© 2000 American Heart Association, Inc.
Vascular Biology |
Accumulation of LDL in Rat Arteries Is Associated With Activation of Tumor Necrosis Factor-
Expression
From the Department of Medicine, Malmö University Hospital, Univerity of Lund, Malmö, Sweden
Correspondence to Jan Nilsson, Department of Medicine, Malmö University Hospital, 205 02 Malmö, Sweden. E-mail jan.nilsson{at}medforsk.mas.lu.se
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Abstract |
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Abstract—Activation of vascular inflammation in response to hyperlipidemia is believed to play an important role during the early stages of atherogenesis. We demonstrate here that exposure of cultured, rat aortic smooth muscle cells to low density lipoprotein (LDL) stimulated tumor necrosis factor-
(TNF-) mRNA and protein expression. Oxidative
modification of LDL resulted in a reduction of this stimulatory effect.
To analyze whether a similar response also occurs in vivo, we
used a recently developed model in which the effects of a rapid
accumulation of human LDL in rat arteries can be studied. As previously
reported, epitopes specific for human apolipoprotein B began to
accumulate in the aorta within 2 to 6 hours after injection of 6 mg of
human LDL. This was followed by expression of oxidized LDL–specific
epitopes after 12 hours. There was no vascular expression of TNF- at
baseline or in phosphate-buffered saline–injected control rats.
However, 24 hours after injection of native LDL, there was a marked
induction of TNF- mRNA and immunoreactivity in the aorta and other
large arteries, whereas injection of oxidized LDL was without effect in
this respect. Preincubation of LDL with the antioxidant probucol before
injection markedly decreased the expression of TNF-
immunoreactivity. The present findings support the notion that LDL
may activate arterial expression of TNF- and
suggest 1 possible mechanism for the inflammatory response in the early
stages of atherosclerosis. The role of LDL oxidation in
this process remains to be fully elucidated.
Key Words: atherosclerosis • oxidized LDL • probucol • TNF-
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Introduction |
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The early stages of atherosclerosis involve increased retention of lipoproteins1 2 in the arterial wall, followed by activation of endothelial expression of leukocyte adhesion molecules, such as vascular cell adhesion molecule-1 and E-selectin,3 4 5 and penetration of mononuclear leukocytes through the endothelium into the intimal layer. Intimal monocytes differentiate into resident macrophages, which then ingest lipoprotein-derived cholesterol and develop into foam cells.6 The fact that macrophages have few receptors for normal LDL but a large capacity to ingest oxidized LDL by the scavenger receptor pathway has led to the conclusion that LDL entrapped in the arterial wall becomes oxidatively modified by reactive oxygen intermediates and/or enzymatically dependent processes.7 8 Oxidative modification of LDL is associated with the formation of proinflammatory substances such as lipid peroxides, platelet-activating factor–like phospholipids, and lysophosphatidylcholine,9 suggesting that lipid oxidation products may also be involved in activation of the inflammatory reaction in atherosclerosis.
The marked inflammatory component of the early atherosclerotic process
has focused attention on the role of cytokines. Tumor necrosis
factor (TNF)- is of particular interest in this respect. It
regulates endothelial expression of leukocyte adhesion
molecules10 11 as well as endothelial
procoagulant and fibrinolytic activity,12
activates the synthesis of growth factors and cytokines
in vascular cells, and stimulates the growth of smooth muscle cells
(SMCs).13 The presence of TNF- has been demonstrated in
human atherosclerotic plaques,14 15 in proliferating SMCs
in the balloon-injured rabbit aorta,16 in balloon-injured
rat femoral arteries,17 and in the media of
coronary arteries during acute rejection of rabbit cardiac
allografts.18 Moreover, circulating TNF- levels are
significantly increased in patients with premature coronary
heart disease compared with age-matched healthy
controls.19 TNF- has also been implicated in the
insulin resistance syndrome, a well-characterized risk factor for
coronary heart disease.20 21 TNF- is also
expressed in adipose tissue and skeletal muscle and is believed to act
locally by regulating the sensitivity of the insulin
receptor.22 23 In these tissues, there is an association
between increased lipid accumulation and TNF-
expression.24 25 We have recently developed an animal
model to study the response of vascular tissue to a sudden and
transient accumulation of lipoproteins in vivo.26 27 The
aim of the present study was to determine whether exposure of
vascular cells to LDL is associated with induction of TNF-
expression.
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Methods |
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Materials
The following mouse monoclonal antibodies were used: anti-human apo B from Boehringer Mannheim; anti-rat monocyte and macrophage antibody (clone ED-1) obtained from Serotec; anti-rat TNF-
supplied by R&D Systems; and anti–-actin (HHF-35)
from Dako. Secondary antibodies were biotinylated horse anti-mouse IgG
supplied by Vector and biotinylated sheep anti-rat IgG from Pierce. The
Limulus amebocyte lysate assay was performed by Microbiology
Reference Laboratory (Cincinnati, Ohio). Male Sprague-Dawley
rats were obtained from B&K Universal AB (Sollentuna, Sweden). Probucol
was kindly provided by Dr Ann-Margret Lindquist, Astra-Hässle
(Mölndal, Sweden). Lipopolysaccharide (LPS) was from
Sigma, and PD-10 columns were from Pharmacia.
Preparation and Oxidation of LDL
Blood samples for LDL preparation were taken from healthy
volunteers after 12 hours of fasting, during which time smokers were
asked to refrain from smoking. All subjects were free from symptoms of
infectious disease at the time of blood sampling. Venous blood was
drawn into precooled Vacutainer (Becton-Dickinson) tubes containing
Na2EDTA (1.4 mg/mL) and placed on ice. Plasma was
then recovered by low-speed centrifugation
(1400g, 20 minutes, 1°C) and kept at this temperature
throughout the preparation procedures. LDL was prepared by
ultracentrifugation in a density
gradient28 as described in detail
earlier.29 LDL was concentrated by pooling LDL prepared
from at least 4 donors, adjusting the density to 1.065 g/L by adding
NaBr containing 10 µmol/L EDTA, and
ultracentrifugation (40 000g, 20 hours,
1°C). The LDL was frozen in 10% sucrose (vol/vol) at -80°C.
Cryopreserving the samples in sucrose did not affect the biological
properties of LDL, as described previously.30 Within
1 hour before the injection of native LDL into the animals, excess salt
and sucrose were removed by running the samples over a PD-10 column
preequilibrated in PBS. The LDL was then sterilized by passing it
through a 0.22-µm filter. The protein content was determined
according to Lowry et al.31 LDL (1 mg/mL) was oxidized by
exposure to 5 µmol/L CuSO4 for 18 hours at
37°C. Compared with native LDL, the oxidized LDL showed increased
electrophoretic mobility on agarose gel,32 increased
amounts of lipid peroxides (
1 µg/mg LDL protein),33
and higher concentrations of thiobarbituric acid–reactive substances
(40 nmol/mg LDL protein).34 Endotoxin levels in both
native and oxidized LDL were <2 ng/mg LDL protein as determined by the
Limulus amebocyte lysate assay.
Cell Culture
SMCs were isolated from rat aorta as described
previously.35 The cells were then grown in Ham’s
F12/Dulbecco’s modified Eagle’s medium (GIBCO BRL) containing 10%
newborn calf serum (GIBCO BRL), 100 U/mL penicillin, and 100 µg/mL
streptomycin at 37°C in an atmosphere of 5%
CO2 in air. The purity of SMC populations was
determined by the presence of smooth muscle–specific -actin
immunoreactivity with HHF-35 antibody. SMCs used for analysis
were seeded in 100-mm plates at 200 000 cells per dish. Subconfluent
cultures were growth-arrested by incubation in serum-free Ham’s F12
medium supplemented with antibiotics for 48 hours. Experiments were
performed on cells cultured for up to 10 passages.
Animal Protocol
Male Sprague-Dawley rats (400 to 500 g) were injected
intraperitoneally with PBS, LPS, or human native or
oxidized LDL while under ether anesthesia . The rats were
killed by intracoronary injection of KCl at 0, 2, 6, 12, 24, or
48 hours while under anesthesia (30 mg ketamine and
3 mg/kg Rompun). Heparin (1000 IU/kg) was injected into the heart,
followed by perfusion with 0.9% saline containing 0.02 mmol/L BHT
for 5 minutes. Rats were perfusion-fixed for 10 minutes with 4%
formaldehyde. Tissues were then placed in 4% formaldehyde for 6 to 12
hours and then transferred to a 15% sucrose solution and kept at 4°C
overnight.
Immunohistochemistry
Tissue sections were deparaffinized with xylene and dehydrated
with graded ethanol. The membranes were permeabilized
in 0.2% Triton X-100. Endogenous peroxidase activity was
quenched by incubating the sections in 0.3%
H2O2, 80% methanol for 30
minutes at room temperature. After being washed, the sections were
blocked with 10% horse serum in PBS for 30 minutes. Primary antibodies
were diluted in PBS and incubated for 18 hours at 4°C in a humidified
chamber. The sections were washed and incubated with the biotinylated
secondary antibody at a dilution of 1:200 for 30 minutes and then
washed. The sections were incubated for 30 minutes with a
peroxidase-labeled avidin-biotin complex and washed again. The sections
were developed with diaminobenzidine (Vector) and counterstained in
hematoxylin. Negative controls included substitution of the primary
antibody with either PBS or an irrelevant antibody.
The TNF- immunoreactivity was graded by a blinded observer according
to a modified version of the technique described by Galis et
al.36 Consistent positive staining involving
>50% of the vascular wall was recorded as (4); positive staining
of 30% to 50% of the area as (3); positive staining of 5% to 30% of
the area as (2); positive staining of 1% to 5% of the area as (1);
and staining of <1% of the area as (0).
RT-PCR Analysis of Rat TNF- mRNA
For each group described above, 3 to 6 male Sprague-Dawley rats
were studied to evaluate the presence of TNF- mRNA. Each animal was
decapitated; their aorta, iliac, and femoral arteries were removed,
rinsed in sterile saline, trimmed of adventitial tissue, and
immediately stored in LN2. Other tissues were
removed and treated similarly. RNA samples prepared from the livers of
oxidized LDL–treated animals served as positive controls for the
polymerase chain reaction (PCR) analysis (data not shown).
Total RNA was purified by using RNAgents total RNA isolation system for
reverse transcription (RT)–PCR (Promega), after which each RNA sample
was treated with DNase (DNase I, Ambion) and repurified a second time.
Two micrograms of total RNA was reverse-transcribed at 42°C for 50
minutes by the random-priming method with Pd(N)6
hexamers (Pharmacia Biotech). Analysis of specific mRNAs by
RT-PCR was accomplished by using a Clontech amplimer set. For our
purpose, the rat TNF- amplimer set (5500-3) and rat ß-actin
amplimer set (5506-1) were used. An aliquot of each reaction was
subjected to electrophoresis on 1.8% agarose gels. Bands were
analyzed by ethidium bromide staining. Quantification of the
TNF- and ß-actin bands was performed with a Fuji Bas 1500
BioImaging analyzer. Results were expressed as the ratio of
intensity of the TNF- to the ß-actin bands.
TNF- ELISA
TNF- levels in culture medium and in rat serum were assayed
by using high-sensitivity rat TNF- immunoassay plates (Amersham,
Life Science).
Statistical Methods
Values are given as mean±SD. Between-group analyses
were made with ANOVA followed by post hoc testing. A value of
P<0.05 was considered significant.
Ethical Considerations
This study was approved by the local institutional Animal Care
and Use Committee.
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Results |
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Expression of TNF-
in Cultured Rat Aortic SMCs Exposed to LDL
and Oxidized LDLCultured, serum-starved rat aortic SMCs did not contain detectable amounts of TNF-
mRNA as assessed by RT-PCR. Exposure of SMCs to 50
µg/mL human LDL for 6 hours resulted in a marked expression of
TNF- mRNA (Figure 1
). Increasing the
LDL level to 100 µg/mL did not result in any further stimulation of
TNF- mRNA expression. Oxidative modification abolished most of the
stimulatory effect of LDL on TNF- mRNA expression. No significant
increase in the release of TNF- was found in SMCs exposed to up to
100 µg/mL LDL or oxidized LDL for 6 hours. Incubation of SMCs with 50
µg/mL LDL for 12 hours resulted in a significant increase in the
amount of TNF- released from the cells (Figure 2). However, increasing the concentration
of LDL, as well as oxidation of LDL, resulted in less activation of
TNF- release. Low concentrations of LDL and oxidized LDL (1 to 10
µg/mL) did not stimulate TNF- mRNA and protein expression in the
cells.
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Vascular TNF- Expression in Response to LDL
There was no vascular TNF- immunoreactivity present in
control rats (Figure 3A), whereas
injection of 100 µg of LPS induced a marked expression of TNF- in
both arteries and veins (data not shown). We have previously shown that
LDL accumulates in the rat vascular wall 6 to 24 hours after
intravenous injection. This LDL accumulation is associated
with modifications that lead to its recognition by antibodies generated
against malondialdehyde- and hydroxynonenal-modified
LDL.26 Injection of 6 mg of human LDL was found to
activate vascular expression of TNF- within 6 to 12 hours,
depending on the size of the vessel. The earliest expression of TNF-
immunoreactivity was found in small arteries and veins (Figure 3B). At 6 hours, some TNF- immunoreactivity was also
present in the endothelial lining of larger
arteries (Figure 3C). Twelve hours after administration of LDL,
TNF- immunoreactivity was present in the media of most arteries
and showed a primarily cellular location (Figure 3D). At 24
hours, TNF- immunoreactivity was abundant throughout the media in
the aorta, iliac, and femoral arteries (Figure 3E). By later
time points, the TNF- staining had faded and completely disappeared
72 hours after injection of LDL (Figure 3F). A summary of the
changes in expression of TNF- immunoreactivity in the aorta, iliac,
and femoral arteries after injection LDL is shown in Figure 4. SMCs in other organs did not express
TNF- after LDL injection. However, an increased expression of
TNF- was observed in the epithelium of the small intestine, adipose
tissue, and the liver (data not shown). The levels of TNF- in the
sera of all animals were below the detection limit (50 pg/mL) of the
rat TNF- ELISA.
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Effect of Injection of Oxidized LDL
Injection of human in vitro–oxidized LDL did not induce vascular
TNF- expression. Earlier studies had shown that preoxidized LDL is
immediately removed by the liver and does not accumulate in the
vascular wall. An increased expression of TNF- was found in the
liver 12 and 24 hours after injection of oxidized LDL (data not
shown).
Effect of Probucol on Vascular TNF- Expression in Response
to LDL
To analyze the possible association between LDL oxidation
in the vascular wall and activation of TNF- expression, plasma was
incubated with 50 µmol/L of the antioxidant probucol before
isolation of LDL. This treatment resulted in a 4-fold increase in the
lag phase for diene formation in response to copper (data not shown).
Injection of probucol-loaded LDL resulted in the vascular accumulation
of apo B comparable with that observed in animals given LDL, but the
accumulation of malondialdehyde and hydroxynonenal antigens (ie,
epitopes specific for oxidized LDL) was markedly
reduced.26 Moreover, pretreatment with probucol resulted
in almost complete inhibition of LDL-induced activation of vascular
TNF- expression (Figure 5). This
effect appeared to be more prominent than that on the accumulation of
epitopes specific for oxidized LDL.
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Activation of Vascular TNF- mRNA Expression in Response to LDL
and Oxidized LDL
To determine whether the vascular expression of TNF- in
response to LDL was associated with a local activation of TNF- gene
transcription, mRNA expression in the aorta and iliac arteries was
analyzed by the RT-PCR technique. Equal loading was assessed by
using primers specific for rat ß-actin. All arteries in animals given
LDL were found to contain TNF- mRNA, whereas no expression of
TNF- mRNA was found in animals given PBS alone or probucol-loaded
LDL (Figure 5
).
One possibility is that the decrease in vascular TNF- expression in
rats given probucol-pretreated LDL is due to a direct
inhibitory effect of probucol on SMC TNF- expression. To
investigate this possibility, interleukin-1–treated, cultured, rat
SMCs were grown with or without 50 µmol/L probucol for 24 hours,
and TNF- mRNA levels were analyzed by RT-PCR. Interleukin-1
treatment resulted in a 3-fold increase in TNF- mRNA levels in the
cells, but this increase was not affected by addition of probucol (data
not shown). To examine the possibility that the effect of LDL was
mediated by factors generated during a cell-mediated oxidation process
but not present in copper-oxidized LDL, cultured SMCs were exposed
to LDL with or without 50 mmol/L probucol for 24 hours. Again,
probucol was without effect on the induction of TNF- mRNA expression
(data not shown). To further analyze whether the induction of
TNF- mRNA expression by LDL was caused by minor oxidative
modifications by iron ions present in Ham’s F12 medium, SMCs were
exposed to LDL in iron-free Eagle’s minimum essential medium. However,
culture of the cells in this medium for 24 hours resulted in cell
damage and activation of TNF- mRNA expression that was even greater
than that induced by LDL (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Activation of vascular inflammation in response to lipid accumulation is generally believed to play a critical role in the initiation of atherosclerosis. However, the mechanisms by which lipids activate the inflammatory response remain to be fully elucidated. Recent studies have shown that the cytokine TNF-
has important functions in the regulation of lipid and glucose
metabolism. It is expressed in skeletal37 and
adipose 22 23 24 25 tissue and induces lipolysis through
inhibition of the insulin receptor.23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 It has been
proposed that TNF- acts as a "sensor" of fat cell lipid content
and protects against lipid overload.20 If TNF- has
similar functions in the vascular wall, it represents 1
possible mechanism by which accumulation of lipids may initiate early
inflammatory lesions.
The present study has demonstrated increased expression of TNF-
in cultured, aortic SMCs grown in the presence of LDL. Accumulation of
LDL in the vascular wall in vivo also resulted in the activation of
TNF- expression. The results show that whereas no TNF- is
expressed in control rat arteries, abundant TNF- immunoreactivity is
induced after injection of human LDL. In the proximal part of the
aorta, this immunoreactivity was predominantly located in the
subendothelial layer, whereas in the distal aorta,
iliac, and femoral arteries and in small, muscular arteries, TNF-
immunoreactivity was encountered throughout the medial layer.
Generally, TNF- staining was stronger in smaller than larger
arteries. Although the endotoxin level of LDL preparations used in the
present study was <2 ng/mg LDL protein, the possibility that the
effect of LDL on vascular TNF- expression was due to LDL endotoxin
contamination should be considered. However, preincubation of LDL with
100 µg/mL LPS diminished LDL-induced TNF- expression and was even
less potent than LPS alone (data not shown).
An increased expression of TNF- in response to lipid accumulation
could play an important role during the early phases of
atherosclerosis. TNF- is a potent
activator of endothelial adhesion molecule
expression.39 TNF- is also known to activate
SMC migration17 and proliferation.40 It is
expressed in migrating SMCs after balloon injury of rat and rabbit
arteries,17 by proliferating SMCs in the coronary
arteries of rabbit rejected cardiac allografts,18 and in
SMCs in human atherosclerotic plaques.14 Accordingly,
induction of SMC production of TNF- by LDL may also be
involved in the activation of SMCs during the formation of
fibromuscular lesions.
Several studies have suggested that oxidation of LDL is involved in the
formation of early atherosclerotic lesions. Oxidized LDL stimulates
endothelial adhesion molecule expression, acts as a
chemoattractant for monocytes, and stimulates the synthesis of other
monocyte chemoattractants, such as monocyte chemoattractant
protein-1. Treatment with antioxidants has also been shown to reduce
plaque formation in hypercholesterolemic animals.
Previous experiments in the same animal model used in this study have
shown that the LDL that has accumulated in the arterial
wall undergoes oxidative modification within 6 to 12 hours and that
this change is accompanied by increased expression of adhesion
molecules. Treatment with the antioxidant probucol partially inhibited
the formation of oxidation products and expression of adhesion
molecules but did not affect the extent of apo B
trapping.26 Oxidized LDL has also been shown to
activate expression of TNF- in adherent
monocytes.13
The present study suggests a complex role for the induction of
TNF- by oxidized LDL. Oxidized LDL injected into rats accumulates in
the liver but not in the arteries. This situation is associated with
marked expression of TNF- in the liver. Injection of native LDL
results in the activation of vascular TNF- expression at the same
time points at which oxidation of LDL occurs in the vessel wall.
Pretreatment of LDL with probucol markedly reduced vascular TNF-
expression in response to LDL injection. In contrast to these findings,
oxidized LDL failed to induce TNF- expression in cultured SMCs. The
observation that probucol failed to inhibit TNF- synthesis induced
by interleukin-1 in cultured SMCs suggests that the in vivo effect of
probucol was not explained by a direct inhibition of TNF- secretion
unrelated to LDL oxidation. In accordance, Ku et al41
found that probucol lacked effect in the LPS-induced TNF- expression
in macrophages.
There is an apparent paradox in these findings. On the 1 hand, the
antioxidant probucol does not inhibit LDL-induced activation of
TNF-, and oxidized LDL fails to stimulate TNF- expression in
vitro, which together strongly suggest that oxidation is not a key
mechanism in the LDL-induced activation of TNF-. On the other hand,
probucol inhibits LDL-induced activation in vivo, and this effect does
not appear to be explained by a direct effect of probucol itself on
TNF- expression. One possible explanation to these discrepant
findings is that oxidation is involved in the LDL-induced expression of
TNF- in the vascular wall but that this oxidation is different from
the modifications of LDL obtained by exposure to copper in vitro.
Alternatively, other mechanisms may be involved in the initial
modifications of LDL trapped in the vascular wall. Electron microscopic
studies have shown aggregation and fusion of LDL particles in the
vascular extracellular matrix.42 This process may involve
phospholipases, leading to the release of phospholipids and fatty
acids.43 These substances represent potential
activators of TNF- in vascular cells, and the oxidative
modifications of LDL may be the consequence rather than the cause of
the subsequent inflammatory reaction. In support of the latter
possibility, activation of the proinflammatory transcription factor
nuclear factor-
B in vascular cells by VLDL has been shown to involve
the release of fatty acids and to be independent of
oxidation.27 Another possibility that should be taken into
account is the involvement of immune reactions. Previous studies have
shown that injection of human LDL into rats initiates the
production of antibodies, some of which form immune complexes
with LDL.44 However, because vascular expression of
TNF- was already evident a few hours after LDL administration in the
present study, this possibility is less likely. The mechanisms
responsible for the increased expression of TNF- in the intestinal
epithelium, adipose tissue, and the liver in response to LDL
injection remain to be clarified. However, it is interesting to
note that all of these tissues have important functions in the
regulation of lipid metabolism.
In summary, the present findings demonstrate that accumulation of
LDL in the vascular wall is associated with an activation of vascular
TNF- expression, which could be involved in the formation of early
inflammatory lesions. This induction may be due to LDL oxidation or an
enzymatic release of phospholipids and fatty acids from aggregated LDL
particles.
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Acknowledgments |
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This study was supported by grants from the Swedish Heart-Lung Foundation, the Swedish Medical Research Council (8311), the Axel and Margareta Ax:son Johnson Foundation, King Gustaf V 80th Birthday Fund, and the Ernhold Lundström Foundation.
Received June 15, 1999; accepted March 17, 2000.
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