(Circulation. 1996;94:1255-1262.)
© 1996 American Heart Association, Inc.
Articles |
Luminal Foam Cell Accumulation Is Associated With Smooth Muscle Cell Death in the Intimal Thickening of Human Saphenous Vein Grafts
the Department of Pathology (M.M.K., H.B., N.deM., J.M.), AZ Middelheim, Antwerp; and the Division of Pharmacology (G.R.Y.DeM., A.H.), Centre for Electron Microscopy (A.B., W.J.), and Department of Chemistry (L.VanV.), University of Antwerp, Wilrijk, Belgium.
Correspondence to Dr M. Kockx, Department of Pathology, AZ Middelheim, Lindendreef, 1, B-2020 Antwerp, Belgium. E-mail mark.kockx@uia.ua.ac.be.
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Abstract |
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Background Occlusion of saphenous vein grafts is a major problem after coronary artery bypass graft surgery. Diffuse intimal thickening develops in all implanted aortocoronary saphenous vein grafts within 6 months to 1 year. In some regions of the thickened intima, foam cells accumulate along the luminal margin. This particular morphology resembles the morphology of unstable atherosclerotic plaques as they occur in coronary arteries. In the present study, we focused on the possible topographic relation between luminal foam cell accumulation and cell death of smooth muscle cells (SMCs) within the adjacent thickened intima.
Methods and Results Segments of occluded and suboccluded implanted human aortocoronary saphenous vein grafts were obtained during reintervention coronary artery bypass graft surgery in 30 patients. In the regions of the vein grafts with luminal foam cell accumulation, the percentage of SMC 
-actin immunoreactive area of the superficial intimal thickening was 6±1.4%, which was different from the 17.6±2.3% of the deep intimal thickening. A strong negative correlation between the number of foam cell nuclei and the percentage of SMC -actin immunoreactive area in the adjacent superficial intimal thickening was present (r=-.77, P<.001). Within the superficial intimal thickening, cytoplasmic and DNA fragmentation could be detected, which points to apoptotic cell death. A fraction of the cytoplasmic fragments fitted the ultrastructural characteristics of matrix vesicles and showed pronounced calcium and phosphorus accumulation as demonstrated with the use of x-ray microanalysis.
Conclusions The close spatial relation among foam cell accumulation, pronounced intimal SMC loss, and cell death suggests the presence of a foam cell–derived factor that can induce cell death in the SMC population of the intimal thickening. The depletion of the intimal SMC population could promote plaque rupture and thrombotic complications in the grafts.
Key Words: apoptosis • smooth muscle cells • vein grafts • atherosclerosis • foam cells • calcium
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Introduction |
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Diffuse intimal thickening develops in all implanted aortocoronary saphenous vein grafts within 6 months to 1 year. The process is regarded as an unavoidable adaptive response of the vein wall to the modified flow conditions.1 The intimal thickening that develops is mainly composed of SMCs.
In some regions of the vein grafts, foam cells accumulate along the luminal margin. The process can be considered as an accelerated form of atherosclerosis2 3 and can precipitate vein graft occlusion.
In a previous study,4 we demonstrated foam cell replication and SMC apoptosis in occluded atherosclerotic human saphenous vein grafts. Foam cell accumulation and replication were mainly present in the innermost layer of the transplanted vessel. Apoptotic cell death was detected within the underlying intimal thickening. Within the same vein graft, regions with pronounced foam cell accumulation alternated with regions without foam cell accumulation.
In the present study, we focused on the possible topographical relation between luminal foam cell accumulation and cell death within the adjacent thickened intima. The regions of the vein grafts that demonstrate only intimal thickening are compared with the regions in which associated luminal foam cell accumulation can be found. A better understanding of the mechanism of SMC loss in the intimal thickening could be important for the prevention of complications such as plaque weakening, rupture, and resulting complications.
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Methods |
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Vein grafts with a
70% reduction in luminal diameter, as demonstrated with preoperative cardiac catheterization, were considered to be significantly stenotic. Segments of occluded and suboccluded implanted aortocoronary saphenous vein grafts were obtained during reintervention coronary bypass operation in 30 patients. The mean time interval between the initial and second intervention was 9.9±2.4 years. The vein grafts were fixed in either formaldehyde or glutaraldehyde.
The formalin-fixed vein grafts (n=16) were processed for light microscopy, immunohistochemistry, and ISEL. Nonimplanted saphenous vein segments (surplus segments) were used as a control. After fixation in 4% formalin, at least three fragments per graft were embedded in paraffin. A Scharlach red fat stain was performed on cryostat sections of the formalin-fixed segments of the vein graft. An alizarin stain was performed to detect calcium deposits.5
The glutaraldehyde-fixed vein grafts (n=14) were processed for TEM.
Immunohistochemistry
The reactions were carried out with the use of the indirect peroxidase antibody conjugate technique. The following monoclonal primary antibodies were used: SMC -actin (dilution 1:2000, Sigma Chemical Co), collagen type IV, macrophages HAM-56 and CD-68 (DAKO), and Ki-67 (Immunotech). All antibodies were diluted in PBS. After three washes with PBS, the sections were incubated with goat anti-mouse peroxidase (Jackson) for 45 minutes. For demonstration of the complex, AEC was used as a chromogen. For negative controls, the primary antibody was omitted. The specificity of the primary antibodies was tested in tissue with known staining patterns. We compared the staining pattern of rabbit intestine by the antibody against Ki-67 (MIB-1) with in vivo bromodeoxyuridine-labeled cells. The distribution of proliferative activity as demonstrated by the antibody against Ki-67 was similar to a 1-day labeling experiment with bromodeoxyuridine. To verify whether the antibody against Ki-67 did not react with apoptotic cell nuclei, we studied macrophages in culture (THP-1 cell line). Apoptotic nuclei, which were easily identified in the cell culture with the use of morphological criteria, were strictly negative.
Quantification
The images were analyzed with the use of a color-image analysis system (PC Image Colour, Foster Findlay Associates). The venous wall was divided into six equal-sized sectors. In the central region of each sector, three adjacent rectangles along the same radius, covering the entire thickness of the atherosclerotic plaque, were measured. The most luminal rectangle was located along the luminal border. This region often contained numerous foam cells; the number of foam cell nuclei was quantified within this rectangle.
For measurement of the second and third rectangles, the frame was moved along the same radius toward the adventitia. The second rectangle was located within the SIT. The third rectangle was located within DIT. The area and percentage of the -actin immunoreactive area within both the superficial and deep intimal rectangles were measured. The decrease in the percentage of immunoreactive area is a measurement of SMC loss and of a decrease in the -actin expression by the remaining SMC population. The segmentation of the -actin immunoreactive area was done through interactive selection of the gray-level zone corresponding with the brown of the immunoreactive regions.
DNA ISEL
For DNA ISEL, we used an adaptation of the technique of Wijsman et al.6 Briefly, after deparaffinization and rehydration tissue sections were incubated with 3% citric acid for 1 hour and then treated with proteinase K 20 µg/mL (Boehringer Mannheim) for 10 minutes at room temperature. Both TUNEL and ISNT were used.
For the TUNEL technique, the ApopTagkit/Oncor (Gaithersburg) was used with minor modifications.
For the ISNT technique, the sections were rinsed in a buffer (50 mmol/L Tris-HCl, 5 mmol/L MgCl2, 0.0005% BSA, pH 7.5) for 10 minutes, dried, and then incubated at 37°C for 1 hour with the same buffer containing 0.01 mmol/L dATP, 0.01 mmol/L dCTP, 0.01 mmol/L dGTP (Sigma), and 0.01 mmol/L biotin-16-dUTP (Boehringer Mannheim) with 20 U/mL of the Klenow fragment of DNA polymerase I (Boehringer Mannheim). Incorporated biotin-16-dUTP was demonstrated through incubation of the sections with a monoclonal antibody against biotin (DAKO) at a dilution of 1:40 for 30 minutes. The antibody was visualized with the use of a goat anti-mouse peroxidase (Jackson) at a dilution of 1:125 for 45 minutes.
With both the TUNEL and ISNT techniques, the labeled antibody was visualized with the use of AEC (Sigma). Sections were lightly counterstained with hematoxylin and mounted in glycerin jelly. Negative controls included omission of terminal deoxynucleotidyl transferase or the Klenow fragment from the labeling mixture.
TEM
The fragments for TEM were fixed for 2 hours in 1% (vol/vol) glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4). They were postfixed for 30 minutes in 1% (vol/vol) osmium tetroxide in 0.1 mol/L sodium cacodylate buffer (pH 7.4). After dehydration in an ethanol gradient, they were embedded in LX-112 (Ladd Research Industries). Selection of the zones most representative for the lesions was made on 2-µm sections oriented in a transverse plane (perpendicular to the bloodstream) and stained with toluidine blue. Fifty-nanometer-thick sections were cut with an Ultratome Nova (Reichert-Jung). They were stained during a period of 30 minutes at 40° with uranyl acetate and during a period of 15 minutes at 20° with lead citrate in an Ultrostainer 2168 (LKB). The sections were examined in a Jeol-1200 EX TEM at 80 kV. Photographs were made with electron microscopy film 4489 Estar Thick Base (Kodak).
Fourier Transform Laser Microprobe Mass Spectrometry
Sections as prepared for light microscopy were analyzed with Fourier transform laser microprobe mass spectrometry with external ion source, which was developed in our laboratory7 and later available as Bruker CMS 47x Microfocus (Bruker Spectrospin). The ionization is performed with a pulsed Nd:YAG frequency quadruple laser (
=266 nm, 
=4 ns), focused on a spot of 5-µm diameter. The sample is irradiated in reflection geometry. Sample observation also occurs at a x700 magnification. The ions are then transferred through static electrical fields to the Fourier transform mass spectrometry Infinity cell8 in a 4.7-T superconducting magnet. Mass resolution routinely exceeds 100 000, at m/z of 1000, and the mass accuracy is >1 ppm.
X-ray Microanalysis
For the study of calcium and phosphorus distribution in the sections, a scanning TEM (Jeol 1200 EX) equipped with an EDS detector (Tracor TN 5500) was used to acquire radiographic mappings.
Statistical Analysis
The number of patients is indicated by n. One vein graft of each patient was examined on different transversal sections. One transverse section per vein graft was examined with six measurements, each consisting of a foam cell–rich area, an SIT, and a DIT. The percentage of immunoreactive area of the superficial intima was compared with the percentage of the deep intima with the use of a Student's paired t test. Regression analysis was done by comparing the number of foam cell nuclei with the percentage of immunoreactive area of both the superficial and deep intima. The SPSS for Windows software package was used for these purposes. A value of P=.05 was considered statistically significant.
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Results |
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Macroscopy
The vein grafts demonstrated a thickened wall that contained foci of yellow-brown friable fat accumulation along the luminal border. These regions were frequently associated with red-brown thrombi that often occluded the remaining vascular lumen.
Light Microscopy
All vein grafts showed a concentric intimal thickening with a thickness of from 1000 to 1500 µm. The intimal thickening consisted of SMCs lying within a dense matrix of collagen fibers. In the regions without luminal foam cell accumulation, no differences between the SIT and DIT were present.
At least one of the transverse sections per vein graft contained a region of foam cell accumulation (Fig 1a
). This region was located along the luminal border of the intimal thickening. The region of foam cell accumulation was often associated with mural thrombi. The combination of a fibrous intimal thickening and foam cell accumulation is comparable with atherosclerotic plaque formation. We distinguish three different layers within the plaque. (1) The luminal region of the plaque contained numerous foam cells and is called the foam cell–rich region. The thickness of this layer was variable within a transversal section and between transverse sections at different levels of the same vein graft. Within the same vein graft, regions with pronounced foam cell accumulation alternated with regions without foam cell accumulation. The regions without foam cell accumulation consisted of only intimal thickening (regions 2 and 3). (2) The mid region of the plaque adjacent to the foam cell–rich region, or the SIT. (3) The deep region of the plaque, or the DIT. This region was mainly composed of SMCs.
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In the foam cell–rich region of the plaque, the majority of lipids were located within the cytoplasm of the cells (Fig 1b
). The adjacent mid region of the plaque (SIT) contained few SMCs and areas of extracellular lipid deposition. A fraction of the SMCs contained discrete lipid droplets within their cytoplasm. The DIT and the media of the vein grafts contained no lipid deposition. In the vein grafts without foam cell accumulation, lipid deposition was absent in the intimal thickening.
An Alizarin stain demonstrated calcium accumulation within the fibrous intimal thickening when luminal foam cell accumulation was present (Fig 1c). The calcium deposits were present as fine granular cytoplasmic or matrix vesicles. The calcium deposits were often localized within the SIT adjacent to the luminal region of foam cell accumulation. Calcium deposits were not present in the foam cell–rich region itself or in the intimal thickening of the vein grafts without foam cell accumulation.
Immunohistochemistry
In the regions without foam cell accumulation, the SIT and DIT were mainly composed of SMCs, which were immunoreactive for SMC -actin. The SMCs were lying in a circular direction (perpendicular to the bloodstream) and had a fusiform shape. The percentage of SMC -actin immunoreactive area of the SIT was not different from that of the DIT (Fig 2). Macrophages were either absent or scarcely present.
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In the region with luminal foam cell accumulation, a clear difference was present between the SIT and DIT. The foam cells were immunoreactive for HAM-56 and CD-68 but negative for SMC -actin, demonstrating their monocytic origin. Numerous foam cell nuclei were immunoreactive for Ki-67 (Fig 1d).
The percentage of SMC -actin immunoreactive area of the SIT was 6±1.4% compared with 17.6±2.3% for the DIT (Figs 2, 3a, and 3b). The number of foam cell nuclei in the luminal region was 1929±248/mm2. A strong negative correlation between the number of foam cell nuclei and the percentage of SMC -actin immunoreactive area in the SIT was present (r=-.77, P<.001).
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DNA ISEL
The TUNEL and ISNT techniques gave a similar staining pattern. The regions without luminal foam cell accumulation did not show labeled nuclei.
The regions with luminal foam cell accumulation demonstrated labeled nuclei and nuclear fragments of fusiform SMCs within the superficial intima (Fig 4a and 4b). Nonimplanted saphenous vein segments (surplus segments) did not show labeled nuclei.
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TEM
The regions without luminal foam cell accumulation were covered with endothelial cells. There was a considerable variation in the ultrastructural characteristics of the endothelial cells. In some endothelial cells, prominent bundles of microfilaments were present. In other endothelial cells, the number of Weibel-Palade bodies was increased. The subendothelial basal lamina was multilaminated and thickened. SMCs of the SIT showed an intact cytoplasm with microfilaments and subplasmalemmal microvesicles. The basal lamina around the SMCs was thickened and multilaminated. The SMCs were lying in small clusters of two to four cells, with contact via appositions or cytoplasmic invaginations. Cell fragmentation or matrix vesicles were not present.
The regions with luminal foam cell accumulation within the SIT demonstrated SMCs with a dropping off and fragmentation of the cytoplasm and condensation of the chromatin, which are ultrastructural characteristics of apoptosis. The cytoplasm of the SMCs in this layer was often fragmented, and it was possible to detect cells that were completely fragmented into small vesicles (Fig 5). Their SMC origin could still be revealed because these vesicles were surrounded by a cage composed of basal laminae. The vesicles had different diameters, and some had the ultrastructural characteristics of matrix vesicles. A dense connective tissue matrix of cross-banded collagen fibers was present between the SMC clusters, surrounded by basal laminae.
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X-ray Microanalysis
We analyzed the cell fragments detected via TEM in the SIT in regions with foam cell accumulation. The cell fragments with a dense staining showed a clear calcium and phosphorus signal (Fig 6A). Adjacent cell fragments with normal cytoplasmic density did not show calcium or phosphorus accumulation.
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Fourier Transform Laser Microprobe Mass Spectrometry
In the vein grafts without luminal foam cell accumulation, a calcium signal was not detectable.
In the vein grafts with luminal foam cell accumulation, a clear calcium signal was detected with this technique. Phosphate was not present (Fig 6B and 6C). This means that calcium is not present as hydroxyapatite. This result, combined with the detected phosphorus signal with the use of x-ray microanalysis and the fat accumulation, suggests a binding of calcium to long-chain phospholipids.
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Discussion |
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In the present study, we demonstrated that all implanted vein grafts developed a pronounced concentric intimal thickening. This layer is composed mainly of SMCs. In some regions of the vein grafts, foam cells accumulate and replicate along the luminal layer of the thickened intima. This is associated with a pronounced cell loss in the adjacent region of the intimal thickening. This association could be of interest in understanding the progressive cell loss that occurs during the progression of atherosclerotic plaques.
The foam cells were found at the luminal side of the thickened intima and within adherent thrombi. The presence of foam cells in the luminal region and an adjacent fibrous hypocellular layer is reminiscent of the fibrolipid lesions of the human aorta described by Bocan and Guyton.9 The foam cells were macrophages, as demonstrated by their cytoplasmic immunoreactivity for HAM-56 and CD-68 and their ultrastructural characteristics. In general, the foci of the foam cells were not covered with a fibrous cap, as seen in most of the arterial atherosclerotic plaques. The present study demonstrates that these foam cells are replicating, as demonstrated by their nuclear immunoreactivity for Ki-67. The antibody recognizes the protein Ki-67, which appears during G1, S, and G2 phases and mitosis but not during the G0 phase of the cell cycle.10 The specificity of the antibody for detection of cell replication was confirmed in a previous study.11 These findings indicate that the foam cell lesions develop not only through monocyte accumulation with subsequent foam cell transformation but also through foam cell replication. We recently demonstrated similar high replication rates in foam cells within foam cell lesions of hypercholesterolemic rabbits.11 The high replication fraction of foam cells in the luminal layer could be the consequence of a gradient of growth factors. It was demonstrated that M-CSF is present at high concentrations in the immediate subendothelial cells in advanced fatty streaks.12 13 Deeper, less cellular layers of the fatty streak contain little or no M-CSF. Previously, we demonstrated that the endothelial cells covering fatty streaks in hypercholesterolemic rabbits demonstrated and enhanced von Willebrand factor content.14 This could be a manifestation of a change in endothelial cell function. Therefore, it is possible that the altered endothelial cells are responsible for a gradient of different growth factors. M-CSF has been demonstrated in endothelial cells of both human and rabbit tissue.12 15
The major part of the vein graft wall was formed by a myointimal thickening, which was almost exclusively composed of SMCs and a dense matrix of collagen fibers. When luminal foam cell accumulations were present, a clear distinction between a superficial cell-poor and a deep cell-rich intima could be detected and quantified. SMC death, reminiscent of apoptosis, could be detected within the superficial cell-poor intimal thickening. Few data exist on the extent and distribution of apoptotic cell death in atherosclerotic blood vessels. Isner et al16 found evidence of apoptotic cell death in primary atherosclerotic and, in particular, restenotic lesions. Apoptotic cell death was positively linked to cell replication. Restenotic lesions, showing high replication rates, also demonstrated more apoptotic nuclei. A similar result was found by Bochaton-Piallat et al17 in the intimal thickening induced after endothelial denudation of the rat aorta. In the present study, a type of accelerated human atherosclerosis was studied. A clear association between foam cell accumulation and cell death in the intimal thickening could be demonstrated. This finding confirms the results of Imai and Thomas18 showing diet-induced lesions in cerebral atherosclerosis in swine in a study 
25 years ago. These authors studied the induced atherosclerotic lesions extensively with TEM and found SMC death in the plaques. The authors described these changes as necrosis of the cells, but their description of the nuclear and cytoplasmic changes fulfills the criteria of apoptotic cell death.19 In the present study, we found cell loss and cell death located in the SIT, adjacent to luminal foam cell accumulations with high replicative activity.
The detection of DNA fragmentation in tissue sections through the use of TUNEL or ISNT has been considered to be a good marker for apoptotic cell death.5 20 However, it is evident from the present study and a previous study that the cytoplasmic changes of apoptosis as detected with TEM were far more frequent than the nuclei labeled with the use of the ISNT or TUNEL technique.11 This finding suggests that the apoptotic cytoplasmic cell fragments can be detected for a longer period of time than the nuclear fragments in the atherosclerotic plaque. This possibility could be a consequence of an impaired phagocytosis process in the plaque. Another explanation is that a fraction of the SMC population may undergo apoptotic cell death without nuclear condensation or DNA fragmentation, a finding that is in agreement with the observation that SMCs in culture may undergo apoptosis without DNA fragmentation.21 Recent data from the study of Jacobson et al22 state that the nucleus is not a prerequisite for apoptotic cell death because cytoplasts (cells without a nucleus) show apoptotic cell death after growth factor deprival and other cell death signals.
Therefore, the use of in situ labeling techniques may underestimate the extent of apoptotic cell death in atherosclerotic plaques. The remaining SMCs within the superficial layer of the intimal thickening were always surrounded by a cage of thickened basal laminae. The cytoplasm of the SMCs in this layer was often fragmented, and it was possible to detect cells that were completely fragmented into small vesicles. Their SMC origin could still be revealed because these vesicles were surrounded by a cage composed of basal laminae. The vesicles had different diameters and had the ultrastructural characteristics of matrix vesicles. A fraction of these vesicles indeed showed a pronounced calcium and phosphorus accumulation as demonstrated through radiographic analysis. These structures are analogous to the vesicles described by Anderson23 during the process of calcification in the epiphyses of long bones. The combination of energy-dispersive x-ray analysis and Fourier transform mass spectrometry led us to conclude that calcium in the vesicles is not bound to phosphate but rather probably to phospholipids. Recently, the presence was demonstrated of cholesterol within the center of calcified granules from human atherosclerotic plaques.24 The association of apoptotic cell death and formation of matrix vesicles could provide an interesting pathway. Apoptotic cell death is associated in a cell line with the appearance of phosphatidylserine on the cell surface.25 The same type of acidic phospholipid is present in matrix vesicles of human chondrocytes.23 Phosphatidylserine was shown to have a strong affinity for ionic calcium, with increased calcium affinity in the presence of phosphate. This indicates that phosphatidylserine and other phospholipids could act as a non–energy-requiring mechanism, localized at the site of mineralization. The possibility that calcification in atherosclerotic plaques is the consequence of apoptotic cell death and subsequent matrix vesicle formation could provide an interesting pathway for pharmacological interventions.
The close topographical relation among lipid accumulation, cell death, and calcium deposition suggests the following hypothesis: A fraction of the SMC population in atherosclerotic plaques, adjacent to regions of foam cell accumulation, is killed by factors that are toxic to the SMCs but not to the foam cells, which are of macrophage origin. The responsible factors are not known at this moment, but ox-LDL and nitric oxide must be considered.26 27 28 Both of these factors are known to induce apoptosis in the macrophages themselves. Apoptosis, however, was not frequent in the foam cell population. Therefore, other or additional factors must be considered. Gleason et al29 demonstrated that excess cholesterol can cause calcium influx into vascular SMCs, an effect that demonstrates sensitivity to the calcium channel–blocking agents diltiazem, verapamil, and nifedipine. Increased cytoplasmic calcium levels are a potent stimulus for cell death in different models.30 31 However, it must be stated that the calcium deposition within the SIT can either be related to the cause or merely a consequence of SMC death.
The luminal accumulation of inflammatory cell infiltrates in human atherosclerotic vein grafts was also described by van der Wal et al.32 These authors state that this particular morphology is reminiscent of that described for unstable atherosclerotic plaques as they occur in coronary arteries.33 In their study, the authors found that the infiltrates at the luminal surface of atherosclerotic vein grafts contained both macrophages and T lymphocytes.32 The T lymphocytes could be a potent source for interferon-
. Hansson et al34 found that interferon- inhibits SMC -actin expression and is an inhibitor of proliferation of vascular SMCs. Furthermore, interferon- is an activator of macrophages and may also regulate lipid metabolism in these cells.35 These findings could give an alternative explanation for the close spatial relation between foam cell accumulation and intimal SMC death.
Another consideration is that the SMCs of the thickened intima could be programmed to die and that a foam cell–derived factor activates the machinery of apoptotic cell death. Han et al36 found that the concentration of apoptotic cells in advanced human coronary atherosclerotic plaques is highest in regions enriched in macrophages. These authors support the idea that within these regions, macrophages are initiating and/or propagating apoptotic cell death events. Schwartz and Bennett37 found that SMCs derived from atherosclerotic plaques show a higher rate of apoptotic cell death in cell culture compared with SMCs derived from the media or normal arteries. Recently, Geng and Libby38 demonstrated interleukin-1ß–converting enzyme expression in human atherosclerotic plaques. This is of considerable interest because of the potential of interleukin-1ß–converting enzyme or related enzymes in the induction of apoptotic cell death.39 The present study indicates that although there are arguments from the literature that some intimal SMCs are programmed to die, an additional foam cell–derived factor could be necessary to induce the cell death and subsequent cell loss in the intimal SMC population. A better understanding of the mechanism of SMC loss in the intimal thickening could be important for the prevention of complications such as plaque weakening, rupture, and resulting complications.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr De Meyer and Dr Van Vaeck are indebted to the National Fund for Scientific Research (Belgium), at which they are a senior research assistant and research director, respectively.
Received January 18, 1996; revision received March 13, 1996; accepted March 26, 1996.
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References |
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