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(Circulation. 2001;104:16.)
© 2001 American Heart Association, Inc.

Brief Rapid Communications

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

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.).

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.

	Abstract

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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-2-nonenal (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 µmol/L), and the intracellular free radical scavenger N-acetylcysteine (400 µmol/L) and was completely inhibited by the c-Src tyrosine kinase inhibitor radicicol (10 µmol/L), the protein kinase C (PKC) inhibitor Ro31-8220 (0.1 µmol/L), and the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 µmol/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.

Key Words: atherosclerosis • hypertension • lipoproteins • muscle, smooth • urotensins

	Introduction

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Urotensin 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 protein-coupled 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-2-nonenal (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

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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.

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.

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 µg/mL BSA, 10 µg/mL insulin, 20 µg/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 µCi/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

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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 µg/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).

View larger version (55K): [in this window] [in a new window]   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 µmol/L (177±7%, P<0.0001 versus control), LPC at 15 µmol/L (156±8%, P<0.0001), and HNE at 1 µmol/L (142%±8%, P<0.005; Figures 1D, 1E, and 1F). When UII (25 nmol/L) was added with H₂O₂ (5 µmol/L), LPC (5 µmol/L), or HNE (1 µmol/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 µL/plate of 2 mL), the epidermal growth factor receptor tyrosine kinase inhibitor erbstatin A (10 µmol/L), the c-Src tyrosine kinase inhibitor radicicol (10 µmol/L), the PKC inhibitor Ro31-8220 (0.1 µmol/L), the MAPK kinase inhibitor PD098059 (10 µmol/L), and the intracellular free radical scavenger NAC (400 µmol/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, 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).

View larger version (45K): [in this window] [in a new window]   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 anti-Gq/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.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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).

View this table: [in this window] [in a new window]   Table 1. [3H]Thymidine Incorporation Into VSMC DNA

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 ß-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 long-standing clinical observations that the interaction of risk factors promotes the development of atherosclerosis.

	Footnotes

 
Guest Editor for this article was Joseph Loscalzo, MD, PhD, Boston University School of Medicine, Boston, Mass.

Received March 26, 2001; revision received May 8, 2001; accepted May 10, 2001.

	References

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2. Ames RS, Sarau HM, Chambers JK, et al. Human urotensin II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature. 1999;401:282–286.[Medline] [Order article via Infotrieve]

3. MacLean MR, Alexander D, Strirrat A, et al. Contractile response to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in the rat. Br J Pharmacol. 2000;130:201–204.[Medline] [Order article via Infotrieve]

4. Opgaard OS, Nothacker HP, Ehlert FJ, et al. Human urotensin II mediates vasoconstriction via an increase in inositol phosphates. Eur J Pharmacol. 2000;406:265–271.[Medline] [Order article via Infotrieve]

5. Watanabe T, Pakala R, Benedict CR, et al. Lysophosphatidylcholine and reactive oxygen species mediate the synergistic effect of mildly oxidized LDL with serotonin on vascular smooth muscle cell proliferation. Circulation. 2001;103:1440–1445.[Abstract/Free Full Text]

6. Watanabe T, Pakala R, Benedict CR, et al. Lipid peroxidation product 4-hydroxy-2-nonenal acts synergistically with serotonin in inducing vascular smooth muscle cell proliferation. Atherosclerosis. 2001;155:37–44.[Medline] [Order article via Infotrieve]

7. Weiss D, Kools JJ, Taylor R. Angiotensin II-induced hypertension accelerates the development of atherosclerosis in apoE-deficient mice. Circulation. 2001;103:448–454.[Abstract/Free Full Text]

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