From the Department of Pathology, A.Z. Middelheim, Antwerp (M.M.K.,
J.M.), and the Division of Pharmacology (UIA) (G.R.Y. De M., H.B., A.G.H.) and
the Centre for Electron Microscopy (W.J.), University of Antwerp, Wilrijk,
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
Methods and Results—Human atherosclerotic plaques were studied by
whole-mount carotid endarterectomy specimens
(n=18). This approach allowed comparison of adaptive intimal
thickenings, fatty streaks, and advanced atherosclerotic plaques of the
same patient. The fatty streaks differed from adaptive intimal
thickenings by the presence of BAX (P<0.01), a
proapoptotic protein of the BCL-2 family. Both regions were
composed mainly of smooth muscle cells (SMCs), and macrophage
infiltration was low and not different. Apoptosis, as detected
by DNA in situ end labeling (terminal
deoxynucleotidyl transferase end labeling [TUNEL]
and in situ nick translation) was not present in these regions.
Apoptosis of SMCs and macrophages, however, was
present in advanced atherosclerotic plaques that were present
mainly in the carotid sinus. A dense infiltration of
macrophages (5.8±3% surface area) was present in these
advanced atherosclerotic plaques. Cytoplasmic remnants of
apoptotic SMCs, enclosed by a cage of thickened basal lamina,
were TUNEL negative and remained present in the plaques as matrix
vesicles.
Conclusions—We conclude that SMCs within human fatty streaks
express BAX, which increases the susceptibility of these cells to
undergo apoptosis. The localization of these susceptible SMCs
in the deep layer of the fatty streaks could be important in our
understanding of the transition of fatty streaks into atherosclerotic
plaques, which are characterized by regions of cell death. Matrix
vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs
that can calcify and may be considered the graves of SMCs that have
died in the plaques.
In the present study, regional differences between the committed
phases and the executive phases of apoptosis were studied in
whole-mount human atherosclerotic plaques and the adjacent
nonatherosclerotic intima. The executive phase of apoptosis was
detected by DNA in situ end labeling. Changes in the expression of
proteins of the BCL-2 family were used to detect the committed
phase.
Immunohistochemistry
The following polyclonal antibodies were used: BAX, ICE, and BCL-2 from
Santa Cruz and fibrinogen from Cappel. 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) for 45 minutes. The polyclonal
antibodies were detected by a PAP complex. For demonstration of the
complex, AEC was used as a chromogen.
The specificity of the immunohistochemical reactions was checked by
omitting the primary antibody and substituting an unrelated antibody at
the same concentration. Both antibodies (monoclonal and polyclonal)
against BAX and BCL-2 gave identical results.
DNA In Situ End Labeling
In both the TUNEL and the ISNT techniques, the labeled antibody was
visualized by AEC. 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 CD-68 and
Definitions
Adaptive Intimal Thickening
Fatty Streaks
Atherosclerotic Plaques
Quantification
Transmission Electron Microscopy
Statistical Analysis
Adaptive Intimal Thickening
The adaptive intimal thickening was composed primarily of
longitudinally oriented SMCs that strongly expressed
Transmission electron microscopy of this region showed SMCs with intact
cytoplasm. The cytoplasm contained microfilaments and
subplasmalemmal vesicles. A thin basal lamina surrounded
each smooth muscle. The extracellular matrix contained cross-banded
collagen fibers. Cytoplasmic remnants were rare or absent.
Fatty Streaks
Transmission electron microscopy showed that most of the SMCs contained
small lipid vacuoles in their cytoplasm. SMCs that were filled with
lipid vacuoles could also be found. Macrophages were
present and were crowded with lipid vacuoles. Cytoplasmic remnants
could be detected in the interstitial matrix, but cell
death (complete disintegration of the cytoplasm) was not
detectable.
Advanced Atherosclerotic Plaques
In the fibrous caps, most of the foam cells were of macrophage
origin and showed granular material within their cytoplasm. These cells
expressed both BAX (Figure 2C
Transmission electron microscopy of plaque tissue confirmed that few
intact smooth muscles remained present. The remaining SMCs were
strangely shaped, with thin elongated cytoplasmic extensions and
prominent cages of hypertrophic, multilamellated basal laminae.
Moreover, small membrane-bound vesicles of varying size were shed from
the SMCs, and often SMCs had died by disintegration into myriad
vesicles (Figure 6
Complete specimens of human atherosclerotic plaques and adjacent
nonatherosclerotic intimal thickenings were obtained during a carotid
endarterectomy. The specimens were examined in toto
with longitudinal whole-mount sections. The same tissue section of a
carotid endarterectomy specimen contained regions
of adaptive intimal thickening, adjacent media, fatty streaks, and
advanced atherosclerotic plaques (for definitions, see the AHA
Medical/Scientific Statement15 ). With this
approach, we could compare the different regions with respect to cell
composition, apoptotic cell death, apoptosis-related
proteins, and cell replication.
Most advanced atherosclerotic plaques were located at the outer
wall of the carotid sinus, opposite the bifurcation flow divider,
confirming the studies of Glagov et al.16 One
difference between regions with minimal atherosclerotic plaque
formation and advanced plaques was the pronounced cell death in the
latter. The focal regions of extensive cell death are known as the
necrotic cores. Discrete necrotic core formation is reported to be
present in early
atherosclerosis.17 The necrotic
cores do not contain SMCs and interstitial collagen
fibers.18 This implies that the plaques can
destabilize by expansion of these regions. Regions of adaptive intimal
thickening and fatty streaks could be found both proximal (in the
communis part) and distal (in the interna) to the advanced
atherosclerotic plaques present in the carotid sinus. The fatty
streaks are considered early atherosclerotic
lesions.15 It is important to note that the term
"fatty streaks" covers a spectrum of lesions from those consisting
almost entirely of macrophages to those composed mostly of
SMCs. The fatty streaks in our study were composed mainly of
lipid-laden SMCs. The lesions were similar to those described in the
aorta of young adults by Katsuda et al.19
Interestingly, the lipid-laden SMCs that were the predominant cells in
the fatty streaks of our study and in the study of Katsuda et al were
found primarily in the deeper layer of the lesions. Foam cells of
macrophage origin were scarce and, if present, located in
the superficial layer.
In the adaptive intimal thickening and fatty streaks, cell replication
and apoptotic cell death were absent. In the advanced
atherosclerotic plaques, apoptotic cell death and cell
replication were present in regions with macrophage
infiltration. The overall number of TUNEL-positive nuclei in the
plaques was low if an optimal enzyme concentration and pretreatment
with a calcium chelating agent were used.11 Foci
of TUNEL-positive nuclei and nuclear fragments could be found around
the necrotic cores and fibrous cap.
Because these labeled nuclei were focally present, an
estimation of percentages of TUNEL-positive nuclei depends completely
on the selected regions and the number of nuclei used in the
denominator. Therefore, we have chosen to separate only regions with
and without TUNEL-positive nuclei. The TUNEL-positive nuclei and
nuclear fragments belong to the macrophages and SMCs, as
demonstrated with TUNEL+CD-68 and TUNEL+
The TUNEL-labeled nuclei were found mainly in the regions of the
plaques that show a dense infiltration by macrophages. This
finding was also noticed by Han et al.3
The topographical relationship between macrophage
infiltration and TUNEL positivity suggests that the inflammatory
reaction described in advanced atherosclerotic
plaques22 23 24 25 can destabilize the plaque in at
least two ways.26 27 The first is that the
accumulated macrophages secrete or activate
metalloproteinases.26 This results in collagen
breakdown.28 Recently, it was demonstrated that reactive
oxygen species derived from foam cells of macrophage origin
could activate latent metalloproteinases of the
interstitium.29 A second way for destabilization
is that a foam cell derived factor could kill the adjacent SMCs in the
plaque.12 30 Increased levels of TNF-
Another consideration is that the SMCs of the atherosclerotic
plaques could be programmed to die and that one of the above-mentioned
macrophage-derived factors is capable of completing the
programmed SMC death. This is suggested by the finding that SMCs
derived from the atherosclerotic plaque but not from the media die when
brought in culture.14 Interestingly, the fatty
streaks in our study differed from adaptive intimal thickenings by the
presence of BAX, a proapoptotic protein of the BCL-2 family. It
was demonstrated that BCL-2 heterodimerizes in vivo with a conserved
homologue, BAX, that accelerates programmed cell
death.38 39 40 Recent data state that BAX
expression can activate a common pathway of apoptosis
both caspase-dependent and -independent.41
However, apoptotic cell death, as detected by DNA in situ end
labeling, is not present in either of these regions. This is a good
example to illustrate that apoptosis is regulated in mammalian
cells by multiple prolife or prodeath factors.42
However, the expression of BAX in the foam cells of SMC origin in the
fatty streaks indicates that these cells become susceptible for
apoptosis. Interestingly, in both our study and the study of
Katsuda et al,19 the lipid-laden SMCs in the
fatty streaks were located mainly in the deep layer of the lesions. It
is known that the regions of cell death in the full-blown
atherosclerotic plaques are present primarily in the deep layer of
the plaques.
The SMCs that showed BAX expression contained lipid vacuoles and
often were transformed in foam cells. This indicates a possible
relationship between the accumulation of lipid in the cytoplasm of SMCs
and a change in the BAX/BCL-2 ratio. It was demonstrated that
apoptosis can be induced by lipid peroxides, particularly
oxysterols, in cultured vascular SMCs through caspase 3 (CPP-32
protease) activation and BCL-2 protein
downregulation.43 In a recent study, caspase 3
was detected in human atherosclerotic plaques, and colocalization of
caspase 3 expression and TUNEL positivity was
found.44
The increased BAX protein expression within the advanced
atherosclerotic plaques was located in the regions around the necrotic
core and the fibrous cap. The increased BAX expression was the
consequence of an increased cytoplasmic immunoreactivity within
remaining SMCs and the strong immunoreactivity of BAX protein
expression within the cytoplasmic remnants of apoptotic SMCs
called the matrix vesicles.11 The vesicles were
enclosed by cages of thickened PAS-positive basal lamina, which was
confirmed by transmission electron microscopy. This provides further
argument that these matrix vesicles are cytoplasmic remnants of SMC
death. The cytoplasmic remnants of SMC and matrix vesicles did not show
ICE expression.
We conclude that SMCs within the fatty streaks increase their
expression of BAX and become susceptible for apoptosis. The
localization of these susceptible SMCs in the deep layer of the fatty
streak could be important to our understanding of the transition of a
fatty streak into an atherosclerotic plaque, which is characterized by
the appearance of focal and diffuse regions of cell death mainly in the
deep layer of the plaques. Cytoplasmic remnants of apoptotic
SMCs, which are TUNEL negative, remain present in the plaques as
matrix vesicles. These vesicles are excessive in regions of the plaque
with pronounced cell loss, possibly by a lack of scavenging. The
vesicles that can calcify can be considered the graves of SMCs that
have died in the plaques.
Received September 29, 1997;
revision received January 16, 1998;
accepted January 27, 1998.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Apoptosis and Related Proteins in Different Stages of Human Atherosclerotic Plaques
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—The transition of a fatty
streak into an atherosclerotic plaque is characterized by the
appearance of focal and diffuse regions of cell death. We have
investigated the distribution of apoptotic cell death and
apoptosis-related proteins in early and advanced
atherosclerotic lesions.
Key Words: atherosclerosis • apoptosis • cholesterol • carotid arteries • muscle, smooth • cells
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Recent work by our
and other laboratories demonstrates the presence of apoptotic
cell death in human and experimental atherosclerotic
plaques.1 2 3 4 5 6 Most of these studies use DNA in
situ end-labeling techniques (TUNEL or ISNT)7 8 9
to detect apoptotic cell death within the plaques. If aspecific
labeling is avoided by optimization of the enzyme
concentration10 and use of a calcium chelating
agent,11 low levels of apoptotic cell
death (never >2%) were found in the
plaques.1 4 10 Human atherosclerotic plaques
display a pronounced morphological variability in the different
regions. Recently, it was demonstrated that apoptosis in
atherosclerotic plaques is associated with macrophage
infiltration,3 6 12 whereas lesions consisting
only of SMCs present very little apoptosis, as demonstrated
by the TUNEL technique. DNA fragmentation is a rather late stage of
apoptotic cell death. Apoptosis occurs in at least two
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 derived from atherosclerotic plaques but not of
the media die when brought in culture. This suggests that the SMCs of
the atherosclerotic plaques but not the medial SMCs are committed to
die.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Human carotid endarterectomy specimens
were obtained from patients (n=18) who had a carotid stenosis
>70% as demonstrated by digital subtraction angiography and Duplex.
The patients (9 women and 9 men) were
normocholesterolemic and had a mean age of 72±5 years.
The specimens were fixed in 4% formalin and opened along their
longitudinal axes, and complete longitudinal sections of the
paraffin-embedded specimens were mounted on APES
(3-aminopropyltriethoxysilane)- precoated slides. All sections were
stained by a trichrome Masson's, Verhoeff's elastin, and PAS stains.
These whole-mount sections contained the inner wall of the distal
common carotid artery, the proximal part of the external carotid
artery, and the carotid sinus. In each section, advanced
atherosclerotic plaques alternated with fatty streaks and adaptive
intimal thickenings (Figure 1
).
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Figure 1. Example of a whole-mount carotid
endarterectomy specimen used in this study. The
specimen, consisting of a complete cast of the atherosclerotic carotid
bifurcation, was projected, and the contours were drawn on paper
(white lines). The specimens contained the inner wall of the distal
common carotid artery (communis), proximal part of the external carotid
artery (externa), and carotid sinus. The specimen also contained
adjacent inner media that was cleaved during the surgical procedure
in the communis region and at the flow divider (blue region).
In each section, advanced atherosclerotic plaques (red regions)
alternate with regions of fibromuscular intimal thickening (green
region) and fatty streaks (orange region).
The following primary monoclonal antibodies were used: 
-SMC
actin from Sigma Chemical Co; CD 68 (anti-macrophage), LCA
(common leukocytic antigen CD-45), and BCL-2 from Dako, Glostrup,
Denmark; BAX from Pharmingen, San Diego, Calif; and Ki67 from
Immunotech, Marseille, France.
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.11 Both
the TUNEL7 and ISNT8 9
techniques were used. For the TUNEL technique, ApopTagkit/Oncor
(Gaithersburg) was used with minor modifications. For the ISNT
technique, the sections were rinsed in a buffer (Tris-HCl 50
mmol/L, MgCl2 5mmol/L, BSA 0.0005%, pH 7.5) for
10 minutes, dried, and later 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 d-UTP (Boehringer Mannheim) with
20 U/mL of the Klenow fragment of DNA polymerase I (Boehringer
Mannheim). Incorporated biotin-16 d-UTP was demonstrated by incubating
the sections with a monoclonal antibody against biotin (Dako, Glostrup)
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. -SMC actin. Therefore, we used DAB instead of AEC as a chromogen.
Moreover, we combined the TUNEL technique with a PAS stain.
The following definitions are based on the definitions of the
American Heart Association Medical/Scientific
Statement.15
We have defined adaptive intimal thickening as the accumulation
of SMCs between the endothelium and the media. The SMCs
are oriented mainly in the longitudinal direction and are
immunoreactive for -SMC actin. The cytoplasm of the smooth muscle
does not show lipid vacuoles. Macrophages, as detected by their
expression of CD-68, are not present in this layer.
This layer was defined as adaptive intimal thickening with lipid
deposition. Lipid deposition was detected by the presence of small
vacuoles in the cytoplasm of the SMCs. This was confirmed on adjacent
sections stained with a Scharlach red fat stain. The percent of the
total area that was immunoreactive for macrophages (CD-68) was
<1%. Regions of pronounced cell loss were not present in this
layer. It should be noted that the term "fatty streak" covers a
spectrum of lesions from those consisting almost entirely of
macrophages to those that are composed entirely of SMCs, with
every gradation in between. The fatty streaks in our study were
composed mainly of SMCs.
These regions were defined by the presence of foci of pronounced
cell loss. These regions were defined as necrotic cores. The cores
could be detected by the presence of fibrin(ogen). The region between
the endothelium and the necrotic core was defined as
the fibrous cap. Unstable atherosclerotic plaques were defined by the
presence of numerous foam cells of macrophage origin within the
fibrous caps.
The images were analyzed with a color image
analysis system (PC Image Color, Foster Findlay Associates).
Each whole-mount carotid endarterectomy specimen,
consisting of a complete cast of the atherosclerotic carotid
bifurcation, was projected, and the contours were drawn on paper.
This allows mapping of the atherosclerotic plaques present in each
specimen (Figure 1
). Subsequently, 15 regions (5 different regions in
triplicate), each 65x100 µm, from each carotid
endarterectomy specimen were quantitatively
analyzed. These 5 different regions were located in the media
(n=3), adaptive intimal thickening (n=3), fatty streak (n=3), fibrous
caps (n=3), and necrotic cores (n=3). For each region, the percent
immunoreactive areas for -SMC actin, CD-68, LCA, and BAX were
measured. The latter variables were expressed as percent of the
total area.
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 for the lesions was made
from 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).
Data are expressed as mean±SEM. The five different regions were
compared for their BAX immunoreactivity by use of one-way ANOVA
followed by the Tamhane's T2 test.
The SPSS package for Windows (SPSS Inc) was applied for these purposes.
A 1% level of significance was selected.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Human Atherosclerotic Plaques (n=18)
The carotid endarterectomy specimens were cut
longitudinally, and whole-mount sections were made of all cases. The
specimens were complete casts of carotid bifurcations and consisted of
intima and atherosclerotic plaques of the communis part, the proximal
part of the external carotid artery, and the carotid sinus. This
approach allows a comparative study of the different stages of
atherosclerotic plaques present in each specimen. A
trichrome-Masson stain showed that the cell density in these different
regions was variable. In general, advanced atherosclerotic plaques
showed a severe loss of SMCs, most extreme in the necrotic core. This
indicates that cell death must have occurred during plaque progression.
In this study, we compared the cell composition of each region,
expression of apoptosis-related proteins, and cell replication
and apoptotic cell death.
This stage was often present in the communis and the distal
part of the interna (distally from the carotid sinus), regions that
also contained adjacent fragments of the inner media. -SMC actin
(10.5±1.1% of the total area; Figure 1). The SMCs showed a morphology
similar to the medial SMCs. Macrophages, as detected by their
immunoreactivity for CD-68, were very rare and occupied <0.1% of the
total area. Scattered rare lymphocytes, detected by their
immunoreactivity for LCA, could be found and covered <1% of the total
area. Fibrinogen and von Willebrand factor depositions were
absent. In general, most of the SMCs within the media and the adaptive
intimal thickenings expressed -SMC actin but not BAX (Figures 2A and 3).
BCL-2 and ICE could not be detected. Cell replication (as demonstrated
by Ki-67) and apoptotic cell death (as demonstrated by DNA in
situ end labeling) could not be detected.
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Figure 2. -SMC actin expression and BAX expression in
four different regions of the carotid
endarterectomy specimen of Figure 1. A, Adaptive
intimal thickening. The SMCs show -SMC actin but do not express BAX.
B, Fatty streak. Most SMCs express -SMC actin and contain lipid
vacuoles. These smooth muscles are immunoreactive for BAX. Scattered
mononuclear cells, which are negative for BAX, are present. C,
Fibrous cap of an unstable atherosclerotic plaque. Residual smooth
muscle can be detected between the foam cells. The foam cells are of
macrophage origin, as detected by their expression for CD-68
(not shown), and are immunoreactive for BAX. D, Region within the
necrotic core. Empty splitlike spaces or spaces filled with cytoplasmic
remnants are present. These cytoplasmic remnants contain remnants
that are immunoreactive for -SMC actin and are often surrounded by a
prominent cage of PAS (see Figure 4), indicating their SMC origin.
These cytoplasmic remnants are associated with matrix vesicles and are
immunoreactive for BAX. Scale bar=30 µm.
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Figure 3. Quantification of BAX expression in five different
regions of the human carotid endarterectomy
specimens. The media and adaptive intimal thickening consist almost
exclusively of SMCs with <0.2% macrophages and were negative
for BAX. The fatty streaks contain foam cells of SMC origin that showed
a strong expression of BAX, although the TUNEL technique was strictly
negative in these regions. The regions that contained numerous
macrophages (CD-68–positive surface area >5.5%) and few
residual SMCs ( -SMC actin surface area <0.2%) were located around
the necrotic cores and fibrous caps of atherosclerotic plaques. These
regions showed a high expression of BAX and nuclei that were labeled by
the TUNEL technique. This indicates apoptotic cell death in
those regions (see also Figure 4). Data are expressed as mean±SEM.
*P<0.01 versus media and intima.
These regions were composed primarily of SMCs that express -SMC
actin (10.0±1.5% of the total area). A significant fraction of these
SMCs showed intracellular fat accumulation (Figure 2B). Moreover,
SMC-derived foam cells were present. Lipid accumulation in these
cells was verified on adjacent cryostat sections that were stained with
Scharlach red fat stain. Scarce macrophages were present
(<0.2% of the total area). Lymphocytes were present in a
variable degree (<0.2% of the total area). Fibrinogen and von
Willebrand factor were not detectable. Interestingly, the
lipid-laden SMCs were most pronounced in the deep layer of the lesions.
Foam cells of macrophage origin were scarce and, if
present, located in the superficial layer. The smooth muscle that
showed intracellular fat accumulation and the smooth muscle–derived
foam cells showed a strong cytoplasmic BAX expression (Figures 2B and 3). The rare macrophages showed foam cell transformation and
BAX expression. BCL-2 was not detectable. ICE could be detected in the
macrophages; the SMCs were negative. Cell replication and
apoptotic cell death could not be detected.
Most of the advanced atherosclerotic plaques were located at the
outer wall of the carotid sinus, opposite the bifurcation flow divider.
The plaques showed a shoulder, a fibrous cap, and a necrotic core. The
core was largely acellular, indicating that cell death must have
occurred. Surrounding the necrotic cores, numerous foam cells of
macrophage origin, as demonstrated by their immunoreactivity
for CD-68, were present (5.8±3.2% of the total area). Lymphocytes
occupied <0.2% of the total area. Multinucleated giant cell
transformation was frequent in this region. However, -SMC
immunoreactivity was nearly absent in these regions (<0.2% of the
total area). This is a consequence of smooth muscle cell loss and the
fact that the remaining SMCs lose their -SMC actin content. ) and ICE. BCL-2 was not detectable. The
foam cells surrounding the necrotic cores and in the fibrous caps
showed nuclei that were labeled with Ki-67 and DNA in situ end
labeling. This indicates that both cell replication and
apoptotic cell death occurred in these regions. Around the
necrotic core, BAX-immunoreactive material and cell remnants could be
detected (Figures 2D and 3). Moreover, with the DNA in situ end
labeling, nuclear fragments could be detected within and around the
necrotic cores, including the fibrous cap. This could indicate an
ongoing process of cell death in this region. Immunohistochemical
staining of adjacent sections and/or double staining with TUNEL and
-SMC actin or CD-68 identified both apoptotic SMCs and
macrophages. Double staining, however, often failed to
establish the identity of apoptotic nuclei and nuclear
remnants. Therefore, we combined the TUNEL technique with a PAS stain.
Foam cells of macrophage origin showed nuclei that were labeled
(Figure 4A). SMCs (Figure 4B, bottom) but
not the macrophages (Figure 4A, bottom) were surrounded by a
cage of PAS-positive basal lamina. The SMCs in the fibrous cap
especially were surrounded by prominent cages of PAS-positive material.
Using this technique, we could detect TUNEL-labeled nuclei and nuclear
fragments that were enclosed by a cage of PAS-positive material,
indicating smooth muscle apoptosis. (Figure 4B, top and
bottom). This indicates that the SMCs die within their cage of
thickened basal laminae. Moreover, we could detect cages of
PAS-positive material that contained small vesicles (1 µm) that
were not labeled by the TUNEL technique (Figure 4C, top and bottom) and
associated labeled vesicles (Figure 5).
Cages of PAS-positive material that were empty were also
present.
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Figure 4. Advanced human atherosclerotic plaque stained by
the TUNEL technique. The same TUNEL-labeled sections were subsequently
stained by PAS stain (TUNEL+PAS technique). A, Foam cell–rich area
around a necrotic core. A labeled nucleus is present (top). Bottom,
The foam cells are not surrounded by a basal lamina, and an adjacent
section showed that these cells express CD-68 (not shown). The
TUNEL-positive nucleus corresponds to a macrophage undergoing
apoptotic cell death. B, Fibrous cap adjacent to the foam
cell–rich area of Figure 4A . A labeled nucleus is present in a
fibrous cell–poor area (top). Bottom, This TUNEL-positive nucleus
belongs to a cell that is surrounded by a prominent cage of
PAS-positive basal laminae. This points to an SMC undergoing
apoptotic cell death. Most of the SMCs in this region do not
express -SMC actin. Adjacent to this cell are PAS-positive empty
cages of thickened basal lamina. C, Fibrous cap adjacent to the region
of Figure 4B. Clusters of small vesicles are very frequent in these
regions. These vesicles are not labeled by the TUNEL technique (top).
Bottom, The clusters of small vesicles are surrounded by cages of
PAS-positive thickened basal lamina, indicating their smooth
muscle origin.
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Figure 5. High-power photomicrograph of a TUNEL-positive
nucleus in an advanced atherosclerotic plaque. This nucleus was also
surrounded by a cage of basal lamina, which points to an SMC undergoing
apoptotic cell death. The labeled nucleus is associated with
numerous small vesicles. Most of the small vesicles are TUNEL negative.
These clusters of vesicles (matrix vesicles) are very frequent in the
regions of the atherosclerotic plaques that demonstrate extensive cell
loss. Scale bar=30 µm. ). These vesicles were
enclosed by prominent cages of basal lamina. These ultrastructural
findings fit with the TUNEL+PAS stain (Figure 4B and 4C, top and
bottom). The basal laminae around the SMCs were irregularly thickened
and multilaminated, and fragments of thickened basal laminae
unassociated with cells could be found. Macrophages, not
enclosed by a basal lamina, completely filled with lipid vacuoles and
myeloid bodies could also be demonstrated.
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Figure 6. Transmission electron microscopy of an advanced
atherosclerotic plaque. Two smooth muscle cells are demonstrated that
are completely disintegrated into myriad vesicles (granulovesicular
degeneration). The prominent basal laminae (bl) around these clusters
of vesicles led us to conclude that the vesicles are of SMC and not of
macrophage origin. This electron microscopy picture is the
explanation for the vesicles of Figure 4B and 4C (bottom) and Figure 5.
Col indicates cross-banded collagen. Scale bar=2 µm.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present study compares the presence of apoptotic
cell death and apoptosis-related proteins in different stages
of atherosclerotic plaque formation. -SMC actin. This confirms
the findings of other groups.1 2 6 A significant
fraction of the labeled nuclei and nuclear fragment could not be
stained by CD-68 or -SMC actin, which could reflect a loss of
specific markers during apoptosis. A feature of smooth muscles
in atherosclerotic plaques is that they are surrounded by cages of
thickened basal lamina (pancakelike SMCs).20
Basal lamina and basement membranes can be stained by a PAS stain. By
combining the TUNEL technique with a PAS stain, we could detect
TUNEL-labeled nuclei and nuclear fragments that were enclosed by a cage
of PAS-positive material, indicating smooth muscle apoptosis.
Moreover, clusters of TUNEL-negative cytoplasmic remnants, which were
enclosed by thickened basal laminae, were present. Transmission
electron microscopy confirmed the presence of small membrane-bound
vesicles of varying size that were shed from SMCs and the SMCs that had
died by disintegration into myriad vesicles. These vesicles were
enclosed by prominent cages of basal lamina. These vesicles are similar
to the granulovesicular degeneration of SMCs present in cerebral
atherosclerosis that was described by
Stehbens21 20 years ago. The vesicles are also
comparable to the matrix vesicles present in epiphysis of long
bones. protein
and mRNA are present in atherosclerotic plaques of
hypercholesterolemic rabbits.31
In addition, in studies in vitro, Morel et al32
and Jimi et al33 demonstrated that oxidized LDL
injured vascular SMCs. The explanation, however, is not so evident
because oxidized LDL is also cytotoxic for the macrophages
themselves.34 35 Moreover, contrary effects of
lightly and strongly oxidized LDL have been
reported.36 A combination of different factors
secreted by macrophages and T-lymphocytes probably is
responsible for the SMC death.37
Selected Abbreviations and Acronyms
AEC
=
3-amino-9-ethyl carbazole
ICE
=
caspase 1
ISNT
=
in situ nick translation
PAS
=
periodic acid–Schiff
SMC
=
smooth muscle cell
TUNEL
=
terminal deoxynucleotidyl transferase end
labeling
Acknowledgments
Dr De Meyer is a research associate of the Flemish Fund for
Scientific Research (FWO). Dr Kockx is a holder of a fund for
fundamental clinical research of the Flemish Fund for Scientific
Research (FWO). We wish to acknowledge the technical support of Ludo
Zonnekeyn and Rita Van Den Bossche.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
, tumor necrosis factor-, and
interleukin-1 ß. Arterioscler Thromb Vasc Biol. 1996;16:19–27.
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