CLINICAL STUDIES
Plaque inflammation in restenotic coronary lesions of patients with stable or unstable angina
Jan J. Piek, MD*  ,
Allard C. Van Der Wal, MD,
Martijn Meuwissen, MD* ,
Karel T. Koch, MD* ,
Steven A. J. Chamuleau, MD* ,
Peter Teeling, RT,
Chris M. Van Der Loos, PhD and
Anton E. Becker, MD, FACC
* Department of Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
Department of Cardiovascular Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
Manuscript received March 4, 1999;
revised manuscript received October 15, 1999,
accepted November 19, 1999.
Reprint requests and correspondence: Dr. Jan J. Piek, Department of Cardiology, B2-108, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands
j.j.piek{at}amc.uva.nl
 |
Abstract
|
|---|
OBJECTIVES
To evaluate immunohistochemically various parameters of inflammation in coronary atherectomy specimens obtained from restenotic culprit lesions of patients presenting with either stable or unstable angina (UA).
BACKGROUND
There is no information regarding the relationship between atherosclerotic plaque inflammation and the severity of the coronary syndromes in patients with restenotic coronary lesions.
METHODS
A total of 37 patients with either stable angina or UA underwent directional coronary atherectomy for restenotic coronary lesions. Cryostat sections of atherectomy specimen were immunohistochemically stained with monoclonal antibodies CD68 (macrophages [MACs]), CD3 (T-lymphocytes) and alpha-actin (smooth muscle cells [SMCs]). Smooth muscle cell contents and MAC contents were planimetrically quantified as the percentage immunopositive tissue area of the total tissue area. T-lymphocytes were counted at 100-x magnification throughout the entire section and expressed as number of cells per mm2.
RESULTS
Restenotic coronary lesions of patients with UA or stable angina showed no significant difference in SMC areas (31.9% ± 16.3% vs. 38.5% ± 18.8%, respectively; p = NS). However, restenotic coronary lesions of patients presenting with unstable angina contained significantly more MACs (24.4% ± 15.1% vs. 10.5% ± 5.8%, p = 0.001) and T-lymphocytes (18.8 cells/mm2 ± 15.1 cells/mm2 vs. 8.6 cells/mm2 ± 9.8 cells/mm2; p = 0.034) than patients with stable angina.
CONCLUSIONS
These results suggested that inflammation appears to affect plaque instability in restenotic coronary lesions resulting in unstable coronary syndromes.
|
Abbreviations and Acronyms
| | CCS | = Canadian Cardiovascular Society | | DCA | = directional coronary atherectomy | | MAC(s) | = macrophage(s) | | SMC(s) | = smooth muscle cell(s) | | REF | = reference diameter | | UA | = unstable angina |
|
Studies evaluating atherosclerotic plaque specimens retrieved by directional coronary atherectomy (DCA) have demonstrated differences in the extent of plaque inflammation between patients with stable and unstable angina (UA) (1–3). These studies provided further support to the contention that infiltration of macrophages (MACs) is crucial for the onset of unstable coronary syndromes (4–6). Thus far, these clinicopathologic relations between the type of angina and the extent of inflammation in culprit lesions are derived from patients with de novo lesions. Recent literature suggests an association between infiltrated plaque MACs and the development of restenosis after DCA (7). Tissues of patients with a restenotic lesion as their culprit have not been studied from this point of view. The aim of the study was to evaluate the relationship between the presence of inflammatory cells in restenotic lesions and the types of evolving ischemic coronary syndromes.
|
Methods
|
|---|
Patient population.
A total of 37 patients with a restenotic culprit lesion that was suitable for DCA were included. The patient cohort consisted of 18 patients with chronic stable angina, classes 1–3 according to the Canadian Cardiovascular Society (CCS) classification (8) and 19 patients with UA, classes I–III according to Braunwald’s classification (9).
Immunohistochemistry.
Immediately after the DCA procedure, the obtained tissue fragments were carefully oriented in embedding fluid (Tissue Tek; Sakura Finitek Europe BV, Zoeterwoude, The Netherlands) in such a way that the sites with the largest surface area were in the plane of cutting the sections. Thereafter, they were frozen in liquid nitrogen and stored at –80°C. Frozen sections were cut at 5 µm, one section was stained with hematoxylin-eosin for morphologic evaluation and adjacent serial sections were mounted for immunohistochemistry. The primary monoclonal antibodies used were anti-CD68 (Dakopatts, Glostrup, Denmark) for the detection of macrophages (MACs), anti-CD3 (Beckton and Dickinson, Mountain View, California) for the detection of T-lymphocytes and anti alpha-actin (SMA-1, Dakopatts, Glostrup, Denmark) for the detection of smooth muscle cells (SMCs). In both cases a three-step indirect peroxidase method was used as previously described (10), and antibody complexes were visualized by 3-amino-ethylcarbazole. No counter stain was used.
Morphometric analysis.
Results of anti-CD 68, anti-CD3 and anti-alpha-actin immunostaining were planimetrically quantified using image analysis software on a personal computer connected with a video-mounted microscope. The total tissue area of the immunostained sections of each atherectomy specimen was outlined manually on the video screen and measured. The tissue areas of the immunostained sections, which were occupied by immunopositive stained cells (MACs or SMCs), were measured automatically using gray scale detection with a fixed threshold. Thereafter, the SMC and MAC areas were calculated as percentages of the total tissue area. T-lymphocytes were counted at 100-x magnification throughout the entire section and expressed as number of cells per mm2. The pathologists were blinded to the results of the clinical classification of anginal symptoms.
Statistical analysis.
Data are expressed as mean ± SD. For comparison of clinical, angiographical and immunohistochemical data, an unpaired Student t test was used for continuous data and a chi-square test for categorical data. A Mann-Whitney U test was performed for nonparametric continuous data and a Fisher’s exact test for categorical data. We used multivariate linear regression to adjust differences in SMC, MAC and T-lymphocytes between patients with stable and unstable angina for difference in baseline characteristics between these patients groups. The SPSS package for Windows version 9.0 (SPSS Inc. 1999, Arlington, Virginia) was applied for these purposes. Values of p < 0.05 were considered statistically significant.
|
Results
|
|---|
Patient population.
Clinical and angiographical characteristics of the study patients are shown in Tables 1 and 2. Patients with UA were significantly older than patients with stable angina (p = 0.02); otherwise, the patient populations were comparable.
Quantitative immunohistochemistry.
Inflammatory cells, MACs (anti-CD68) and T-lymphocytes (anti-CD3) were found in the specimen of all patients but in highly variable amounts. Moreover, their tissue localization showed two distinct patterns. First, the inflammatory cells could be observed scattered or in small clusters in tissue fragments imposed of loosely arranged myxoid tissue also containing large amounts of alpha-actin positive SMC ("neointimal tissue"). Second, they were present in atheromatous tissue fragments as tight infiltrates. In this type of tissue, the CD68-positive MACs often had a foam cell appearance. A representative example of this feature is shown in Figures 1–3.
View larger version (101K):
[in this window]
[in a new window]
|
Figure 1 Part of an atherectomy specimen derived from a restenotic culprit coronary lesion of a patient with unstable angina, H&E (haematoxylin & eosin) stained frozen section. a: overview, boxed areas are shown in b and c. b: detail of hypercellular myxoid area showing stellate-shaped cells amidst abundant extracellular matrix. c. Detail of atheromatous tissue showing a rim of mononuclear cells near to atheroma (upper part). Magnification 22.5x, reduced by 70% (a) and 172.5x, reduced by 70% (b,c).
|
|
View larger version (105K):
[in this window]
[in a new window]
|
Figure 2 Serial section adjacent to Figure 1, immunostained with anti-alpha-SMC actin. b: detail of myxoid tissue showing a large amount of young stellate SMCs. c: detail of atheromatous tissue containing only a few SMCs. Magnification 22.5x (a) and 172.5x (b,c). SMC = smooth muscle cell.
|
|
View larger version (102K):
[in this window]
[in a new window]
|
Figure 3 Serial section adjacent to Figure 1, immunostained with anti-CD 68. Figure 3a: overview. b: detail of myxoid tissue showing low numbers of diffusely spread small MACs. c: detail of atheromatous tissue showing rim of closely packed immunostained MACs. Magnification 22.5x, reduced by 70% (a) and 172.5x, reduced by 70% (b,c). MAC(s) = macrophage(s).
|
|
Quantitative immunohistochemistry of restenotic coronary lesions of patients with unstable or stable angina showed no significant difference in SMC areas (31.9% ± 16.3% vs. 38.5% ± 18.8%, respectively; p = NS). However, restenotic coronary lesions of patients presenting with UA contained significantly more MACs (24.4% ± 15.1% vs. 10.5% ± 5.8%, p = 0.001) and T-lymphocytes (18.8 cells/mm2 ± 15.1 cells/mm2 vs. 8.6 cells/mm2 ± 9.8 cells/mm2; p = 0.034) than patients with stable angina (Fig. 4). We corrected these differences for the uneven age distribution between patients with stable and UA. This correction did not influence the observed differences.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 4 Percentages of immunostained smooth muscle cell areas and macrophage areas and number of T-lymphocytes/mm2 in restenotic coronary lesions underlying stable angina (CCS 1–3) and unstable angina (Braunwald I–III). Data are expressed as mean ± SD. CCS = Canadian Cardiovascular Society. Open bar = stable angina; closed bar = unstable angina.
|
|
|
Discussion
|
|---|
The results of this study demonstrated that there is a positive association between the clinical manifestation of coronary syndromes and the extent of inflammation (MAC and T-lymphocytes) in atherectomy specimens of patients with a restenotic lesion. This association could not be statistically attributed to the age difference between patients with stable and UA.
A study by Moreno et al. (1) on atherectomy specimens obtained from de novo lesions revealed that MAC infiltration of plaque tissue represents a marker for plaque instability. Subsequent studies of atherectomy specimens confirmed the relationship between plaque instability and unstable coronary syndromes by demonstrating increased amounts of T-lymphocytes and expression of human leukocyte antigen-DR molecules on cells (3), MACs producing the proteolytic enzyme gelatinase B (11) and MACs producing the thrombosis initiator tissue factor (12). In contrast, there is no information in the literature regarding the role of inflammation in restenotic coronary lesions. Thus far, clinicopathological studies, evaluating tissue specimens obtained by DCA, revealed SMC proliferation with concomitant extracellular matrix synthesis and marked expression of growth factors as principal features of restenotic lesions (2,12–16). The principal role of SMCs is in accordance with autopsy based studies, demonstrating the local wall response to mechanical injury after percutaneous coronary interventions (17–19). Indeed, it has been postulated that massive SMC proliferation with abundant extracellular matrix production could give rise to plaque expansions, which eventually could lead to symptoms of UA (13,15). This concept was based on similarities in SMC proliferation and the expression of the SMC growth factors aFGF and bFGF between lesions of patients with UA and patients with restenotic coronary lesions. Moreover, Chen et al. (15) found a similar phenotypic modulation of SMCs in patients with UA and postangioplasty restenosis using transmission electron microscopy. However, these patients with restenosis were not divided in the subgroups of stable and UA. More recent insight suggests that excessive SMC growth results in a preference for presentation of stable, rather than a presentation of unstable, coronary syndromes (20,21). Be that as it may, the fact remains that this study showed no relationship between the content of SMCs and the clinical presentation of anginal symptoms. Moreover, none of the previous reports quantified MAC density as a marker of plaque inflammation in patients with stable or UA as part of their restenosis. Our finding of large concentrations of inflammatory cells in atheromatous tissue and relatively small amounts in the classical type restenosis tissue suggested that the inflammatory activity results from preexisting plaque at the site of restenosis (plaque burden). One could hypothesize that these inflammatory cells destabilize the tissue of restenotic coronary lesions in a similar way as has been described for unstable primary coronary lesions.
In conclusion, our findings suggest that plaque instability relates to inflammation rather than to the extent of SMC proliferation. This observation provides a novel insight into pathophysiology of restenosis supporting the contention that plaque inflammation is pivotal in destabilization of restenotic coronary lesions resulting in unstable coronary syndromes.
Study limitations.
This study suggested that inflammation of restenotic lesions and, hence, its clinical expression relates to pre-existent de novo atherosclerotic plaque tissue before intervention. However, it cannot be excluded that the atheromatous tissue with inflammatory cells has developed after the interventional procedure, particularly not in patients with a long time interval between the initial intervention and the atherectomy procedure. Nevertheless, this limitation does not alter the conclusion regarding the positive association between the extent of MAC infiltration in restenotic coronary lesions and its clinical presentation.
|
Acknowledgments
|
|---|
The authors acknowledge the expert statistical assistance of Jan G. P. Tijssen (PhD, Department of Clinical Epidemiology and Biostatistics) and the skilled assistance of the technical and nursing staff of the Cardiac Catheterization Laboratory (chief: M. Meesterman).
|
Footnotes
|
|---|
Dr. J. J. Piek is clinical investigator for the Netherlands Heart Foundation (grant # D96.020).
|
References
|
|---|
1. Moreno PR, Falk E, Palacios IF, et al. Macrophage infiltration in acute coronary syndromes. Implications for plaque rupture. Circulation. 1994;90:775–778[Abstract/Free Full Text]
2. Arbustini E, De Servi S, Bramucci E, et al. Comparison of coronary lesions obtained by directional coronary atherectomy in unstable angina, stable angina and restenosis after either atherectomy or angioplasty. Am J Cardiol. 1995;75:675–682[CrossRef][Medline]
3. van der Wal AC, Becker AE, Koch KT, et al. Clinically stable angina pectoris is not necessarily associated with histologically stable atherosclerotic plaques. Heart. 1996;76:312–316[Abstract/Free Full Text]
4. Davies MJ, Richardson PD, Woolf N, et al. Risk of thrombosis in human atherosclerotic plaques: role of extracellular lipid, macrophage and smooth muscle cell content. Br Heart J. 1993;69:377–381[Abstract/Free Full Text]
5. van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994;89:36–44[Abstract/Free Full Text]
6. Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1996;91:2844–2850
7. Moreno PR, Bernardi VH, Lopez-Cuellar J, et al. Macrophage infiltration predicts restenosis after coronary intervention in patients with unstable angina. Circulation. 1996;94:3098–3102[Abstract/Free Full Text]
8. Campeau L. Grading of angina pectoris. Circulation. 1976;54:522–533[Medline]
9. Braunwald E. Unstable angina: a classification. Circulation. 1989;80:410–414[Free Full Text]
10. van der Loos CM, Das PK, van den Oord JJ, Houthoff HJ. Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies. J Immun Meth. 1989;117:45–52
11. Brown DL, Hibbs MS, Kearney M, et al. Identification of 92-kD gelatinase in human coronary atherosclerotic lesions. Association of active enzyme synthesis with unstable angina. Circulation. 1995;91:2125–2131[Abstract/Free Full Text]
12. Moreno PR, Bernardi VH, Lopez-Cuellar J, et al. Macrophages, smooth muscle cells and tissue factor in unstable angina. Implications for cell-mediated thrombogenicity in acute coronary syndromes. Circulation. 1996;94:3090–3097[Abstract/Free Full Text]
13. Flugelman MY, Virmani R, Correa R, et al. Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina. A clue to the mechanism of transformation from the stable to the unstable clinical state. Circulation. 1993;88:2493–2500[Abstract/Free Full Text]
14. Nikol S, Isner JM, Pickering JG, et al. Expression of transforming growth factor-beta 1 is increased in human vascular restenosis lesions. J Clin Invest. 1992;90:1582–1592[Medline]
15. Chen YH, Chen YL, Lin SJ, et al. Electron microscopic studies of phenotypic modulation of smooth muscle cells in coronary arteries of patients with unstable angina pectoris and postangioplasty restenosis. Circulation. 1997;95:1169–1175[Abstract/Free Full Text]
16. Taylor JA, Farb AA, Angello DA, et al. Proliferative activity in coronary atherectomy tissue. Clinical, histopathologic and immunohistochemical correlates. Chest. 1995;108:815–820[Abstract/Free Full Text]
17. Essed CE, van den Brand M, Becker AE. Transluminal coronary angioplasty and early restenosis. Fibrocellular occlusion after wall laceration. Br Heart J. 1983;49:393–396[Abstract/Free Full Text]
18. Ueda M, Becker AE, Fujimoto T. Pathological changes induced by repeated percutaneous transluminal coronary angioplasty. Br Heart J. 1987;58:635–643[Abstract/Free Full Text]
19. Glagov S. Intimal hyperplasia, vascular modeling and the restenosis problem. Circulation. 1994;89:2888–2891[Free Full Text]
20. Weissberg PL, Clesham GJ, Bennett MR. Is vascular smooth muscle cell proliferation beneficial? Lancet. 1996;347:305–307[CrossRef][Medline]
21. Lafont A, Libby P. The smooth muscle cell: sinner or saint in restenosis and the acute coronary syndromes? J Am Coll Cardiol. 1998;32:283–285[Free Full Text]
22. ACC/AHA Task ForceRyan TJ, Faxon DP, Gunnar RP. Guidelines for percutaneous transluminal angioplasty. J Am Coll Cardiol. 1988;12:529–545[Medline]
This article has been cited by other articles:
|
|
|
|
 
D. B Panagiotakos, K. Dimakopoulou, K. Katsouyanni, T. Bellander, M. Grau, W. Koenig, T. Lanki, R. Pistelli, A. Schneider, A. Peters, et al.
Mediterranean diet and inflammatory response in myocardial infarction survivors
Int. J. Epidemiol.,
June 1, 2009;
38(3):
856 - 866.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Wang, Z. Liu, Q. Li, M. Karpurapu, V. Kundumani-Sridharan, H. Cao, N. Dronadula, F. Rizvi, A. K. Bajpai, C. Zhang, et al.
An Essential Role for gp130 in Neointima Formation Following Arterial Injury
Circ. Res.,
March 30, 2007;
100(6):
807 - 816.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Nishihira, T. Imamura, A. Yamashita, K. Hatakeyama, Y. Shibata, Y. Nagatomo, H. Date, T. Kita, T. Eto, and Y. Asada
Increased expression of interleukin-10 in unstable plaque obtained by directional coronary atherectomy
Eur. Heart J.,
July 2, 2006;
27(14):
1685 - 1689.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M Meuwissen, A C van der Wal, H W M Niessen, K T Koch, R J de Winter, C M van der Loos, S Z H Rittersma, S A J Chamuleau, J G P Tijssen, A E Becker, et al.
Colocalisation of intraplaque C reactive protein, complement, oxidised low density lipoprotein, and macrophages in stable and unstable angina and acute myocardial infarction
J. Clin. Pathol.,
February 1, 2006;
59(2):
196 - 201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Zampelas, D. B Panagiotakos, C. Pitsavos, C. Chrysohoou, and C. Stefanadis
Associations between coffee consumption and inflammatory markers in healthy persons: the ATTICA study
Am. J. Clinical Nutrition,
October 1, 2004;
80(4):
862 - 867.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Z.H. Rittersma, R. J. de Winter, K. T. Koch, C. E. Schotborgh, M. Bax, G. S. Heyde, J. P. van Straalen, K. J. Mulder, J. G.P. Tijssen, G. T. Sanders, et al.
Preprocedural C-Reactive Protein Is Not Associated with Angiographic Restenosis or Target Lesion Revascularization after Coronary Artery Stent Placement
Clin. Chem.,
September 1, 2004;
50(9):
1589 - 1596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Chrysohoou, D. B. Panagiotakos, C. Pitsavos, U. N. Das, and C. Stefanadis
Adherence to the Mediterranean diet attenuates inflammation and coagulation process in healthy adults: The Attica study
J. Am. Coll. Cardiol.,
July 7, 2004;
44(1):
152 - 158.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-P. Yang, L.-J. Ho, Y.-L. Lin, S.-M. Cheng, T.-P. Tsao, D.-M. Chang, Y.-L. Hsu, C.-Y. Shih, T.-Y. Juan, and J.-H. Lai
Carvedilol, a new antioxidative {beta}-blocker, blocks in vitro human peripheral blood T cell activation by downregulating NF-{kappa}B activity
Cardiovasc Res,
September 1, 2003;
59(3):
776 - 787.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Soejima, A. Irie, S. Miyamoto, I. Kajiwara, S. Kojima, J. Hokamaki, T. Sakamoto, T. Tanaka, M. Yoshimura, Y. Nishimura, et al.
Preference Toward a T-Helper Type 1 Response in Patients With Coronary Spastic Angina
Circulation,
May 6, 2003;
107(17):
2196 - 2200.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. V. Zalai, M. D. Kolodziejczyk, L. Pilarski, A. Christov, P. N. Nation, M. Lundstrom-Hobman, W. Tymchak, V. Dzavik, D. P. Humen, W. J. Kostuk, et al.
Increased circulating monocyte activation in patients with unstable coronary syndromes
J. Am. Coll. Cardiol.,
November 1, 2001;
38(5):
1340 - 1347.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Meuwissen, J. J. Piek, A. C. van der Wal, S. A. J. Chamuleau, K. T. Koch, P. Teeling, C. M. van der Loos, J. G. P. Tijssen, and A. E. Becker
Recurrent unstable angina after directional coronary atherectomy is related to the extent of initial coronary plaque inflammation
J. Am. Coll. Cardiol.,
April 1, 2001;
37(5):
1271 - 1276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Stefanadis, K. Toutouzas, E. Tsiamis, C. Stratos, M. Vavuranakis, I. Kallikazaros, D. Panagiotakos, and P. Toutouzas
Increased local temperature in human coronary atherosclerotic plaques: an independent predictor of clinical outcome in patients undergoing a percutaneous coronary intervention
J. Am. Coll. Cardiol.,
April 1, 2001;
37(5):
1277 - 1283.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Methods