© 1998 American Heart Association, Inc.
Original Contributions |
Cell Composition, Replication, and Apoptosis in Atherosclerotic Plaques After 6 Months of Cholesterol Withdrawal
From the Department of Pathology (M.M.K.), A.Z. Middelheim, Antwerp, Belgium; the Division of Pharmacology (G.R.Y. De M., N.B., H.B., A.G.H.), University of Antwerp, Wilrijk, Belgium; and Applied Cardiovascular Morphology (M.W.M.K.), Antwerp, Belgium.
Correspondence to Dr M. Kockx, Department of Pathology, A.Z. Middelheim, Lindendreef, 1, B-2020 Antwerp, Belgium. E-mail mark.kockx{at}uia.ua.ac.be
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Abstract |
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Abstract—Unstable human atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophagic origin. Apoptosis of SMCs in the fibrous cap could destabilize the plaque and promote plaque rupture. In an experimental approach, we have studied apoptotic cell death and related proteins in atherosclerotic plaques of cholesterol-fed rabbits and examined the effects of cholesterol withdrawal. The induced atherosclerotic plaques at the thoracic aorta were composed of both fibromuscular tissue and foam cells. The presence of SMCs overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques. The plaques showed signs of cell replication and apoptotic cell death (1.8±0.5% terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei). Cell replication was confined mostly to the macrophages, whereas 34% of the TUNEL-labeled cells were SMCs. Both the macrophages and SMCs in the plaques expressed BAX, a proapoptotic protein of the BCL-2 family. After 6 months of cholesterol withdrawal, the thickness of the plaques in all localizations of the aorta was unchanged, but apoptosis was nearly absent (<0.1% of nuclei). Moreover, macrophages disappeared from the plaques, whereas the SMCs that remained present lost their lipid accumulation and strongly reduced their BAX expression. These changes were associated with a reduction of cell replication and increased deposition of fibrillar collagen fibers in the plaques, which pointed to plaque stabilization. In conclusion, the cell composition but not the thickness of atherosclerotic plaques was profoundly altered after a 6-month cholesterol withdrawal period. These changes were associated with a strong reduction of cell replication and apoptotic cell death. Moreover, the expression of the proapoptotic factor, BAX, was reduced in the remaining cells, which were mainly SMCs. These findings could help to explain the benefit of lipid-lowering therapy on plaque stabilization.
Key Words: atherosclerosis • apoptosis • smooth muscle cell • BAX • terminal deoxynucleotidyl transferase nick end-labeling
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophage accumulation is linked to plaque destabilization and can lead to acute coronary syndromes.1 2 3 4 In a previous study involving the occurrence of cholesterol-induced atherosclerotic plaques of the rabbit aorta, we were struck by the high cell replication of macrophages and the presence of apoptotic cell death in the plaques.5
Apoptosis is also present in human atherosclerotic plaques6 7 8 9 10 11 12 and is pronounced in regions with macrophage infiltration, whereas lesions consisting only of smooth muscle cells exhibit very little apoptosis. Most of these studies detect apoptotic cells by the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) technique, which uses DNA fragmentation as a marker of apoptotic cell death.
DNA fragmentation is a rather late stage of apoptotic cell death. Apoptosis occurs in at least 2 stages.13 After a signal, which may be either intrinsic or extrinsic to the cell, the cell enters a committed phase. This is terminated in cell-autonomous fashion by a transition to a final execution phase. The latter, which includes DNA fragmentation, is brief and decisive. Bennet et al14 have found that most smooth muscle cells derived from atherosclerotic plaques but not those from the media die when brought in culture. This suggests that the smooth muscle cells of the atherosclerotic plaques but not the medial smooth muscle cells are committed to die. In the present study, cell replication and the execution and commitment phases of apoptotic cell death were quantified in experimental atherosclerotic plaques after 26 weeks of cholesterol feeding and after a period of durable normalization of the serum cholesterol levels. The execution phase of apoptosis was detected by DNA in situ end-labeling.15 16 17 18 Changes in the expression of proteins of the BCL-2 family were used to detect the commitment phase.
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Materials and Methods |
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Male New Zealand White rabbits (2.8 to 3.5 kg) were fed a diet supplemented with low-dose cholesterol (0.3%) for 26 weeks (n=20). Ten randomly selected animals were killed after this period (26w chol group); the other animals were fed a normal diet for another 26 weeks (26w chol wd group). After this period, these animals were also killed for study. A control group of 10 animals was included. Five control animals were killed after 26 weeks and 5 were killed after 52 weeks. Serum was stored at -20°C, and serum total cholesterol, free cholesterol, serum triglyceride, phospholipid, LDL, HDL, and VLDL levels were determined. For histological examination, the complete aorta was dissected and fixed in situ in 4% formalin. Transversal sections (5 µm thick) of paraffin-embedded tissues were mounted on slides precoated with 3-aminopropyltriethoxysilane (Sigma). Masson's trichrome stain, sirius red hematoxylin, and Verhoeff's elastic stain were used on all sections. A Sudan IV (Scarlet Red Michaelis, BDH) fat stain was used on frozen sections of formalin-fixed segments.
Immunohistochemistry
The following primary monoclonal antibodies were used:
-smooth muscle actin (Sigma Chemical Co), MO/RAM-11 (anti-rabbit
macrophages) and BCL-2 (Dako), BAX (rat monoclonal,
Pharmingen), and Ki-67 (MIB-1, Immunotech).
All antibodies were diluted in PBS. The monoclonal antibodies were detected by an indirect peroxidase antibody conjugate technique. The sections were incubated with a goat anti-mouse peroxidase antibody (Jackson) and a rabbit anti-rat antibody (Dako) for 45 minutes. For demonstration of the complex, 3-amino-9-ethylcarbazole was used as a chromogen. The specificity of the immunohistochemical reactions was checked by omitting the primary antibody and substituting the antibody by an unrelated antibody at the same concentration.
To demonstrate the colocalization of BAX and -smooth muscle actin,
we used a stain and washout technique that was recently developed in
our laboratory. A classical double immunohistochemical stain was
difficult to interpret because of the color overlap and the problem of
mixed colors. Therefore, we first stained the sections with an antibody
against -smooth muscle actin and made digital photomicrographs This
was followed by an antigen-retrieval step (trypsin digestion and
citrate buffer treatment in a microwave oven), which was necessary to
destroy the residual antibodies of the first stain and retrieve the
antigen for the immunohistochemical stain for BAX. The same cells that
were photographed for -smooth muscle actin were relocated in the
section, and digital pictures were taken. Controls for this stain and
washout technique included omitting the primary antibody against BAX.
This resulted in a complete disappearance of the staining. This
indicates that the antibodies used for the -smooth muscle actin
stain (first stain) did not interfere with the secondary antibody used
to detect BAX (second stain).
DNA In Situ End-Labeling
After deparaffinization and rehydration, tissue sections were
incubated with 3% citric acid. This step removes all small
calcium-containing vesicles that can be responsible for aspecific
binding of the nucleotides.18 Both
TUNEL15 and in situ nick translation
(ISNT)16 17 were used. For the TUNEL technique,
the ApopTag kit (Oncor) 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, and 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, dCTP, and 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 by incubating the sections with a monoclonal antibody
against biotin (Dako) at a dilution of 1:40 for 30 minutes. The
antibody was visualized by a goat anti-mouse peroxidase (Jackson) at a
dilution of 1:125 for 45 minutes.
In both the TUNEL and the ISNT techniques, the labeled antibody was visualized by 3-amino-9-ethylcarbazole. 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. Tonsils were used as a positive control.
To identify cell types undergoing apoptosis, double staining
was performed by combining TUNEL and immunohistochemistry for RAM-11
and -smooth muscle actin.
Quantification
The images were analyzed using a color image
analysis system (PC Image Color, Foster Findlay Associates).
The atherosclerotic plaques were divided into rectangular areas located
in the superficial and deep part of the plaques. The area and
percentage of immunoreactive areas within each rectangle were measured.
The segmentation of the immunoreactive area was performed by
interactive selection of the gray level zone corresponding
with the brown color of the immunoreactive regions.
In the atherosclerotic plaques of the cholesterol-fed rabbits, 20 rectangular areas per transversal section were counted along both the superficial and the deep parts of the plaques. The areas were randomly chosen. If the intimal thickness exceeded 200 µm, then a luminal rectangle (base, 90 µm along the endothelial cell layer; height, 100 µm into the intima) and a deep rectangle (base, 90 µm along the internal elastic lamina; height, 100 µm into the plaque) were measured. If the intimal thickness was <200 µm, the intima was split into a superficial and a deep half. The side along the endothelial and the internal elastic membrane was again 90 µm. The mean percentage of the total plaque area per transversal section that was covered by the 20 rectangular areas was 15±2%.
For each region, the total area, the total number of nuclei, the
numbers of Ki-67 and DNA in situ end-labeled nuclei, and the percentage
of immunoreactive areas for RAM-11, -smooth muscle actin, and BAX
were measured. The latter variables were expressed as percentage of
the total area.
The plaque area of the different parts of the aorta (ascending, arch, thoracic, abdominal proximal, and distal) was measured by tracing the internal elastic lamina and the luminal circumference.
Statistical Analysis
Cell Number per Area, Cell Replication, and Apoptosis of
Atherosclerotic Plaques
The total number and the number of labeled nuclei per fixed
cross-sectional area in superficial and deep layers was calculated. The
data for the deep versus the superficial layer were statistically
evaluated using the Wilcoxon signed rank test. To compare the
26w chol group with the 26w chol wd group, the Mann-Whitney
U test was applied.
Plaque Area and Thickness in Different Parts of the Aorta
The 26w chol and 26w chol wd groups were compared using an
unpaired Student t test.
Serum Lipid Values
The values between the 2 groups (26w chol versus 26w chol wd)
were compared using the unpaired Student t test. To compare
the values between the time points (week 0, week 26, and week 52),
ANOVA (3 time points) or the paired Student t test (2 time
points) was applied.
The SPSS package for Windows (SPSS Inc) was applied for these purposes. A 5% level of significance was selected.
Transmission Electron Microscopy
The fragments for transmission electron microscopy 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 of the lesions was made
on 2-µm sections oriented in a transverse plane (perpendicular to the
bloodstream) and stained with toluidine blue. Sections (50 nm thick)
were cut on an Ultratome Nova (Reichert-Jung). They were stained for 30
minutes at 40°C with uranyl acetate and for 15 minutes at 20°C with
lead citrate in an Ultrostainer 2168 (LKB). The sections were examined
in a Jeol-1200 EX transmission electron microscope at 80 kV.
Photographs were made with electron microscopy film (4489 Estar Thick
Base, Kodak).
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Results |
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Serum Lipid Values
The details of the serum lipid values at the start of the experiment, after 26 weeks of cholesterol supplement, and after the cholesterol withdrawal period are shown in the Table
. The diet with a cholesterol
supplement of 0.3% induced a pronounced
hypercholesterolemia at week 26, which was
completely reversed after 26 weeks of cholesterol
withdrawal.
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Atherosclerotic Plaques of Cholesterol-Fed Rabbits at
26 Weeks (n=10)
The induced atherosclerotic plaques at the thoracic aorta were
composed of both fibromuscular tissue and foam cells. The presence of
smooth muscle cells overlying macrophage accumulation was
reminiscent of the structure of human atherosclerotic plaques (Figure 1). However, an important difference was
the absence of plaque rupture and thrombosis.
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The atherosclerotic plaques showed a clear distinction between a
superficial region composed of numerous foam cells and a deep layer
that was fibrous and contained few cells. In the superficial layer, the
majority of lipid was located within the cytoplasm of the cells (foam
cells) (Figure 2A), and
interstitial collagen fibers were nearly absent in this
layer (Figure 2B). Most of these foam cells were of macrophagic origin,
as indicated by their cytoplasmic immunoreactivity for RAM-11 (Figure 2C). The smooth muscle cells were filled with lipid vacuoles, and foam
cells of smooth muscle origin were also present in this layer
(Figure 2D).
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The deeper layer contained areas of extracellular lipid and
interstitial collagen fibers, as demonstrated by sirius red
polarization microscopy (Figure 2B
).
The nuclear density and the percent RAM-11 and -smooth muscle actin
immunoreactive areas were higher in the superficial layer (Figure 3). Although the size of the plaques
decreased toward the bifurcation, the composition and distribution of
the cells in the plaques was rather consistent.
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Cell Replication
Within the superficial region, 11.2% of the nuclei demonstrated
immunoreactivity for Ki 67 (Figure 3). This indicates that the cell
replication in this region is high. A double immunohistochemical stain
for Ki-67 and RAM-11 (Figure 4A) or
-smooth muscle actin (Figure 4B) demonstrated that 95% of the
Ki-67–labeled cells were macrophages and 5% were smooth
muscle cells.
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The deeper layer of the atherosclerotic plaques was cell poor, with low
replication rates, which were significantly different from the
superficial layer. This deeper layer contained macrophages,
cellular remnants, and smooth muscle cells with a decreased -smooth
muscle actin content. These cells were negative for RAM 11.
Apoptotic Cell Death/BAX Expression
Both the superficial and the deep layer of the atherosclerotic
plaques showed apoptotic nuclei, as visualized by TUNEL and
ISNT. The percentages of labeled nuclei in the deep layer were not
different from the percentages in the superficial regions (Figure 3).
The labeled nuclei were either condensed or enlarged. A significant
fraction of the TUNEL-labeled nuclei and nuclear remnants could not be
stained by RAM-11 or -smooth muscle actin, which could reflect a
loss of specific markers during the execution phase of
apoptosis. A feature of smooth muscle cells but not of
macrophages in atherosclerotic plaques is that they are
surrounded by cages of thickened basal lamina.5 8
Basal laminae and basement membranes can be stained by a periodic
acid–Schiff's (PAS) stain. By combining the TUNEL technique with a
PAS stain, we could detect TUNEL-labeled nuclei and nuclear fragments
that were not enclosed by PAS-positive laminae, indicating
apoptosis of macrophages (Figure 4C), and labeled
nuclei that were enclosed by a cage of thickened basal laminae, which
points to smooth muscle cell apoptosis (Figure 4D). Moreover,
clusters of TUNEL-negative cytoplasmic remnants, which were enclosed by
thickened basal lamina, were present, which can be considered as
cytoplasmic remnants of apoptotic smooth muscle cells. By use
of this technique, we have found that 34% of all TUNEL-labeled cells
were smooth muscle cells and 66% were macrophages.
The cellular origin of the TUNEL-labeled nuclei was also studied by
immunohistochemical double stains. Immunohistochemical staining of
adjacent sections and double stainings of TUNEL with RAM-11 or
-smooth muscle actin identified both apoptotic smooth muscle
cells (Figure 4E) and macrophages.
All endothelial cells, a significant proportion of
macrophage-derived foam cells, and many smooth muscle
cells, particularly in superficial layer of the plaque, expressed BAX.
The smooth muscle cells in the adjacent media did not express BAX. A
stain and washout technique for -smooth muscle actin and BAX
demonstrated that the lipid-laden smooth muscle cells expressed BAX
(Figure 4F and 4G); BCL-2 could not be detected. By combining the BAX
immunohistochemical stain with a PAS stain (Figure 4H), we demonstrated
that 47% of all BAX immunoreactive cells in the plaques were smooth
muscle cells; the others were macrophages. Interestingly, 35%
of all TUNEL-labeled smooth muscle cells were also BAX immunoreactive,
whereas only 8% of all TUNEL-labeled macrophages expressed
BAX.
The arteries of the age-matched control animals (n=5) were without
intimal thickening and did not show TUNEL-labeled nuclei. The
endothelial cells expressed BAX, whereas the smooth
muscle cells of the media did not (Figure 4L).
Transmission Electron Microscopy
Transmission electron microscopy of the plaques showed an
uninterrupted layer of endothelial cells (Figure 5A). Underneath the
endothelial cells, numerous foam cells were
present. The foam cells were crowded with large nonmembrane-bound
vacuoles, presenting ramified lamellipodia, containing
lysosomes and nondescript cellular debris, all characteristics
of monocyte-derived macrophages. The smooth muscle cells
contained large dilated profiles of rough endoplasmic reticulum,
medium-sized optically empty vacuoles not bounded by a membrane, few
filaments, few pinocytotic vesicles, and a fragmented basal lamina
(Figure 5A), all stigmata of cells with the synthetic phenotype
and fat accumulation. The thickened basal laminae surrounding these
smooth muscle cells are the ultrastructural equivalent of PAS-positive
cages around the smooth muscle cell (see Figure 4D and 4H). Smooth
muscle cells showing dropping off and fragmentation of cytoplasm and
condensation of the chromatin, which are ultrastructural
characteristics of apoptosis, could be demonstrated. The deep
layer contained cross-banded collagen fibers among irregular moderately
electron-dense granular material and smooth muscle cells.
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Atherosclerotic Plaques in Rabbits After 26 Weeks of
Cholesterol Supplement Followed by 26 Weeks of
Cholesterol Withdrawal (n=10)
The lipid values showed a normalization after the
cholesterol withdrawal period (Table). The cell composition
of the atherosclerotic plaques after a period of
cholesterol withdrawal was profoundly altered (Figure 2A, chol wd; Figure 2B, chol wd). The distinction between a superficial
foam cell–rich layer and a deep fibrous region was lost. Most plaques
were transformed into fibrous collagen-rich plaques, although residual
fat deposits could still be detected. The thickness of the plaques was
not significantly different from the plaque thickness of the 26-week
group, and the areas of the plaques in the different parts of the aorta
were not decreased (Figure 6). The cell
density was decreased (Figure 7).
Macrophages (as demonstrated by a RAM-11 stain) disappeared
almost completely from the plaques (Figure 2C, chol wd; Figure 3). Most
of the remaining cells were smooth muscle cells. The smooth muscle
cells were spindle-shaped and expressed -smooth muscle actin (Figure 2D, chol wd). A superficial band of -smooth muscle actin–expressing
smooth muscle cells was often present and was reminiscent of a
fibrous cap present in human atherosclerotic plaques.
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Cell Replication
The plaques showed a strong reduction in nuclei that were labeled
by the antibody against Ki-67. This indicates a strong reduction of
cell replication within the plaques (Figure 3).
Apoptotic Cell Death/BAX Expression
The plaques showed also a strong reduction in the nuclei that were
labeled by the TUNEL and ISNT techniques (Figure 3). Moreover, a strong
reduction in the expression of BAX was noticed and quantified (Figures 4I, 4J, 4K, and 7). This was a consequence of both the disappearance of
the BAX-expressing macrophages and a loss of the BAX expression
in the smooth muscle cells. BCL-2 could not be detected. We have
calculated RAM-11, -smooth muscle actin, and BAX expression per cell
before and after cholesterol withdrawal (Figure 7).
Interestingly, the BAX immunoreactive area expressed per cell was
significantly decreased after cholesterol withdrawal.
The arteries of the age-matched control animals (n=5) did not develop
atherosclerotic plaques. The smooth muscle cells in the media of these
control animals remained strictly negative for BAX (Figure 4L). The
endothelial cells of the aorta in these control animals
showed BAX expression that was independent of the age of the
animals.
Transmission Electron Microscopy
The plaques were covered by an uninterrupted layer of
endothelial cells, which had become flatter. The
plaques were mainly composed of smooth muscle cells that showed a
contractile phenotype. Few lipid vacuoles remained in the
cytoplasm of the cells. Between the smooth muscle cells, numerous
cross-banded collagen fibers were present (Figure 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates that a cholesterol withdrawal period of 6 months does not induce a regression of experimentally induced atherosclerotic plaques. This finding was reported by Anitschkow19 and subsequently confirmed by other authors.20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 The atherosclerotic plaques in the present study were induced by giving rabbits a cholesterol supplement of 0.3% for 6 months. This resulted in atherosclerotic plaques that contained both foam cells of macrophagic origin, smooth muscle cells, and collagen fibers. The presence of smooth muscle cells overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques.
The plaques after 26 weeks of cholesterol supplement fulfill some criteria of unstable human atherosclerotic plaques, since most of the superficial area was occupied by foam cells of macrophagic origin, and interstitial collagen fibers were absent or scarce in the superficial region of the plaque.1 35 However, an important difference was the absence of plaque rupture and thrombosis. Before the cholesterol withdrawal period, the plaques showed a high cell replication, which was mainly limited to the macrophage population, and demonstrated apoptotic cell death. Moreover, both the macrophages and the smooth muscle cells expressed BAX, a proapoptotic protein of the BCL-2 family.36 37 38 39 This protein was only weakly expressed in the adjacent media and was absent in the aorta of age-matched normocholesterolemic rabbits.
Although the plaques did not regress and even tended to extend into the arch and thoracic and abdominal aorta after the cholesterol withdrawal period, the plaques showed numerous qualitative changes. The distinction between a superficial foam cell–rich layer and a deep fibrous region was lost. Most plaques were transformed into fibrous collagen-rich plaques that showed few residual fat deposits, confirming the study of Aikawa et al.28 Birefringent polarizing fat, which at 26 weeks is mostly globular and intracellular, is present after the cholesterol withdrawal period as needlelike crystals in an extracellular position in the deep layer of the plaques. This confirms the study of Small et al.40 It cannot be excluded that some of the fibrous transformation of the plaques is merely related to the aging of the plaques, since we did not include a group of animals that received the cholesterol supplement for 52 weeks. Some authors have indeed demonstrated that the total amount of collagen of experimental atherosclerotic plaques increased during plaque progression.40 However, we and others have not observed a complete fibrous transformation of the superficial layers and the edges of the plaques when the cholesterol supplement was not stopped.5 40
Most of the changes after 6 months of cholesterol withdrawal are the result of pronounced changes in the cell composition, cell replication, and apoptosis within the plaques during the cholesterol withdrawal period. A strong reduction or even disappearance of the macrophages from the plaques was a major feature. The mechanism and complete time course by which the macrophages disappear from the plaques was beyond the scope of the present study. Stary41 has demonstrated that the foam cells of macrophagic origin in atherosclerotic plaques of cholesterol-fed monkeys disappeared by cell death in the first weeks after cholesterol withdrawal, whereas death of smooth muscle cells became infrequent. In the present study, it is possible that the macrophages disappeared by apoptosis in the early period after cholesterol withdrawal. However, further studies with tissues collected and examined at several time points after the cholesterol withdrawal could reveal interesting data concerning the fate of the disappearing macrophages. After the 26-week cholesterol withdrawal period, the plaques are mainly composed of smooth muscle cells; cell replication and apoptotic cell death are no longer detectable in these plaques. This is another argument for the hypothesis that the macrophages are responsible for the induction of apoptotic cell death and cell replication in the plaques. A double immunohistochemical stain demonstrated that 95% of the Ki-67–labeled cells were macrophages, whereas only 5% of the labeled cells were smooth muscle cells. These data are comparable to the findings of Rekhter and Gordon42 in human atherosclerotic plaques. A major difference is the absence of neovascularization and thrombosis in the present study. In the study of Rekhter and Gordon, a significant fraction of the proliferating cell nuclear antigen–labeled cells were endothelial cells. These authors found an overall proliferative activity of 2% in the plaques, which is less than the values we found in the plaques at 26 weeks. The difference in the percentage of labeled cells in the present study could be a consequence of the denominator used to calculate the fraction of labeled nuclei. In a previous study involving human atherosclerotic plaques in human saphenous vein grafts and carotid atherosclerotic plaques, we found that cell replication was mainly present in the foam cells of macrophagic origin.8 Regions around the necrotic core and in the cap of unstable atherosclerotic plaques contained high percentages of Ki-67–labeled nuclei. However, it remains to be proven whether these cells are dividing or arrested in the G1 phase of the cell cycle.
Apoptotic cell death was studied by the detection of DNA fragmentation by use of the TUNEL technique. Low but reproducible values were detected in the plaques. The values of 0.5% to 2% were in the same range as those found and reported in atherosclerotic plaques of cholesterol-fed rabbits in a previous study by our group.5 The occurrence of DNA cleavage in a cell is also called the final or executive phase of the apoptotic cascade. This phase is short (6 hours) and irreversible. The short duration of this phase could explain the low values that we have found in the plaques. The execution phase is preceded by a phase of commitment. During this phase, the cells increase their susceptibility for apoptosis. During this phase, the cell increases different proapoptotic factors; however, the cells remain TUNEL-negative. The increased BAX expression can be considered as a marker of the commitment phase of apoptosis. The length of this phase is not known. There exists a correlation between the BAX/BCL-2 ratio and the susceptibility for cell death. For example, Purkinje cells of the cerebellum show a very high BAX expression39 and are believed to be one of the most sensitive subpopulations of the brain for ischemic cell death.43 In contrast to Isner et al,12 we could not detect BCL-2 in the normal media or the plaques. Other authors have also reported that normal arteries of mice contain little or no BCL-2 protein and mRNA.39 It is not clear from the present study which factor is a BAX heterodimer in the smooth muscle cells and macrophages, since BCL-2 was not detectable in either cell type. However, it is possible that BCL-2–related proteins like BCL-xl44 or Mcl-1 are involved.
Another indication that smooth muscle cells in the plaques can be committed for apoptotic cell death is coming from the caspase 8 system by Fas-Fas ligand interaction.45 Geng et al46 found that smooth muscle cells in atherosclerotic plaques showed increased Fas expression, which could be another argument that smooth muscle cells in the plaques are committed for apoptosis. The commitment phase can be followed by the final execution phase if other factors are also present. The TUNEL technique detects only the final execution phase of apoptosis. This could explain the fact that numerous cells are BAX-positive in the plaque but that only a small fraction is TUNEL-positive. The relationship between BAX and TUNEL is obvious for the smooth muscle cells but less clear for the macrophages: 35% of all TUNEL-labeled smooth muscle cells were also BAX immunoreactive before the lipid lowering, whereas only 8% of all TUNEL-labeled macrophages were BAX immunoreactive. After a period of cholesterol withdrawal, BAX expression in the atherosclerotic plaques was greatly decreased. This was a consequence of both the disappearance of the macrophages and a loss of the lipid accumulation in the smooth muscle cells. Interestingly, the BAX-immunoreactive area expressed per cell was significantly decreased after cholesterol withdrawal. This indicates that after the cholesterol withdrawal period, the remaining smooth muscle cells decrease their susceptibility to undergo apoptotic cell death.
The changes in the cell composition of plaques after cholesterol withdrawal is in agreement with the study of Shiomi et al.47 The authors found a significant decrease in the macrophage and lipid deposits and an increase in the collagen content in atherosclerotic plaques of Watanabe heritable hyperlipemic rabbits treated with pravastatin. Moreover, pravastatin treatment suppressed the decrease in the smooth muscle cell area during lesion progression. This indicates that the benefit of lipid lowering in human atherosclerotic plaques could be plaque stabilization caused by decreasing the macrophage content, resulting in less smooth muscle cell death8 and collagen breakdown. Macrophages are reported to be present in unstable atherosclerotic plaques and at sites of plaque ruptures.3 4 48 A major benefit of lipid lowering could be the reduction of the macrophage infiltration in the plaque, a phenomenon that is not always associated with a reduction of the plaque thickness.
A problem in the evaluation of lipid-lowering trials is that the clinical symptoms and angiographical data are not always related to the changes that occur in the plaques. In the present experimental study, we have determined that the thickness and area of the plaques does not change after cholesterol withdrawal. Moreover, we have found that the lesions tended to increase in the aortic arch and thoracic and abdominal aortas. This indicates that an angiographical study performed on these animals would possibly not have shown a beneficial effect of the cholesterol withdrawal.
The detection of macrophage infiltration in atherosclerotic plaques in vivo would be the ideal technique for evaluating the beneficial effect of lipid-lowering drugs on plaque stability. An interesting approach could be the thermal detection of cellular infiltrates in living atherosclerotic plaques by a thermistor.49 Understanding the molecular mechanism of plaque progression, combined with modern medical imaging techniques, could help to explain the benefit of lipid-lowering therapy on plaque stabilization.
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Acknowledgments |
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Dr De Meyer is a research associate of the Flemish Fund for Scientific Research. Dr Kockx is a holder of a fund for fundamental clinical research of the Flemish Fund for Scientific Research. The authors want to acknowledge the technical support of Johannes Muhring, Ludo Zonnekeyn, Rita VandenBossche, and Christel Van Campenhout.
Received September 4, 1997; accepted June 8, 1998.
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