimens. PCNA-positive cells and cells with evidence for apoptosis were commonly observed in the same specimen. Because PCNA and TUNEL immunostaining was performed on two different pieces of a potentially heterogeneous lesion, however, it seems prudent to defer attempts to attach any further interpretation to these observations.

Identifying Apoptosis
Apoptosis has been characterized biochemically by activation of an endonuclease that cleaves the DNA of the cell at the linker regions between nucleosomes, yielding small double-stranded fragments of DNA 180 to 200 bp long. These fragments can be visualized as a series of bands ("oligosomal ladder") by agarose gel electrophoresis⁴⁸ ; such a ladder (as opposed to a continuous "smear") pattern has in fact been regarded as the most characteristic hallmark of apoptosis.^{3 23} Several laboratories, however, have now established that the classic DNA ladder may be delayed or absent in cell death that appears by other criteria to be apoptotic,^{3 24 25 49 50 51 52 53 54} leading to the conclusion that ". . . DNA degradation may be characteristic but not necessary [or sufficient] for the sequence of events leading to apoptosis."⁵⁴ These discrepant observations may relate in part to evidence that apoptosis in cells of epithelial mesenchymal origin may not involve DNA degradation into oligonucleosome-sized multimers and/or that the endonuclease(s) responsible for the 185-bp fragments vary from one cell type to another.⁴⁸ In fact, the only two previously published reports documenting apoptosis in vascular SMCs^{5 25} failed to identify DNA ladders in normal (ie, nontransformed) vascular SMCs, despite the fact that both studies involved cultured cells. This led Leszczynski et al²⁵ to speculate that ". . . several independent/interdependent pathways regulating apoptosis may exist, and the activation of some of these pathways may lead to DNA fragmentation, whereas activation of others may not."

An additional technical issue complicating the identification of DNA ladders is that agarose gel electrophoresis is not capable of detecting low-frequency breaks in low numbers of apoptotic cells.⁴⁸ This is a particular problem for the analysis of atherectomy specimens in which the average amount of DNA we can extract from these specimens is typically <10 µg and often <5 µg and the number of apoptotic cells is low.

Apoptotic bodies constitute the most characteristic morphological feature of apoptosis.^{3 55} Although apoptotic bodies have been recognized frequently in cell cultures, identification of apoptotic bodies in tissue section is limited.^{3 56} It is possible that the apparent difficulty of identifying apoptotic bodies in tissue sections is related to the fact that apoptotic bodies that arise in tissues are rapidly ingested by phagocytes and then the bodies undergo lysosomal degradation; in contrast, apoptotic bodies formed in cell cultures typically escape phagocytosis.³

For all these reasons, we sought to take advantage of the histochemical technique developed by Gavrieli et al⁷ to identify evidence of apoptosis in a series of prospectively retrieved atherectomy specimens. Although this technique has achieved acceptability as a marker of apoptosis,⁵⁵ it is fair to note⁴⁹ that the sensitivity and specificity of this technique remain to be further defined.

Study Implications
These preliminary findings establish that apoptosis is a feature of human vascular pathology. The finding that apoptosis is found more frequently in restenosis than primary human vascular lesions is consistent with the notion that restenotic lesions are typically more proliferative than primary lesions. Given that the light microscopic appearance of apoptotic cells is limited to only a few minutes, even the frequency of apoptotic cells detected in the current series may represent significant cell loss. It would thus appear that apoptosis might indeed act to modulate the potential contribution of cellular proliferation to lesion development, as Clowes et al⁴ implied and Bennett and coworkers⁵ suggested.

Furthermore, attempts to augment apoptosis after a balloon injury constitute a theoretical approach to the prevention of restenosis. For example, one such strategy currently under investigation in our laboratory is intended to transfect SMCs with the so-called ICE⁵⁷ gene as a means of expediting programmed cell death.

	Acknowledgments

 
This work was supported in part by grant HL-40518 and an Academic Award in Vascular Medicine (HL-02824) from the NHLBI, NIH, Bethesda, Md. We are indebted to Douglas Losordo, Gina Schatteman, Ken Walsh, and Lawrence Weir for helpful discussions regarding the work described in this article.

Received January 30, 1995; revision received March 9, 1995; accepted March 19, 1995.

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