Involvement of reactive oxygen species in urotensin II-induced proliferation of cardiac fibroblasts
Introduction
Urotensin II is a cyclic dodecapeptide synthesized by proteolytic cleavage from a precursor molecule, prepro-urotensin II, and has been identified as a potent vasoconstrictor (Ames et al., 1999). Urotensin II has been identified within the heart (Matsushita et al., 2003), which is also a site with an abundant expression of urotensin II receptor (Ames et al., 1999). Acute urotensin II infusion into nonhuman primates results in severe myocardial depression, characterized by bradycardia and reduced stroke volume and contractility (Ames et al., 1999), suggesting a functional role for this peptide within the heart. It is now well established that urotensin II levels are increased in several cardiovascular diseases (Bousette and Giaid, 2006). For example, in patients with coronary artery disease, it has been shown that urotensin II plasma levels are significantly greater than the levels in normal patients and the severity of the disease is increased proportionally to the urotensin II plasma levels (Papadopoulos et al., 2008). Moreover, recent studies in end-stage renal disease further suggest that, perhaps because of the interference with sympathetic and nitric oxide systems, urotensin may mediate the favorable echocardiographic profile of patients with high circulating urotensin (Zoccali et al., 2008). In addition to its clinical significance, both in vitro and in vivo studies further suggest the role of urotensin II in the development of cardiac fibrosis (Zhang et al., 2007). Bousette et al. demonstrated that selective block of urotensin II reduces diastolic dysfunction by decreasing myocardial fibrosis post-coronary ligation in vivo, and inhibits urotensin II-mediated fibroblast proliferation in vitro (Bousette et al., 2006). However, little is known about the urotensin II-mediated intracellular signaling pathways related to proliferation of cardiac fibroblasts.
The aim of the present study was to clarify the regulation of proliferation by urotensin II in rat cardiac fibroblasts. We further determined the intracellular signal transduction pathways focusing especially on reactive oxygen species production involved in this process. This study shows that reactive oxygen species production is involved in urotensin II-induced cell proliferation, the tyrosine phosphorylation of epidermal growth factor receptors, and extracellular signal-regulated kinase phosphorylation in rat cardiac fibroblasts.
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Materials
Dulbecco's modified Eagle's medium, fetal calf serum and tissue culture reagents were from Life Technologies, Inc. (Grand Island, NY, U.S.A.). 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126) was obtained from Tocris Cookson Ltd. (Bristol, U.K.). AG 1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) was obtained from Calbiochem (La Jolla, CA, USA.). Human recombinant urotensin II and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies
Urotensin II stimulates cardiac fibroblast proliferation
To evaluate whether urotensin II can regulate proliferation of cardiac fibroblasts, cells were stimulated with urotensin II, and the proliferative activity of cardiac fibroblasts was determined by cell counting and BrdU incorporation. Stimulation with urotensin II (30–100 nM) for 24 h significantly increased the cell number and BrdU incorporation in cardiac fibroblasts (Fig. 1A, B). Urotensin II (30 nM) also significantly increased the proliferative activity of cardiac fibroblasts in a
Discussion
In the present study, we have shown that reactive oxygen species production is involved in urotensin II-induced cell proliferation, extracellular signal-regulated kinase phosphorylation and epidermal growth factor receptor transactivation in rat cardiac fibroblasts.
The heart is composed of not only cardiac myocytes but also non-myocytes, particularly fibroblasts (Eghbali, 1992). Unlike cardiac myocytes, cardiac fibroblasts can proliferate and increase the deposition of extracellular matrix
Acknowledgement
This study was supported with grants from National Science Council (NSC 95-2314-B-039-048-MY2), and Grant No CMU-96-202 from the China Medical University, Taichung, Taiwan, ROC We are gratefully acknowledged to the ePoster presentation of this study in the “Signaling in the heart” session at the “ESC Congress 2007”, Vienna, Austria. The authors wish to thank Miss Sherry Lo for assistance in manuscript editing and revision.
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These authors contributed equally to this work.