Author + information
- Received July 10, 2000
- Revision received November 14, 2000
- Accepted December 15, 2000
- Published online April 1, 2001.
- Martijn Meuwissen, MD∗,
- Jan J Piek, MD∗,1,* (, )
- Allard C van der Wal, MD†,
- Steven A.J Chamuleau, MD∗,
- Karel T Koch, MD∗,
- Peter Teeling, RT†,
- Chris M van der Loos, PhD†,
- Jan G.P Tijssen, PhD∗ and
- Anton E Becker, MD, FACC†
- ↵*Reprint requests and correspondence
: Dr. Jan J. Piek, Department of Cardiology, B2-108, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
This study was performed to evaluate the relationship between plaque inflammation of the initial culprit lesion and the incidence of recurrent angina for one year after directional coronary atherectomy (DCA).
A positive correlation between coronary plaque inflammation and angiographic restenosis has been reported.
A total of 110 patients underwent DCA. Cryostat sections were immunohistochemically stained with monoclonal antibodies CD68 (macrophages), CD-3 (T lymphocytes) and alpha-actin (smooth muscle cells [SMCs]). The SMC and macrophage contents were planimetrically quantified as a percentage of the total tissue area. T lymphocytes were counted as the number of cells/mm2. The patients were followed for one year to document recurrent unstable angina pectoris (UAP) or stable angina pectoris (SAP).
Recurrent UAP developed in 16 patients, whereas recurrent SAP developed in 17 patients. The percent macrophage areas were larger in patients with recurrent UAP (27 ± 12%) than in patients with recurrent SAP (8 ± 4%; p = 0.0001) and those without recurrent angina (18 ± 14%; p = 0.03). The number of T lymphocytes was also greater in patients with recurrent UAP (25 ± 14 cells/mm2) than in patients with recurrent SAP (14 ± 8 cells/mm2; p = 0.02) and those without recurrent angina (14 ± 12 cells/mm2; p = 0.002). Multiple stepwise logistic regression analysis identified macrophage areas and T lymphocytes as independent predictors for recurrent UAP.
There is a positive association between the extent of initial coronary plaque inflammation and the recurrence of unstable angina during long-term follow-up after DCA. These results underline the role of ongoing smoldering plaque inflammation in the recurrence of unstable angina after coronary interventions.
The significance of atherosclerotic plaque-associated inflammation remains controversial in the context of restenosis after coronary interventional procedures. The neointima, considered the dominant tissue component in restenosis lesions, generally contains few, if any, inflammatory cells. Instead, the tissue is composed mainly of smooth muscle cells (SMCs) and related extracellular matrix components and, in fact, has been promoted as having a stabilizing effect on restenosis lesions (1). However, clinical experiences have shown that recurrences due to restenosis, after coronary interventional procedures, still may manifest as unstable angina pectoris (UAP), although stable angina pectoris (SAP) is more frequent (2,3). Because UAP is usually associated with intraplaque inflammation, surface erosions and mural thrombotic events (4,5), there may be a persistent role for a lesion-associated inflammatory process. Indeed, we have recently shown that restenosis lesions from patients presenting with UAP contain more inflammatory cells than those obtained from patients with SAP (6). Moreno et al. (7)provided data showing a positive association between the amount of macrophages present in the atherectomy specimens of the initial culprit lesions and the occurrence of angiographically documented restenosis. Again, data suggest that inflammation has a role, although the understanding of this phenomenon remains unresolved.
The observations alluded to earlier provide the background for an analysis of patients who underwent directional coronary atherectomy (DCA) to see whether the extent of coronary plaque inflammation of the initial culprit lesion had a positive correlation with recurrent angina.
Directional coronary atherectomy was performed in 130 consecutive patients between 1993 and 1995 who met the following inclusion criteria: 1) the coronary lesion was identified as the “culprit lesion” on the basis of clinical and electrocardiographic findings; 2) the culprit lesion was considered suitable for DCA (i.e., proximal and eccentric); 3) the DCA procedure was successful, resulting in a coronary plaque specimen >1.5 mm2and a residual diameter stenosis <35% of the initial culprit lesion. Twenty patients were excluded because of 1) an inability to cross the lesion with the atherectomy device (n = 3); acute coronary occlusion (n = 2); stent implantation (n = 4); incomplete follow-up due to noncardiac death (n = 2); and insufficient amounts of plaque material for analysis (n = 9). Thus, 110 patients were identified as eligible for this study; 64 patients were treated for UAP (Braunwald’s classes I–III; UAP group) (8)and 46 patients for chronic SAP (Canadian Cardiovascular Society (CCS) classes I–III; SAP group) (9).
Hypertension was defined as diastolic blood pressure >95 mm Hg and/or systolic blood pressure >160 mm Hg. Patients with hypercholesterolemia were defined as those with total cholesterol >230 mg/dl or those already taking cholesterol-lowering agents. The Institutional Ethics Committee approved the study protocol. All patients gave written, informed consent.
The retrieved coronary plaque specimens were immediately frozen in liquid nitrogen and stored at −80°C. The specimens were serially sectioned at 5-μm thickness and mounted for immunohistochemistry. The primary monoclonal antibodies used were anti-CD68 (Dakopatts, Glostrup, Denmark) for macrophages, anti-CD3 (Becton and Dickinson, Mountain View, California) for T lymphocytes and anti–alpha-smooth muscle actin (SMA-1; Dakopatts) for SMCs. In all cases, a three-step indirect peroxidase method was used as previously described (10), and antibody complexes were visualized by 3-amino-ethylcarbazole. No counterstain was used.
The results of immunostaining for macrophages, T lymphocytes and SMCs were planimetrically quantified with the use of image analysis software (version 1.6065, National Institutes of Health) on a personal computer connected to a video-mounted microscope. The total tissue area of each atherectomy section was outlined manually on the video screen and measured. In these areas, the immunostained areas (macrophages or SMCs) were measured automatically using gray-scale detection with a fixed threshold. Immunopositive areas (macrophages or SMCs) were calculated as a percentage of the total tissue area. T lymphocytes were counted at 100× magnification throughout the entire section and expressed as number of cells/mm2. The pathologists had no knowledge of the results of the clinical classification of anginal symptoms.
Patients were followed for one year for the development of recurrent SAP or recurrent UAP. In those patients with a clinical manifestation of angina, coronary angiography was performed to verify whether the recurrences of angina were related to restenosis at the initial site of intervention. Restenosis was defined as a diameter stenosis (DS) >50% by quantitative coronary angiography (QCA) of the initial treated lesion.
Patients with angina due to a new lesion and patients without angina were classified in the “no angina” group (Fig. 1). Two cardiologists independently performed the classification of anginal symptoms. A third interview followed in case of disagreement on the clinical data and consensus was reached.
Quantitative measurements of coronary stenosis
Quantitative coronary angiography was performed immediately before and after DCA, preceded by an intracoronary bolus (0.1 mg) of nitroglycerin; the same procedure was done in patients who presented with recurrent symptoms of angina in the follow-up period. Percent DS, reference diameter (RD) in millimeters and minimal lumen diameter (MLD) in millimeters were measured by QCA. Analysis was performed using one end-diastolic cine frame from the angiogram showing the severest degree of the coronary narrowing, using the CMS MEDIS system, version 3.2 (Leiden, The Netherlands) (11).
Data are expressed as the mean value ± SD. For comparison of clinical, angiographic and immunohistochemical data, the unpaired Student ttest was used for continuous data and the Mann-Whitney Utest for nonparametric continuous data. For comparison of continuous data within the patient subgroups (no angina, SAP or UAP), one-way analysis of variance and a post hoc multiple comparisons test (Scheffé) was used. The chi-square or Fisher exact test was performed for categorical data. A p value < 0.05 was considered statistically significant.
For univariate and multivariate analyses, clinical characteristics (age, gender, clinical risk factors and medications), angiographic data (DS, MLD and RD before and after DCA, the Ambrose classification and the American College of Cardiology/American Heart Association classification ) and immunohistochemical data (macrophages, T lymphocytes and SMCs) were analyzed by using stepwise logistic regression to determine the independent predictors of recurrent UAP. We tested for a trend to assess any relation of increasing extent of T lymphocytes and macrophages with risk of future recurrent UAP, after dividing the extent of initial plaque inflammation (macrophages and T lymphocytes) into tertiles, defined by the distribution of the inflammatory cells of the group with recurrent UAP.
One-year clinical follow-up was completed in all 110 patients. A flow chart of these patients is shown in Figure 1. Recurrent angina, related to >50% diameter restenosis of the initially treated lesion, occurred in 33 patients (30%). Of these 33 patients, 17 presented with SAP at a mean interval of 5.7 ± 2.8 months (range 3 to 12) and 16 presented with UAP at a mean interval of 4.9 ± 3.7 months (range 2 to 11). The differences between these two groups are not statistically significant.
The clinical baseline characteristics of the 110 patients included in this study are shown in Table 1. We distinguished patients without angina, with recurrent SAP and with recurrent UAP during the one-year follow-up period. Similarly, the angiographic characteristics are shown in Table 2.
Inflammation and recurrent unstable angina
Table 3shows that the immunopositive areas of macrophages and the number of T lymphocytes in the initial atherectomy specimens were significantly larger in patients eventually developing recurrent UAP than in those presenting with recurrent SAP (p = 0.02 and p = 0.0001, respectively) and in those without recurrent angina during follow-up (p = 0.002 and p = 0.03, respectively; using analysis of variance and post hoc Scheffé test).
Univariate analysis showed a positive and significant correlation between the number of T lymphocytes/mm2atherectomy tissue (p = 0.002) and percent macrophage areas (p = 0.005) with recurrent UAP.
Multivariate logistic regression analysis identified percent macrophage areas (odds ratio [OR] 1.04, 95% confidence interval [CI] 0.996 to 1.087, p = 0.079) and T lymphocytes/mm2(OR 1.055, 95% CI 1.008 to 1.103, p = 0.020) as independent predictors of recurrent UAP.
The relationship between the recurrence of UAP within one year after DCA and the distribution of patients divided into tertiles according the extent of macrophage areas and the number of T lymphocytes/mm2is shown in Figure 2. Patients in the higher tertile of macrophages (≥31%) had significantly more risk of recurrent UAP as compared with those in the middle (21% to 31%) and lower (≤21%) tertiles (relative risk 3.5 and 3.9, respectively). Patients in the higher tertile of T lymphocytes (≥33 cells/mm2) had significantly more risk of recurrent UAP as compared with those in the middle (18 to 33 cells/mm2) and lower (≤18 cells/mm2) tertiles (relative risk 4.6 and 6.8, respectively).
This study shows that the extent of coronary plaque inflammation of the initial culprit lesion, as documented by immunohistochemistry of the atherectomy specimens, is positively associated with recurrent UAP during long-term follow-up after DCA.
Initial coronary plaque inflammation and recurrent unstable angina
Two inflammatory variables were quantified in the patients’ atherectomy tissue: the number of T lymphocytes, which have important immunoregulatory functions in the inflammatory response, and the extent of infiltration by macrophages, which are cells with secretory effector functions in plaque inflammation. A significant difference in plaque inflammation (i.e., related to higher numbers of T lymphocytes/mm2and percentage macrophage areas) was noted between patients who presented with recurrent unstable angina at follow-up and those who did not.
Clinical studies have suggested a relationship between the presentation of de novo coronary syndromes and the recurrent clinical manifestation of angina due to restenosis (2,14). Foley et al. (2)reported that half of the patients with initially unstable angina presented their restenosis also as unstable angina. In contrast, patients with initially stable angina who developed restenosis presented their symptoms, in most cases, again, as stable angina. In general, coronary restenosis is considered to present more often with a stable pattern of angina (3).
However, we are unaware of any studies evaluating the relationship between plaque inflammation of the initial culprit lesion and the subsequent manifestation of coronary syndromes. The present study shows that high numbers of macrophages and T lymphocytes in the initial coronary plaque correlate with recurrent UAP at follow-up.
In our previous studies, we have demonstrated an association between the extent and activation of plaque inflammation in primary coronary lesions and the clinical presentation of coronary syndromes (5,15). The culprit lesions of patients with stable (CCS classes I–III) and unstable (Braunwald’s classes I–III) angina were examined. The extent of plaque inflammation, as determined by macrophages and T lymphocytes, and activation of these inflammatory cells by expression of human leucocyte antigen class II (DR) molecules and interleukin-2 receptors, increased from stable angina to the severest form of unstable angina. In contrast, the dominant tissue components in restenosis after coronary interventional procedures are the SMCs and their related extracellular matrix (6,16). Indeed, this type of tissue repair has been suggested to stabilize coronary plaques. Lafont and Libby (17)hypothesized that percutaneous transluminal coronary angioplasty may have a beneficial effect, because SMC proliferation may lead to stabilization of coronary plaques, which had been shown to be prone to rupture. However, a number of restenotic lesions are still responsible for unstable coronary syndromes (2,6). Our findings indicate that initial plaque inflammation contributes to the severity of recurrent angina despite proliferation of SMCs. This finding may be explained by the fact that after DCA, a substantial amount of the initial atherosclerotic plaque may still persist; hence, the persistence of a smoldering inflammatory process at the site of intervention cannot be dismissed (18,19). Macrophages and T lymphocytes, which produce numerous growth factors and cytokines, activate the ongoing inflammation. A well-known effect is the potent inhibitory effect of the T cell cytokine interferon-gamma on SMC proliferation and collagen production by SMCs. In laboratory animals, administration of interferon-gamma inhibits the neointimal response of SMC growth and matrix production (20,21). One could speculate that an excess of T lymphocytes in the initial plaque inhibits the repair process of SMCs after interventional procedures, with clinically unstable angina as a result. In this context, it is worthwhile to reiterate that atherosclerotic plaques that underlie SAP are not completely devoid of inflammatory cells and recent-onset activation of T lymphocytes (15). In a previous study, we demonstrated that in unstable restenotic plaque, the amount of inflammatory cells (macrophages and T lymphocytes) is significantly higher than that in stable restenotic plaque (6). The present findings indicate that the extent of plaque inflammation is relevant not only for the clinical manifestation of primary lesions, but also for the presentation of recurrent angina. Moreover, these observations strongly suggest that the inflammatory process at the site of the culprit lesion is not eradicated by atherectomy, so that the smoldering effects of the inflammation again may destabilize the repair tissues.
Follow-up coronary angiography was only performed in those patients with recurrent complaints of angina (30%). Therefore, it is difficult to compare this study with other reports on this subject, that relates to angiographic restenosis (7). The potential influence of the anti-inflammatory effects of lipid-lowering therapy on modification of the presentation of recurrent angina remains unknown, because only a limited number of patients were treated with lipid-lowering agents.
The present findings strongly suggest that the chronic inflammatory process at the site of the culprit lesion is not eradicated by atherectomy, so that the smoldering effects of the inflammation may again destabilize the repair tissues after coronary interventions. This emphasizes the significance of therapeutic strategies aimed at reducing plaque inflammation to avoid recurrence of unstable coronary syndromes.
The authors acknowledge the expert statistical assistance of M. Merkus, MD (Department of Clinical Epidemiology and Biostatistics); the skilled assistance of the technical and nursing staff of the Cardiac Catheterization Laboratory; and the secretarial assistance of Marsha Schenker.
↵1 Dr. Piek is a clinical investigator sponsored by the Netherlands Heart Foundation (grant no. D96.020).
- Canadian Cardiovascular Society
- directional coronary atherectomy
- diameter stenosis
- minimal lumen diameter
- quantitative coronary angiography
- reference diameter
- stable angina pectoris
- smooth muscle cell
- unstable angina pectoris
- Received July 10, 2000.
- Revision received November 14, 2000.
- Accepted December 15, 2000.
- American College of Cardiology
- Libby P
- Lehmann K.G,
- Maas A.C,
- van Domburg R,
- de Feyter P.J,
- van den Brand M,
- Serruys P.W
- Moreno P.R,
- Falk E,
- Palacios I.F,
- Newell J.B,
- Fuster V,
- Fallon J.T
- van der Wal A.C,
- Becker A.E,
- Koch K.T,
- et al.
- Piek J.J,
- van der Wal A.C,
- Meuwissen M,
- et al.
- Moreno P.R,
- Bernardi V.H,
- Lopez-Cuellar J,
- et al.
- Braunwald E
- van der Zwet P.M.J,
- Reiber J.H.C
- Ryan T.J,
- Faxon D.P,
- Gunnar R.M,
- et al.
- Bauters C,
- Lablanche J.M,
- McFadden E.P,
- Leroy F,
- Bertrand M.E
- van der Wal A.C,
- Piek J.J,
- de Boer O.J,
- et al.
- Ueda M,
- Becker A.E,
- Tsukada T,
- Numano F,
- Fujimoto T
- Lafont A,
- Libby P
- Waller B.F,
- Pinkerton C.A
- Hansson G.K,
- Holm J