Synergistic Effect of Urotensin II With Mildly Oxidized
LDL on DNA Synthesis in Vascular Smooth Muscle Cells
Takuya Watanabe, MD; Rajbabu Pakala, PhD; Takashi Katagiri, MD; Claude R. Benedict, MD, DPhil
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
Background—The urotensin II (UII) found in coronary atheroma is the most potent vasoconstrictor known to date. Mildly
oxidized LDL (moxLDL) contributes to atherogenesis and plaque formation. We assessed the effect of UII and its
interaction with moxLDL and the oxidative components of moxLDL on vascular smooth muscle cell (VSMC)
proliferation.
Methods and Results—Growth-arrested VSMCs were incubated in serum-free medium with different concentrations of
LDL, moxLDL, oxLDL, hydrogen peroxide, lysophosphatidylcholine, or 4-hydroxy-2-nonenal, with or without UII.
[3H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. UII stimulated [3H]thymidine
incorporation in a dose-dependent manner, with a maximal effect at a concentration of 50 nmol/L (161%). Low
concentrations of UII potentiated the mitogenic effect of LDL (108% to 242%), oxLDL (129% to 302%), moxLDL
(120% to 337%), hydrogen peroxide (177% to 226%), lysophosphatidylcholine (115% to 332%), and 4-hydroxy-2nonenal (142% to 299%). The synergistic interaction between UII and moxLDL was partially inhibited by anti-Gq/11␣
antibody, the epidermal growth factor receptor tyrosine kinase inhibitor erbstatin A (10 ␮mol/L), and the intracellular
free radical scavenger N-acetylcysteine (400 ␮mol/L) and was completely inhibited by the c-Src tyrosine kinase
inhibitor radicicol (10 ␮mol/L), the protein kinase C (PKC) inhibitor Ro31-8220 (0.1 ␮mol/L), and the mitogenactivated protein kinase (MAPK) kinase inhibitor PD098059 (10 ␮mol/L).
Conclusions—Our results suggest that UII acts synergistically with moxLDL in inducing VSMC proliferation via the
c-Src/PKC/MAPK pathway, which may explain the relatively rapid progression of atherosclerosis in patients with
hypertension and hypercholesterolemia. (Circulation. 2001;104:16-18.)
Key Words: atherosclerosis 䡲 hypertension 䡲 lipoproteins 䡲 muscle, smooth 䡲 urotensins
U
rotensin II (UII) is a cyclic 12-amino acid peptide
detected in fish spinal cords and plasma (⬇30 pmol/L)
that was recently cloned from the spinal cord of humans.1
Human UII is found in vascular and cardiac tissue (including
coronary atheroma) and shows a contractile effect on many
arteries from nonhuman primates, including coronary, pulmonary and carotid arteries, suggesting a hypertensive response.2,3 The potency of the vasoconstriction of UII is an
order of magnitude greater than that of endothelin-1, making
UII the most potent mammalian vasoconstrictor identified
thus far.2 Recent studies have shown that the vasoconstrictive
effect of UII is mediated via GPR14, an orphan G proteincoupled receptor that may couple to the Gq/11␣ pathway.4
However, the downstream signaling pathway remains
unclear.
Oxidized LDL (oxLDL) is a well-established risk factor
for atherosclerosis that stimulates vascular smooth muscle
cell (VSMC) differentiation and proliferation. In particular, mildly oxidized LDL (moxLDL) interacting with other
vasoactive agents and growth factors present in the vasculature may play an important role in the development of
atherosclerosis and hypertension.5 Recently, we showed
that the mitogenic effect of moxLDL is mediated by its
oxidative components, such as reactive oxygen species
(ROS), lysophosphatidylcholine (LPC), and 4-hydroxy-2nonenal (HNE).5,6
This study sought to examine the proliferative effect of UII
on VSMCs and its interaction with moxLDL and to demonstrate the mechanism responsible for synergistic interaction
between these 2 agents in inducing VSMC proliferation.
Methods
Materials
Human UII, LDL, LPC, HNE, hydrogen peroxide (H2O2), anti-Gq/
11␣ antibody, erbstatin A, radicicol, Ro31-8220, PD098059, and
N-acetylcysteine (NAC) were purchased from Sigma. [3H]Thymidine (specific activity, 20 Ci/mol) was obtained from DuPont-NEN.
Received March 26, 2001; revision received May 8, 2001; accepted May 10, 2001.
From the Department of Internal Medicine, Division of Cardiology, University of Texas–Houston Health Science Center (T.W, R.P., C.R.B.), and the
Third Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan (T.K.).
Guest Editor for this article was Joseph Loscalzo, MD, PhD, Boston University School of Medicine, Boston, Mass.
Correspondence to Dr Claude R. Benedict, Department of Internal Medicine, Division of Cardiology, The University of Texas–Houston Health Science
Center, 6431 Fannin, MSB 6.039, Houston, TX 77030.
© 2001 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
16
Watanabe et al
Urotensin II and MoxLDL Induce VSMC Proliferation
17
LDL Oxidation
MoxLDL and oxLDL were prepared as described previously.5
Lipoprotein concentrations are expressed as protein concentrations.
Even at a concentration of 10 mg/mL, native LDL showed no
development of thiobarbituric acid–reactive substances (TBARs),
whereas moxLDL showed a slight increase in TBARs formation (2
to 4 nmol/mg protein), with no change in the electrophoretic
mobility. In contrast, oxLDL showed a significant increase in
TBARs formation (35 nmol/mg protein) and an increase in the
electrophoretic mobility.
Cell Culture
VSMCs were isolated from the thoracic aortas of male New Zealand
White rabbits (body weight, ⬇3 kg, n⫽35) by the explant method
and were cultured in a humidified atmosphere (5% CO2/95% air) at
37°C.5,6 After ⬇3 weeks, the tissue blocks were removed, and the
migrated VSMCs were cultured; this was followed by a subculture
with trypsinization. The identity of the VSMCs was confirmed by
morphological examination and by staining for ␣-actin.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
DNA Synthesis
DNA synthesis was examined by measuring [3H]thymidine incorporation into the cellular DNA, as described previously.5,6 After
synchronization or growth arrest of VSMCs, medium was replaced
with Dulbecco’s modified Eagle’s medium containing 500 ␮g/mL
BSA, 10 ␮g/mL insulin, 20 ␮g/mL transferrin, and 25 ng/mL
selenium. VSMCs were incubated with different concentrations of
UII and with LDL, moxLDL, oxLDL, H2O2 (a donor of ROS), LPC,
or HNE for 48 hours. Otherwise, VSMCs were incubated with
indicated concentrations of UII and moxLDL and with the epidermal
growth factor receptor tyrosine kinase inhibitor erbstatin A, the c-Src
tyrosine kinase inhibitor radicicol, the protein kinase C (PKC)
inhibitor Ro31-8220, the mitogen-activated protein kinase (MAPK)
kinase inhibitor PD098059, or the intracellular free radical scavenger
NAC for 48 hours. Anti-Gq/11␣ antibody was added 1 hour before
the addition of UII and moxLDL. VSMCs were exposed to [3H]thymidine at a concentration of 1 ␮Ci/plate for the last 5 hours of the 48
hour incubation period, and [3H]thymidine incorporation into VSMC
DNA was measured. All the experiments were performed in quadruplicate, and each experiment was repeated a minimum of 3 times.
Statistical Analysis
All values are expressed as mean⫾SEM. The data were compared by
2-tailed unpaired Student’s t test between 2 groups and by 1-way
ANOVA followed by Bonferroni test when ⬎2 groups were involved. Differences were considered statistically significant at
P⬍0.05.
Results
Effect of UII With LDL, MoxLDL, or OxLDL on
VSMC DNA Synthesis
UII significantly increased [3H]thymidine incorporation in a
concentration-dependent manner, with a maximal effect at a
concentration of 50 nmol/L (161⫾10%, P⬍0.0001 versus
control; Figure 1). LDL, moxLDL, and oxLDL had a maximal effect at a concentration of 5 ␮g/mL (data not shown).
When tested in combination, UII (50 nmol/L) significantly
increased the mitogenic effect of LDL (500 ng/mL; by
242⫾23%; P⬍0.005; Figure 1A). When added together, even
nonmitogenic concentrations of UII (10 or 25 nmol/L) acted
synergistically with moxLDL (100 ng/mL) or oxLDL (50
ng/mL) in inducing [3H]thymidine incorporation (337⫾35%,
P⬍0.0001; 302⫾36%; P⬍0.001, respectively; Figures 1B
and 1C).
Figure 1. Effect of UII with LDL, moxLDL, oxLDL, H2O2, LPC, or
HNE on VSMC DNA synthesis. Growth-arrested VSMCs were
stimulated with given concentrations of UII with LDL (A), moxLDL (B), oxLDL (C), H2O2 (D), LPC (E), or HNE (F) in serum-free
medium for 48 hours, and amount of [3H]thymidine incorporation
was measured. Data are shown as mean⫾SEM (n⫽12). Control
value (223⫾12 counts per minute) was 100%. *P⬍0.0001,
⫹P⬍0.0005, and #P⬍0.05 vs control; @ P⬍0.0001, **P⬍0.0005,
⫹⫹P⬍0.001, ##P⬍0.005, and @@ P⬍0.05 vs corresponding
controls.
Effect of UII With ROS, LPC, or HNE on VSMC
DNA Synthesis
H2O2, LPC, or HNE significantly increased [3H]thymidine
incorporation in a concentration-dependent manner. H2O2 had
a maximal effect at a concentration of 5 ␮mol/L (177⫾7%,
P⬍0.0001 versus control), LPC at 15 ␮mol/L (156⫾8%,
P⬍0.0001), and HNE at 1 ␮mol/L (142%⫾8%, P⬍0.005;
Figures 1D, 1E, and 1F). When UII (25 nmol/L) was added
with H2O2 (5 ␮mol/L), LPC (5 ␮mol/L), or HNE (1 ␮mol/L),
there were synergistic rather than an additive effects on
[3H]thymidine incorporation (226⫾7%, 332⫾25%, and
299⫾59%; P⬍0.0001, respectively; Figures 1D, 1E, and 1F).
Effect of Anti-Gq/11␣ Antibody, Erbstatin A,
Radicicol, Ro31-8220, PD098059, or NAC on
Mitogenic Interaction Between UII and MoxLDL
To discover how UII and moxLDL exert their synergistic
interaction, we assessed the effects of anti-Gq/11␣ antibody
(2.5 ␮L/plate of 2 mL), the epidermal growth factor receptor
tyrosine kinase inhibitor erbstatin A (10 ␮mol/L), the c-Src
tyrosine kinase inhibitor radicicol (10 ␮mol/L), the PKC
inhibitor Ro31-8220 (0.1 ␮mol/L), the MAPK kinase inhibitor PD098059 (10 ␮mol/L), and the intracellular free radical
scavenger NAC (400 ␮mol/L) on the interaction between UII
(10 nmol/L) and moxLDL (100 ng/mL) in inducing [3H]thymidine incorporation. Anti-Gq/11␣ antibody, erbstatin A,
18
Circulation
July 3, 2001
[3H]Thymidine Incorporation Into VSMC DNA
Control
100⫾5%
UII (10 nmol/L)
100⫾3%
Endothelin-1 (10 nmol/L)
139⫾21%
Angiotensin II (10 nmol/L)
136⫾16%
MoxLDL (100 ng/mL)
114⫾7%
UII (10 nmol/L)⫹moxLDL (100 ng/mL)
345⫾37%*
Endothelin-1 (10 nmol/L)⫹moxLDL (100 ng/mL)
287⫾12%*
Angiotensin II (10 nmol/L)⫹moxLDL (100 ng/mL)
277⫾21%*
Values are mean⫾SEM. 100⫾5%⫽222⫾10 counts per minute.
*P⬍0.0001 vs moxLDL.
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Figure 2. Effect of anti-Gq/11␣ antibody, erbstatin A, radicicol,
Ro31-8220, PD098059, or NAC on mitogenic interaction
between UII and moxLDL. Growth-arrested VSMCs were stimulated with UII and/or moxLDL in serum-free medium with antiGq/11␣ antibody, erbstatin A, radicicol, Ro31-8220, PD098059,
or NAC for 48 hours, and amount of [3H]thymidine incorporation
was measured. Data are shown as mean⫾SEM (n⫽12). Control
value (238⫾11 counts per minute) was 100%. *P⬍0.0001 vs
untreated control; ⫹P⬍0.0001, #P⬍0.005, and @P⬍0.05 vs
UII⫹moxLDL.
radicicol, Ro31-8220, PD098059, or NAC by themselves had
no significant effect on [3H]thymidine incorporation (Figure
2). Anti-Gq/11␣ antibody, erbstatin A, and NAC partially
inhibited the synergistic interaction between UII and moxLDL (P⬍0.05), whereas radicicol, Ro31-8220, and
PD098059 completely abolished the synergistic interaction
between the two (P⬍0.0001).
Discussion
It is well established that hypertension and hypercholesterolemia enhance the development of atherosclerosis in human
and animal studies.7 Vasoconstrictors such as endothelin-1,
angiotensin II, and oxLDL accelerate atherosclerosis by
inducing VSMC proliferation. The extent of changes in the
LDL particle induced by oxidation (moxLDL to oxLDL)
depends on the pro-oxidant conditions and the length of time
the particle is exposed to these conditions. Under physiological conditions, which include the presence of various antioxidants in the plasma, complete oxidation of LDL may not
be feasible; instead, such conditions are more likely to result
in partial oxidation of LDL, with production of moxLDL,
which was shown to be the most potent of the 3 forms of
LDL.5 In this study, the combination of UII (10 nmol/L) with
moxLDL (100 ng/mL) resulted in the highest induction of
VSMC proliferation among cells incubated with UII and LDL
or oxLDL (Figure 1) and endothelin-1 or angiotensin II with
moxLDL (Table).
The increased atherogenic effect of moxLDL is attributed
to the chemical changes brought about by the oxidation
processes. During the early stages of oxidation, there is a
significant accumulation of peroxides and other ROS. As
oxidation proceeds, phosphatidylcholine is converted to
LPC.5 More or less at the same time, unsaturated aldehydes,
such as HNE, are generated by ␤-scission of alkoxyl radicals
in the polyunsaturated fatty acids that are present in LDL.6
Several studies from our laboratory and others have shown
that moxLDL and oxLDL and their oxidative components
(ie, ROS, LPC, and HNE) induce VSMC proliferation via
the redox-sensitive pathway and the extracellular signal–
regulated kinase (ERK) 1/2 MAPK pathway.5,6
Like most vasoactive agents, such as angiotensin II,
endothelin-1, and serotonin, UII may also induce VSMC
proliferation via the activation of the G protein-coupled
receptor or the PKC/c-Src tyrosine kinase/MAPK pathway. In
this study, the synergistic interaction between UII and moxLDL was abolished by the PKC inhibitor Ro31-8220, the
c-Src tyrosine kinase inhibitor radicicol, and the MAPK
kinase inhibitor PD098059, suggesting that the amplification
of the PKC/c-Src tyrosine kinase/MAPK pathway may play a
key role in inducing synergistic interaction between the two
agents. These findings provide an understanding of the
potential molecular mechanisms responsible for the longstanding clinical observations that the interaction of risk
factors promotes the development of atherosclerosis.
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Synergistic Effect of Urotensin II With Mildly Oxidized LDL on DNA Synthesis in Vascular
Smooth Muscle Cells
Takuya Watanabe, Rajbabu Pakala, Takashi Katagiri and Claude R. Benedict
Circulation. 2001;104:16-18
doi: 10.1161/hc2601.092848
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