Adenovirus-mediated delivery of relaxin reverses cardiac fibrosis

https://doi.org/10.1016/j.mce.2007.09.008Get rights and content

Abstract

We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to β2-adrenergic receptor (β2AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in β2AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis.

Introduction

Cardiac fibrosis is a process of myocardial remodeling where there is abnormal expansion of the cardiac extracellular matrix (ECM) due to fibroblast activation and excessive accumulation of collagen and other components (Weber, 2000, Tyagi et al., 1996). Fibrosis is a common hallmark of cardiovascular diseases and interferes with normal myocardial architecture and function leading to a variety of pathophysiological problems such as diastolic and systolic dysfunction, increased risk of myocardial ischemia, arrhythmias, sudden cardiac death, and worsening of heart failure (Weber, 2000). Current therapies such as angiotensin-converting enzyme inhibitors and aldosterone inhibitors (Lijnen and Petrov, 2003, Mandarim-de-Lacerda and Pereira, 2003) are effective but slow-acting with undesirable side-effects and thus novel specific and effective therapies, with shorter treatment periods, to abolish or attenuate the development of fibrosis are highly desired.

In recent years, the polypeptide hormone relaxin has surfaced as a potent regulator of collagen in the ECM of multiple organs including reproductive organs, skin, kidney, lung, liver, and heart (Samuel et al., 2003, Samuel et al., 2004a, Samuel et al., 2004b, Garber et al., 2001, Williams et al., 2001, Unemori et al., 1996, Unemori and Amento, 1990). Investigations into the cardiac phenotype of relaxin gene-knockout mice (RLX−/−) have revealed that there is an age-related development of cardiac fibrosis, leading to altered diastolic function (Du et al., 2003). More importantly, several studies have demonstrated that relaxin delivery via osmotic mini-pumps reverses fibrotic cardiomyopathy in three rodent models of established cardiac fibrosis: (i) in mice lacking the relaxin gene (Samuel et al., 2004a); (ii) in transgenic (TG) mice with cardiac-restricted overexpression of β2-adrenergic receptors (β2AR) (Samuel et al., 2004a); and (iii) in spontaneously hypertensive rats (Lekgabe et al., 2005). Together, these studies have established that relaxin is an innate regulator of collagen deposition.

Recombinant adenovirus-mediated gene delivery has been utilized for experimental gene therapy in a variety of heart disorders (Hajjar et al., 2000, Hammond and McKirnan, 2001, Jones and Koch, 2005) and pilot clinical trials have also been initiated (Relph et al., 2005). In almost all studies, intra-coronary or intra-myocardial injection of adenovirus was adopted. Furthermore, few studies have tested interventions that specifically target myocardial fibrosis. In this study, we have directly investigated the feasibility of systemic adenoviral-mediated relaxin delivery. We have characterized adenoviruses producing bioactive rat and mouse relaxin peptides and evaluated the effectiveness of intravenous injection of these viruses in a transgenic murine model of cardiac fibrosis. The results demonstrate that systemic relaxin gene delivery can address the complicating factor of cardiac fibrosis associated with cardiovascular diseases, without any notable side-effects.

Section snippets

Adenovirus construction

The pAd-Easy-1 virus and shuttle plasmid, pAd-TrackCMV, were obtained from Dr. B. Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD (He et al., 1998)). Rat and mouse relaxin full length cDNAs were generated from pregnant ovary cDNA using RT-PCR and ligated into pBS-KS using KpnI and NotI linkers. The constructs were sequenced on both strands to verify the sequence and then subcloned into the shuttle vector, pAdTrack-CMV to yield pAdTrack-rRLN (rat relaxin) and pAdTrack-mRLN (mouse

In vitro characterization of Ad-rRLN and Ad-mRLN

Full-length cDNAs for mouse and rat relaxin were cloned from pregnant ovaries and inserted into the pAd-TrackCMV vector and sequenced. Before Ad-rRLN and Ad-mRLN were used in in vivo studies they were first tested for their ability to produce immunoreactive relaxin peptide in HEK-293T cells. Ad-GFP infected cells were used as a negative control.

The presence of relaxin peptide was first tested in Ad-rRLN infected cells and media using a rat relaxin antibody and Western blotting. As an antibody

Discussion

In this study, we employed a somatic gene therapy approach to explore the therapeutic potential of relaxin on cardiac fibrosis in mice. We have used a model of cardiac fibrosis, cardiac-restricted overexpression of β2AR, in which we have previously reversed fibrosis using H2 relaxin peptide delivered via osmotic mini-pumps implanted subcutaneously (Samuel et al., 2004a). As relaxin cannot be delivered orally, and has a short plasma half-life, continuous infusion is the only viable form of

Acknowledgements

The studies in this paper were supported by a National Health and Medical Research (NH&MRC) grant to RADB (300012), NHMRC Fellowships to RDH, WGT and X-JD, and a National Heart Foundation grant (G 04M 1524) to CSS and X-JD. EDL is a recipient of a scholarship from the Jenny Ryan Foundation. The authors would like to thank Prof Geoffrey Tregear for advice and encouragement.

References (44)

  • Y. Brandenburger et al.

    Increased expression of UBF is a critical determinant for rRNA synthesis and hypertrophic growth of cardiac myocytes

    FASEB J.

    (2001)
  • M.M. Burcin et al.

    Adenovirus-mediated regulable target gene expression in vivo

    Proc. Natl. Acad. Sci. U.S.A.

    (1999)
  • X.J. Du et al.

    Beta(2)-adrenergic receptor overexpression exacerbates development of heart failure after aortic stenosis

    Circulation

    (2000)
  • X.J. Du et al.

    Preserved ventricular contractility in infarcted mouse heart overexpressing beta(2)-adrenergic receptors

    Am. J. Physiol. Heart Circ. Physiol.

    (2000)
  • X.J. Du et al.

    Age-dependent cardiomyopathy and heart failure phenotype in mice overexpressing beta(2)-adrenergic receptors in the heart

    Cardiovasc. Res.

    (2000)
  • X.J. Du et al.

    Increased myocardial collagen and ventricular diastolic dysfunction in relaxin deficient mice: a gender-specific phenotype

    Cardiovasc. Res.

    (2003)
  • X.M. Gao et al.

    Sex hormones and cardiomyopathic phenotype induced by cardiac beta 2-adrenergic receptor overexpression

    Endocrinology

    (2003)
  • T.G. Golos et al.

    Control of corpus luteum function during the second half of pregnancy in the rat: a direct relationship between conceptus number and both serum and ovarian relaxin levels

    Endocrinology

    (1982)
  • R.J. Hajjar et al.

    Prospects for gene therapy for heart failure

    Circ. Res.

    (2000)
  • M.L. Halls et al.

    Multiple binding sites revealed by interaction of relaxin family peptides with native and chimeric relaxin family peptide receptors 1 and 2 (LGR7 and LGR8)

    J. Pharmacol. Exp. Ther.

    (2005)
  • H.K. Hammond et al.

    Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials

    Cardiovasc. Res.

    (2001)
  • T.C. He et al.

    A simplified system for generating recombinant adenoviruses

    Proc. Natl. Acad. Sci. U.S.A.

    (1998)
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    These authors contributed equally to this work.

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