Background Apoptosis that has been reported in human atherosclerosis may contribute to the remodeling of atherosclerotic plaques. The identification of specific markers for apoptosis in these plaques would permit the development of specific therapeutic strategies to limit their progression. Cysteine protease CPP-32 is essential for apoptotic death in mammalian cells and appears to be an attractive candidate.
Methods and Results We studied 12 atherosclerotic plaques from 12 patients who underwent carotid endarterectomy. Apoptosis was analyzed by in situ end labeling of fragmented DNA (TUNEL method) and corroborated by the presence of DNA fragmentation in agarose gel electrophoresis. CPP-32 was detected with the use of a specific monoclonal antibody, and its expression was compared with that of interleukin-1β–converting enzyme (ICE). We showed that CPP-32 was highly expressed in 10 of 12 atherosclerotic plaques and that it colocalized with apoptotic cells. Expression of ICE generally paralleled that of CPP-32, but ICE was also detected in plaques negative for CPP-32 and showing no apoptosis.
Conclusions CPP-32 is highly expressed within human atherosclerotic plaques and is closely related to apoptosis. This finding suggests that CPP-32 may be the ICE-like enzyme responsible for apoptosis in human atherosclerosis and opens new perspectives for the development of therapeutic strategies to alter the progression of this disease.
Apoptosis is a recognized pathophysiological phenomenon that contributes to the remodeling of human atherosclerotic plaques.1 2 3 4 5 6 Identification of specific markers for apoptosis in human atheroma is critical to the development of specific therapeutic strategies to control the progression of these lesions. Studies on the nematode Caenorhabditis elegans have identified a number of cysteine proteases, particularly CED-3, as key proteins for the induction of the apoptotic process.7 8 The cysteine protease interleukin-1β–converting enzyme (ICE), which is related to CED-3, has recently been detected in human atheroma,3 and it has been suggested that ICE, or an ICE-like enzyme, may be responsible for apoptotic cell death in human atherosclerotic lesions.3 However, interventions on ICE would also modulate the production of interleukin-1β, a key cytokine of the immunoinflammatory response, and would therefore not be specific to the apoptotic process.
Recent results9 10 in mice with targeted disruption of ICE indicate that in vivo apoptosis is unaffected in these mice and that only in vitro apoptosis in response to the selective Fas-mediated pathway is altered. CPP-32 has been reported to be the ICE-like enzyme responsible for the initiation of apoptotic cell death in mammalian cells.11 12 This cysteine protease, which may be the human equivalent of CED-3, cleaves and inactivates with high efficiency and specificity poly(ADP-ribose) polymerase, an enzyme required for DNA repair and genome integrity.11 12 13 14 If CPP-32 is implicated in apoptotic cell death in human atherosclerotic plaques, specific pharmacological modulation may be of clinical importance in human atherosclerosis. Therefore, we analyzed human atherosclerotic plaques for the presence of apoptosis and the expression of CPP-32.
Twelve human atherosclerotic plaques removed from 12 patients undergoing carotid endarterectomy were collected. The arterial specimens were immediately placed for 2 hours in fresh 4% paraformaldehyde, then transferred to a 30% sucrose-PBS solution before being embedded in paraffin. Other adjacent arterial specimens were snap-frozen in optimal-cutting-temperature tissue-processing medium (OCT compound, Miles Inc Diagnostics Division) with liquid nitrogen and stored at −80°C for cryostat sectioning. For each specimen, several 5-μm sections were obtained for classic histological analysis, in situ detection of apoptosis, and immunohistochemical studies.
In Situ Detection of Apoptotic Cell Death
In situ detection of apoptotic cells (in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of fragmented DNA, or TUNEL) was performed according to Gavrieli et al.15 When paraffin sections were used, they were first deparaffinized, transferred to xylene, and rehydrated in descending alcohol series (100%, 95%, 70%, 50%, and 0%). After rehydration, the slides were incubated with 20 μg/mL proteinase K in PBS. When frozen sections were used, they were fixed for 15 minutes in 10% neutral buffered formalin at room temperature, washed once in PBS, and then transferred for 5 minutes in 0.1% Triton X-100 in 0.1% sodium citrate. For both paraffin and frozen sections, endogenous peroxidase was inactivated by treating the slides with 3% hydrogen peroxide. Tissue sections were stained by use of the ApopDETEK Cell Death In Situ Labeling and Detection System (Enzo Diagnostics, Inc) to detect cells showing internucleosomal DNA fragmentation. The procedure was performed according to the supplier’s instructions. The method is based on the preferential binding of terminal deoxynucleotidyl transferase (TdT) to 3′-OH ends of DNA.15 Briefly, residues of biotinylated dUTP were catalytically added to the ends of DNA fragments with the use of the enzyme TdT. For negative controls, deionized water was used instead of TdT. After end labeling, the sections were incubated with an avidin-biotin horseradish peroxidase and stained with diaminobenzidine to detect the biotin-labeled nuclei. Apoptotic bodies were identified by the presence of brown staining. Tissue sections of rat mammary glands obtained at the fourth day after weaning, purchased from Oncor, were used as positive controls. At least four sections from each specimen were analyzed. Ten random fields per section were examined at high magnification (×400) to determine the percentage of nuclei positively labeled for DNA fragmentation. In general, apoptotic cells were more easily detected in paraffin sections.
DNA Extraction and Electrophoresis
Fresh material from the 12 atherosclerotic plaques studied was used for DNA extraction. Five specimens of normal human internal mammary artery obtained during coronary bypass surgery were used as controls. For DNA extraction, tissues were homogenized in a solution of TRIzol reagent (1 mL/mg of tissue) (Life Technologies, Inc) and mixed with chloroform (0.2 mL/mL of TRIzol reagent). After phenol/chloroform extraction, DNA was precipitated with 100% ethanol, washed in a solution containing 0.1 mol/L sodium citrate in 10% ethanol, and then suspended in 75% ethanol. After centrifugation, the DNA pellet was briefly dried and dissolved in 8 mmol/L NaOH, then the pH was adjusted to 8 and the EDTA concentration to 1 mmol/L. Five micrograms from each sample was loaded into 1.5% agarose gel containing 0.5 μg/mL ethidium bromide. Electrophoresis was then conducted at 80 V for 2 hours.
Frozen sections were incubated with 1:50 vol/vol normal horse serum or 2% BSA in PBS for 30 minutes at room temperature, washed once in PBS, then incubated with either a primary mouse monoclonal antibody against CD68 for macrophage identification (DAKO-CD68, KP1), a primary mouse monoclonal antibody against human smooth muscle α-actin (HHF35, DAKO) for identification of smooth muscle cells, or a rabbit polyclonal primary antibody against T-cell CD3 (DAKO) for identification of T lymphocytes. These antibodies were used at a dilution of 1:200. To identify CPP-32 within atherosclerotic plaques, a specific mouse monoclonal antibody (Transduction Laboratories) was used at a dilution of 1:1000. To examine differences in expression between ICE and CPP-32, adjacent serial sections were stained with two rabbit polyclonal antibodies (Santa Cruz Biotechnology): one antibody specific for the precursor form of ICE (pro-ICE), ICE (A-19), and another antibody specific for the 10-kD subunit of ICE, ICEp10 (G-20). These antibodies were unlikely to recognize CPP-32. They were used at a dilution of 1:1000. After washing in PBS, the slides were incubated with secondary biotinylated antibodies: a biotinylated horse anti-mouse IgG (Vector Laboratories, Inc) at a dilution of 1:200 for detection of stains with antibodies against CD68, smooth muscle α-actin, or CPP-32 and a biotinylated goat anti-rabbit IgG at a dilution of 1:200 for the stains of anti-CD3, anti-ICE, and anti-ICEp10. Immunostains were visualized with the use of avidin-biotin horseradish peroxidase (brown staining) or alkaline phosphatase (red color) visualization systems (Vectastain ABC Kit standard and elite, Vector Red, Vector Laboratories). Irrelevant mouse IgG and normal rabbit serum were used for negative controls.
Frozen sections gave the highest quality for colocalization of CPP-32 within apoptotic cells. The samples were first immunostained with the anti–CPP-32 monoclonal antibody, and the stains were visualized by use of the alkaline phosphatase substrate system. The slides were then processed for TUNEL detection by use of the horseradish peroxidase substrate system. At least four sections from each specimen were analyzed. Ten random fields per section were examined at high magnification (×400) to calculate the percentage of stained cells.
Detection of Apoptosis and Protease CPP-32 in Human Atherosclerotic Plaques
Vascular cells of underlying normal arterial walls did not show TUNEL staining, and there was no CPP-32 expression in these regions. Conversely, significant apoptotic cell death (in >10% of cells) was observed in 10 of 12 atherosclerotic plaques (Table 1⇓; Fig 1A⇓). Apoptotic cells were preferentially located in the lipid core, in the fibrous cap, and at the junction with the normal media (shoulder of the plaque). CPP-32 expression was observed in the same plaques within the same regions and correlated with the presence of apoptosis (Table 1⇓; Fig 1B⇓).
The in situ detection of apoptosis was associated with the presence of oligonucleosomal DNA fragments on agarose gel electrophoresis (Fig 2⇓).
Colocalization of CPP-32 With Apoptotic Cells
To examine whether apoptotic cells expressed CPP-32 and vice versa, double staining was performed in the different atherosclerotic plaques. As shown in Fig 3⇓, immunoreactive CPP-32 was colocalized with apoptotic (positive TUNEL staining) macrophages (Fig 3A⇓), smooth muscle cells (Fig 3B⇓), and T lymphocytes (Fig 3C⇓), suggesting that CPP-32 may be responsible for apoptotic cell death in human atherosclerotic plaques.
Cell-Type Determination in Human Plaques
Analysis of serial sections and sections with double staining revealed that almost 50% of macrophages were apoptotic, whereas only a small percentage of smooth muscle cells were apoptotic (Table 2⇓). Apoptosis occurred frequently in T lymphocytes (Table 2⇓), particularly in case of massive infiltration of these cells.
We observed a number of nonapoptotic cells (TUNEL−) that stained positive for CPP-32. Adjacent sections showed that the majority of these cells were T lymphocytes and a few were macrophages (Table 2⇑). The TUNEL+ cells negative for CPP-32 were cell debris.
Expression of ICE in Human Atherosclerotic Plaques
Pro-ICE was expressed in both apoptotic and nonapoptotic cells in all specimens examined (data not shown). A more restricted expression of ICEp10 was observed, although it was detected in all 12 plaques. The distribution of ICEp10 paralleled that of CPP-32 in 9 of 12 atherosclerotic plaques. In 3 plaques, however, there were evident discrepancies between ICEp10 and CPP-32 expression (Fig 4⇓). Two plaques showed high levels of ICEp10 expression but were negative for CPP-32 expression (patients 3 and 7 in Table 1⇑). Interestingly, no significant apoptosis was present in these 2 plaques. In the remaining plaque (patient 8 in Table 1⇑), we also noted ICEp10+/CPP-32−/TUNEL− areas in conjunction with ICEp10+/CPP-32+/TUNEL+ areas.
Apoptotic cell death in human atherosclerotic plaques probably plays an important role in plaque remodeling, although it is not yet possible to conclude whether this process is beneficial or detrimental. The increased susceptibility to apoptosis in plaque-derived cultured smooth muscle cells is probably due to an intrinsic property of the cells and suggests that apoptosis is indeed the result of programmed cell death.1 5 The expression of ICE in advanced human atheroma also favors this hypothesis.3
Previous studies2 3 4 6 have shown that macrophages and smooth muscle cells make up the bulk of the apoptotic cells. In the present study, apoptotic cell death occurred mainly in macrophages and also involved a large percentage of T lymphocytes, particularly when major infiltrates of these cells were encountered. Although we found a number of apoptotic smooth muscle cells, the large majority of these cells were nonapoptotic, in agreement with a recent report by Björkerud and Björkerud.6 This finding may be related to loss of Fas expression in smooth muscle cells in human atherosclerotic plaques.16
Identification of key pathways leading to apoptosis in human atherosclerotic plaques is critical for the development of specific therapeutic strategies to modulate this process. It has been shown that an ICE-like enzyme, CPP-32/apopain, rather than ICE itself, is responsible for the initiation of apoptotic cell death in mammalian cells.12 Moreover, a recent report17 demonstrated that Fas induces a sequential activation of ICE-like and CPP-32-like proteases leading to apoptosis and that downstream CPP-32 is sufficient to cause apoptosis, even in the absence of ICE.
In the present study, we have shown that CPP-32 was highly expressed in human atherosclerotic plaques but was undetectable in the underlying normal arterial wall. CPP-32 was expressed in macrophages, T lymphocytes, and smooth muscle cells of human atheroma and was colocalized with apoptotic cells. The anti-CPP32 antibody used in the present study was directed against amino acids 1 to 219 of human CPP-32, covering both p17 and p12 subunits of CPP-32.12 Therefore, this antibody may recognize both activated and unactivated forms of the protease. However, several lines of evidence indicate that we may have detected CPP-32 under its active form: (1) CPP-32 was not detected in the underlying normal (nonapoptotic) arterial wall; (2) almost all apoptotic cells were positive for CPP-32; and (3) almost all nonapoptotic macrophages and smooth muscle cells and the majority of nonapoptotic T lymphocytes were negative for CPP-32. It is noteworthy, however, that a significant number of nonapoptotic T lymphocytes stained positive for CPP-32. Therefore, we cannot exclude the possibility that in these cells, a part of immunoreactive CPP-32 was inactive. This finding might be accounted for either by the presence of unactivated CPP-32 in these cells, the expression of anti-apoptotic proteins, or different kinetics of the apoptotic process.
The expression of CPP-32 generally paralleled that of ICEp10. However, ICEp10 was also detected in regions with no CPP-32 expression and no apoptosis, indicating that CPP-32 may be required for ICE-mediated apoptosis in this disease.
In conclusion, we have shown that CPP-32 is highly expressed in apoptotic cells within human atherosclerotic plaques and that this protease may be the ICE-like enzyme responsible for apoptosis in human atherosclerosis. These findings should open new perspectives for the development of therapeutic strategies to alter the progression of the disease.
This work was supported by grant CNAMTS/INSERM No. 4API12. Dr Mallat was supported by Assistance Publique, Hôpitaux de Paris, and by Institut Electricité Santé, Paris.
- Received December 9, 1996.
- Revision received February 3, 1997.
- Accepted February 7, 1997.
- Copyright © 1997 by American Heart Association
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