- Email: [email protected]
Evidence of apoptosis in human carotid atheroma
Evidence of apoptosis in human carotid atheroma M. M. Konstadoulakis, MD, PhD, G. D. Kymionis, MD, PhD, M. Karagiani, MD, PhD, V. Katergianakis, N. Doundoulakis, MD, PhD, V. Pararas, A. Koutselinis, MD, PhD, M. Sehas, MD, and P. Peveretos, MD, Athens, Greece Purpose: Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cellular development and homeostasis. In this study, we examined the role that apoptosis may have in the pathogenesis of atherosclerosis. Methods: We examined immunohistochemically 20 normal carotid arteries and the carotid arteries of 86 patients (mean age, 68 years; range, 52 to 80 years) who underwent atherectomy for primary atherosclerosis. The expression of two genes, BCL2, which inhibits apoptosis, and BAX, which induces apoptosis, was examined and correlated to the presence of risk factors that included hypertension, smoking, hyperlipidemia, and diabetes mellitus. Results: BAX expression was found in 26 of 86 cases (30%), and no immunoreactivity was found in the normal carotid specimens. BCL2 expression was not seen in any examined tissues (atherosclerotic or normal carotid arteries). Of the 26 patients who expressed the BAX gene, 22 were hypertensive (85%), and hypertension (>160/95 mm Hg) was present in 25 of 60 patients (41%) who did not express the BAX gene (p < 0.01). No significant correlation was found between the expression of the BAX gene and other risk factors (smoking, hyperlipidemia, diabetes mellitus) or presenting symptoms. Conclusions: In a significant number of stenosed carotid arteries (30%), we found no evidence of apoptosis suggested by the presence of BAX expression. Hypertension was more prevalent in those patients with BAX gene expression than in those patients without BAX gene expression. BCL2 expression, which inhibits apoptosis, was not found. Further study of this phenomenon may contribute to the discovery of new treatments for atherosclerosis. (J Vasc Surg 1998;27:733-9.)
Apoptosis plays an important role in the regulation of tissue development, differentiation, and homeostasis.1 Apoptosis differs morphologically from necrosis because cells exhibit cytoplasmic and nuclear condensation and then fragment into apoptotic bodies without membrane disruption. The apoptotic bodies then undergo phagocytosis by neighboring cells or macrophages without inducing an inflammatory response.2,3 Apoptosis has been From the Molecular Immunology Laboratory (Drs. Konstadoulakis, Kymionis, and Karagiani), the First Department of Propedeutic Surgery (Katergianakis, Pararas, and Peveretos), and the Third Department of Surgery (Sehas), Medical School of Athens, Hippokration Hospital; the Department of Vascular Surgery, Red Cross Hospital of Athens (Dr. Doundoulakis); and the Department of Forensic Medicine, University of Athens (Koutselinis). Reprint requests: M. M. Konstadoulakis, MD, PhD, Kalvou 24, 15452 Psyhiko, Athens, Greece Copyright © 1998 by The Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter. 0741-5214/98/$5.00 + 0 24/1/87947
studied extensively for its role in the immune response, tumor cell growth and regulation, and many other physiologic processes, such as the development of cartilage bone and gut and the remodeling of limb buds.4,5 Only a few studies have linked apoptosis to the cardiovascular system. Excessive accumulation of cells in the intima is believed to be a major cause of disease progression in atherosclerosis.6,7 This cell accumulation is attributed to an increased migration, to a proliferation of smooth muscle cells (SMCs), monocytes/macrophages, and T lymphocytes,8 or to both. Within the sclerotic region of the human atheromatous plaque, a low density of cells with some appearance of cellular debris is found. The process by which a cellular lesion evolves into an acellular sclerotic lesion is poorly understood but presumably involves extracellular matrix synthesis and cell deletion and could be initiated by a process caused by apoptosis. Apoptosis requires the expression of a specific set of genes, such as BCL2 and related genes.9,10 The product of the BCL2 gene, when elevated in cells, in vivo or in vitro, prevents the normal course of cell 733
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death that is induced by trophic factor deprivation or other stimuli without altering proliferation.11 Recently several other proteins that are homologous to BCL2 have been isolated, and their complementary DNAs have been cloned.10 One of these proteins is encoded by BAX and counteracts the effect of BCL2 on cell survival. The BCL2 to BAX ratio has been proposed to determine whether a cell undergoes apoptosis.12,13 In this study, we examined the BAX and BCL2 expression in patients with atherosclerosis to establish a possible role for these genes in the pathogenesis of this disease. MATERIALS AND METHODS Arterial specimens. Atherosclerotic plaques from human carotid arteries with atherosclerosis were obtained from patients who underwent carotid endarterectomy. All patients had stenosis >75%. The degree of stenosis was estimated directly with duplex examination. Peak systolic velocity that was more than 250 cm/sec, end diastolic peak velocity that was more than 100 cm/sec, and internal carotid/common carotid peak velocity ratio that was more than 1.8 were used to determine a >75% stenosis. Additional angiography was performed in several patients to ensure the result of duplex examination. Patient records were reviewed to identify a variety of information that included age, gender, presence of risk factors (hypertension, smoking, hyperlipidemia, and diabetes mellitus), and presenting symptoms. Twenty samples of normal carotid arteries, which were obtained from the Department of Forensic Medicine at the University of Athens, were used as controls. All of the samples were retrieved from persons with an average age of 18 years (range, 17 to 19 years) who died in car accidents with no medical history and no atherosclerotic changes in autopsy. All specimens were placed in fresh 4% (wt/vol) paraformaldehyde immediately after retrieval. After 2 hours the tissue was transferred to a 30% sucrosephophate buffer solution and then embedded in paraffin. For each tissue specimen, several 4 µm sections were cut. One section from each specimen was stained with hematoxylin and eosin for conventional light microscopic analysis. Immunohistochemical analysis. Paraffin tissue sections (5 µm) were mounted on polylysine-coated glass slides. Paraffin was removed from the slides when heated at 60° C for 10 minutes and then washed three times in xylene for 10 minutes each. After gradual hydration through graded alcohols, the sections were rinsed in distilled water for 5 min-
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utes. Slides then were incubated for 30 minutes in 0.3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. The sections were incubated in citrate buffer (0.01 mol/L: pH 6.0) for two 5-minute cycles in a microwave oven for antigen retrieval. For localization of the BCL2 and BAX protein, the sections were incubated for 60 minutes at room temperature with a 1:40 dilution and 1:1000 dilution, respectively, of the monoclonal antibody (Dakopatts, Glostrup, Denmark) and visualized by the indirect avidin-biotin procedure with 3,3diaminobenzidine tetrahydrochloric as the substrate chromogen. The immunostained slides were counterstained with hematoxylin for 1 minute. Normal lymph node tissue represented positive control for BCL2 immunostaining, and sections of normal breast epithelium that were positive for BAX expression were included as positive controls for BAX. Each positive control was consistently positive. Negative controls for BCL2 and BAX were obtained by substituting the primary antibody with the immunoglobulin fraction of nonimmune mouse serum. Negative control sections were unstained. Light microscopic analysis. On conventional hematoxylin-eosin stained sections, apoptotic cells were identified by the presence of pyknotic nuclei and nuclear fragments.14 The cells were isolated from neighboring cells as a result of the disruption of intercellular interactions. To verify that BAX gene had been expressed in apoptotic cells and to correlate the extent of apoptosis to the expression of BAX gene, sections that were consecutive to those stained with BAX antibody were stained with conventional hematoxylin-eosin stained sections (Fig. 1, B) and compared with sections that expressed the BAX gene (Fig. 1, A). Histologic analysis for immunostaining. All slides were evaluated for immunostaining in a blind fashion by a pathologist without knowledge of other clinopathologic data. Staining for both BCL2 and BAX was classified visually into a grading system on the following four-point scale: no staining present in any of the cells (-), weak staining in less than 20% of SMCs and macrophages (+), moderate staining in 20% to 50% of SMCs and macrophages (++), and intense staining in more than 50% of the cells (+++). To test the repeatability of this classification, another investigator evaluated the same slides without knowledge of the former classification or other data. Most of the slides (97%) were classified similarly by both investigators. When the investigators disagreed on a section, the views of a third pathologist were taken into consideration.
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A
B
Fig. 1. A, Expression of BAX gene (arrows) in a section of atheromatous carotid artery (×10). B, Consecutive section stained with haematoxylin-eosin (×10). A correlation exists between expression of BAX gene and identification of apoptotic morphologic changes (arrows).
From the atherectomy specimens, comparable areas were selected by cell type and cell number, which distinguished the following areas: macrophage/monocyte–enriched areas that displayed macrophage cells, foam cells, and other inflammatory cells; myxomatous areas that contained SMCs; and collagen-enriched sclerotic areas of low cellular density that were enriched with a dense sclera-like collagenous matrix.
Because atherosclerotic lesions are extremely heterogenous in nature, we selected these regions for comparative analysis. Statistical analysis. Statistical comparison of the immunostaining results with the presence of risk factors (hypertension, smoking, hyperlipidemia, and diabetes mellitus) and the presenting symptoms was performed by χ2 analysis.
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Fig. 2. Expression of BAX gene in smooth muscle cells (brown cytoplasm) of atheromatous carotid artery (×40).
Table I. Correlation between the extent of BAX gene expression and the risk factors Extent of BAX expression Weak (+) Moderate (++) Intense (+++)
No (26) 9 16 1
Hypertension (22) 6 15 1 p = 0. 18
RESULTS We found that 26 of 86 cases (30%) expressed the BAX gene. Of these 26 cases, 9 cases (34.6%) had slight staining (+), 16 cases (61.5%) had moderate staining (++), and 1 case (3.9%) had intense staining for BAX protein. A correlation between the expression of BAX gene (Fig. 1, A) and the identification of apoptotic morphology changes, pyknotic nuclei and nuclear fragments (Fig. 1, B) was found in all sections and regions that expressed the BAX gene. In sections that were unstained for BAX expression, both normal and atherosclerotic arteries, we found no apoptotic morphology in cells. Quantitative analysis revealed that the myxomatous area and the macrophage-enriched regions represented 30% of the lesion area and the remaining 70% of the hypocellular sclerotic area. The expression of BAX gene was area-dependent. In myxomatous areas, the expression of BAX gene was approximately 10% to 30% of SMCs (Fig. 2), and in macrophage-
Smoking (12)
Diabetes (3)
3 9 0 p = 0. 39
1 2 0 p = 0. 93
Hyperlipidemia (4) 1 3 0 p = 0. 80
enriched regions, the BAX gene expression was observed in 30% to 40% of macrophages (Fig. 3). None of the 20 samples of normal carotid arteries were BAX immunopositive. Not one of the examined tissues, both normal and atherosclerotic, was BCL2 positive. A comparison of these findings with the presence of risk factors, including hypertension, smoking, hyperlipidemia, and diabetes mellitus, showed a significant correlation only between BAX expression and hypertension. In our sample, 47 patients (55%) were hypertensive (>160/95 mm Hg), 40 (47%) were smokers, 11 (13%) had diabetes mellitus, and 15 (17%) had hyperlipidemia. Of the 26 patients who expressed the BAX gene, 22 were hypertensive (85%), and hypertension was present in 25 of 60 patients (41%) who did not express the BAX gene (p < 0.01). No significant correlation was found between the expression of the BAX gene and the other risk factors of atherosclerosis. Table I shows the correlation between the extent
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Fig. 3. Expression of BAX gene in macrophages (brown cytoplasm) of atheromatous carotid artery (×40).
Table II. Correlation between the extent of BAX gene expression and the symptoms Extent of BAX expression Weak (+) Moderate (++) Intense (+++)
No (26) 9 16 1
TIA (13)
AF (10)
CVA (2)
ASX (4)
5 7 1 p = 0. 14
2 7 1 p = 0. 28
1 1 0 p = 0. 23
2 2 0 p = 0. 59
of BAX expression and the risk factors. No significant correlation was found between the extent of BAX gene expression (weak, moderate, intense) and risk factors, such as hypertension (p = 0.18), smoking (p = 0.39), diabetes mellitus (p = 0.93), and hyperlipidemia (p = 0.80). The following presenting symptoms preceded each operation: transient ischemic attack in 40 patients (47%), amaurosis fugax in 22 patients (26%), cerebrovascular accident in 9 patients (10%), and no symptoms in 15 patients (17%). The expression of BAX gene was correlated with these presenting symptoms, and no significant correlation was found (Table II). DISCUSSION Intimal thickening has an important role in the proliferation of SMCs.15 In the last decade, we have learned a great deal about growth factors and cytokines that promote SMC proliferation.16 Yet advanced atherosclerotic lesions, such as atheroma,
contain few proliferating cells.17 Analysis of proliferation markers reveals a low rate of SMC proliferation that is similar to that of macrophages, which are the major cellular components of human atherosclerotic lesions. The initial cell migration and proliferation that is a result of injury to the vessel is believed to be a major cause of vascular disease.6 However, the accumulation of cells in the intima theoretically is the sum of cell migration and cell growth. Cell death and alteration of any of these events could affect the disease process. Apoptosis has been seen, for instance, in normal endothelial cells. The cycle of growth, remodeling, regression, and decay has been documented in aortic endothelial cells and SMCs of the incisal artery during tooth development. Recently the regulation of c-myc expression has been found to induce apoptosis in rat vascular SMCs.18 The BCL2 proto-oncogene was identified originally from a human chromosomal translocation that predisposed affected individuals to malignant trans-
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formation of immune cells. Increased expression of BCL2 enhances the cellular survival of immune cells,19,20 hematopoetic cells,21 and neurons.22 Bennett et al.23 showed that human vascular SMC apoptosis is suppressed by the expression of the BCL2 gene. The BCL2 product is a membrane-associated protein that suppresses apoptosis in several different cell types. The mechanism of BCL2 action is unknown, but BCL2 expression can inhibit apoptosis because of free radicals and lipid peroxidation, both of which are implicated in atherogenesis.24 Recently several other proteins that are homologous to BCL2 protein have been isolated, and their complementary DNAs have been cloned.9,10 As one of these proteins, BAX product is induced by growth factor depletion10 and promotes apoptosis. The homology among the BCL2-related proteins is concentrated in two domains, BH1 and BH2, that are required for their dimerization and function.25 In this study, we examined the possibility that apoptosis, in addition to cell proliferation, contributes as an important independent factor to the pathologic changes of human atheromatous lesions. We came to this conclusion by studying the expression of two genes, BCL2, which inhibits apoptosis, and BAX, which induces apoptosis. BAX expression was found in 26 of the 86 cases (30%). Depending on the type of the lesion (macrophage-rich or myxomatous area), 10% to 30% of SMCs (Fig. 2) and 30% to 40% of macrophages (Fig. 3) expressed the BAX gene. No immunoreactivity was seen in the normal carotid specimens. On the contrary, BCL2 expression was not seen in atherosclerotic arteries or normal carotid arteries. Han et al.26 reported similar results that showed that 10% of the cells in sclerotic lesions and 46% of the cells in the macrophage-rich area are TUNEL+ as found by in situ DNA endlabeling method. Geng and Libby27 reported evidence for apoptosis in coronary and carotid atheroma at the same rates. Also with an in vitro approach, Bennett et al.23 reported a higher rate of apoptosis in SMCs that were derived from human atheroma as compared with those from normal arteries. Isner et al.28 demonstrated that apoptosis is found more frequently in restenosis than in primary human vascular lesions. This finding is consistent with the notion that the restenotic lesions are typically more proliferate than primary lesions. In our study, similar findings were not seen because the rate of restenosis in carotid arteries is very small. Furthermore, attempts to augment apoptosis after a balloon injury constitute a theoretical approach to the prevention of restenosis. One such strategy that is currently under investigation
involves transfecting SMCs with the so-called ICE gene, which increases apoptosis in cells, or with other genes to expedite programmed cell death. This phenomenon may contribute to the discovery of new treatments for atherosclerosis. We were able to demonstrate that apoptosis is present in 30% of human atherosclerotic lesions and that the major cell types that undergo apoptosis are apparently SMCs and macrophages. These findings suggest that, in addition to cell migration and proliferation, apoptotic cell death also influences the progression of atherosclerosis. The identification of factors that influence the level of apoptosis in the human atherosclerotic lesion should contribute to a better understanding of vascular disease progression. In our study, a significant correlation (p < 0.01) between expression of the BAX gene and patients with hypertension was found. No similar correlation was observed between the BAX gene and the other risk factors. Atherosclerotic lesions that contain a high level of apoptotic cells, such as the areas enriched in macrophages within granulation tissues, may also represent regions of active vascular wound remodeling. Apoptosis is possibly a part of normal vascular wound healing, whereas dysregulated apoptosis and inefficient removal of apoptotic bodies may contribute to the progression of the atherosclerotic plaque and increase the severity of the disease. Thus excessive apoptosis without appropriate phagocytosis may contribute to additional macrophage recruitment and secretion of inflammatory cytokines. This contribution may induce increased cellular migration, proliferation, and other disease-complicating factors, such as the release of oxidized lipids that exacerbate the severity of atherosclerotic lesions. These data highlight a role for apoptosis in human atherosclerosis. Our results and those results that emerge from other laboratories broaden the traditional view that emphasizes the role of cell proliferation in atheromatosis. Apoptosis may have an important and independent role in the contribution of those pathologic changes that are characteristic of advanced atherosclerotic lesion. The pathobiology of human atherosclerosis should consider apoptosis as a novel mechanism that participates in plaque evolution. Further study of this phenomenon may contribute to the discovery of new treatments, such as gene therapy, for atherosclerosis. REFERENCES 1. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991;7:663-98.
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2. Kerr JFR, Harmon BV. Definition and incidence of apoptosis: an historical perspective. In: Tomes LD, Cope FO, editors. Apoptosis: the molecular basis of cell death. Plainview, NY: Cold Spring Harbor Laboratory Press; 1991. p.5-29. 3. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251-306. 4. Williams L, Bell LA. Synchronous development of the rat colon. Anat Embryol 1991;183:573-8. 5. Lewinson D, Silbermann M. Chondroblasts and endothelial cells collaborate in the process of cartilage resorption. Anat Rec 1992;233:504-14. 6. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990’s. Nature 1993;362:801-9. 7. Munro JM, Cotran RS. Biology of disease the pathogenesis of atherosclerosis: atherogenesis and inflammation. Lab Invest 1988;58:249-61. 8. Jonasson L, Holm J, Skalls O, Bondjercs G, Hansson GK. Regional accumulation of T cells, macrophages, and SMCs in the human atherosclerotic plaque. Atherosclerosis 1986; 6:131-8. 9. Boise LH, Gonzales-Garcia M, Postema CE, et al. Bcl-x, a bcl-2 related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993;74:597-608. 10. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programmed cell death. Cell 1993;74:609-19. 11. Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol 1994;124:1-6. 12. Barinaga M. Cell suicide: by ICE not fire. Science 1994;263:754-6. 13. Williams GT, Smith CA. Molecular regulation of apoptosis: genetic controls of cell death. Cell 1993;74:774-9. 14. Sugamura K, Makino M, Shirai H, Kimura O, Maeta M, Itoh H, et al. Enhanced induction of apoptosis of human gastric carcinoma cells after preoperative treatment with 5Fluorouracil. Cancer 1997;79:12-7. 15. Thyberg J, Hedin U, Sjolund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arterioscler Thromb 1990; 10:966-90. 16. Libby P, Clinton SK. Cytokines as mediators of vascular pathology. Nouv Rev Fr Hematol 1992;34:547-53.
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17. Gordon D, Reidy MA, Benditt EP, Shwartz SM. Cell proliferation in human coronary arteries. Proc Natl Acad Sci U S A 1990;87:4600-4. 18. Bennett MR, Evan GI, Newby AC. Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-γ, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ Res 1994;74:525-36. 19. Sentman CL, Shutter JR, Hochenberry D, Kanagawa O, Korsmeyer SJ. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 1991;67:879-88. 20. Henderson S, Rowe M, Gregory C, Croom Courter D, Warry F, Longnecker R, et al. Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 1991;65:1107-15. 21. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haematopoietic cell survival and cooperates with c-myc to immortalize pre B-cells. Nature 1988;335:440-2. 22. Garcia I, Martiinou I, Tsujimoto Y, Martinou JC. Prevention of programmed cell death of sympathetic neurons by the bcl2 protooncogene. Science 1992;258:302-4. 23. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest 1995; 95:2266-74. 24. Esterbauer H, Wag G, Puhl H. Lipid peroxidation and its role in atherosclerosis. Br Med Bull 1993;49:566-76. 25. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994;369:321-3. 26. Han DKM, Handenshild CC, Hong MK, Tjurmin A, Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol 1995;147:267-77. 27. Geng Y-L, Libby P. Evidence for apoptosis in advanced human atheroma: localization with interleucin - 1β - converting enzyme. Am J Pathol 1995;147:251-66. 28. Isner JM, Kearney M, Bortman S, Passer J. Apoptosis in human atherosclerosis and restenosis. Circulation 1995;91:2703-14.
Submitted June 12, 1997; accepted Nov. 28, 1997.
BOUND VOLUMES AVAILABLE TO SUBSCRIBERS Bound volumes of the Journal of Vascular Surgery for 1998 are available to subscribers only. They may be purchased from the publisher at a cost of $108.00 for domestic, $134.82 for Canadian, and $126.00 for international subscribers for Vol. 27 (January to June) and Vol. 28 (July to December). Price includes shipping charges. Each bound volume contains a subject and author index, and all advertising is removed. Copies are shipped within 60 days after publication of the last issue in the volume. The binding is durable buckram with the journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Subscription Services, Mosby, Inc., 11830 Westline Industrial Dr., St. Louis, MO 63146-3318, USA. In the United States call toll free 800-325-4177, ext. 4351. In Missouri or foreign countries call 314-453-4351. Subscriptions must be in force to qualify. Bound volumes are not available in place of a regular Journal subscription.
Apoptosis plays an important role in the regulation of tissue development, differentiation, and homeostasis.1 Apoptosis differs morphologically from necrosis because cells exhibit cytoplasmic and nuclear condensation and then fragment into apoptotic bodies without membrane disruption. The apoptotic bodies then undergo phagocytosis by neighboring cells or macrophages without inducing an inflammatory response.2,3 Apoptosis has been From the Molecular Immunology Laboratory (Drs. Konstadoulakis, Kymionis, and Karagiani), the First Department of Propedeutic Surgery (Katergianakis, Pararas, and Peveretos), and the Third Department of Surgery (Sehas), Medical School of Athens, Hippokration Hospital; the Department of Vascular Surgery, Red Cross Hospital of Athens (Dr. Doundoulakis); and the Department of Forensic Medicine, University of Athens (Koutselinis). Reprint requests: M. M. Konstadoulakis, MD, PhD, Kalvou 24, 15452 Psyhiko, Athens, Greece Copyright © 1998 by The Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter. 0741-5214/98/$5.00 + 0 24/1/87947
studied extensively for its role in the immune response, tumor cell growth and regulation, and many other physiologic processes, such as the development of cartilage bone and gut and the remodeling of limb buds.4,5 Only a few studies have linked apoptosis to the cardiovascular system. Excessive accumulation of cells in the intima is believed to be a major cause of disease progression in atherosclerosis.6,7 This cell accumulation is attributed to an increased migration, to a proliferation of smooth muscle cells (SMCs), monocytes/macrophages, and T lymphocytes,8 or to both. Within the sclerotic region of the human atheromatous plaque, a low density of cells with some appearance of cellular debris is found. The process by which a cellular lesion evolves into an acellular sclerotic lesion is poorly understood but presumably involves extracellular matrix synthesis and cell deletion and could be initiated by a process caused by apoptosis. Apoptosis requires the expression of a specific set of genes, such as BCL2 and related genes.9,10 The product of the BCL2 gene, when elevated in cells, in vivo or in vitro, prevents the normal course of cell 733
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death that is induced by trophic factor deprivation or other stimuli without altering proliferation.11 Recently several other proteins that are homologous to BCL2 have been isolated, and their complementary DNAs have been cloned.10 One of these proteins is encoded by BAX and counteracts the effect of BCL2 on cell survival. The BCL2 to BAX ratio has been proposed to determine whether a cell undergoes apoptosis.12,13 In this study, we examined the BAX and BCL2 expression in patients with atherosclerosis to establish a possible role for these genes in the pathogenesis of this disease. MATERIALS AND METHODS Arterial specimens. Atherosclerotic plaques from human carotid arteries with atherosclerosis were obtained from patients who underwent carotid endarterectomy. All patients had stenosis >75%. The degree of stenosis was estimated directly with duplex examination. Peak systolic velocity that was more than 250 cm/sec, end diastolic peak velocity that was more than 100 cm/sec, and internal carotid/common carotid peak velocity ratio that was more than 1.8 were used to determine a >75% stenosis. Additional angiography was performed in several patients to ensure the result of duplex examination. Patient records were reviewed to identify a variety of information that included age, gender, presence of risk factors (hypertension, smoking, hyperlipidemia, and diabetes mellitus), and presenting symptoms. Twenty samples of normal carotid arteries, which were obtained from the Department of Forensic Medicine at the University of Athens, were used as controls. All of the samples were retrieved from persons with an average age of 18 years (range, 17 to 19 years) who died in car accidents with no medical history and no atherosclerotic changes in autopsy. All specimens were placed in fresh 4% (wt/vol) paraformaldehyde immediately after retrieval. After 2 hours the tissue was transferred to a 30% sucrosephophate buffer solution and then embedded in paraffin. For each tissue specimen, several 4 µm sections were cut. One section from each specimen was stained with hematoxylin and eosin for conventional light microscopic analysis. Immunohistochemical analysis. Paraffin tissue sections (5 µm) were mounted on polylysine-coated glass slides. Paraffin was removed from the slides when heated at 60° C for 10 minutes and then washed three times in xylene for 10 minutes each. After gradual hydration through graded alcohols, the sections were rinsed in distilled water for 5 min-
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utes. Slides then were incubated for 30 minutes in 0.3% hydrogen peroxide in methanol to quench endogenous peroxidase activity. The sections were incubated in citrate buffer (0.01 mol/L: pH 6.0) for two 5-minute cycles in a microwave oven for antigen retrieval. For localization of the BCL2 and BAX protein, the sections were incubated for 60 minutes at room temperature with a 1:40 dilution and 1:1000 dilution, respectively, of the monoclonal antibody (Dakopatts, Glostrup, Denmark) and visualized by the indirect avidin-biotin procedure with 3,3diaminobenzidine tetrahydrochloric as the substrate chromogen. The immunostained slides were counterstained with hematoxylin for 1 minute. Normal lymph node tissue represented positive control for BCL2 immunostaining, and sections of normal breast epithelium that were positive for BAX expression were included as positive controls for BAX. Each positive control was consistently positive. Negative controls for BCL2 and BAX were obtained by substituting the primary antibody with the immunoglobulin fraction of nonimmune mouse serum. Negative control sections were unstained. Light microscopic analysis. On conventional hematoxylin-eosin stained sections, apoptotic cells were identified by the presence of pyknotic nuclei and nuclear fragments.14 The cells were isolated from neighboring cells as a result of the disruption of intercellular interactions. To verify that BAX gene had been expressed in apoptotic cells and to correlate the extent of apoptosis to the expression of BAX gene, sections that were consecutive to those stained with BAX antibody were stained with conventional hematoxylin-eosin stained sections (Fig. 1, B) and compared with sections that expressed the BAX gene (Fig. 1, A). Histologic analysis for immunostaining. All slides were evaluated for immunostaining in a blind fashion by a pathologist without knowledge of other clinopathologic data. Staining for both BCL2 and BAX was classified visually into a grading system on the following four-point scale: no staining present in any of the cells (-), weak staining in less than 20% of SMCs and macrophages (+), moderate staining in 20% to 50% of SMCs and macrophages (++), and intense staining in more than 50% of the cells (+++). To test the repeatability of this classification, another investigator evaluated the same slides without knowledge of the former classification or other data. Most of the slides (97%) were classified similarly by both investigators. When the investigators disagreed on a section, the views of a third pathologist were taken into consideration.
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A
B
Fig. 1. A, Expression of BAX gene (arrows) in a section of atheromatous carotid artery (×10). B, Consecutive section stained with haematoxylin-eosin (×10). A correlation exists between expression of BAX gene and identification of apoptotic morphologic changes (arrows).
From the atherectomy specimens, comparable areas were selected by cell type and cell number, which distinguished the following areas: macrophage/monocyte–enriched areas that displayed macrophage cells, foam cells, and other inflammatory cells; myxomatous areas that contained SMCs; and collagen-enriched sclerotic areas of low cellular density that were enriched with a dense sclera-like collagenous matrix.
Because atherosclerotic lesions are extremely heterogenous in nature, we selected these regions for comparative analysis. Statistical analysis. Statistical comparison of the immunostaining results with the presence of risk factors (hypertension, smoking, hyperlipidemia, and diabetes mellitus) and the presenting symptoms was performed by χ2 analysis.
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Fig. 2. Expression of BAX gene in smooth muscle cells (brown cytoplasm) of atheromatous carotid artery (×40).
Table I. Correlation between the extent of BAX gene expression and the risk factors Extent of BAX expression Weak (+) Moderate (++) Intense (+++)
No (26) 9 16 1
Hypertension (22) 6 15 1 p = 0. 18
RESULTS We found that 26 of 86 cases (30%) expressed the BAX gene. Of these 26 cases, 9 cases (34.6%) had slight staining (+), 16 cases (61.5%) had moderate staining (++), and 1 case (3.9%) had intense staining for BAX protein. A correlation between the expression of BAX gene (Fig. 1, A) and the identification of apoptotic morphology changes, pyknotic nuclei and nuclear fragments (Fig. 1, B) was found in all sections and regions that expressed the BAX gene. In sections that were unstained for BAX expression, both normal and atherosclerotic arteries, we found no apoptotic morphology in cells. Quantitative analysis revealed that the myxomatous area and the macrophage-enriched regions represented 30% of the lesion area and the remaining 70% of the hypocellular sclerotic area. The expression of BAX gene was area-dependent. In myxomatous areas, the expression of BAX gene was approximately 10% to 30% of SMCs (Fig. 2), and in macrophage-
Smoking (12)
Diabetes (3)
3 9 0 p = 0. 39
1 2 0 p = 0. 93
Hyperlipidemia (4) 1 3 0 p = 0. 80
enriched regions, the BAX gene expression was observed in 30% to 40% of macrophages (Fig. 3). None of the 20 samples of normal carotid arteries were BAX immunopositive. Not one of the examined tissues, both normal and atherosclerotic, was BCL2 positive. A comparison of these findings with the presence of risk factors, including hypertension, smoking, hyperlipidemia, and diabetes mellitus, showed a significant correlation only between BAX expression and hypertension. In our sample, 47 patients (55%) were hypertensive (>160/95 mm Hg), 40 (47%) were smokers, 11 (13%) had diabetes mellitus, and 15 (17%) had hyperlipidemia. Of the 26 patients who expressed the BAX gene, 22 were hypertensive (85%), and hypertension was present in 25 of 60 patients (41%) who did not express the BAX gene (p < 0.01). No significant correlation was found between the expression of the BAX gene and the other risk factors of atherosclerosis. Table I shows the correlation between the extent
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Fig. 3. Expression of BAX gene in macrophages (brown cytoplasm) of atheromatous carotid artery (×40).
Table II. Correlation between the extent of BAX gene expression and the symptoms Extent of BAX expression Weak (+) Moderate (++) Intense (+++)
No (26) 9 16 1
TIA (13)
AF (10)
CVA (2)
ASX (4)
5 7 1 p = 0. 14
2 7 1 p = 0. 28
1 1 0 p = 0. 23
2 2 0 p = 0. 59
of BAX expression and the risk factors. No significant correlation was found between the extent of BAX gene expression (weak, moderate, intense) and risk factors, such as hypertension (p = 0.18), smoking (p = 0.39), diabetes mellitus (p = 0.93), and hyperlipidemia (p = 0.80). The following presenting symptoms preceded each operation: transient ischemic attack in 40 patients (47%), amaurosis fugax in 22 patients (26%), cerebrovascular accident in 9 patients (10%), and no symptoms in 15 patients (17%). The expression of BAX gene was correlated with these presenting symptoms, and no significant correlation was found (Table II). DISCUSSION Intimal thickening has an important role in the proliferation of SMCs.15 In the last decade, we have learned a great deal about growth factors and cytokines that promote SMC proliferation.16 Yet advanced atherosclerotic lesions, such as atheroma,
contain few proliferating cells.17 Analysis of proliferation markers reveals a low rate of SMC proliferation that is similar to that of macrophages, which are the major cellular components of human atherosclerotic lesions. The initial cell migration and proliferation that is a result of injury to the vessel is believed to be a major cause of vascular disease.6 However, the accumulation of cells in the intima theoretically is the sum of cell migration and cell growth. Cell death and alteration of any of these events could affect the disease process. Apoptosis has been seen, for instance, in normal endothelial cells. The cycle of growth, remodeling, regression, and decay has been documented in aortic endothelial cells and SMCs of the incisal artery during tooth development. Recently the regulation of c-myc expression has been found to induce apoptosis in rat vascular SMCs.18 The BCL2 proto-oncogene was identified originally from a human chromosomal translocation that predisposed affected individuals to malignant trans-
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formation of immune cells. Increased expression of BCL2 enhances the cellular survival of immune cells,19,20 hematopoetic cells,21 and neurons.22 Bennett et al.23 showed that human vascular SMC apoptosis is suppressed by the expression of the BCL2 gene. The BCL2 product is a membrane-associated protein that suppresses apoptosis in several different cell types. The mechanism of BCL2 action is unknown, but BCL2 expression can inhibit apoptosis because of free radicals and lipid peroxidation, both of which are implicated in atherogenesis.24 Recently several other proteins that are homologous to BCL2 protein have been isolated, and their complementary DNAs have been cloned.9,10 As one of these proteins, BAX product is induced by growth factor depletion10 and promotes apoptosis. The homology among the BCL2-related proteins is concentrated in two domains, BH1 and BH2, that are required for their dimerization and function.25 In this study, we examined the possibility that apoptosis, in addition to cell proliferation, contributes as an important independent factor to the pathologic changes of human atheromatous lesions. We came to this conclusion by studying the expression of two genes, BCL2, which inhibits apoptosis, and BAX, which induces apoptosis. BAX expression was found in 26 of the 86 cases (30%). Depending on the type of the lesion (macrophage-rich or myxomatous area), 10% to 30% of SMCs (Fig. 2) and 30% to 40% of macrophages (Fig. 3) expressed the BAX gene. No immunoreactivity was seen in the normal carotid specimens. On the contrary, BCL2 expression was not seen in atherosclerotic arteries or normal carotid arteries. Han et al.26 reported similar results that showed that 10% of the cells in sclerotic lesions and 46% of the cells in the macrophage-rich area are TUNEL+ as found by in situ DNA endlabeling method. Geng and Libby27 reported evidence for apoptosis in coronary and carotid atheroma at the same rates. Also with an in vitro approach, Bennett et al.23 reported a higher rate of apoptosis in SMCs that were derived from human atheroma as compared with those from normal arteries. Isner et al.28 demonstrated that apoptosis is found more frequently in restenosis than in primary human vascular lesions. This finding is consistent with the notion that the restenotic lesions are typically more proliferate than primary lesions. In our study, similar findings were not seen because the rate of restenosis in carotid arteries is very small. Furthermore, attempts to augment apoptosis after a balloon injury constitute a theoretical approach to the prevention of restenosis. One such strategy that is currently under investigation
involves transfecting SMCs with the so-called ICE gene, which increases apoptosis in cells, or with other genes to expedite programmed cell death. This phenomenon may contribute to the discovery of new treatments for atherosclerosis. We were able to demonstrate that apoptosis is present in 30% of human atherosclerotic lesions and that the major cell types that undergo apoptosis are apparently SMCs and macrophages. These findings suggest that, in addition to cell migration and proliferation, apoptotic cell death also influences the progression of atherosclerosis. The identification of factors that influence the level of apoptosis in the human atherosclerotic lesion should contribute to a better understanding of vascular disease progression. In our study, a significant correlation (p < 0.01) between expression of the BAX gene and patients with hypertension was found. No similar correlation was observed between the BAX gene and the other risk factors. Atherosclerotic lesions that contain a high level of apoptotic cells, such as the areas enriched in macrophages within granulation tissues, may also represent regions of active vascular wound remodeling. Apoptosis is possibly a part of normal vascular wound healing, whereas dysregulated apoptosis and inefficient removal of apoptotic bodies may contribute to the progression of the atherosclerotic plaque and increase the severity of the disease. Thus excessive apoptosis without appropriate phagocytosis may contribute to additional macrophage recruitment and secretion of inflammatory cytokines. This contribution may induce increased cellular migration, proliferation, and other disease-complicating factors, such as the release of oxidized lipids that exacerbate the severity of atherosclerotic lesions. These data highlight a role for apoptosis in human atherosclerosis. Our results and those results that emerge from other laboratories broaden the traditional view that emphasizes the role of cell proliferation in atheromatosis. Apoptosis may have an important and independent role in the contribution of those pathologic changes that are characteristic of advanced atherosclerotic lesion. The pathobiology of human atherosclerosis should consider apoptosis as a novel mechanism that participates in plaque evolution. Further study of this phenomenon may contribute to the discovery of new treatments, such as gene therapy, for atherosclerosis. REFERENCES 1. Ellis RE, Yuan JY, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991;7:663-98.
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2. Kerr JFR, Harmon BV. Definition and incidence of apoptosis: an historical perspective. In: Tomes LD, Cope FO, editors. Apoptosis: the molecular basis of cell death. Plainview, NY: Cold Spring Harbor Laboratory Press; 1991. p.5-29. 3. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251-306. 4. Williams L, Bell LA. Synchronous development of the rat colon. Anat Embryol 1991;183:573-8. 5. Lewinson D, Silbermann M. Chondroblasts and endothelial cells collaborate in the process of cartilage resorption. Anat Rec 1992;233:504-14. 6. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990’s. Nature 1993;362:801-9. 7. Munro JM, Cotran RS. Biology of disease the pathogenesis of atherosclerosis: atherogenesis and inflammation. Lab Invest 1988;58:249-61. 8. Jonasson L, Holm J, Skalls O, Bondjercs G, Hansson GK. Regional accumulation of T cells, macrophages, and SMCs in the human atherosclerotic plaque. Atherosclerosis 1986; 6:131-8. 9. Boise LH, Gonzales-Garcia M, Postema CE, et al. Bcl-x, a bcl-2 related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993;74:597-608. 10. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programmed cell death. Cell 1993;74:609-19. 11. Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol 1994;124:1-6. 12. Barinaga M. Cell suicide: by ICE not fire. Science 1994;263:754-6. 13. Williams GT, Smith CA. Molecular regulation of apoptosis: genetic controls of cell death. Cell 1993;74:774-9. 14. Sugamura K, Makino M, Shirai H, Kimura O, Maeta M, Itoh H, et al. Enhanced induction of apoptosis of human gastric carcinoma cells after preoperative treatment with 5Fluorouracil. Cancer 1997;79:12-7. 15. Thyberg J, Hedin U, Sjolund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arterioscler Thromb 1990; 10:966-90. 16. Libby P, Clinton SK. Cytokines as mediators of vascular pathology. Nouv Rev Fr Hematol 1992;34:547-53.
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17. Gordon D, Reidy MA, Benditt EP, Shwartz SM. Cell proliferation in human coronary arteries. Proc Natl Acad Sci U S A 1990;87:4600-4. 18. Bennett MR, Evan GI, Newby AC. Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-γ, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ Res 1994;74:525-36. 19. Sentman CL, Shutter JR, Hochenberry D, Kanagawa O, Korsmeyer SJ. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 1991;67:879-88. 20. Henderson S, Rowe M, Gregory C, Croom Courter D, Warry F, Longnecker R, et al. Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 1991;65:1107-15. 21. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haematopoietic cell survival and cooperates with c-myc to immortalize pre B-cells. Nature 1988;335:440-2. 22. Garcia I, Martiinou I, Tsujimoto Y, Martinou JC. Prevention of programmed cell death of sympathetic neurons by the bcl2 protooncogene. Science 1992;258:302-4. 23. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest 1995; 95:2266-74. 24. Esterbauer H, Wag G, Puhl H. Lipid peroxidation and its role in atherosclerosis. Br Med Bull 1993;49:566-76. 25. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994;369:321-3. 26. Han DKM, Handenshild CC, Hong MK, Tjurmin A, Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol 1995;147:267-77. 27. Geng Y-L, Libby P. Evidence for apoptosis in advanced human atheroma: localization with interleucin - 1β - converting enzyme. Am J Pathol 1995;147:251-66. 28. Isner JM, Kearney M, Bortman S, Passer J. Apoptosis in human atherosclerosis and restenosis. Circulation 1995;91:2703-14.
Submitted June 12, 1997; accepted Nov. 28, 1997.
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