Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:19-27
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:19-27.)
© 1996 American Heart Association, Inc.
Apoptosis of Vascular Smooth Muscle Cells Induced by In Vitro Stimulation With Interferon-
, Tumor Necrosis Factor
, and Interleukin-1ß
Yong-Jian Geng;
Qi Wu;
Maria Muszynski;
Göran K. Hansson;
Peter Libby
From the Vascular Medicine and Atherosclerosis Unit, Department of
Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston,
Mass, and the King Gustaf V Research Institute (G.K.H.), Karolinska Institute,
Karolinska Hospital, Stockholm, Sweden.
Correspondence to Dr Peter Libby, Vascular Medicine and Atherosclerosis
Unit, Department of Medicine, Brigham and Women's Hospital, Harvard
Medical School, 221 Longwood Ave, Boston, MA 02115.
 |
Abstract
|
|---|
Abstract Recent studies have documented evidence for the
death
of smooth muscle cells (SMCs) within advanced human
atheroma.
These lesions contain macrophages and T
lymphocytes in addition
to SMCs. We therefore investigated whether
interferon-

(IFN-

),
a cytokine secreted by T lymphocytes,
or interleukin-1ß
(IL-1ß) and tumor necrosis factor

(TNF-

),
two cytokines
characteristically produced by activated
macrophages, can trigger
apoptosis of vascular SMCs.
Simultaneous treatment with IFN-
and TNF-

and/or
IL-1ß but not with each cytokine alone
promoted death of
human and rat SMCs. Exposure for 48 hours
to a combination of IFN-

(400 U/mL), TNF-

(400 U/mL), and IL-1ß
(100 U/mL) significantly
(
P<.001) increased the accumulation
of oligonucleosomes
comprising DNA fragments and histones in
human SMCs. Electrophoresis of
genomic DNA showed internucleosomal
fragments of genomic DNA isolated
from the cytokine-cotreated
SMCs of both humans and rats.
These cells exhibited morphological
changes typical of
apoptosis, including cell shrinkage, membrane
blebbing,
chromatin condensation, and nuclear fragmentation.
In situ 3' end
labeling of DNA fragments with terminal transferase
confirmed the
fragmentation of genomic DNA in these cells. Simultaneous
treatment
with IFN-

and TNF-

or IL-1ß induced elaboration of
nitrite,
an end product of nitric oxide, in rat but not human SMCs.
NG-monomethyl-
L-arginine
inhibited
nitrite accumulation and also partly blocked
cytokine-induced
apoptosis of rat SMCs but had
little effect on human SMCs, suggesting
operation of both nitric
oxidedependent and independent
mechanisms for
cytokine-induced apoptosis in vascular SMCs.
Production
of immune cytokines by vascular cells and/or
infiltrating leukocytes
may regulate apoptotic death of SMCs
during atherogenesis.
Key Words: smooth muscle cells cell death nitric oxide cytokines atherosclerosis
 |
Introduction
|
|---|
In addition to
lipids and connective tissue, atherosclerotic
lesions consist of SMCs,
macrophages, and T lymphocytes.
1 During
the
prolonged process of atherogenesis, complex interactions
between the
vascular cells and infiltrating immune cells yield
local
production of a great variety of biologically active substances
such
as cytokines and growth factors.
2 3 These
bioactive substances
may in turn regulate cell functions, such as
replication, differentiation,
and death, in a paracrine or autocrine
fashion and consequently
influence cellularity and morphogenesis of the
vessel wall.
The cytokines IFN-
4 5 (secreted
mainly by activated T cells) and IL-16 7 and
TNF-
8 (products of activated
macrophages as well as SMCs themselves) can regulate gene
expression, differentiation, and growth of vascular SMCs in vitro and
in vivo. Although IL-17 and TNF-
8
stimulate but IFN-
inhibits SMC
proliferation,4 9 these
cytokines share many biological actions. For example, at least
in rodent cells, IFN-
, TNF-
, and IL-1, together or alone, can
induce expression of NOS.10 11 NO produced by the
cytokine-inducible form of NOS exerts many well-known
effects on cells, including the ability to kill microbial pathogens and
tumor cells.12
Cell death occurs in advanced atherosclerotic lesions, often resulting
in formation of hypocellular fibrous zones and a lipid-rich
"necrotic" core. Recent reports provide evidence that
apoptosis, a form of programmed cell death,13 can
mediate some of the cell death in human atherosclerotic
lesions.14 15 16 SMCs isolated from
atherosclerotic arteries
undergo apoptosis more frequently than do cells from normal
vessels.17 In situ detection of DNA fragments demonstrates
that many SMCs in plaques15 16 and in balloon-injured
arteries16 18 bear this marker of apoptosis.
The mechanisms that trigger apoptosis in atherosclerotic
lesions remain unknown. Cytokines can promote apoptosis
in some cell types: IL-1 induces apoptosis of pancreatic
cells19 and chondrocytes20 in an NO-dependent
pattern; TNF-
promotes apoptosis of
endothelial cells,21 and IFN-
triggers
apoptosis of vascular SMCs, which overexpress functional
c-myc after transfection with c-myc
cDNA.22 Since cytokine-producing cells (eg,
macrophages and T lymphocytes) abound in advanced
atherosclerotic lesions,2 23 we hypothesized that
cytokines might contribute to apoptosis of vascular
SMCs during atherogenesis. The present study tested whether
cultured SMCs undergo apoptosis when exposed to recombinant
cytokines.
 |
Methods
|
|---|
Reagents
The recombinant cytokines used in this study were
rat
IFN-

(Holland
Biotechnology), human IFN-

(Genzyme Corp), murine
TNF-

(Genentech
Inc), and human TNF-

and IL-1ß (Endogen, Inc).
Sulfanilamide
and
N-(1-naphthyl)ethylenediamine
hydrochloride were obtained
from Merck. L-NMMA was from Calbiochem
Corp. Acridine orange,
ethidium bromide, and propidium iodide were
purchased from Sigma
Chemical Co, and YOYO-1, a sensitive DNA-binding
fluorochrome,
was from Molecular Probes, Inc.
Vascular SMC Isolation and Culture
Human24 and
rat4 vascular SMCs were
isolated from the tunica media of aortas of 35- to 45-year-old
organ donors without overt atherosclerosis and
6-week-old Sprague-Dawley rats, respectively. The cells
were cultured in Dulbecco's minimal essential medium (GIBCO)
supplemented with 10% FCS and antibiotics.24 Cells were
identified as vascular SMCs by their characteristic
hillsand-valleys growth pattern and by
immunofluorescence with anti
smooth
muscle actin monoclonal antibody. They were passed by trypsinization,
plated at a density of 2x104 cells/mL, and used for
experiments at passages 2 through 7. Cells at a subconfluent density
were treated with IFN-
, IL-1ß, and TNF-
alone or together. In
some experiments, L-NMMA (500 µmol/L) was added together with the
cytokines to block endogenous NO synthesis.
Cell Viability
Cell viability was determined by fluorochrome
staining.
SMCs (104 cells/chamber) were cultured in eight-chamber
slides. The cells were treated with 400 U/mL IFN-
, 400 U/mL TNF-
,
and 100 U/mL IL-1ß for 24, 48, 72, or 96 hours. At the end of
incubation, the nucleic acidbinding fluorescent dyes
acridine orange and ethidium bromide (10 µg/mL each in culture
medium) were added to the cultures. After the slides were stained, they
were coverslipped and observed by fluorescent microscopy, and
viable (green fluorescent nuclei) and nonviable (red or orange
fluorescent nuclei) cells were counted. For each sample, at
least 200 cells were counted in different high-power fields. The
percentage of viable cells was determined by the following formula:
percent cell viability=100x(number of viable cells)/(total number
of
cells).
Cell Morphology and Nuclear Staining
Cell morphology was
examined by phase-contrast microscopy
and recorded by microphotography during incubation with
cytokines. For visualization of nuclei, cells were cultured in
eight-chamber slides and treated with or without cytokines.
At the end of incubation, cells were fixed in ice-cold 4%
formaldehyde. After washing with PBS containing Tween 20 (0.5%), cells
were stained with 1 µg/mL YOYO-1 in PBS for 5 minutes, mounted in
70% glycerol in PBS, and observed under an Olympus fluorescent
microscope.
Enzyme Immunoassay of Oligonucleosomes
Internucleosomal DNA
fragmentation characteristic of
apoptosis occurs several hours before breakdown of the plasma
membrane of apoptotic cells. Enrichment of mononucleosomes or
oligonucleosomes in cytokine-treated and untreated SMCs was
quantitatively determined by sandwich-enzyme immunoassay with a
cell-death detection ELISA kit purchased from Boehringer
Mannheim Corp. Briefly, 5x104 cells cultured in 24-well
plates were treated with IFN-
, TNF-
, and IL-1ß for 48 hours. At
the end of the incubation, culture medium was removed to an Eppendorf
tube and centrifuged at 20 000g. The resulting
pellets contained oligonucleosomal fragments. Meanwhile, adherent SMCs
were permeabilized in the sample buffer containing
Tween 20. The supernatants containing the cytoplasmic oligonucleosomes
released from SMC nuclei were combined with the oligonucleosomal
fragments in medium. The combined supernatants (100 µL) were
transferred to a 96-well microplate precoated with monoclonal antibody
against histone-4. After incubation for 1 hour at room temperature and
washing, oligonucleosomal DNA fragments bound to the microplate were
detected by using monoclonal anti-DNA antibody conjugated with the
enzyme peroxidase. 2,2'-Azinodi-[3-ethylbenzthiazoline
sulfonate] was
used as the enzyme substrate, and colored product was measured by
spectrophotometry at 405 nm.
In Situ Detection of Apoptotic Cells
Cells undergoing
apoptosis can accumulate
internucleosomal DNA fragments in their nuclei. In situ detection of
apoptotic cells was performed by using TUNEL with an ApoTag in
situ apoptosis detection kit (Oncor Inc). Briefly, cells were
cultured in eight-chamber slides and treated with or without
cytokines for 72 hours. After cytokine treatment, cells
were washed in PBS, fixed, incubated with 5 µg/mL proteinase K for 10
minutes, and then labeled with digoxigenin-conjugated dUTP and the
enzyme terminal deoxyribonucleotide transferase.
Labeled DNA fragments were stained with anti-digoxigenin monoclonal
antibody linked with peroxidase. The chromogenic substrate
diaminobenzidine was used as the substrate for peroxidase.
Flow Cytometry
SMCs were cultured in six-well plates in
Dulbecco's minimal
essential medium containing 10% FCS. After incubation with or without
cytokines and trypsinization, cells were suspended in fresh
medium containing 50 µg/mL propidium iodide for 5 minutes at 37°C
and then subjected to flow cytometry on a
fluorescence-activated cell sorter flow cytometer
immediately after incubation. A light-scatter gate was set up to
eliminate cell debris from analysis. Cellular propidium iodide
fluorescence signal was recorded on the FL2 channel and
analyzed by using Lysys II software.
DNA Fragmentation Analysis
Cells (5x106)
were lysed in 1 mL DNA
extraction solution containing 20 mmol/L Tris-HCl, pH 7.4, 0.1 mol/L
NaCl, 5 mmol/L EDTA, and 0.5% sodium dodecyl sulfate. The
lysates were incubated with 100 µg/mL proteinase K at 37°C for 16
hours. After incubation, 1 mL of phenol/chloroform (1:1) was mixed well
with the enzyme-digested cell lysates, and the mixture was
centrifuged at 20 000g for 20 minutes. DNA in the
upper (aqueous) phase was incubated with 5 µg/mL DNase-free RNase
A at 37°C for 1 hour and extracted with phenol/chloroform again. DNA
was collected by precipitation with 1 mL isopropanol and 0.1 mL of 5
mol/L NaCl at -20°C overnight. After centrifugation,
the resulting DNA pellets were washed with 75% ethanol and air dried.
DNA was dissolved in 10 mmol/L Tris-HCl and 1 mmol/L EDTA, and its
concentration was determined at 260 nm by spectrophotometry. DNA
electrophoresis was carried out in 1.5% agarose gels containing 1
µg/mL ethidium bromide, and DNA fragments were visualized by exposing
the gel to UV light.
Nitrite Assay
In cell cultures, the majority of NO produced
by
cytokine-stimulated SMCs is converted into nitrite by
reaction with ambient oxygen. We therefore measured nitrite
accumulation in SMCs by using the Griess reagent, composed of 2.9
mmol/L sulfanilic acid and 0.2 mmol/L
N-(1-naphthyl)ethylenediamine hydrochloride in 5%
phosphatic acid.10 11 Briefly, culture medium was
incubated with an equal volume of the Griess reagent at room
temperature for 30 minutes. The colored product of the
diazotization reaction was spectrophotometrically quantified at 540 nm
by using a microplate ELISA reader. Sodium nitrite dissolved in the
same medium was used as standard.
Statistical Analysis
Differences between means were evaluated
by using two-tailed
Student's t tests. ANOVA in cell viability was carried out
by using the ANOVA program in Excel (Microsoft Co). Significance was
established when the probability value was less than .05.
 |
Results
|
|---|
Death of Vascular SMCs Exposed to Cytokines
We examined the
viability of cultured human and rat vascular
SMCs
by staining the cells with the fluorochromes acridine orange
and
ethidium bromide. Viable cells excluded ethidium bromide
but were
permeable to acridine orange, which reacts with DNA
to yield green
nuclear fluorescence and with RNA to produce
red
fluorescence in the cytoplasm (Fig 1

). Nonviable
cells show
red ethidium bromide fluorescence in their nuclei
due to ethidium
bromide entry (Fig 1d

). Under basal conditions,
cultures of
both human and rat SMCs contained few nonviable cells (Fig
1a
and 1e

). Treatment with either IFN-

(400
U/mL), TNF-

(400
U/mL),
or IL-1ß (100 U/mL) alone for 24 hours did not increase
the
percentage of dead cells, and prolongation of the cytokine
incubation
time to 48 and 72 hours slightly increased the number of
dead
cells in the single cytokinetreated SMCs (Figs 1

and 2

).
However, a combination of these cytokines
induced much higher
levels of cell death compared with the control
cultures or those
treated with a single cytokine.
Simultaneous exposure to IFN-

,
TNF-

, and IL-1ß
caused higher levels of cell death than
did the double treatments (Fig
2

).
Flow cytometric determination of cell viability with
the DNA-binding
dye propidium iodide further demonstrated similar cytokine
effects on rat SMCs, which are more easily detached during
apoptosis than are human SMCs. Few rat SMCs died when cultured
in normal medium containing either no cytokines or an
individual cytokine (Fig 3
). More than 95% of
unstimulated rat SMCs excluded propidium iodide, and exposure to either
IFN-
or TNF-
alone did not reduce cell viability (Fig
3
).
However, simultaneous treatment with both IFN-
and
TNF-
at 400 U/mL each for 72 hours reduced cell viability from
96±1% to 48±8% (P<.01), indicating the synergy in
induction of cell death between the two cytokines (Fig 3
).
Nonviable cells exhibited high but varying levels of propidium iodide
fluorescent intensity (Fig 3
), suggesting that DNA degradation
might occur in these nonviable cells, leading to a wide range of their
cellular DNA content.
Morphological Characterization of Vascular SMCs Undergoing
Apoptosis
Phase-contrast microscopy showed morphological changes
characteristic of apoptosis in cytokine-treated
human and rat SMCs. When exposed to IFN-
, TNF-
, and IL-1ß, some
human SMCs shrank and retracted from their neighbor cells. Their
membranes appeared blebbed, and the cytoplasm condensed (Fig
4
). The surviving cells also showed certain degrees of
morphological change, with an elongated and bipolar appearance (Figs
1
and 4
). We used the ultrasensitive DNA-binding
dye YOYO-1, which
discloses nuclear morphology better than does acridine orange or
ethidium bromide, to examine human SMCs treated with or without the
cytokines. Staining with YOYO-1 clearly visualized fragmented
nuclei with condensed chromatin, and some nuclei fragmented and formed
apoptotic bodies (Fig 5
). The morphological
changes induced in SMCs by cytokine stimulation were
distinguishable from those caused by treatment with high
concentrations of H2O2, which kills SMCs
very rapidly. In contrast to the cells treated with cytokines,
SMCs exposed to H2O2 appeared swollen rather
than shrunken, but their connections with adjacent cells remained
intact (Fig 4h
). In contrast to cytokine-treated cells,
H2O2-treated SMCs showed a relatively normal
nuclear morphology, as illustrated by YOYO-1 staining (Fig 5d
).
These
observations suggest that cytokine treatment induces SMC death
via apoptosis, whereas at high concentrations,
H2O2 causes SMC death via primary necrosis or
oncosis, a form of nonprogrammed cell death.25 Rat SMCs
similarly treated developed pyknotic nuclei as well (Fig
5f
).
Accumulation of Oligonucleosomes in Vascular SMCs Undergoing
Apoptosis
Cells undergoing apoptosis can release mononucleosomes or
oligonucleosomes comprising DNA fragments and histones from their
nuclei into the cytoplasm or even into the extracellular
compartment.26 27 We examined the accumulation of
oligonucleosomes in the cytokine-treated cells by using
ELISA with anti-histone and anti-DNA antibodies to verify the
occurrence of apoptosis. We observed a significantly increased
accumulation of oligonucleosomes in human SMCs treated
simultaneously with IFN-
, TNF-
, and IL-1ß for 48
hours (Fig 6
). In contrast, few oligonucleosomes
accumulated in the untreated cultures (Fig 6
). In the presence
of
TNF-
(400 U/mL) and IL-1ß (100 U/mL), IFN-
increased
accumulation of oligonucleosomes in human SMCs in a
concentration-dependent manner (Fig 7
).
We further
visualized DNA fragments in situ by using the TUNEL
technique. Few cells (<5%) in the cultures with or without each
cytokine alone showed TUNEL staining (Fig 8
).
However, 34±4% of human SMCs in the cultures cotreated with IFN-
,
TNF-
, and IL-1ß for 48 hours showed positive staining with TUNEL
(P<.05 versus untreated controls, n=3) (Fig
8
). Some cells
with TUNEL-positive nuclei also showed TUNEL staining in their
cytoplasm, indicating the presence of cytoplasmic DNA fragments in the
cells undergoing apoptosis (Fig 8
).
We also evaluated
internucleosomal DNA fragmentation, a biochemical
marker for apoptosis, by assessing the size of genomic DNA
isolated from the cytokine-treated and untreated SMCs by
using agarose gel electrophoresis (Fig 9
). Most DNA
extracted from both human (Fig 9A
) and rat (Fig
9B
) SMCs showed a high
molecular weight (>20 kb). Internucleosomal DNA fragments at 180 to
200 bp or multiples appeared in SMCs treated simultaneously
with IFN-
, TNF-
, and/or IL-1ß (Fig 9
), indicating
the
occurrence of apoptosis in these cells. In contrast, no
appreciable levels of DNA fragmentation existed in the untreated
control cells (Fig 9
).
NO Synthesis and Apoptosis in Vascular SMCs Stimulated
With Cytokines
The cytokines IFN-
, TNF-
, and IL-1ß can
together elicit expression of the inducible form of NOS by vascular
SMCs.10 Such augmented NO synthesis can mediate
apoptosis of murine macrophages28 and
human chondrocytes.29 We therefore tested whether
enzymatically synthesized NO mediates cytokine-induced
apoptosis in vascular SMCs. In rat SMCs, stimulation with a
combination of IFN-
(400 U/mL) and TNF-
(400 U/mL) induced high
levels of nitrite accumulation (Fig 10
). However, human
SMCs so treated did not produce substantial amounts of NO in response
to the same cytokines (Fig 10
).
In rat SMCs cotreated
with IFN-
and TNF-
, the addition of the NOS
inhibitor L-NMMA (500 µmol/L) significantly increased the
number of viable cells as demonstrated by flow cytometry (Fig
3
) and
fluorescent microscopy (Table
) but diminished
nitrite accumulation (Fig 10
). L-NMMA also inhibited
internucleosomal
DNA fragmentation in the cytokine-cotreated rat SMCs (Fig 9B
).
However, this NOS inhibitor did not completely block
the cytokine-induced apoptosis. Furthermore,
L-NMMA did not appear to alter the viability (Table
) of the
cytokine-treated human vascular SMCs, which produced little
nitrite. This result suggests that cytokines induce
apoptosis of vascular SMCs by both NO-dependent and
-independent pathways.
 |
Discussion
|
|---|
Apoptosis appears to require an exogenous signal and
a complex
endogenous gene-controlled
apoptotic machinery.
30 31 We sought
here to
test whether cytokines produced by inflammatory cells
trigger
death of vascular SMCs. By using a combination of techniques
for
analyzing markers for apoptosis, we now demonstrate that
when
combined, the cytokines IFN-

, TNF-

, and IL-1ß can
induce
apoptosis of cultured human and rat SMCs. The data
support the
notion that SMCs in atherosclerotic lesions may undergo
apoptosis
in response to the proinflammatory cytokines
produced locally
by activated macrophages and T
lymphocytes as a consequence
of the ongoing local immune and
inflammatory response characteristic
of
atherogenesis.
15
Vascular SMCs exhibit heterogeneity both in vivo and in
vitro. Normal arterial intima contain very small numbers of
SMCs. During atherogenesis, increased numbers of SMCs exist in the
thickening intima; these SMCs show substantial differences in gene
expression and function from medial SMCs. However, many in vitro and in
vivo studies2 3 argue that in in vitro culture,
medial
SMCs can undergo modification during passages and gain some of the
biological properties of neointimal SMCs during
atherogenesis; thus they are widely used as target cells in in vitro
experiments.
It is likely that different subpopulations of SMCs may respond
differently to factors such as cytokines. We found that some
SMCs survived cytokine treatment even at high concentrations
while their neighbor cells died and that the surviving cells showed a
morphology distinct from that of untreated cells. Recent studies on the
effects of IFN-
on c-myctransfected rat SMCs
demonstrate that both apoptotic and mitotic figures can exist
in the same cell cultures.22 These findings indicate that
cytokines may selectively act on certain subpopulations of
SMCs, thus leading to depletion of these cells from tissues or
modulating their phenotype.
Prolonged exposure to IL-1 alters the morphology and growth pattern of
human SMCs, resulting in accumulation of cells with an elongated,
bipolar shape.7 Treatment with IFN-
suppresses
expression of
smooth muscle actin in rat SMCs9
while augmenting the expression of major histocompatibility complex
class II genes in both rat4 and human32 SMCs.
In atherosclerosis, SMCs in the intimal lesions differ
from those of the tunica media in phenotype, but the underlying
mechanism remains uncertain. Cytokines produced from and evoked
by infiltrating immune cells are likely involved in phenotypic change
of SMCs by inducing apoptosis of certain subtypes of SMCs that
are sensitive to the cytokines.
Our experimental results indicate that the individual cytokines
alone do not suffice to cause apoptosis of either rat or human
SMCs. Exposure to either IL-16 7 or
TNF-
8
can stimulate SMC proliferation, whereas
IFN-
4 5 9 inhibits SMC
proliferation. These
findings suggest that these cytokines may activate
different signal transduction pathways that synergize or antagonize
each other in regulation of cell turnover.
Several different signal transduction pathways may mediate the
induction of apoptosis by these proinflammatory
cytokines. Among them, the L-arginine/NOS pathway
may serve as a potential trigger of apoptosis, as many studies
have demonstrated that IFN-
, TNF-
, and IL-1, alone or
synergistically, induce synthesis of NO from L-arginine in
various types of rodent cells, including vascular SMCs.12
We observed that cytokine-stimulated, NO-generating rat
SMCs died via apoptosis, and addition of the NOS
inhibitor L-NMMA could partially block these
cytokine effects. This result agrees well with the data from
recent studies showing that NO mediates apoptosis of murine
macrophages28 33 and their tumor target
cells,34 rat pancreatic beta-cells,19 and
human chondrocytes.29
However, it remains unclear how activation of NO synthesis leads to
apoptosis in the cytokine-treated cells. We have
shown that NO can form iron (II)nitrosyl complexes with respiratory
enzymes containing nonheme ironsulfur clusters in mitochondria of
rat SMCs and consequently inhibit respiration and ATP synthesis in
cytokine-treated SMCs.10 11 NO also
inactivates enzymes important for DNA synthesis and
repair.12 Recently, NO generated by
cytokine-inducible NOS was found to stimulate the
expression of the tumor suppressor gene p53 in RAW 264.7
macrophages and pancreatic RINm5F cells before
apoptosis.35 Expression of p53 can lead to cell
death via apoptosis in various normal and malignant cell
types.35 These observations suggest that high levels of NO
production catalyzed by cytokine-inducible NOS may
promote apoptosis by multiple mechanisms.
Under the same experimental conditions, however, human vascular SMCs do
not produce any appreciable levels of NO in response to IFN-
,
TNF-
, and IL-1ß, in accord with recent work from other
laboratories.36 A similar situation exists in cultured
human macrophages, which, unlike rodent macrophages,
produce little NO when exposed to the
cytokines.37 38 39 However, human
hepatocytes40 and chondrocytes41
can express inducible NOS and synthesize considerable amounts of NO in
response to cytokine stimulation. Apparently, differences in
cytokine induction of NO exist between species and cell types.
The relatively high capacity for NO synthesis in rodent SMCs may partly
explain why the rodent cells die via apoptosis when exposed to
cytokines more readily than do human SMCs, which require more
stringent conditions for NO production. Nonetheless,
stimulation with the cytokines can provoke apoptosis of
human SMCs even in the absence of NO production, and blocking
of NO synthesis with L-NMMA cannot completely abolish apoptosis
of rat SMCs induced by cytokines. It is likely that both
NO-dependent and -independent mechanisms mediate the
cytokine-induced apoptosis of vascular
SMCs.
Much prior work on the cell biology of atherosclerosis
has focused on SMC proliferation.3 During atherogenesis,
arterial SMCs migrate from the tunica media to the intima,
where they proliferate and synthesize extracellular matrix,
contributing to focal thickening of the intima. In this context,
apoptosis may serve as an adaptive mechanism to limit excessive
cell replication and thickening of the intima. However, in advanced
atheroma, SMC synthesis of extracellular matrix may
actually stabilize the plaque structure. A high level of
apoptotic cell death might impair maintenance of this
matrix scaffolding of the fibrous cap of the plaque and thus predispose
to plaque rupture, a major cause of acute atherosclerotic syndromes
such as acute myocardial infarction or unstable angina. Therefore,
dysregulation of apoptosis may influence both formation and
complication of atherosclerosis and thereby provide a
new therapeutic target.
 |
Selected Abbreviations and Acronyms
|
|---|
| ELISA |
= |
enzyme-linked immunosorbent
assay |
| FCS |
= |
fetal calf
serum |
IFN- |
= |
interferon- |
| IL |
= |
interleukin |
| L-NMMA |
= |
NG-monomethyl-L-arginine |
| NO |
= |
nitric
oxide |
| NOS |
= |
nitric oxide
synthase |
| PBS |
= |
phosphate-buffered
saline |
| SMC |
= |
smooth muscle
cell |
TNF- |
= |
tumor necrosis
factor |
| TUNEL |
= |
terminal
deoxyribonucleotide transferasemediated
dUTP nick end
labeling |
| YOYO-1 |
= |
benzoxazolium-4-quinolinium
dimer |
|
 |
Acknowledgments
|
|---|
This work was supported by National Institutes of Health grant
HL-34636,
the Swedish Medical Research Council (project No. 6816),
and
the Swedish Medical Heart-Lung Foundation. Yong-Jian Geng is
a
recipient of the ICRETT Award granted by the International
Union
Against Cancer, Geneva, Switzerland.
Received July 7, 1995;
accepted September 15, 1995.
 |
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