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Circulation. 1997;96:3360-3368

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(Circulation. 1997;96:3360-3368.)
© 1997 American Heart Association, Inc.

Articles

Topographic Analysis of Proliferative Activity in Carotid Endarterectomy Specimens by Immunocytochemical Detection of the Cell Cycle–Related Antigen Ki-67

Richard Brandl, MD; Thomas Richter, MD; Klaus Haug, MD; Manfred G. Wilhelm, PhD; Peter C. Maurer, MD; ; Walter Nathrath, MD

From the Department of Vascular Surgery (R.B., K.H., P.C.M.), the Institute for Pathology and Pathological Anatomy (T.R., W.N.), and the Institute for Medical Statistics and Epidemiology (M.G.W.), Rechts der Isar Medical School, Technical University of Munich (Germany).

Correspondence to Richard Brandl, MD, Department of Vascular Surgery, Technical University of Munich Medical School, Rechts der Isar Medical Center, Ismaninger Str. 22, 81675 Munich, Germany.


*    Abstract
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 *Abstract
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Background On the basis of contradictory results found in animal experiments and coronary atherectomy tissue, there is an ongoing debate about the significance of cellular proliferation in human atherosclerosis. In the present prospective study, the cell cycle–related antigen Ki-67 was detected for topographic determination of cell turnover in distinct regions of human carotid endarterectomy specimens harvested en bloc by surgical biopsy.

Methods and Results After en bloc resection, serial sections of 26 consecutive carotid lesions were analyzed by histomorphological examination and immunohistochemistry. Thereby, 319 high-power fields were attributed to separate plaque regions defined as follows: distal boundary of the lesion with normal intima, plaque shoulder, core region, and diffuse intimal thickening. Endothelial cells, smooth muscle cells, T cells, and macrophages were identified by immunostaining of factor VIII–related protein, {alpha}-actin, CD68, and CD45R0. An overall proliferation index of 0.49±1.05% was yielded by positive anti–Ki-67 immunolabeling, predominantly in macrophage-rich areas characterized by high cell density (>1000 cells/mm2) as well as in reparative sites in the perimeter of atheroma, intramural thrombosis, plaque hemorrhage, and neovascularization (P<.01). Few or no signs of proliferation activity were found in normal intima, in areas of dense {alpha}-actin positivity, or adjacent media. As shown by double immunostaining, macrophages and unspecified mesenchymal cells represented the prevailing proliferating cell type.

Conclusions Our results suggest that proliferation in advanced human carotid lesions is confined to the intima and focally concentrated in central plaque regions negative for {alpha}-actin. Furthermore, it apparently occurs primarily as part of inflammatory processes and structural repair predominantly involving macrophages, as well as unspecific mesenchymal cells.


Key Words: arteriosclerosis • carotid arteries • cells • immunohistochemistry • muscle, smooth • plaque


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Proliferative activity of smooth muscle cells (SMCs) has been documented in various animal experiments as an essential determinant of atherosclerotic formations.1 2 3 4 In atherosclerosis in humans the role of proliferation in distinct states of the disease and in heterogeneous plaque compositions is undefined. Hitherto, proliferation has been assessed by proliferating cell nuclear antigen (PCNA) detection in particles of plaque tissue retrieved by catheter atherectomy devices, rendering randomly selected specimens of uncertain anatomic and geometric reference.5 6 7 8 9 Plaque tissue harvested en bloc by surgical endarterectomy under precautions of meticulous biopsy may be useful in coping with this shortcoming. Arteriosclerotic tissue originating from the carotid bifurcation appears to be particularly suitable for detailed analysis of the plaque architecture. A stereotyped sequence of distinct pathological conditions can be observed in these lesions originating from an anatomically defined location.10 In the longitudinal extension, the carotid lesion emerges from a diffuse intimal thickening in the common carotid artery and is usually limited to a short segment at the origin of the internal carotid artery. A spectrum of human atherosclerotic formations is focused in this area regularly, including a tapering from core and shoulder regions to morphologically normal intima.11 12 These conditions were used to prospectively examine the presence and topographic distribution of proliferative activity in standardized sections of carotid endarterectomy specimens retrieved by en bloc biopsy. Cell turnover was determined by immunocytochemical detection of the nuclear antigen Ki-67, a protein of 345 kD and 395 kD expressed in close relation to the cell cycle.13 14 It may be recognized by the monoclonal antibody MIB1,15 which recently has been introduced in routine surgical pathology as a reliable proliferation marker and prognostic indicator for several malignant tumors.16


*    Methods
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 up arrowIntroduction
 *Methods
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The tissue specimens were obtained from 25 consecutive patients operated on for high-grade carotid stenosis. Before surgery, the degree of stenosis was determined by ultrasound and biplanar angiography, following the criteria of the NASCET investigators.17 According to patient history and computed tomography scan, 9 stenoses were categorized as asymptomatic and 17 as symptomatic. One female patient (M.J.) presented with bilateral symptomatic lesions; both were enrolled. The patients were burdened by a mean of 2.4 (1 to 4) risk factors. There were no statistical differences between symptomatic and asymptomatic patients with reference to age, sex, risk factors, medication, or degree of stenosis listed in Table 1Down. All operations were performed with the patient under general anesthesia with fentanyl and halothane as well as systemic heparin (100 U/kg) during clamping time. The patient-specific medication including antiplatelet agents was continued perioperatively. The informed consent of the patients as well as the approval of the protocol by the Ethics Committee of the Faculty of Medicine were obtained.


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  		Table 1. Patient Characteristics

Tissue Preparation and Light Microscopy
Intraoperatively, the carotid plaque tissue was endarterectomized by a careful operative technique preserving the en bloc plaque structure (see Fig 1Down, A through C). The samples were fixed in formalin immediately upon removal for 24 hours. Before paraffin embedding, the samples were dissected transluminally and longitudinally in the bifurcation level to obtain two homologous halves. Histologically, the lesions were classified according to the recommendations of the Committee on Vascular Lesions of the Council on Arteriosclerosis of the American Heart Association.18 All the lesions were judged as type V and type VI in their clinically most significant region (Table 1Up). Thereby disruption of the lesion surface was not scored as a complicating feature because an artifact by surgical manipulation could not be thoroughly excluded. Thus only intraluminal thrombus or plaque hemorrhage was pertinent to the diagnosis of a type VI lesion. There was no statistical difference in the prevalence of a lesion type in symptomatic and asymptomatic stenoses.



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  		Figure 1. Angiographic, macroscopic, and histological aspect of a typical example of the carotid lesions studied. A, Angiography showing a high-grade stenosis of the right internal carotid artery at its origin (arrow). The external carotid artery is occluded in this case. B, Digitalized photograph of the en bloc atherectomy sample of the same carotid bifurcation. The proximal margin of the sample consists of diffuse intimal thickening and was dissected from the tissue resting in situ. Distally, it is limited by the natural extent of the plaque that may be removed by peeling off the thickened intima that in most cases dissects spontaneously from the outer media and more distal parts of normal intima. C, Same sample after longitudinal and transluminal dissection in the bifurcation level. D, Histological section after paraffin embedding showing an excentric lesion in the internal carotid artery (elastica van Gieson stain). The core region is filled with masses of atheromatous debris. At the origin of the internal carotid artery corresponding to the angiographic maximum of the stenosis, an adherent thrombus is visible (arrows), classifying the lesion as type VI according to American Heart Association criteria.18 Normal intima extends as a thin rim at the lateral side distal to the stenosis. The tangential section of the vessel wall proximal to the bifurcation mimics an occlusion of the common carotid artery that was not real as documented by angiography (magnification x10). DIT indicates diffuse intimal thickening; CR, core region; PS, plaque shoulder; NI, normal intima; CCA, common carotid artery; ICA, internal carotid artery; and ECA, external carotid artery.

The plaque regions were differentiated according to the criteria listed in Table 2Down and categorized as follows: distal boundary of the lesion and transition to normal intima (NI), plaque shoulder (PS), plaque center with fibrous cap and core region (CR), and prestenotic plane atherosclerosis (DIT) (see Fig 1DUp). At least 12 randomly selected high-power fields (HPF) in vital areas of each lesion were chosen for histological evaluation. Histomorphological characteristics were described on hematoxylin and eosin (HE)-stained and elastica van Gieson (EvG)-stained specimens at x400 magnification, with observers blinded to clinical data.


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  		Table 2. Definition of Intimal Regions

Definition of Histomorphological Characteristics
The total cell number was determined by the number of nuclei present in the HPF. Areas of hypocellular fibrosis showed predominantly collagen with an average of <50 cells per HPF. Foam cells, lymphocytes, and hemosiderin-laden macrophages were discriminated in HE-labeled, EvG-labeled, and immunocytochemically labeled sections. A dense focal accumulation of lymphatic cells was defined as lymphatic infiltration. An intramural thrombosis and in vivo plaque hemorrhage were discriminated from artificial hemorrhage due to surgical manipulation by accompanying signs of organization in varying stages. Plaque hemorrhage without reparative reaction was not considered for evaluation. Extracellular cholesterol was stated positive in the case of dissociation of fibrillar structures by light substance or cholesterol clefts. Calcification was determined as dispersed granular or bulky deposition of basophil amorphous material. Neovascularization was regarded as present if the HPF contained at least one capillary vessel with positive staining for factor VIII.

Immunocytochemistry
Single and double immunocytochemistry were performed on serial sections for identification of proliferating cells. Endothelial cells, SMCs, T cells, and macrophages were identified by use of antibodies to factor VIII–related antigen, smooth muscle {alpha}-actin, CD45R0, and CD68, respectively. Proliferative activity of plaque cells was detected by use of the antibody MIB1 targeting the Ki-67 antigen. The embedded tissue was cut into 3-µm-thick sections and desiccated overnight at 48°C. After deparaffinization, the specimens were microwaved in citrate buffer for 15 minutes. Endogenous peroxidase was blocked by 3% H2O2 followed by incubation in a moist chamber with the primary antibody (clone MIB1, lot No. 7000/96, 1:20 in 1% BSA, Dianova; Hamburg, FRG) at room temperature for 2 hours after a PBS rinse for 5 minutes. Subsequently, incubation with the secondary antibody (anti-mouse IgG, 1:200, ABC Elite Kit, Vector Laboratories; Burlingame, Calif) was performed for 30 minutes, followed by avidin-biotin amplification (ABC kit) for 30 minutes. The detection reaction was carried out with 0.1% 3'3'diaminobenzidine (DAB) and counterstaining with hematoxylin.

Anti–{alpha}-actin (clone HHF 35, code No. M 0635) 1:50 in BSA for 2 hours at room temperature and a secondary antibody (rabbit anti-mouse IgG, 1:50 in 1:5 human serum for 30 minutes, Dianova; Hamburg, FRG) were used to identify SMC. The incubation with the APAAP complex for 30 minutes (1:50, Dianova) was followed by development with fast red (Naphtol-AS-MX-Phosphat/Chromogen fast red TR, Serva). For single and double immunostaining controls for {alpha}-actin and Ki-67, human small intestine was used with every run (Fig 2Down), showing positivity for {alpha}-actin in the tunica muscularis and for Ki-67 in the basal crypt epithelium. For identification of endothelial cells, anti–factor VIII monoclonal antibodies (clone F8/86, code No. M616) were used as described for {alpha}-actin with protease pretreatment. Macrophages were identified by anti-CD68 labeling (clone PG-M1 code No. U 7029) using an EPOS system (DAKO). Controls for anti-CD68 immunostaining exhibited predominant positivity in the follicles of lymphatic tissue. Additional controls with normal mouse serum and application of the secondary antibody were negative.



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  		Figure 2. Light photomicrograph of a section of human intestine double-immunostained with anti–{alpha}-actin and anti–Ki-67 antibodies. A brown nuclear staining identifies numerous proliferating cells displaying anti–Ki-67 reactivity in the basal crypt epithelium (diaminobenzidine). Smooth muscle cells in the tunica muscularis are labeled red as the result of positive staining for {alpha}-actin (APAAP); counterstaining is with hematoxylin. Original magnification x200.

On the MIB1 single-labeled slides, each field was scored for location in intima or media, total number of nuclei, histomorphological characteristics, and presence of cell-type–specific markers. Thereby CD68-positive cells were determined as a percentage of total cell number. The number of MIB1-positive cells in relation to the total cell number was expressed as proliferative index.

Double Immunostaining
For identification of cells, serial sections of 11 patients were double-immunostained with MIB1 combined with each cell-specific antibody ({alpha}-actin, CD68, CD45R0, factor VIII), and vimentin. Thereby anti–Ki-67 immunostaining preceded the cell-type–specific immunoreaction. For characterization of macrophages in double-immunolabeled slides, an APAAP system was used as described for {alpha}-actin (clone KP1, code No. M 0814). The number of double-stained cells for each cell-type–specific marker was determined in corresponding microscopic fields containing at least three MIB1-positive cells. Primary anti-CD45R0 antibody ligation (clone UCHL1, code No. M 0742) was detected using a biotin-streptavidin amplified detection system (SuperSensitive Kit, BioGenex) for 20 minutes at room temperature. Slides were again washed in PBS and reacted with alkaline phosphatase–conjugated streptavidin for 20 minutes at room temperature and developed with fast red. Antibodies to the intermediate filament protein vimentin were used to document the mesenchymal origin of the cells. The primary antibody (clone V9, code No. M725) 1:7000 in BSA for 2 hours at room temperature and the DAKO ChemMate detection kit (code No. K 5005) including the secondary antibody and the APAAP complex were used for the detection of vimentin. All primary antibodies for single and double immunolabeling, except MIB1, were obtained from DAKO.

Statistical Analysis
Data are expressed as mean±SD. The relationship between histomorphological characteristics and proliferation was described by multivariate analysis. The parameters were considered as interdependent, according to the frequent colocalization in the plaque tissue. Thus a generalized linear model for correlated data was applied and evaluated with a generalized estimation equation (GEE) approach. Differences between groups were considered significant at a probability value of <.05.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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In all cases, the evaluated HPFs could be clearly attributed to the plaque regions as defined. Fig 3Down illustrates the separate plaque regions as shown in the distal parts of a high-grade lesion. Notably, the distal boundary of all samples consisted of an adherent lip of morphologically regular and normal-size intima. In the proximal part of the sections, a region of diffuse intimal thickening could be clearly separated from the core region. The overall intimal cell density varied within a wide range from 5 to 800/HPF (mean, 160.2±126.2), yet there was no significant difference in cellularity in the plaque regions as defined (NI, 122.3±72.3; PS, 131.7±72.3; CR, 176.2±153.1; DIT, 154.9±95.3 cells/HPF; NS). NI was characterized by preponderantly laminar and {alpha}-actin–positive cell adjustment without signs of advanced plaque degeneration. In the shoulder region, foam cells and extracellular lipid deposits represented the predominant features. CR exhibited heterogenous cellularity in addition to sparse {alpha}-actin positivity and was populated by fibroblasts and CD68-positive macrophages as preponderant cell types. Characteristics of tissue degeneration such as atheroma, calcification, plaque hemorrhage, intramural thrombosis, and neovascularization were frequent in these areas. DIT predominantly showed extended lipid deposits with evidence of plaque degeneration and secondary reorganization that were less common than in CR. Adjacent layers of media were characterized by laminar adjusted spindle-shaped cells, most of which ({approx}90%) were identified as SMCs by positive staining for {alpha}-actin.



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  		Figure 3. Light photomicrograph of the distal branch of a high-grade carotid lesion (longitudinal section). Note the adherent rim of undiseased intima at the distal boundary. NI indicates normal intima; PS, plaque shoulder; CR, core region with necrotic core, and fc, fibrous cap. The borders of the plaque regions are marked by dotted lines. Original magnification x40; hematoxylin and eosin stain.

Cell Proliferation in Plaque Regions and Histomorphological Associations
Positive staining for at least one Ki-67–positive cell was found in 118 of 319 (37.0%) HPFs. A total of 250 of 51252 cells were scored positive, resulting in an overall proliferation index (PI) of 0.49±1.05%. Cell proliferation was more distinct in CR (PI, 0.81±1.37%, peak PI, 8.3%) compared with DIT (PI, 0.26±0.46%, P<.0003) and PS (PI, 0.12±0.41%, P<.0001; see Fig 4Down). No proliferating cells were observed in normal-sized intima (NI) as well as in adjacent media. In 8 samples, tissue extended over a necrotic core was preserved in the sections and could be clearly discriminated as intact fibrous cap. The thickness of these fibrous caps ranged from 150 to 400 µm. Immunolabeling exhibited no proliferative activity in the corresponding HPFs.



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  		Figure 4. Ki-67 proliferation index in distinct carotid plaque regions. NI indicates normal intima; PS, plaque shoulder; CR, core region; and DIT, diffuse intimal thickening. * P<.001, ** P<.003.

Proliferating cells were found exclusively in the intima distributed in a single cell pattern, but focal accumulation was more common. Groups of 2 to 5 positive cells were seen most often in a hypercellular environment (>1000 cells/mm2, P<.001). Fig 5ADown shows a typical accumulation of Ki-67–positive cells in a hypercellular area of the deep intima. There was a significant association with histomorphological characteristics of plaque degeneration and inflammatory tissue repair. Frequently, colocalizations with atheromatous lipid deposits (P<.05) (Fig 5BDown), lymphatic infiltration (P<.01), intramural thrombosis (P<.01), plaque hemorrhage (P<.001), or neovascularizations (P<.001) were observed (Fig 6ADown). Furthermore, there was a significant association with foam cells (P<.01), hemosiderin-laden macrophages (P<.005), and lymphocytes (P<.01) (see Table 3Down).



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  		Figure 5. Light photomicrographs of sections immunolabeled with anti–Ki-67 antibody MIB1 (original magnification x200). A, Ki-67–positive cells in a cell-rich area in the stenotic plaque center (core region, lipid core not visible). Note that proliferation is strictly limited to the intima and concentrated in a baseline opposite to the media (lamina elastica interna not visible). There is no evidence of anti–Ki-67 reactivity in the adjacent media (right). B, Focal accumulation of Ki-67–positive cells in the perimeter of a lipid core (intima, core region).



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  		Figure 6. Light photomicrographs of double-immunolabeled sections. A, Proliferating cells positive for Ki-67 (brown nuclei, black arrows) colocalized with intimal neovascularizations as shown by positive immunolabeling of the endothelium by anti–factor VIII antibody. Note traces of a local plaque hemorrhage in the proximity of the microvessels (right). The extravasation of red blood cells is accompanied by hypercellular tissue reaction with one cell displaying anti–Ki-67 reactivity (light arrow, x400). B, Double immunolabeling for Ki-67 and {alpha}-actin. Anti–Ki-67 reactivity of nuclei is present in the intima of the core region. All cells in this area are negative for {alpha}-actin. However, dense positive staining for {alpha}-actin (red) is found in an adjacent rim of media, which, on the other hand, is devoid of anti–Ki-67 reactivity (x100). C, High-power field of the same intimal region double immunolabeled for Ki-67 and CD68, demonstrating predominantly foam cells. The majority of the cells exhibits anti-CD68 reactivity (red). Seven of 10 proliferating cells present in this area display a positive double immunolabeling for Ki-67 and CD68, indicating proliferating mac- rophages (x400). D, Macrophage foam cell–rich lipid core, double immunolabeled for vimentin and Ki-67. The cytoplasm of all cells is positive for vimentin (red). Brown nuclei indicating anti–Ki-67 reactivity are found predominantly in macrophage foam cells without evidence of advanced transformation by confluent intracellular lipid droplets.


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  		Table 3. Histological Characteristics and Anti–Ki-67 Reactivity in Microscopic Fields

Differentiation of Proliferating Cells
Double immunostaining for vimentin and Ki-67 revealed a mesenchymal character of all proliferating cells. As shown by double immunostaining for Ki-67 and {alpha}-actin, plaque regions characterized predominantly by regular smooth muscle cell population were commonly free of any proliferating activity (Fig 6BUp). {alpha}-Actin expression was a strong negative predictor of anti–Ki-67 reactivity (P<.001). In contrast, macrophages (37.5%) and unspecified mesenchymal cells (47.1%) constituted the predominant proliferating populations, in particular in areas of structural reorganization characterized by dense CD68 reactivity (Fig 6CUp). Occasionally, sprouting capillaries were identified by combined positivity for factor VIII and Ki-67 located in the endothelium of intimal microvessels (3.1%). CD45R0-positive T cells represented 9.6% of all positive double-immunostained cells.

Clinicohistopathological Correlations
There was no association evident between patient-related proliferation indices and clinical parameters including age, sex, and degree of stenosis. Furthermore, proliferation characteristics showed no significant correlation to neurological symptoms, risk factors, or medication.


*    Discussion
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 up arrowResults
 *Discussion
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This report analyzes topographic aspects of cell replication in distinct plaque components that have been preserved by complete biopsy of heterogeneous and complex carotid lesions. Previous immunocytochemical studies of human atherosclerotic tissue have been performed on plaque tissue resected at random during routine operative intervention or by catheter atherectomy.5 6 7 8 9 In contrast, meticulous operative biopsy according to standardized conditions offers the opportunity to preserve the topographical structure of fresh tissue, including short segments of normal intima. This methodological approach was a prerequisite in the present investigation of proliferation characteristics in defined regions of human carotid lesions. In a sense, our technique of en bloc endarterectomy enabled us to establish a new model for the study of human atherosclerosis.

On the basis of animal experiments of injury-induced atherosclerosis, proliferation of SMCs is considered a key event in atherogenesis.1 19 20 In human vascular pathology, SMC proliferation has been shown to be crucial in neointima formation,21 22 AV shunt stenosis,23 24 and transplant atherosclerosis,25 26 with homogeneous myocellular fibrosis as the prevailing histological feature. In the chronic type of human atherosclerosis, information regarding the extent and distribution of cellular proliferation hitherto is limited. For coronary atherectomy tissue, conflicting results of PCNA proliferation indices have been reported ranging from almost 0% to 3.6%.6 7 9 27 Although this is a small difference in terms of figures, the clinical result of low rate of proliferation with respect to the temporal multiplication factor of years or even decades may be substantially different. The reason for the reported contradictory findings is unclear but may most probably be explained by the random fashion of tissue sampling by the coronary atherectomy device with uncertain pathoanatomic orientation. The interpretation that different mixtures of different lesion types may have been sampled is supported by the present observation that the proliferation activity in different intimal regions varied within a wide range (0% to 8.3%), depending on plaque complexity.

Another possible explanation may be found in biological and immunocytochemical properties of PCNA, which has been used in all the previous work dealing with proliferation in human atherosclerosis.6 7 9 27 PCNA positivity becomes very low at mitosis and depends in a wide range on the conditions of tissue fixation,28 a possible source of inconsistant and less distinct labeling results. Compared with PCNA, which has been shown to give positive results in nonreduplicative DNA synthesis as well,29 there is a high specificity of Ki-67 expression for cycling cells, as documented by autoradiographic 3H-thymidine incorporation in animals.14 27 30 In our hands, labeling for PCNA in serial sections showed a distribution similar to that of Ki-67 but was less distinct, with a slightly lower overall proliferation index (data not shown). In conclusion, we feel that in comparison to PCNA, the monoclonal antibody MIB1 targeting the antigen Ki-67 may be the more reliable tool for proliferation studies in human atherosclerotic tissue.

In our study, we focused on cells kept in the context of the specific site in the plaque texture. In general, the estimation of a dynamic process on the basis of static observations may be difficult. On the other hand, this shortcoming may be compensated for by the fact that the specific carotid plaque regions, as defined, simultaneously present characteristics of early and advanced stages of the disease in a geometric order. The localization of proteins specific to cell types and their interaction with the surrounding matrix in transition zones of normal intima to early intimal thickening may help to explain pathogenetic mechanisms in the initiation and progression of atherosclerotic processes in humans. From another viewpoint, the obligatory limitation of carotid stenoses in their distal longitudinal extension raises interest in possible factors involved in the local termination of plaque growth.

In the carotid series presented herein, the overall Ki-67 reactivity corresponds with previous studies of Gordon et al27 and O'Brien et al6 reporting low overall proliferation rates in human atherosclerotic tissue. However, the mean proliferation index of 0.49% appears exceedingly high compared with baseline replication rates of 0% to 0.009%, which have been determined for normal human arteries31 as well as control animals.32 While in the rat carotid artery model proliferation in the media is the initial event in injury induced arteriosclerosis,1 no proliferative activity was found in adjacent media layers in any region. Furthermore, the absence of proliferative activity in NI, a region morphologically consistent with normal intima or minimal intimal thickening, indicates that proliferation is not the initial pathophysiological process leading to longitudinal growth of advanced human lesions. Proliferative activity was found in small amounts in the shoulder region known as the "classic" growth area but was present predominantly among cells populating the plaque center. The peak of proliferative activity concentrated in this plaque region (PI, 0.81±1.37%, with a maximum of 8.3%) was significantly elevated compared with prestenotic (P<.003) and shoulder regions (P<.001), which both were characterized by a lower complexity of the histological structure.

Proliferative activity was tightly correlated with sites of plaque tissue degeneration and structural repair. The most preponderant concentration of proliferating cells was observed focally in hypercellular areas of the deep intima as well as in the perimeter of atheromas, plaque hemorrhages, and intramural thrombi, obviously being part of an ongoing process of reorganization. The latter is confirmed by the frequent colocalization of Ki-67 reactivity with neovasculature, which has been discussed as a reparative plaque component analogous to wound angiogenesis.33 There are several possible explanations for the association of proliferative activity and neovascularization. First, it might be argued that proliferating cells represent sprouting endothelial cells. Second, the focal accumulation of Ki-67–positive cells in the proximity of established microvessels suggests that inflammatory bloodborne cells have immigrated through the capillaries, attracted by mediators such as monocyte chemotactic protein-1,34 35 transforming growth factor-ß,36 and oxidized low-density lipoprotein.37 38 Third, resident plaque cells may be stimulated to proliferate, since intraplaque microvessels are a potential paracrine source of growth factors and cytokines.20 Alternatively, microlesions or frank rupture of the capillaries may cause intraplaque hemorrhage, which in turn may induce local cellular repair activities including proliferation of fibroblasts and macrophages mediated by a number of potent growth regulatory blood components. In summary, the tight correlation of Ki-67 reactivity with morphological characteristics of plaque degeneration and complexity documents that proliferation contributing to plaque progression in carotid artery disease is primarily related to repair mechanisms displaying a secondary response to injury.

Remarkably, {alpha}-actin expression was a strong negative predictor of proliferative activity (P<.001). Areas exhibiting {alpha}-actin expression typical of contractile SMCs were excluded from Ki-67 positivity, with very few exceptions. This observation, which to some extent is in contrast to our current understanding of SMC proliferation in human atherosclerosis, may refer to the phenotype modulation of these cells. SMC-derived cells have been shown to reduce their myofilaments along with their modulation to a metabolically active phenotype in plaque tissue.39 Since a number of intermediary or synthetic SMCs may not be recognized as {alpha}-actin–bearing cells by routine immunocytochemical detection, proliferating cells not amenable to a certain cell type in the present series may, to some extent, represent metabolically modified SMCs. Alternatively, SMCs in their contractile state may be separated from or refractory to local proliferative stimuli.

As shown by double immunostaining, CD68-positive macrophages constituted the prevailing proliferating cell type in carotid lesion independent of plaque region. There are several lines of evidence indicating that the presence of macrophages in the plaque tissue results not only from transendothelial migration but also from a local proliferation process as well. 3H-thymidine labeling in HE-stained sections has been almost exclusively attributed to cells appearing as foam cells and monocytes.40 Findings in arteriosclerotic tissue in animals indicate that reduplicating foam cells are derived from macrophages.41 In human arteriosclerotic lesions, proliferating macrophages have been described exhibiting a proliferative activity similar to that of SMCs.27 Macrophage-derived foam cells are thought to be responsible for repair mechanisms in the borders around pooled extracellular lipid deposits.42 Similarly, in the present study a significant colocalization was found between proliferative activity and macrophage foam cells in serial sections, but in foam cell rich–lipid cores, proliferating cells were preferably identified as macrophages whose cytoplasm was not extensively transformed by confluent lipid. There are two possible explanations for this observation. First, proliferation of macrophages may represent a process preceding their transformation into foam cells. Second, macrophages populating inflammatory foam cell–rich regions may release growth factors and cytokines20 and thereby stimulate the proliferation of other cell types such as modified SMCs, unspecified mesenchymal cells,27 and T cells.43 Lymphocytes as single cells, as well as part of dense lymphocytic infiltrations, were significantly colocalized with Ki-67 reactivity in serial sections, suggesting that besides tissue repair, inflammatory processes condition the plaque milieu in which proliferation occurs.

The proliferation of SMCs has been discussed as an important mechanism involved in biomechanical plaque stability.44 For the carotid lesion, aspects of plaque composition and stability are of special interest, since angioplasty currently is being discussed as a possible interventional treatment of high-grade stenoses.45 Fibrous plaques are considered stable, whereas lesions rich in soft extracellular lipids and macrophages are thought to be more fragile.46 47 In this context it may be of interest that proliferative activity of SMCs or any other cell type was not present in the arch of the fibrous caps, which, according to their reduced thickness, must be considered as particularly prone to rupture. The observation that proliferation in carotid plaque tissue is predominantly related to macrophage reduplication gives rise to a reevaluation of proliferative processes in this particular vascular region. Macrophages are known to produce matrix-degrading proteases in remarkable amounts.48 49 50 An intense expression of the matrix metalloproteinase-1 by macrophages has been shown in the margin of the core region of carotid lesions in colocalization with circumscript plaque hemorrhage.51 Therefore, proliferation of macrophages as a protease-producing population may imply an increased level of matrix degradation and, in consequence, a decrease of plaque stability in advanced carotid lesions.

There were no correlations found between clinical characteristics and individual Ki-67 reactivity. However, information about the exact duration of exposure to the corresponding risk factors and medications for each patient was lacking. Much larger series may be required to achieve reliable data on this issue. In particular, the statistical analysis of the angiographic severity of the stenoses in correlation to the proliferation indices was compromised by the fact that in our consecutive series, all stenoses were high-grade according to the NASCET criteria except one. Admittedly, the correlation of the angiographic NASCET graduation with the actual percentage of lumen obliteration at the level of the maximum of the stenosis may be poor.52 On the other hand, longitudinal sections as used in the present series may not be suitable for determining the exact degree of stenosis histomorphometrically. Further detailed studies will be required to selectively sort out proliferation characteristics refering to individual risk factors, actual intimal thickness, neurological symptoms, or clinical progression. Because of the paucity of anamnestic data on plaque progression, no conclusions can be drawn from our immunocytochemical proliferation studies refering to actual lesion growth. Immunohistochemical proliferation markers are accepted as independent prognostic factors in a variety of benign and malignant tumors (for survey see Reference 1616 ). In atherosclerosis research, little is known about clinicohistopathological correlations between proliferation markers and long-term results after interventional or surgical treatment. In a follow-up study conducted by Taylor and coworkers,9 the clinical result 6 months after coronary catheter atherectomy was found to be independent of labeling indices for PCNA and basic fibroblast growth factor (bFGF). Follow-up ultrasound investigations of the present series evaluating postoperative neointimal formations in correlation to immunocytochemical proliferation characteristics are in progress.


*    Acknowledgments
 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Br 1583/1-1). The authors thank Renate Hegenloh, Sabine Noll, and Birgit Geist for their technical assistance and expertise with double immunocytochemistry. We are indebted to Dr Sharon Page and Catherine Röh for correcting and preparing the manuscript.

Received December 16, 1996; revision received June 30, 1997; accepted July 15, 1997.


*    References
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  1. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle cell growth in the absence of endothelium. Lab Invest.. 1983;49:327-333.[Medline] [Order article via Infotrieve]
  2. Kim DN, Schmee J, Lee KT, Thomas WA. Atherosclerotic lesions in the coronary arteries of hyperlipidemic swine, I: cell increases, divisions, losses and cells of origin in first 90 days on diet. Atherosclerosis.. 1987;64:231-242.[Medline] [Order article via Infotrieve]
  3. Stemerman M, Ross R. Experimental arteriosclerosis, I: fibrous plaque formation in primates, an electron microscopic study. J Exp Med.. 1972;136:769-789.[Abstract]
  4. Zeymer U, Fishbein MC, Forrester JS, Cercek B. Proliferating cell nuclear antigen immunohistochemistry in rat aorta after balloon denudation. Am J Pathol.. 1992;141:685-690.[Abstract]
  5. Barbieri E, Tanganelli P, Taddei G, Attino V, Molinari G, Mirandola G, Cosimi A, Destro G. I1 ruolo dell'aterectomia direzionale nello studio della ristenosi postangioplastica. Cardiologia.. 1991;36:461-467.[Medline] [Order article via Infotrieve]
  6. O'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N, Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM. Proliferation in primary and restenotic coronary atherectomy tissue: implications for antiproliferative therapy. Circ Res.. 1993;73:223-231.[Abstract/Free Full Text]
  7. Pickering JG, Weir L, Jekanowski J, Kearney MA, Isner JM. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest.. 1993;91:1469-1480.
  8. Strauss BH, Umans VS, van Suylen RJ, de Feyter PJ, Marco J, Robertson GC, Renkin J, Hendricks G, Vuzevski VD, Bosman FT, Serruys PW. Directional atherectomy for the treatment of restenosis within coronary stents: clinical angiographic and histologic results. J Am Coll Cardiol.. 1992;20:1465-1473.[Abstract]
  9. Taylor AJ, Farb AA, Angello DA, Burwell LR, Virmani R. Proliferative activity in coronary atherectomy tissue: clinical, histopathologic, and immunohistochemical correlates. Chest.. 1995;108:815-820.[Abstract/Free Full Text]
  10. Imparato AM. The carotid bifurcation plaque: a model for the study of atherosclerosis. J Vasc Surg.. 1986;3:249-255.[Medline] [Order article via Infotrieve]
  11. Bassiouny HS, Davis H, Massawa N, Gewertz BL, Glagov S, Zarins CK. Critical carotid stenoses: morphologic and chemical similarity between symptomatic and asymptomatic plaques. J Vasc Surg.. 1989;9:202-212.[Medline] [Order article via Infotrieve]
  12. Imparato AM, Riles TS, Gorstein F. The carotid bifurcation plaque: pathologic findings associated with cerebral ischemia. Stroke.. 1979;10:238-245.[Abstract/Free Full Text]
  13. Gerdes J, Li L, Schlueter C, Duchrow M, Wohlenberg C, Gerlach C, Stahmer I, Kloth S, Brandt E, Flad HD. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol.. 1991;138:867-873.[Abstract]
  14. Scott RJ, Hall PA, Haldane JS, van Noorden S, Price Y, Lane DP, Wright NA. A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction. J Pathol.. 1991;165:173-178.[Medline] [Order article via Infotrieve]
  15. Cattoretti G, Becker MHG, Key G, Duchrow M, Schlüter C, Galle J, Gerdes J. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB1 and MIB3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol.. 1992;168:357-363.[Medline] [Order article via Infotrieve]
  16. Hofstädter F, Knüchel R, Rüschoff J. Cell proliferation assessment in oncology. Virchows Arch.. 1995;427:323-341.[Medline] [Order article via Infotrieve]
  17. North American Symptomatic Carotid Endarterectomy Trial (NASCET) Steering Committee. North American Symptomatic Carotid Endarterectomy Trial: methods, patient characteristics, and progress. Stroke.. 1991;22:711-720.[Abstract/Free Full Text]
  18. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. Circulation.. 1995;92:1355-1374.[Abstract/Free Full Text]
  19. Forrester JS, Fishbein M, Helfant R, Fagin J. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol.. 1991;17:758-769.[Abstract]
  20. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature.. 1993;362:801-809.[Medline] [Order article via Infotrieve]
  21. Davies MG, Hagen PO. Pathophysiology of vein graft failure: a review. Eur J Vasc Endovasc Surg.. 1995;9:7-18.[Medline] [Order article via Infotrieve]
  22. Sottiurai VS, Yao JST, Batson RC, Sue SL, Jones R, Nakamura YA. Distal anastomotic intimal hyperplasia: histopathologic character and biogenesis. Ann Vasc Surg.. 1989;3:26-33.[Medline] [Order article via Infotrieve]
  23. Rekhter M, Nicholls S, Ferguson M, Gordon D. Cell proliferation in human arteriovenous fistulas used for hemodialysis. Arterioscler Thromb.. 1993;13:609-617.[Abstract/Free Full Text]
  24. Swedberg SH, Brown BG, Sigley R, Wight TN, Gordon D, Nicholls SC. Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients: clinical, immunocytochemical, light and electron microscopic assessment. Circulation.. 1989;80:1726-1736.[Abstract/Free Full Text]
  25. Hayry P, Isoniemi H, Yilmaz S, Mennander A, Lemstrom K, Raisanen-Sokolowski A, Koskinen P, Ustinov J, Lautenschlager I, Taskinen E. Chronic allograft rejection. Immunol Rev.. 1993;134:33-81.[Medline] [Order article via Infotrieve]
  26. Ventura HO, Mehra MR, Smart FW, Stapleton DD. Cardiac allograft vasculopathy: current concepts. Am Heart J.. 1995;129:791-799.[Medline] [Order article via Infotrieve]
  27. Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell proliferation in human coronary arteries. Proc Natl Acad Sci U S A.. 1990;87:4600-4604.[Abstract/Free Full Text]
  28. Rowlands DC, Brown HE, Barber PC, Jones EL. The effect of tissue fixation on immunostaining for proliferating cell nuclear antigen with the monoclonal antibody PC10. J Pathol.. 1991;165:356-357.[Medline] [Order article via Infotrieve]
  29. Shivji KK, Kenny MK, Wood RD. Proliferating cell nuclear antigen (PCNA) is required for DNA excision repair. Cell.. 1992;69:367-374.[Medline] [Order article via Infotrieve]
  30. Yu CCW, Woods AL, Levison DA. The assessment of cellular proliferation by immunohistochemistry: a review of currently available methods and their applications. Histochem J.. 1992;24:121-131.[Medline] [Order article via Infotrieve]
  31. Spagnoli L, Villaschi S, Neri L, Palmiera G, Taurino M, Faraglia V, Fiorani P. Autoradiographic studies of the smooth muscle cells in human arteries. Arterial Wall.. 1981;7:107-112.
  32. Stary HC, McMillan GC. Kinetics of cellular proliferation in experimental atherosclerosis. Arch Pathol.. 1970;89:173-183.[Medline] [Order article via Infotrieve]
  33. O'Brien ER, Garvin MR, Dev R, Stewart DK, Hinohara T, Simpson JB, Schwartz SM. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol.. 1994;145:883-894.[Abstract]
  34. Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest.. 1991;88:1121-1127.
  35. Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D. Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A.. 1991;88:5252-5256.[Abstract/Free Full Text]
  36. Sporn MB, Roberts AB, Wakefield LM, deCrombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol.. 1987;105:1039-1045.[Free Full Text]
  37. Quinn MT, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis. Proc Natl Acad Sci U S A.. 1988;85:2805-2809.[Abstract/Free Full Text]
  38. Rosenfeld ME, Palinski W, Yla-Herttuala S, Carew TE. Macrophages, endothelial cells, and lipoprotein oxidation in the pathogenesis of atherosclerosis. Toxicol Pathol.. 1990;18:560-571.[Medline] [Order article via Infotrieve]
  39. Campbell GR, Campbell JH, Manderson JA, Horrigan S, Rennick RE. Arterial smooth muscle: a multifunctional mesenchymal cell. Arch Pathol Lab Med.. 1988;112:977-986.[Medline] [Order article via Infotrieve]
  40. Villaschi S, Spagnoli L. Autoradiographic and ultrastructural studies on the human fibro-atheromatous plaque. Atherosclerosis.. 1983;48:95-100.[Medline] [Order article via Infotrieve]
  41. Rosenfeld ME, Ross R. Macrophage and smooth muscle cell proliferation in atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis.. 1990;10:680-687.[Abstract/Free Full Text]
  42. Gerrity RG. The role of the monocyte in atherogenesis, II: migration of foam cells from atherosclerotic lesions. Am J Pathol.. 1981;103:191-200.[Abstract]
  43. Rekhter MD, Gordon D. Active proliferation of different cell types, including lymphocytes, in human atherosclerotic plaques. Am J Pathol.. 1995;147:668-677.[Abstract]
  44. Falk E. Why do plaques rupture? Circulation. 1992;86(suppl III):III-30-III-42.
  45. Iyers S, Roubin GS, Yadav JS, Diethrich EB, Wholey MH, Shawl FA, Satler LF, Leon MB, Dorros G, Ramee SR. Angioplasty and stenting for extracranial carotid stenosis: multicenter experience. Circulation. 1996;94(suppl I):I-58. Abstract.
  46. Davies MJ, Woolf N, Rowles P, Richardson PD. Lipid and cellular constituents of unstable human aortic plaques. Basic Res Cardiol. 1994;89(suppl 1):33-39.
  47. Moreno PR, Falk E, Palacios IF, Newell JB, Fuster V, Fallon JT. Macrophage infiltration in acute coronary syndromes: implications for plaque rupture. Circulation.. 1994;90:775-778.[Abstract/Free Full Text]
  48. Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Acad Natl Sci U S A.. 1991;88:8154-8158.[Abstract/Free Full Text]
  49. Mitchinson MJ, Ball RY. Macrophages and atherogenesis. Lancet.. 1987;2:146-149.[Medline] [Order article via Infotrieve]
  50. Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation.. 1995;92:1565-1569.
  51. Nikkari ST, O'Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE, Clowes AW. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation.. 1995;92:1393-1398.[Abstract/Free Full Text]
  52. Alexandrov AV, Bladin CF, Maggisano R, Norris JW. Measuring carotid stenosis: time for a reappraisal. Stroke.. 1993;24:1292-1296.[Abstract/Free Full Text]



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