Expression of interleukin-1 beta and interleukin-1 receptor antagonist in oxLDL-treated human aortic smooth muscle cells and in the neointima of cholesterol-fed endothelia-denuded rabbits.
ABSTRACT The migration of vascular smooth muscle cells (VSMCs) from the media to the intima and the proliferation of intimal VSMCs are key events in restenotic lesion development. These events, which are preceded and accompanied by inflammation, are modulated by the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), which induces vascular smooth muscle cells to express adhesion molecules and to proliferate. IL-1 beta action is complex and regulated, in part, by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). Whether there was a temporal and spatial correlation between IL-1 beta and IL-1ra expression in, and release by, oxidized low density lipoproteins (oxLDL)-stimulated human aortic smooth muscle cells (HASMCs) was determined by using ELISA and Western blot. In addition, IL-1 beta and IL-1ra expression was detected in the neointima of endothelia-denuded cholesterol-fed New Zealand white rabbits by immunohistochemistry and Western blot. In HASMCs, oxLDL induced IL-beta and IL-1ra expression and release in a dose- and time-dependent manner. Treatment with 20 microg/ml oxLDL resulted in increased IL-1 beta release after 6 h, which peaked at 24 h, and in increased IL-1ra release, first seen after 12 h, but continuing to increase for at least 48 h. In the cells, IL-beta expression showed a similar pattern to release, whereas IL-1ra expression was seen in unstimulated cells and was not increased by oxLDL treatment. Confocal microscopy showed colocalization of IL-beta and IL-1ra expression in oxLDL-stimulated HASMCs. oxLDL caused significant induction of nuclear factor kappa B and activator protein-1 DNA binding activity in HASMCs (6.6- and 3.3-fold, respectively). In cholesterol-fed endothelia-denuded rabbits, the notably thickened intima showed significant IL-1 beta and IL-1ra expression. These results provide further support for the role of IL-1 system in the pathogenesis of restenosis. This is the first demonstration of IL-1 beta and IL-1ra expression and secretion of oxLDL-treated HASMCs and their expression in the rabbit neointima, suggesting that the smooth muscle cells of the intima are an important source of these factors.
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ABSTRACT: As angiogenic growth factors can stimulate the development of collateral arteries, a concept called therapeutic angiogenesis, we performed a phase I/IIa open-label clinical trial using intramuscular injection of naked plasmid DNA encoding hepatocyte growth factor (HGF). We reported long-term evaluation of 2 years after HGF gene therapy in 22 patients with severe peripheral arterial disease. Twenty-two patients with peripheral arterial disease or Buerger disease staged by Fontaine IIb (n=7), III (n=4), and IV (n=11) were treated with HGF plasmid, either 2 mg or 4 mg ×2. Increase in ankle-branchial pressure index >0.1 was observed in 11 of 14 patients (79 %) at 2 years after gene therapy and in 11 of the 17 patients (65%) at 2 months. Reduction in rest pain (>2 cm in visual analog scale) was observed in 9 of 9 patients (100%) at 2 years and in 8 of 13 (62%) patients at 2 months. At 2 years, 9 of 10 (90%) ischemic ulcers reduced by >25%, accompanied by a reduction in the size of ulcer. Severe complications and adverse effects caused by gene transfer were not detected in any patient throughout the period up to 2 years. Overall, the present study demonstrated long-term efficacy of HGF gene therapy up to 2 years. These findings may be cautiously interpreted to indicate that intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs as sole therapy.Arteriosclerosis Thrombosis and Vascular Biology 08/2012; 32(10):2503-9. · 6.34 Impact Factor
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ABSTRACT: Recent clinical evidence has failed to demonstrate the benefits of elevation of serum high-density lipoprotein (HDL), suggesting potential loss of protective effects of HDL at high concentrations. This study aimed to investigate the concentration-related effects of HDL on in vitro and in vivo functions of human endothelial progenitor cells (EPCs) and related angiogenesis. Early and late outgrowth EPCs were generated from human circulating mononuclear cells. Oxidized low-density lipoprotein reduced viability of late outgrowth EPCs, which was reversed dose dependently by HDL. In the absence of oxidized low-density lipoprotein, HDL at low concentrations (5-50 μg/mL, equal to 0.5-5 mg/dL in human) enhanced EPC tube formation by activating phosphatidylinositol-3 kinase/Akt/endothelial NO synthase pathways. Moderate to high concentrations (400-800 μg/mL) of HDL paradoxically enhanced EPC senescence and impaired tube formation by activating Rho-associated kinase (ROCK) and inhibiting phosphatidylinositol-3 kinase/Akt and p38 mitogen-activated protein kinase pathways. Rho-associated kinase inhibitors, either Y27632 or statins, prevented high HDL-induced EPC senescence and improved in vitro tube formation, as well as in vivo capacity of angiogenesis of EPCs. While protecting EPCs from the injury of oxidized low-density lipoprotein, moderate to high concentrations of HDL paradoxically impaired EPCs and related angiogenesis in the absence of oxidized low-density lipoprotein by activating Rho-associated kinase pathways, providing mechanistic evidence of potential hazard effects of HDL.Arteriosclerosis Thrombosis and Vascular Biology 08/2012; 32(10):2405-17. · 6.34 Impact Factor
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ABSTRACT: Proliferation of vascular smooth muscle cells (VSMCs) triggered by inflammatory stimuli and oxidative stress contributes importantly to atherogenesis. The association of green tea consumption with cardiovascular protection has been well documented in epidemiological observations, however, the underlying mechanisms remain unclear. This study aimed to elucidate the effects of the most active green tea catechin derivative, (-)-epigallocatechin-3-gallate (EGCG), in human aortic smooth muscle cells (HASMCs), focusing particularly on the role of a potent anti-inflammatory and antioxidative enzyme heme oxygenase-1 (HO-1). We found that pretreatment of EGCG dose- and time-dependently induced HO-1 protein levels in HASMCs. EGCG inhibited interleukin- (IL-)1β-induced HASMC proliferation and oxidative stress in a dose-dependent manner. The HO-1 inducer CoPPIX decreased IL-1β-induced cell proliferation, whereas the HO-1 enzyme inhibitor ZnPPIX significantly reversed EGCG-caused growth inhibition in IL-1β-treated HASMCs. At the molecular level, EGCG treatment significantly activated nuclear factor erythroid-2-related factor (Nrf2) transcription activities. These results suggest that EGCG might serve as a complementary and alternative medicine in the treatment of these pathologies by inducing HO-1 expression and subsequently decreasing VSMC proliferation.Mediators of Inflammation 01/2014; 2014:523684. · 2.42 Impact Factor
Journal of Cellular Biochemistry 88:836–847 (2003)
Expression of Interleukin-1b and Interleukin-1 Receptor
Antagonist in oxLDL-Treated Human Aortic Smooth
Muscle Cells and in the Neointima of Cholesterol-Fed
Shing-Jong Lin,1,2,4Hui-Tzu Yen,3Yung-Hsiang Chen,1Hung-Hai Ku,3Feng-Yen Lin,3
and Yuh-Lien Chen3*
1Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
2Cardiovascular Research Center, National Yang-Ming University, Taipei, Taiwan
3Institute of Anatomy and Cell Biology, National Yang-Ming University, Taipei, Taiwan
4The Division of Cardiology, Taipei Veterans General Hospital, Taipei, Taiwan
inflammation, are modulated by the proinflammatory cytokine, interleukin-1b (IL-1b), which induces vascular smooth
muscle cells to express adhesion molecules and to proliferate. IL-1b action is complex and regulated, in part, by its
naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). Whether there was a temporal and spatial correlation
between IL-1b and IL-1ra expression in, and release by, oxidized low density lipoproteins (oxLDL)-stimulated human
aortic smooth muscle cells (HASMCs) was determined by using ELISA and Western blot. In addition, IL-1b and IL-1ra
expression was detected in the neointima of endothelia-denuded cholesterol-fed New Zealand white rabbits by
immunohistochemistry and Western blot. In HASMCs, oxLDL induced IL-b and IL-1ra expression and release in a dose-
24 h, and in increased IL-1ra release, first seen after 12 h, but continuing to increase for at least 48 h. In the cells, IL-b
expression showed a similar pattern to release, whereas IL-1ra expression was seen in unstimulated cells and was not
increased by oxLDL treatment. Confocal microscopy showed colocalization of IL-b and IL-1ra expression in oxLDL-
stimulated HASMCs. oxLDL caused significant induction of nuclear factor kappa B and activator protein-1 DNA binding
pathogenesis of restenosis. This is the first demonstration of IL-1b and IL-1ra expression and secretion of oxLDL-treated
HASMCs and their expression in the rabbit neointima, suggesting that the smooth muscle cells of the intima are an
important source of these factors. J. Cell. Biochem. 88: 836–847, 2003.
? 2003 Wiley-Liss, Inc.
Key words: IL-1b; IL-1ra; inflammation; smooth muscle cells; restenosis
The migration of smooth muscle cells into the
intima, followed by their proliferation and
matrix deposition (neointimal hyperplasia), is
a central theme of atherosclerosis and rest-
enosis. Many lines of research suggest that
these events are preceded and accompanied by
inflammation [Ross, 1999; Simon et al., 2000].
a pivotal role in regulating immunoinflamma-
performed to determine whether IL-1 modifies
the inflammatory response [Dinarello, 1996;
Oemar, 1999; Wang et al., 2000]. IL-1b induces
a substantial increase in the expression of
? 2003 Wiley-Liss, Inc.
Grant sponsor: National Science Council, Taiwan; Grant
numbers: NSC 90-2320-B-010-034, NSC 90-2314-B-010-
020; Grant sponsor: Medical Research and Advancement
Foundation (in Memory of Dr. Chi-Shuen Tsou).
*Correspondence to: Dr. Yuh-Lien Chen, Institute of
Anatomy and Cell Biology, National Yang-Ming Univer-
sity, No. 155, Section 2, Li-Nong Street, Shih-Pai, Taipei
112, Taiwan. E-mail: firstname.lastname@example.org
Received 3 September 2002; Accepted 16 October 2002
adhesion molecules by vascular smooth muscle
cells (VSMCs) in vitro, and these factors pro-
the arterial wall [Wang et al., 1994, 1995; Wu
et al., 1999] and stimulate VSMC proliferation
[Nilsson, 1993; Dinarello, 1996]. Recent in vivo
studies have shown increased levels of IL-1b
mRNA in human atherosclerotic lesions [Wang
et al., 1989] and of IL-1b mRNA and protein in
VSMCs, endothelium, and macrophages in
atherosclerotic arteries from nonhuman pri-
mates [Ross et al., 1990; Moyer et al., 1991] and
in coronary arteries of patients with ischemic
heart disease [Galea et al., 1996]. Treatment of
porcine arteries with IL-1b induces intimal
lesions [Ito et al., 1996]. Although the relation-
ship between IL-1b and cardiovascular disease
has been extensively studied, IL-1b expression
in oxidized low density lipoprotein (oxLDL)-
induced human aortic smooth muscle cells
(HASMCs) and in restenotic lesions has not
been studied in detail.
IL-1b production may be an important initi-
ating factor in the cascade of events resulting
in inflammation and vascular disease. It is
specifically inhibited by natural antagonists,
including the IL-1 receptor antagonist (IL-1ra)
[Granowitz et al., 1991], the secreted protein
product of a gene adjacent to the IL-1B gene
which binds to IL-1 type I and II receptors
without producing a signal [Dewberry et al.,
2000]. Recent studies support the hypothesis
that an imbalance between IL-1b and IL-1ra
production at the tissue level is pathogenically
important in chronic inflammatory bowel dis-
ease [Casini-Raggi et al., 1995], in rheumatoid
arthritis [Chomarat et al., 1995], and in chronic
hepatitis [Gramantieri et al., 1999]. Some pa-
tients with acute or chronic liver disease have
elevated serum levels of both IL-1b and IL-1ra
closely involved in neointimal formation in the
rat carotid artery subjected to balloon angio-
plasty [Wang et al., 2000]. However, the tem-
poral expression and cellular source of IL-1b
and IL-1ra in oxLDL-stimulated HASMCs and
during neointimal hyperplasia have not been
measure IL-1b and IL-1ra expression and re-
lease in oxLDL-stimulated HASMCs and their
expression in the neointima of cholesterol-
fed endothelia-denuded rabbits. Our results
show that vascular IL-1b and IL-1ra expression
is upregulated in both cases, consistent with
their playing a role in modulating neointimal
Preparation and Oxidation of LDL
Human LDL (d¼1.019–1.063 g/ml) was iso-
lated by sequential ultracentrifugation of fast-
ing plasma samples from healthy adult males
[Winocour etal.,1992] andextensivelydialyzed
under nitrogen for 24 h at 48C against phos-
phate-buffered saline (PBS, 5 mM phosphate
buffer and 125 mM NaCl, pH 7.4). The LDL
represents the native LDL (nLDL) used in the
current study. LDL was oxidized by dialysis for
24 h at 378C against 10 mM CuSO4in PBS, as
described by Steinbrecher et al. [Steinbrecher
dialyzed for 24 h at 48C against PBS containing
0.3 mM EDTA. The extent of oxidation was
monitored by measuring thiobarbituric acid-
reactive substance (TBARS) and agarose gel
electrophoresis. The cholesterol content of
nLDL and oxLDL was determined using a cho-
lesterol enzymatic kit (Merck).
Human Aortic Smooth Muscle
Cell (HASMCs) Cultures
HASMCs, purchased as cryopreserved ter-
tiary cultures from Cascade Biologics (OR,
USA), were grown in culture flasks in smooth
muscle cell growth medium (Cascade Biologics,
Inc., OR, USA) supplemented with fetal bovine
serum (FBS, 5%), human epidermal growth
factor (3 ng/ml), insulin (10 mg/ml), penicillin
(100 units/ml), streptomycin (100 pg/ml), and
Fungizone (1.25 mg/ml) at 378C in a humidified
5% CO2atmosphere. The growth medium was
changed every other day until confluence. Cells
of HASMC cultures was verified by immuno-
staining with a monoclonal antibody against
smooth muscle-specific a-actin. Before oxLDL
or nLDL treatment, the cells were first serum-
starved for 24 h.
MTT Assay of Cell Viability
As an index of cell viability, mitochondrial
zolium bromide (MTT) assay described by Chen
et al. . The absorbance after nLDL or
oxLDL treatment, normalized to that for cells
IL-1b and IL-1ra Expression in oxLDL-Treated HASMCs and in the Neointima837
incubated in control medium, was used as a
measurement of cell viability, the control cells
being considered 100% viable.
Foam Cell Formation
Foam cell formation was identified by oil red
O staining of oxLDL-incubated HASMCs.
Briefly, cells (5?104) were suspended in 1.5 ml
of medium containing 5% FBS and placed on a
coverslip in a 35-mm dish for overnight, then
before being treated with various doses of
oxLDL. After incubation for 8 h at 378C, the
cells were fixed for 15 min at room temperature
for 2 min at RT with propylene glycol, and
stained for 15 min at RT with oil red O (5 mg/ml
in propylene glycol), followed by hematoxylin
counterstaining. The coverslips were then
washed in tap water. The number of lipid drop-
lets per HASMC, with or without oxLDL treat-
ment, was measured quantitatively using
the WIPLabTMAnalysis Program (Foreseen
Science & Technology); four randomly-selected
regions on each coverslip were systematically
of lipid droplets in HASMCs in each scanned
Quantification of IL-1b and IL-1ra
in Culture Supernatants
An aliquot (200 ml) from three independent
samples of supernatant was added to IL-1b (IL-
1b ELISA kit, R&D systems, Inc., Minneapolis,
MN) and IL-1ra (IL-1ra ELISA kit, Endogen,
plates, processed according to the manufac-
turer’s protocol, read at 450 nm after 30 min.
The sensitivity of each assay was 1 pg/ml.
Western Blots of HASMCs
The cells were washed with PBS, then lysed
mM EDTA, 0.5 % Triton X-100, 1 mM phe-
nylmethylsulfonyl fluoride (PMSF), and cen-
trifuged at 4,000g for 30 min at 48C. All
of the supernatants (15 mg of protein) were
subjected to 12% SDS–PAGE, then electro-
membranes (NEN), which were then incubated
for 2 h with PBS, 0.1% Tween-20, 5% skim milk
before incubation for 1 h with goat antibodies
against humanIL-1b(1:1,000 dilution, R&D)or
human IL-1ra (1:1,000 dilution, R&D). Bound
horseradish peroxidase-conjugated rabbit anti-
goat secondary antibodies (1:3,000 dilution,
followed by exposure to Biomax MR film
(Kodak, Rochester, NY). The intensity of the
band was quantified using a densitometer. Anti
a-tubulin antibodies (1: 1,000 dilution, Onco-
gen) were used to quantify a-tubulin as an
of IL-1b and IL-1ra
The topographical relationship between IL-
1b and IL-1ra was studied by double immuno-
fluorescence labeling in conjunction with con-
focal microscopy. Cells, cultured on coverslips,
were treated with 20 mg/ml of oxLDL for 12 h,
dehyde in PBS. After washing with PBS, the
cells were treated for 5 min with 80% methanol
at ?208C, then for 1 h at RT with PBS contain-
ing 3% skim milk. The coverslips were then
human IL-b antibody (1:100 dilution, R&D) or
goat anti-human IL-1ra antibody (1:100 dilu-
tion, R&D). After rinsing with PBS, the cells
were incubated for 1 h at 378C with FITC-
conjugated horse anti-mouse IgG antibody
(1:300 dilution, Vector, USA) and TRITC-con-
jugated rabbit anti-goat IgG antibody (1:
300 dilution, Sigma). After washing with PBS,
ting medium (Vector) and viewed with a Leica
confocal laser-scanning microscope. In controls
in which the primary antibodies were omitted,
negligible immunofluorescence was seen.
Nuclear Extract Preparation and
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear protein extracts were prepared as
method. The 22-mer synthetic double-stranded
oligonucleotides used as NF-kB and AP-1
probes in the gel shift assay were (50-AGTTG-
CCTGAAAGGGTCCG-50) and (50-ATTCGATC-
CCGCCCCGCTCG-50), respectively. Doubled-
stranded DNA was end-labeled with g-32P-
838Lin et al.
unincorporated nucleotides being removed by
gel filtration on a Sephadex G-25 column (BM-
Quick Spin columns DNA G25, Boehringer-
Mannheim). The DNA-binding reaction was
performed for 20 min at room temperature in a
volume of 20 ml containing 2 mg of nuclear
extract, ?1 ng of
105cpm/ng), 10 mg of salmon sperm DNA
(Sigma-Aldrich), and 15 ml of binding buffer
(20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M
KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM
bound oligonucleotide was separated from un-
5% polyacrylamide gel (acrylamide/bisacryla-
mide 29:1) in 0.25? TBE (1? TBE: 89 mM Tris
base, 89 mM boric acid, 2 mM EDTA buffer, pH
8.0). The gels were vacuum-dried and subjected
toautoradiographyand thefilmsscanned using
a UMAX scanner.
32P-labeled NF-kB (2–5?
Immunohistochemistry of Aortic Specimens
From Control and Cholesterol-Fed
This investigation conformed to the ‘‘Guide
for the care and use of laboratory animals’’ pub-
lished by the US National Institute of Health.
Ten male New Zealand white rabbits, 3 months
of age and weighing 2.5–3.0 kg, were fed for
6 weeks with a 2% high cholesterol diet (Purina
Mills, Inc., St. Louis, MO) to induce hypercho-
lesterolemia. The animals were bled periodi-
cally to measure plasma cholesterol level and
liver and renal function. At the end of the 3rd
week of the high cholesterol diet, the animals
were fasted for 12 h and anesthetized by
intramuscular injection of xylazine (5 mg/kg)
and ketamine hydrochloride (35 mg/kg), then
surgery was performed as previously described
[Chen et al., 2001]. Ten aged-matched male
rabbits on regular diet chow without balloon
6th week of the experiment, the animals were
anesthetized by intravenous injection of 35–
40 mg/kg sodium pentobarbital and sacrificed.
One segment of the abdominal aorta was rinsed
with ice-cold PBS, immersion-fixed with 4%
tochemistry, while the remaining portion was
immediately frozen in liquid nitrogen for pro-
tein extraction. The tissue sections (5–6 mm
thick) were mounted on poly-L-lysine coated
slides, deparaffinized, rehydrated, and washed
with PBS. To study cellular expression and
localization of IL-1b and IL-1ra, serial sections
were incubated with 1% hydrogen peroxide in
methanol for 10 min to block endogenous
peroxidase activity and to permeabilize the
cells, then nonspecific binding was blocked by
incubation for 1 h at RT with PBS containing 5
mg/ml of bovine serum albumin. In the primary
antibody step at 378C for 1 h, the first serial
1b antibody (1:100, R&D), the second with
mouse anti-a-smooth muscle actin (1:400, 1A4,
Sigma), and the third with goat anti-human IL-
1ra antibody (1:100, R&D). The second section
was then incubated for 1.5 h at RT with FITC-
(1:400, Sigma Chemical Co., St. Louis, MO) and
observed by fluorescent microscopy, while
bound antibodies on the first and third slides
were localized by an indirect immunoperoxi-
dase technique (avidin-biotin-horseradish per-
oxidase complex) employing diaminobenzidine
(Vector) as chromogen. Each incubation was
followed by three times 5 min washes in PBS.
Negative controls were performed by omitting
the primary antibody.
Western Blots of Aortal Specimens
The frozen abdominal aortas of control and
were pulverized in liquid nitrogen, then lysed
for 1 h at 48C in 0.5 M NaCl, 50 mM Tris, 1 mM
EDTA, 1% SDS, 10 mg/ml leupeptin, 1 mM
PMSF, and centrifuged at 13,500g for 10 min
at 48C. All subsequent stages were at RT.
Samples of the supernatants (15 mg protein)
were applied to 8% SDS–PAGE and electro-
transferred to PVDF membranes (NEN), which
were then treated for 1 h with PBS containing
0.05% Tween-20 and 2% skim milk, and incu-
bated for 1 h with goat anti-human-IL-1b
(1:1,000; R&D). After washing, the membranes
were incubated for 1 h with horseradish perox-
idase-conjugated donkey anti-goat monoclonal
antibodies (1:3,000; Bethyl), then bound anti-
body was detected using Chemiluminescence
Reagent Plus (NEN) and exposure to Biomax
actin (1:1000;Sig) were used as an internal
IL-1b and IL-1ra Expression in oxLDL-Treated HASMCs and in the Neointima839
Values are expressed as the mean?SEM.
way ANOVA followed by the Dunnett test, with
a P value<0.05 being considered significant.
Effect of nLDL and oxLDL
on HASMC Viability
Figure 1 shows the effect of different periods
of incubation with nLDL or oxLDL on HASMC
viability. Treatment with 20 mg/ml oxLDL did
not result in cell cytotoxicity (115.1?7.6% at
3h, 106.6?5.1% at 6h,98.0?2.2% at 12h, and
97.0?3.6% at 24 h), whereas exposure to 40 or
60 mg/ml oxLDL significantly reduced cell
viability with time (108.0?9.3% and 100.3?
9.3% at 3h, 82.2?7.2% and 39.8?8.3%*at 6h,
17.4?4.3%* and 2.8?1.1%* at 12 h, 23.4?
7.4%* and 1.1?0.4%* at 24 h, respectively,
*P<0.05 vs. the control at the indicated time).
In contrast, treatment with 60 mg/ml nLDL
resulted in a slight increase in viability with
time (99.1?6.0% at 3 h, 102.5?2.2% at 6 h,
112.2 ?5.3% at 12 h, and 123.8?4.0%* at 24 h,
*P<0.05 vs. the control at the indicated time).
Foam Cell Formation by HASMCs
Induced by oxLDL
Treatment of HASMCs for 8 h with different
concentration of oxLDLs induced the formation
loading between untreated HASMCs (Fig. 2A)
and those treated with 20 mg/ml of oxLDL
(Fig. 2B). When measured quantitatively, the
number of lipid droplets per HASMC treated
with oxLDL (7.71?0.44) was significantly
higher than in control cells (0.23?0.11).
IL-1b and IL-1ra Release
by oxLDL-Stimulated HASMCs
HASMCs were incubated for 3–48 h with
various concentrations of oxLDL, then IL-1b
and IL-1ra levels in the culture medium were
measured by ELISA.
Figure 3A shows the time-dependent and
dose-dependent effects of oxLDL on IL-1b re-
on IL-1b release was seen after 6 h, which
increased up to 24 h (269.5?56.1 pg/ml com-
decreased at 48 h (220.1?28.2 pg/ml; data not
shown). Treatment with 40 mg/ml of oxLDL
resulted in a significant increase in IL-1b re-
lease after 6 h, which plateaued at 12–24 h
(454.3?11.1 pg/ml vs. 14.8?8.1 pg/ml), while
treatment with 60 mg/ml of oxLDL resulted in a
significant increase after 3 h, which peaked at
12 h (480.3?17.6 pg/ml vs. 17.4?5.2 pg/ml),
then declined. Native LDL had no effect on IL-
1b release at any time-point.
in the absence or presence of either 60 mg/ml of nLDL or the
indicated concentrations of oxLDL. The values are the mean?
SEM for three experiments, each in triplicate.
foam cells (A), whereas those incubated with 20 mg/ml of oxLDL
in the online issue, which is available at www.interscience.
Identification of foam cell formation by HASMCs using
840 Lin et al.
IL-1ra release. Using 20 mg/ml of oxLDL, the
amount of secreted IL-1ra first showed a signi-
ficantly increase after 12 h, and continued to
increase at 24 h (1,226.3?288.2 pg/ml vs.
380.6?72.2 pg/ml in the untreated control)
and 48 h (1,381.9?246.2 pg/ml; data not
then a further increase, plateauing at 12–24 h
respectively, for 40 and 60 mg/ml of oxLDL vs.
340.6?72.2 pg/ml for the control). nLDL treat-
ment had no effect on IL-1ra release.
Using 20 mg/ml of oxLDL, the fold increase in
IL-b and IL-1ra release seen with treated cells
compared to untreated cells was, respectively,
1.5?0.3 and 1.0?0.1 at 3 h, 3.6?0.5* and
1.1?0.3 at 6 h, 8.9?1.1* and 2.0?0.4* at 12 h,
18.2?3.1* and 3.0?0.7* at 24 h, and 14.6?
3.9* and 3.6?0.9* at 48 h (*P<0.05 vs. the
control at the indicated time, n¼3).
Western Blot Analysis of IL-1b and IL-1ra
Expression in oxLDL-Stimulated HASMCs
HASMCs at 6 h and expression peaked at 24 h,
at which time it was approximately 17-fold
higher than in unstimulated cells (Fig. 4A). In
IL-1ra expression which was not increased by
oxLDL treatment (Fig. 4B).
Indirect Immunofluorescence of IL-1b
and IL-1ra Expression in
The effects of oxLDL were studied by immu-
nofluorescence confocal microscopy. In untreat-
ed cells, IL-1b expression was weak (Fig. 5A),
whereas IL-1ra was present in the cytoplasm
(Fig. 5D) and IL-1ra was present diffusely
throughout the cytoplasm (Fig. 5E); these two
induced by oxLDL or nLDL. (A) IL-1b; (B) IL-1ra. HASMCs were
at the indicated concentration. The data are representative
of triplicate determinations. *P<0.05 vs. controls at the
Time-course of IL-1b and IL-1ra release from HASMCs
1ra expression in oxLDL-treated HASMCs. (A) IL-1b; (B) IL-1ra.
HASMCs were treated with 20 mg/ml oxLDL. The figure is a
representative example of three separate experiments. a-tubulin
was used as an internal control.
Western blot analysis of the time-course of IL-1b and IL-
IL-1b and IL-1ra Expression in oxLDL-Treated HASMCs and in the Neointima841
images are superimposed in Figure 5F. IL-1b
in most cells after oxLDL stimulation.
Effects of oxLDL on NF-kB and AP-1
Activity in HASMCs
Since transcriptional regulation involving
NF-kB or AP-1 activation has been implicated
in the oxLDL-induced expression of inflamma-
tory cytokines, gel-shift assays were performed
to determine whether oxLDL induced NF-kB or
AP-1 activation. As shown in Figure 6A, low
levels of basal NF-kB binding activity were
cells, and treatment with oxLDL (20 mg/ml for
30 min) resulted in a 6.6-fold increase in NF-kB
binding activity; this was specific for NF-kB, as
it was undetectable inthepresenceofa100-fold
not shown). Serum-starved HASMCs also con-
tained small or undetectable amounts of active
AP-1 (Fig. 6B), which was increased 3.3-fold by
Expression of IL-1b and IL-1ra in Smooth
Muscle Cells From Cholesterol-Fed
Immunohistochemistry was used to examine
the cellular expression and localization of IL-1b
and IL-1ra during neointimal hyperplasia.
In the control group, no IL-1b was detected
(Fig. 7A), and smooth muscle cells were only
detected in the tunica media (Fig. 7B), which
showed faint staining for IL-1ra (Fig. 7C). The
treated group showed a markedly thicken-
ed intima which stained strongly for IL-1b
(Fig. 7D), whereas no IL-1b was detected in
the underlying media of the lesioned area. The
cells in the thickened intima were mainly
thickened intima and a few smooth muscle
cells in the media stained strongly for IL-1ra
Western Blotting of Abdominal Aorta Tissue
As shown in Figure 8 and in accordance with
the immunohistochemical results, IL-1b was
microscopy of IL-1b and IL-1ra in HASMCs with or without
weak (A, green fluorescence), whereas IL-1ra was found in the
cytoplasm (B, red fluorescence); the superimposed images only
Immunocytochemical detection by laser confocal
show IL-1ra expression (C). oxLDL-treated HASMCs showed a
dramatic increase in IL-1b expression (D), whereas IL-1ra
expression was unchanged (E). The superimposed images show
that IL-1b and IL-1ra were colocalized in most cells (F). [Color
figure can be viewed in the online issue, which is available at
842Lin et al.
scarcely detectable in the control group, but IL-
1rawas present, whereas,in the cholesterol-fed
endothelia-denuded group, expression of both
IL-1b and IL-1ra was relatively high.
This study demonstrates that IL-1b and IL-
muscle cells in the neointima of cholesterol-fed
endothelia-denuded rabbits. A concomitant in-
crease in IL-1b and IL-1ra release was also
detected in culture media from HASMCs after
oxLDL stimulation in vitro. These results sug-
gest a role for IL-1b and IL-1ra in the devel-
opment of restenotic lesions.
Immunohistochemical results from serial
sion occurred together in both smooth muscle
cells and in the neointima. IL-1b has previously
been detected in diet-induced atherosclerotic
lesions of the iliac artery in monkeys [Moyer
et al., 1991] and in coronary arteries of humans
with ischemic heart disease [Galea et al., 1996]
or with restenosis [Clausell et al., 1995].
Transgenic mice lacking functional IL-1 do not
show the neointimal hyperplasia which is
induced in wild-type mice by low shear stress
[Rectenwald et al., 2000]. In the present study,
the fact that IL-1b synthesis was restricted to
sites of lesion development suggests that IL-1b
plays an important role in the pathogenesis
IL-1b has previously been found in luminal
and adventitial vessel endothelial cells and in
with ischemic heart disease [Galea et al., 1996],
in foam cells, smooth muscle cells, and the
endothelium of diet-induced iliac artery athero-
sclerotic plaques in monkeys [Moyer et al.,
1991], in the endothelium and neointima in a
rat vein graft model [Faries et al., 1996], and in
neointimal cells in balloon injured porcine
coronary arteries [Chamberlain et al., 1999],
phages cultured from human carotid arteries
30 min with or without 20mg/ml of oxLDL, then NF-kB and AP-1 binding activity in nuclear extracts was
analyzed by EMSA using a NF-kB or AP-1 consensus oligonucleotide as probe. The arrows indicate the
positions of the NF-kB- or AP-1-specific complexes.
Autoradiographs showing activation of NF-kB and AP-1 by oxLDL. HASMCs were incubated for
IL-1b and IL-1ra Expression in oxLDL-Treated HASMCs and in the Neointima843
provided direct evidence that IL-1b expression
was localized in smooth muscle cells of the
neointima, a result consistent with a role in
promoting smooth muscle cell migration into
the intima and their subsequent proliferation
and activation in the thickened intima.
Several studies have demonstrated that IL-
et al., 1991], epithelial cells [Haskill et al.,
1991], endothelial cells [Dewberry et al., 2000],
we found, for the first time, that oxLDL is an
effective stimulus for IL-1ra secretion from
HASMCs. This is an interesting result consid-
ering that IL-1b and IL-1ra are coexpressed in
both oxLDL-stimulated HASMCs and in the
neointima. In addition, we demonstrated that,
following oxLDL treatment, the increase in IL-
IL-1b secretion. Expression of IL-1b and IL-1ra
in both HASMCs and cholesterol-fed endothe-
lia-denuded rabbits appeared to differ, with IL-
1ra being detectable before oxLDL treatment or
balloon injury, whereas basal IL-1b expression
was low, and only markedly increased after
muscle cells, and IL-1ra in serial sections of abdominal aortas
from control and cholesterol-fed endothelia-denuded rabbits.
staining. The internal elastic membrane is indicated by double
arrows. The arrowheads and single arrows indicate smooth
muscle cells overlapping with IL-1b or IL-1ra expression,
respectively. In the control group, IL-1b was not detected (A),
Immunohistochemical staining for IL-1b, smooth
whereas low amounts of IL-1ra were present in the media (C);
smooth muscle cells were only detected in the tunica media (B).
In cholesterol-fed endothelia-denuded rabbits, strong IL-1b
expression was only detected in the thickened intima (D),
whereas strong IL-1ra expression was seen in the thickened
intima and in a few smooth muscle cells in the media (F); the
thickened intima and media were mainly composed of smooth
muscle cells (E). [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
844 Lin et al.
in untreated cells is probably due to a cellular
storage phenomenon, as recently shown for IL-
1ra in human umbilical vein endothelial cells
[Dewberry et al., 2000]. Since up to a 100-fold
molar excess of IL-1ra may be required to
counteract the action of IL-1 in a disease situa-
tion [Dewberry et al., 2000], we suggest that de
novo synthesis would not be very efficient in
meeting this requirement, and that storage of
IL-1ra within the cell would be preferable so as
to avoid a long delay in the action of IL-1ra.
IL-1ra is a naturally occurring inhibitor that
counterbalances the biological activity of IL-1b,
acting by binding competitively to IL-1 recep-
tors without inducing signal transduction [Gra-
mantieri et al., 1999]. Increased serum levels of
IL-1ra have been detected in the acute phase of
meningococcal disease and in rheumatoid arth-
ritis, and are supposed to reflect an antiinflam-
matory reaction [van Deuren et al., 1994;
Barrera et al., 1995]. Induction of IL-1ra,
coupled with IL-1b induction, has been demon-
strated in healthy subjects in whom endotox-
emia was produced by injection of Escherichia
coli endotoxin [Granowitz et al., 1991], and IL-
ra induction is seen in cancer patients receiving
IL-1b as a therapeutic agent [Kopp et al., 1996].
are seen in lipopolysaccharide (LPS)-treated
whole blood from both patients with diabetes
mellitus and cigarette smokers, both of which
are risk factors for cardiovascular disease [Mol
et al., 1997]. Allele 2 of the IL-1ra gene is
associated with a lower incidence of restenosis
after coronary stenting [Kastrati et al., 2000].
The biological relevance of IL-1ra to the normal
physiology of IL-1 or to the possible role of IL-1
in pathophysiology remains to be established.
However, it is worth noting that macrophages,
which are involved in the atherogenic process
[Ross, 1993], simultaneously produce IL-1 and
IL-1ra [Mikuniya et al., 2000]. IL-1ra can con-
1993], again demonstrating its anti-inflamma-
tory properties. In this context, the increased
IL-1ra levels seen in cholesterol-fed endothelia-
denuded rabbits were closely associated with
inflammation. The increased IL-ra expression
may be due to increased oxidative stress and
hyperlipidemia, but may also reflect inflam-
mation related to cardiovascular disease. The
overall balance between IL-1b and IL-1ra may
determine the severity of restenotic lesions in
response to the level of smooth muscle cell pro-
liferation. Thus, the IL-1ra level results in our
study could indicate increased inflammation.
Much attention has been paid to the role of
LDL oxidation in the development of cardiovas-
cular disease and, in particular, to the possibi-
lity that LDL oxidation in the arterial wall
activates the inflammatory process that char-
acterizes lesion progression. Activation of the
redox-regulated transcriptional factors, NF-kB
and AP-1, has been suggested to play a key role
in this process, as they bind to the promoters of
many genes involved in inflammation. In the
of HASMCs for 30 min with 20 mg/ml of oxLDL
activated NF-kB and AP-1, suggesting that IL-
1b and IL-1ra upregulation in response to
oxLDL is mediated by these transcriptional
factors. However, NF-kB is a ubiquitously ex-
pressed multiunit transcription factor which is
activated by diverse signals, possibly via phos-
phorylation of the IkB subunit and its dissocia-
tion from the inactive cytoplasmic complex,
followed by translocation of the active dimer,
p50 and p65, to the nucleus [Ghosh and
Baltimore, 1990]. In a recent study, treatment
of human arterial smooth muscle cells for 4 h
with 50 mg/ml of oxLDL was shown to activate
AP-1, but not NF-kB [Ares et al., 1995]. In
contrast, treatment of HUVECs for 48 h with
220 mg/dl of oxLDL activates AP-1 [Lin et al.,
abdominal aorta of normal and cholesterol-fed endothelia-
denuded rabbits. N, normal rabbit; CE, cholesterol-fed endothe-
lia-denuded rabbit. The result is representative of three separate
experiments. In the normal rabbits, although IL-1b was scarcely
detectable, IL-1ra was present, whereas, in the CE rabbits, the
expression of both IL-1b and IL-1ra was relatively high. b-actin
was used as an internal control.
Western blot analysis of IL-1b and IL-1ra in the
IL-1b and IL-1ra Expression in oxLDL-Treated HASMCs and in the Neointima845
NF-kB [Cominacini et al., 2000]. Depending on
the cell type, extent of oxLDL oxidation, and
tory effects of oxLDL on NF-kB and AP-1 acti-
vation. In the present study, we did not prove
conclusively that the oxLDL-induced NF-kB
and AP-1 activation in HASMCs is directly
linked to the role of HASMCs in neointimal
lator in neointimal hyperplasia is fairly com-
plex,there is increasingevidence thatIL-1bisa
potent stimulator of the synthesis of adhesion
molecules, including VCAM-1 and ICAM-1, in
endothelial cells [Wu et al., 1999] and vascular
smooth muscle cells [Wang et al., 1994; Rolfe
et al., 2000]. Other in vitro studies support the
idea that IL-1b may contribute to the migration
of leukocytes into the inflammatory tissue and
to the cellular interaction with other inflamma-
tory cells by upregulating adhesion molecules
[Wang et al., 1995]. It is conceivable that
increased IL-1b expression in the thickened
intima could promote the migration and pro-
liferation of smooth musclecells into the lesions
and facilitate the chronic inflammatory re-
sponse during intimal hyperplasia.
In conclusion, this study has demonstrated
that IL-1b and IL-1ra expression was increased
in smooth muscle cells in the neointima of
cholesterol-fed endothelia-denuded rabbits. A
concomitant increased release of IL-1b and IL-
1ra was also detected in oxLDL-stimulated
HASMCs. These results suggest a role for IL-
of circulating monocytes, the activation of in-
timal macrophages and smooth muscle cells,
and the stimulation of the expression of other
cytokines during the development of restenotic
We thank Mr. Tang-Hsu Chao and Ms. Shu-
Feng Tsai for technical assistance in manu-
Medical Research and Advancement Founda-
tion in Memory of Dr. Chi-Shuen Tsou.
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