Adenovirus-mediated delivery of relaxin reverses 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.
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These authors contributed equally to this work.