The involvement of transforming growth factor-β1 secretion in Urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts

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Abstract

As the most potent vasoconstrictor in mammals, urotensin II (U II) has recently been demonstrated to play an important role in adverse cardiac remodeling and fibrosis. However, the mechanisms of U II-induced myocardial fibrosis remain to be clarified. We postulated that U II alters transforming growth factor-β1 (TGF-β1) expression, and thereby modulates cardiac fibroblast collagen metabolism. Experiments were conducted using cardiac fibroblast from neonatal Wistar rats to determine the expression of TGF-β1, and the role of U II receptor UT in this process. The functional role of TGF-β1 and UT in modulating U II effects on type I, III collagen mRNA expression and 3H-proline incorporation was also analyzed. TGF-β1 gene and protein expression were consistently identified in quiescent cardiac fibroblasts. U II increased the expression of TGF-β1 mRNA and protein in a time-dependent manner. This effect was UT mediated, because UT antagonist urantide abolished U II-induced TGF-β1 expression. U II-induced increase in type I, III collagen mRNA expression and 3H-proline incorporation were both inhibited by a specific TGF-β1 neutralizing antibody and UT receptor antagonist urantide. Hence, our results indicate that TGF-β1 is upregulated in cardiac fibroblasts by U II via UT and modulates profibrotic effects of U II. These findings provide novel insights into U II-induced cardiac remodeling.

Introduction

Urotensin II (U II) is a somatostatin-like cyclic peptide synthesized by proteolytic cleavage from a precursor molecule, prepro-U II, and has been identified as the most potent vasoconstrictor in mammals [1]. In humans, U II binds to a 389 amino acid G-protein coupled receptor termed UT [1]. The G-protein associated with the UT receptor is of the Gq class, which is the same class of G-proteins that bind to angiotensin, endothelin, and α-adrenoceptors. Recently, U II has emerged as a likely contributor to cardiovascular physiology and pathology. U II induced both endothelium-independent vasoconstriction and endothelium-dependent vasorelaxation, the order and magnitude of which were dependent on the species tested and anatomical location [2], [3]. U II also exerted inotropic effects on the isolated human atrial trabecular muscle [4]. Bolus injection of U II into cynomolgus monkeys resulted in the development of cardiovascular collapse [1].

In addition to its short-term roles which include vasoconstriction and chronotropic and inotropic effects on cardiac muscle, U II has also been implicated in the long-term regulation of growth in cardiovascular system recently. A number of studies have suggested an important role of U II in the development of cardiac remodeling. Plasma levels of U II were elevated in patients with congestive heart failure (CHF) compared with control subjects, and this increased plasma level of U II was significantly correlated with left ventricular end-diastolic pressure [5], [6], [7], [8]. In vivo studies found that expression of U II and its receptor was increased in the myocardium of patients with end-stage CHF [9], in the myocardium of rats with myocardial infarction [10] and in the myocardium of rats with chronic hypoxia induced-right ventricular hypertrophy [11]. Blockage of the UT receptor reduced mortality and improved cardiac function in the rat model of myocardial infarction and CHF [12]. Moreover, Tzanidis et al. demonstrated that U II promoted collagen synthesis of cardiac fibroblasts independently and stimulates cellular hypertrophy of cardiac myocytes in conditions of UT upregulation [10], and the UT receptor antagonist BIM-23127 inhibited U II-induced hypertrophy in H9c2 cardiomyocytes [13]. This U II-induced hypertrophy of cardiac myocytes is promoted by ERK1/2 and p38 signaling pathways in an epidermal growth factor receptor-dependent manner [14]. However, the mechanisms of action of U II in cardiac remodeling, especially in cardiac fibrosis, is still incompletely understood. Further insights into the mechanisms of its effects may have important therapeutic implications.

In the current study, the signaling pathways that control U II-mediated cardiac fibrosis were assessed in our in vitro model. This has allowed us to identify a key role for the production of TGF-β1 by cardiac fibroblasts in U II-mediated fibrosis.

Section snippets

Materials

U II was purchased from Sigma (Saint Louis, MO), TGF-β1 specific neutralizing antibody was purchased from R&D Systems (Minneapolis, MN, USA), transforming growth factor-β1 (TGF-β1) ELISA kit was purchased from Bioasource (Camarillo, CA), urantide was purchased from Peptides International (Louisville, Kentucky, USA), Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA).

Cell culture

Neonatal rat cardiac fibroblasts were prepared by the following

TGF-β1 expression in cardiac fibroblasts and its upregulation by U II

Cardiac fibroblasts consistently expressed small amounts of TGF-β1. Both mRNA and protein were detected in cultured cardiac fibroblasts. To study the effect of U II on TGF-β1 mRNA expression in cardiac fibroblasts, the cells were stimulated with the exogenous U II (100 nM) for 4 h to 48 h. Total RNA was extracted and real-time PT-PCR for TGF-β1 was performed. TGF-β1 mRNA expression was significantly increased by U II (Fig. 1a). This effect of U II on TGF-β1 mRNA expression began to increase at

Discussion

Cardiac remodeling denotes alterations in the structural components of the myocardium involving both myocytes and nonmyocyte compartments, a process which underlies progressive heart failure [15]. Recently, U II has been demonstrated to play an important role in this process [5], [9], [10], [12], but the precise mechanisms of U II-induced cardiac remodeling, especially myocardial fibrosis, have not been elucidated.

TGF-β1 is a profibrotic cytokine that stimulates the production of extracellular

Acknowledgments

I want to thank Mr. Jin-Bo Feng, Mrs. Hong Jiang, Miss Xu-Ping Wang, Mrs. Chun-Xi Liu and Mrs. Rong Wang for the technical work in this research.

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