© 1997 American Heart Association, Inc.
Articles |
DNA Fragmentation and Ultrastructural Changes of Degenerating Cells in Atherosclerotic Lesions and Smooth Muscle Cells Exposed to Oxidized LDL in Vitro
From the King Gustaf V Research Institute, Department of Medicine, Karolinska Hospital (S.J., M.C., J.N.) and the Department of Cell and Molecular Biology, Karolinska Institute (J.T.), Stockholm, Sweden.
Correspondence to Dr Stefan Jovinge, King Gustaf V Research Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden. Email Stefan.Jovinge{at}medks.ki.se
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Abstract |
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Abstract Degeneration of smooth muscle cells in the fibrous cap of atherosclerotic lesions is an important factor in plaque rupture. Recent studies have suggested that many plaque cells are in a process of apoptosis as determined by positive deoxyribonucleotide-transferase-mediated dUTP end labeling. In this study, we demonstrate the existence of a colocalization between deoxyribonucleotide-transferase-mediated dUTP end labeling-positive smooth muscle cells and oxidized LDL immunoreactivity in human carotid plaques. Oxidized LDL was found to induce deoxyribonucleotide-transferase-mediated dUTP end labeling positivity in cultured human smooth muscle cells, but only in the presence of tumor necrosis factor-
and interferon-
. Electron
microscopic analysis of cultured smooth muscle cells exposed to
oxidized LDL in the absence of cytokines demonstrated
cytoplasmic swelling and disruption of the plasma membrane, suggesting
cell death by oncosis. Cells exposed to both oxidized LDL and
cytokines were characterized by chromatin and cytoplasmic
condensation compatible with cell death by apoptosis. These
findings further support the notion that oxidized lipids play a role in
plaque cell death.
Key Words: apoptosis • cell death • oxidized LDL • TNF • IFN
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The transformation of a stable fibromuscular atherosclerotic plaque into an unstable, complicated lesion susceptible to rupture is characterized by a degenerative process involving cell death and degradation of extracellular matrix.1 2 Although this process has a critical role in the etiology of acute ischemic events, little is still known regarding the factors responsible for its induction. Considerable interest has focused on the possible role of apoptosis in plaque cell death. Using TUNEL, some investigators have reported signs of DNA fragmentation in up to 40% of macrophage-enriched cell regions in atherosclerotic plaques.3 Other investigators have shown an increased tendency to undergo apoptosis in cells derived from human atheromas.4 The presence of nuclear DNA fragmentation has been interpreted as an indication of cell death by apoptosis, a conclusion that has been supported by the demonstration of 180 to 200 base pair fragments after electrophoretic separation of DNA from plaque SMC.4 However, TUNEL does not fully discriminate between cell death by apoptosis and oncosis (primary necrosis).5 The hypothesis that apoptosis is the major mechanism responsible for cell death in atherosclerotic plaque is also challenged by electron microscopic studies showing that although a large fraction of the cells display signs of degenerative changes, only a few express the nuclear and cytoplasmic condensation characteristic of apoptosis.6
LDL entrapped in the extracellular matrix of the vessel wall is known
to become oxidatively modified in response to reactive oxygen
intermediates and/or enzymes released by surrounding
cells.7 8 9 This process leads to the generation of
proinflammatory phospholipids10 and to the formation of
foam cells through uptake of oxidized LDL by intimal
macrophages and is believed to play a key role in the
atherosclerotic process.11 12 13 14 Oxidized LDL is highly
cytotoxic for cultured endothelial and
SMC,15 16 suggesting that it may be involved also in
plaque cell death. Cytokines released from activated
macrophages represent another possible source of
cytotoxic factors for plaque cells, and Geng and coworkers recently
demonstrated that SMC simultaneously exposed to TNF and
-IFN undergo cell death by apoptosis.17
The objective of the present study was to analyze the pattern of DNA fragmentation and degenerative ultrastructural changes in human cultured vascular SMC exposed to LDL or oxidized LDL. Studies were also performed to compare the localization between oxidized LDL and cells with DNA fragmentation in human atherosclerotic plaque.
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Methods |
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Reagents
Recombinant TNF
and IFN were purchased from R&D systems
(Minneapolis, Minn). Low endotoxin bovine serum albumin and MTT
were from Sigma Chemical (St Louis, Mo). Proteinase K was purchased
from Boehringer Mannheim (Mannheim, Germany). All other cell
culture reagents were obtained from GIBCO BRL (Paisley,
Scotland).
Tissue Preparation
Carotid artery plaque tissue was obtained from 13 patients with
symptomatic ipsilateral carotid artery stenosis
undergoing carotid endarterectomy. A distal section
of a left internal mammary artery and parts from saphenous veins were
obtained after coronary artery bypass procedures. After
removal, all tissues were cut into three parts, the first immediately
snap-frozen in liquid nitrogen and stored -70°C, the second fixed in
4% formalin/50 mM butylated hydroxytoluene/0.2% EDTA, and the third
fixed in 3% glutaraldehyde in 0.1 M sodium
cacodylate-HCl buffer (pH 7.3) with 0.05 M sucrose. For
immunohistochemistry, the frozen specimens were cut in 14-µm-thick
sections on a cryostat, mounted on superfrost glass slides,
acetone-fixed, and stored at -20°C. Formalin-fixed specimens were
embedded in paraffin.
TUNEL
The TUNEL procedure was performed using the In Situ
Apoptosis Detection Kit (Apo-Tag, Oncor). SMC were fixed in
methanol/acetone (3:1) for 10 minutes and then washed in PBS.
Cells or cryostat sections were permeabilized by
incubation with 0.1% Triton X-100 for 5 minutes on ice, incubated with
TdT enzyme for 2 hours at 37°C, and then incubated with
antidigoxigenin peroxidase solution for 30 minutes. The slides were
then rinsed three times in PBS, exposed to diaminobenzidine substrate
for 5 minutes and counterstained by hematoxylin.
Immunohistochemistry
The sections were rinsed for 2x5 minutes in PBS. Butylated
hydroxytoluene (50 µM) was added to all solutions to prevent in vitro
oxidation of LDL. Endogenous peroxidase was blocked with
0.3% H2O2 in methanol for 30 minutes, followed
by washing for 20 minutes in PBS. The slides were then covered with
normal horse serum for 20 minutes and washed in PBS for 10 minutes.
Subsequently, the slides were incubated with the antibodies against
oxidized LDL (NA 59, kindly provided by Dr J. Witztum, San Diego,
Calif), -actin (HHF-35 DAKO,1:50), TNF (Boehringer
Mannheim), and INF (The Binding Site, UK) for 1 hour at room
temperature and rinsed in PBS for 10 minutes. Control slides were
incubated without primary antibody and with an antibody against
glucagon as irrelevant primary antibody. The specimens were then
incubated with biotinylated antimouse immunoglobulin G as the secondary
antibody for 30 minutes, washed with PBS for 10 minutes, further
stained with avidin-biotin for 30 minutes, and rinsed in PBS for 10
minutes. The slides were then exposed to alkaline phosphatase for 2
minutes, counterstained with hematoxylin-eosin for 20 minutes, and
mounted. To study colocalization between cell-specific
immunostaining and TUNEL reactivity, acetone-fixed
sections were first stained by the TUNEL technique followed by
immunohistochemical staining as described above.
A semiquantitative analysis of the immunohistochemistry staining was performed using a modified version of the technique described by Galis et al.18 Consistent positive staining covering more than 50% of the matrix and/or cells was recorded as number 3, consistent positive staining covering less than 50% of the matrix and/or cells was recorded as number 2, variable or weak staining of matrix and/or staining of occasional cells as number 1, and no staining as zero (0). Results are presented as the average score.
Isolation and Culture of SMC
SMC were isolated from the media of saphenous veins obtained at
bypass surgery as described. Briefly, the specimens were cut into small
pieces and allowed to attach to the surface of six-well multiplates by
drying for 15 minutes. The explants were then cultivated in F-12 medium
containing 10% newborn calf serum, 100 U/mL of penicillin, and 100
µg/mL of streptomycin at 37°C in an atmosphere of 5%
CO2 in air. Cells began to migrate out from the explants
within 1 to 2 weeks and reached confluence within another 2 weeks.
Secondary cultures were established by trypsinization and cell
specificity determined using immunohistochemical staining with the
-actin antibody. Experiments were performed in RPMI 1640 medium with
0.1% bovine serum albumin, 100 U/mL penicillin, and 10
µg/mL of streptomycin. For the MTT-method, RPMI without phenol
red was used.
LDL Preparation and Oxidation
LDL was prepared as described by Redgrave and
Carlsson19 using an EDTA concentration of 10 µM. EDTA
was removed before oxidation and for control LDL before addition to
cell cultures, by filtration on a Econo-Pac® 10 DG gel column (Bio
Rad, Hercules, Calif). Protein content was determined according to
Lowry et al.20 Oxidation of LDL (300 µg/mL) was
done by incubation in 10 µM CuSO4/RPMI 1640 at 37°C for
18 hours and confirmed by agarose gel electrophoresis.
MTT Assay of Cell Viability
Cell viability was determined using the MTT method, which
measures the ability of mitochondrial dehydrogenases to form
formazan.21 Briefly, cells were exposed to experimental
substances in 96-well plates containing 100 µL of medium/well. After
the appropriate incubation time, 10 µL of MTT dissolved in RPMI
without phenol red (5 mg/mL) were added to each well for 4 hours
at 37°C. The formed formazan crystals were solubilized by the
addition of 100 µL of 0.1 M HCl/10% Triton X-100 in anhydrous
isopropanol. The absorbance was measured at 570 nm.
Electron Microscopy
Primary fixation of the samples was done in 3%
glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer
(pH 7.3) with 0.05 M sucrose. After rinsing, the specimens were
postfixed in 1% osmium tetroxide in cacodylate buffer with 0.5%
potassium ferrocyanate for 2 hours at 4°C, dehydrated in graded
ethanol (70 to 100%), stained with 2% uranyl acetate in ethanol for
30 minutes, and embedded in Spurr low viscosity epoxy resin. Thin
sections were cut with a diamond knife using an LKB Ultratome IV,
picked up on carbon-coated formvar films, stained with alkaline lead
citrate, and finally examined in a JEOL EM 100CX operated at 60 kV.
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Results |
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Localization of Oxidized LDL and TUNEL-Positive Cells in Human Atherosclerotic Plaques
We have previously reported presence of DNA fragmentation in 9 to 20% of cells present in human carotid plaques obtained at carotid endarterectomy.6 A similar frequency of TUNEL positivity was observed in the lesion analyzed in the present study. TUNEL-positive cells were found mainly in the fibrous cap region and in the core regions of the plaques. Immunoreactivity for oxidized LDL was present in most parts of the lesions but was particularly prominent in the superficial region and in the core of the plaques. TUNEL-positive cells were almost exclusively found in or very close to regions with strong immunoreactivity for oxidized LDL, whereas oxidized LDL also could be found in regions with no or little TUNEL positivity. The latter regions were frequently very cell poor (Fig. 1
, A-C). One normal
internal mammary artery and two saphenous veins were also stained for
oxidized LDL but showed no positive immunostaining.
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Occasional clusters of TNF--expressing cells were found in the
fibrous cap region. These did not show any clear colocalization with
TUNEL-labeled cells or oxidized LDL immunoreactivity (Fig 1D
). No
staining for IFN could be detected. Staining of parallel sections
with antibodies against SMC -actin suggested that most
TUNEL-positive cells were SMC and that most of these were in
contractile phenotype (Fig 1, E and F). A semiquantitative
comparison of plaques with a low (<5%), intermediate (5 to 20%), and
high (>20%) frequency of TUNEL-positive cells did not show any clear
difference in the intensity of oxidized LDL, -actin, or TNF-
immunostaining (Table 1).
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Effect of LDL and Oxidized LDL on Cell Viability and
Fragmentation
Incubation of SMC in serum-free medium supplemented with 100
µg/mL of LDL increased the absorbance in the MTT assay,
whereas the absorbance in the MTT assay in cells incubated with 100
µg/mL of oxidized LDL decreased with time (Fig 2). Addition of 20 ng/mL of
TNF, 40 U/mL of IFN, or both did not influence the cell survival
in serum-free medium (data not shown), in medium containing 100
µg/mL of LDL (data not shown), or in medium containing 100
µg/mL of oxidized LDL (Fig 3).
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) or 100 µg/mL of oxidized LDL
(
) at 24, 48, and 72 hours. Values represent mean±SEM of
three experiments.
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), in combination with 400 U/mL of 
), or in cell cultures treated with 100 µg/mL of oxidized LDL
in combination with both 20 ng/mL of TNF
). Values represent mean±SEM of three
experiments.
Incubation of SMC in serum-free medium with or without addition of LDL
(Fig 4A) or oxidized LDL (Fig 4B) in
concentrations up to 100 µg/mL for 72 hours did not induce DNA
fragmentation as determined by the TUNEL technique. A minor fraction of
the cells incubated in medium containing IFN, IFN/TNF, or
IFN/TNF/LDL for 72 hours were TUNEL positive. In contrast,
incubation of cells in the presence of oxidized LDL together with both
IFN and TNF was found to induce DNA fragmentation in 38.1%±8.2 of
the cells (P<.05 as compared with controls), whereas cells
exposed to oxidized LDL in the presence of only one of these
cytokines showed no TUNEL positivity (Fig 4B).
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), in
combination with 400 U/mL of gIFN (
), or in combination with both
20 ng/mL of TNF
). B, percentage of
TUNEL+ cells at 24, 48, and 72 hours in cell cultures
treated with 100 µg/mL of oxidized LDL alone (
Ultrastructure of SMC Exposed to LDL and Oxidized LDL in
Vitro
Cultured control SMC were characterized by nuclei with smooth
contours, evenly dispersed chromatin, and a thin peripheral
rim of heterochromatin. The cytoplasm of the cells contained abundant
Golgi complexes and rough endoplasmic reticulum, mitochondria, and some
lysosomes (Fig 5A). Exposure of
cells to TNF and/or IFN resulted in some distention of the
endoplasmic reticulum but was otherwise without effect on cell
morphology. The general ultrastructure of the nucleus and cytoplasmic
organelles was not changed in cells incubated with 100 µg/mL
of LDL for 72 hours. However, many cells contained cytoplasmic lipid
droplets, and some lysosomes were filled with degraded
cytoplasmic organelles and other dense material (Fig 5B). Culture in
medium containing oxidized LDL was associated with significant
disruption of morphological integrity. In cultures exposed to 50
µg/mL of oxidized LDL for 24 hours, mitochondrial swelling,
disruption of the endoplasmic reticulum, and Golgi membranes were seen
in many cells. The chromatin appeared more condensed than in control
cells (Fig 5C). Incubation of SMC with 100 µg/mL of oxidized
LDL for 72 hours was associated with major breakdown of cell integrity,
severe cytoplasmic swelling, disruption, and degeneration of the plasma
membrane and other cell organelles. The chromatin was evenly dispersed
without peripheral condensation (Fig 5D). Similar changes
were also observed in many cells exposed to TNF and IFN in
addition to oxidized LDL. However, this treatment was also found to
induce condensation of chromatin and cytoplasmic organelles in many of
the cells. Large vesicles containing cytoplasmic organelles and
surrounded by an intact bilayer membrane were sometimes seen in the
close vicinity of such cells (Fig 5E). Many of these cells were also in
a state of secondary necrosis demonstrating more severe degenerative
changes, including lack of membrane integrity (Fig 5F).
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Discussion |
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Degeneration of SMC in the fibrous cap of complicated lesions is an important factor in plaque rupture and therefore also in the clinical manifestations of atherosclerosis.1 2 Recent studies have shown that a large fraction of cells in human atherosclerotic lesions are in a state of DNA fragmentation and as a consequence are destined for degeneration and death.3 4 22 Several factors such as ischemia, inflammatory cytokines, and accumulation of toxic substances may contribute to the increased rate of cell death in atherosclerotic plaques. Oxidized LDL accumulates in atherosclerotic lesions10 23 and is highly toxic for cultured vascular cells,15 16 suggesting that it may be one of the most important factors involved in plaque cell death. The results of this study show that regions of lesions containing large amounts of oxidized LDL are either cell poor or contain frequent SMC with DNA fragmentation as assessed by TUNEL technique. Moreover, we demonstrate that oxidized LDL induce DNA fragmentation in cultured human SMC, but only in the presence of cytokines. Finally, we show that exposure of cultured human SMC to oxidized LDL results in ultrastructural changes similar to those observed in degenerating SMC in human atherosclerotic lesions. These observations add some support to the notion that oxidized lipids have a role in plaque cell death and destabilization. There was, however, no clear association between the extent of oxidized LDL accumulation in plaques (as assessed by immunohistochemistry) and the frequency of TUNEL-positive cells. There was also no clear colocalization between TNF
immunoreactivity and TUNEL-positive cells
as would have been expected if the combined effect of oxidized LDL and
cytokines was a key mechanism in induction of
apoptosis. The latter findings argue against a major role for
oxidized LDL and cytokines in plaque cell
apoptosis. Several angiographic trials have shown that lipid-lowering therapy inhibits progression of coronary atherosclerosis. However, a common finding in these studies is that a very modest increase in lumen diameter in response to treatment is associated with a dramatic decrease in the incidence of clinical events.24 25 26 This observation has led to the conclusion that the most important effect of lipid-lowering therapy is plaque stabilization rather than regression of plaque size. Since there is no evidence to suggest that native lipoprotein affects matrix integrity and plaque stability, these findings are also in good agreement with the hypothesis that oxidized lipids play an important role in plaque rupture.
The question of whether apoptosis is involved in plaque cell
death has received increasing attention during the last few years.
Using TUNEL to detect cells undergoing apoptotic cell death,
some investigators have reported the presence of up to 40% positive
cells in macrophage-enriched parts of atherosclerotic
plaques.3 This report may represent an
overestimation, as TUNEL has been found to detect cells in a late stage
of primary necrosis (oncosis) and also to label matrix vesicles formed
as a result of cell disintegration.27 Other studies
suggest an apoptotic frequency of about 10% in atherosclerotic
lesions, which is similar to that found in our study.22 28
The notion that apoptosis indeed occurs in atherosclerotic
plaques is also strongly supported by the demonstration of ladders of
multiples of 180 to 200 base pairs after agarose gel electrophoresis of
DNA from plaque-derived cells.4 However, using the
techniques available today, it is difficult to determine the relative
importance of apoptosis versus oncosis in plaque cell death.
Previous studies from our group have shown that although some
degenerating SMC in human lesions have an ultrastructure characteristic
of apoptosis, including chromatin and cytoplasmic condensation,
most of the dying cells show chromatin dispersion, cytoplasmic
swelling, and plasma membrane disruption characteristic of
oncosis.6 The present study shows that SMC death in
response to toxic concentrations of oxidized LDL alone does not result
in TUNEL positivity or chromatin and cytoplasmic condensation
characteristic of apoptosis. However, if SMC are exposed to
oxidized LDL in the presence of TNF and IFN, a large fraction of
the cells become TUNEL positive and demonstrate chromatin and cytoplasm
condensation. Previous electron microscopic evaluation of human lesions
demonstrated presence of both degenerating SMC characterized by
chromatin and cytoplasmic condensation (presumably apoptotic)
as well as swollen cells with dispersed chromatin and disintegrated
cytoplasmic organelles and plasma membrane (presumably in
oncosis).6 The latter cells were more common, indicating
that cell death by oncosis is more frequent than cell death by
apoptosis. Our findings suggest that oxidized LDL may be
involved in both types of cell death and that cells exposed to oxidized
lipids in the absence of an inflammatory process degenerate by oncosis
whereas cells exposed simultaneously to oxidized LDL and
multiple cytokines are directed toward apoptosis.
Geng and coworkers17 recently reported activation of
apoptosis in human cultured SMC simultaneously
exposed to TNF and IFN, an effect further stimulated by the
addition of interleukin-1. In our study, the combination of TNF and
IFN was not sufficient to induce apoptosis in the absence of
oxidized LDL. The reason for this discrepancy is unclear but may
involve differences in cell populations and/or culture conditions.
However, both studies underline the importance of inflammatory cells in
the regulation of SMC death and the mechanism by which it occurs.
The cytotoxic nature of oxidized LDL is well characterized and has been
attributed mainly to its keto- and oxysterol content.16 In
contrast, there is relatively little information concerning the
mechanism of death in cells exposed to oxidized LDL. Several compounds
in oxidized LDL have been shown to induce apoptosis. Nishio et
al demonstrated that 7-ketocholesterol induces
apoptosis in vascular SMC.29 Moreover, studies in
our laboratory have shown that 25-hydroxycholesterol
activates apoptosis in cultured human SMC and that this
effect is potentiated by the addition of TNF and IFN.
Accordingly, keto- and oxysterols may be involved in the
apoptosis induced by oxidized LDL and cytokines found
in the present study. Using light microscopic analysis of
gallocyanin chrome-stained cells, Björkerud and
Björkerud30 found apoptotic-like chromatin
condensation after incubation of human SMC with oxidized LDL. This,
however, was not accompanied by an increased frequency of TUNEL
positivity. Blebbing and morphological changes compatible with
apoptosis in the absence of detectable DNA fragmentation has
previously been reported in other studies on cultured
SMC.31 Oxidized LDL has also been found to induce
apoptotic degeneration of lymphoblast cells,32 of
the P388D1 macrophage cell-line,33 and
in mouse peritoneal macrophages.34
Oxidized LDL is known to accumulate in atherosclerotic plaques. This is believed to occur as a result of oxidative modification of LDL entrapped by vessel wall proteoglycans in response to reactive oxygen intermediates and/or enzymes released by the cells of the vascular wall.7 8 9 During the early stages of atherosclerosis, the amount of oxidized LDL is likely to be low and to have a mainly proinflammatory effect.35 36 37 38 By this mechanism, LDL oxidation may play an important role in plaque growth. At more advanced stages of the disease, increased accumulation of oxidized LDL containing more severely modified lipids may generate a general toxic effect contributing to plaque degeneration and rupture.
In summary, the present study demonstrates that oxidized LDL induces degenerative changes in SMC that are similar to those observed in dying SMC in human atherosclerotic lesions. They also show that oxidized LDL may induce both oncosis and apoptosis in SMC depending on the presence of cytokines. The results further support the hypothesis that modified lipids have an important role in plaque degeneration.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Swedish Medical Research Council (8311 and 6537), the Swedish Heart and Lung Foundation, King Gustaf V 80th Birthday Fund, Prof Nanna Svartz' Fund, the Swedish Society of Medicine, the Sigurd & Elsa Goljes Foundation, and Förenade Liv Mutual Group Life Insurance Company in Stockholm. We thank Bao Wenjie for expert technical assistance.
Received December 3, 1996; accepted March 5, 1997.
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