Elsevier

Peptides

Volume 22, Issue 11, November 2001, Pages 1731-1737
Peptides

Human adrenomedullin gene delivery protects against cardiovascular remodeling and renal injury

https://doi.org/10.1016/S0196-9781(01)00510-1Get rights and content

Abstract

We investigated the potential roles of adrenomedullin (AM) in cardiovascular and renal function by somatic gene delivery. We showed that a single intravenous injection of the human AM gene under the control of cytomegalovirus promoter/enhancer induces a prolonged delay in blood pressure rise for several weeks in spontaneously hypertensive rats, Dahl salt-sensitive, DOCA-salt, and two-kidney one-clip hypertensive rats as compared to their respective controls injected with a reporter gene. Expression of the human AM transcript was identified in the heart, kidney, lung, liver and aorta of the rat after adenovirus-mediated AM gene delivery by RT-PCR followed by Southern blot analysis. Immunoreactive human AM levels were measured in rat plasma and urine following AM gene delivery. AM gene delivery induced significant reduction of left ventricular mass in these hypertensive animal models. It also reduces urinary protein excretion and increases glomerular filtration rate, renal blood flow and urinary cAMP levels. AM gene transfer attenuated cardiomyocyte diameter and interstitial fibrosis in the heart, and reduced glomerular sclerosis, tubular disruption, protein cast accumulation and renal cell proliferation in the kidney. In the rat model with myocardial ischmia/reperfusion injury, AM gene delivery significantly reduced myocardial infarction, apoptosis, and superoxide production. Furthermore, local AM gene delivery significantly inhibited arterial thickening, promoted re-endothelialization and increased vascular cGMP levels in rat artery after balloon angioplasty. Collectively, these results indicate that human AM gene delivery attenuates hypertension, myocardial infarction, renal injury and cardiovascular remodeling in animal models via cAMP and cGMP signaling pathways. These findings provide new insights into the role of AM in cardiovascular and renal function.

Introduction

Adrenomedullin (AM) was first isolated from human pheochromocytoma tissue in 1993 [32] and was identified in the adrenal medulla, kidney, heart, aorta, lung and brain [23], [32], [46], [47]. AM is a potent vasodilator as evidenced by producing hypotensive effect along with a marked reduction of total peripheral resistance in rats, cats and humans after i.v. administration of the AM peptide [5], [26], [42]. In addition to influencing the contractile state of blood vessels, AM also inhibits protein synthesis and thus cardiac hypertrophy in cultured cardiomyocytes [50]. Furthermore, AM peptide inhibited DNA synthesis and collagen production in cardiac fibroblasts, which is mediated through a cAMP-dependent pathway [21]. These combined findings suggest that AM, produced in both cardiac myocytes and fibroblasts, may act as an autocrine/paracrine modulator in the process of cardiac remodeling by suppressing mitogen and collagen synthesis in the heart. Moreover, direct intra-renal arterial infusion of AM peptide in dogs produced diuretic, natriuretic effects as well as increases of renal blood flow, glomerular filtration rate [12], [27]. These findings imply that AM is involved in the regulation of renal function, tubular ion and water transport [15], [19]. AM may also participate in the regulation of glomerular diseases as evidenced by inhibiting PDGF-induced mesangial cell proliferation and migration, which are likely to be mediated via cAMP-dependent process [7], [36]. Studies of an NO-cGMP-dependent pathway for the action of AM have also been reported [17], [48].

Many studies have reported specific AM receptors while others have shown that AM shares a receptor with calcitonin gene-related peptide (CGRP) [9], [13], [14], [22], [29], [30]. Recent findings have indicated the involvement of receptor activity-modifying proteins (RAMPs) in determining AM binding [4]. A recent study demonstrated that a core seven transmembrane protein referred to as calcitonin-receptor-like receptor (CRLR) binds to either AM or CGRP depending on which RAMP is present [37]. In addition, RAMP and CRLR interactions have been elucidated. When CRLR interacts with RAMP2, an AM receptor is created but when CRLR interacts with RAMP1, a CGRP receptor is created [2], [4], [28], [41]. In the rat ischemic heart failure model, both left ventricle AM and RAMP2 mRNA levels were elevated while RAMP1 mRNA levels remained unchanged [43]. This understanding of AM receptor provides insights for examining the role of AM as a cardiac autocrine/paracrine factor.

Abnormalities of AM levels have been reported in the pathophysiology of human diseases. For example, plasma AM levels are increased in patients with essential hypertension, cardiac hypertrophy, heart failure and renal disease [20], [25], [34]. Elevated AM production could be a biological attempt to compensate for cardiac and renal damages. A recent report showed that transgenic mice overexpressing the adrenomedullin gene under the control of preproendothelin-1 promoter have decreased mean blood pressure and no change in heart rate [49]. Nitric oxide synthase (NOS) inhibition normalized blood pressure in these mice, supporting a role of nitric oxide (NO) in mediating AM effects. The AM transgenic mice are less susceptible to the detrimental effects of lipopolysaccharide-mediated septic shock [49]. Embryos of AM deficient mice die at midgestation with extreme hydrops fetalis and cardiovascular abnormalities, including overdeveloped venricular trabeculae and underdeveloped arterial walls [3]. These findings indicate important roles of AM in the development, cardiovascular function. In order to achieve long-term expression of AM, we delivered the human AM gene in an adenoviral vector via systemic and local routes and evaluated the role of AM in volume-and pressure-overload hypertensive animals, in cardiac ischemic and balloon-injured artery models. Using somatic gene transfer approaches, we showed that AM has multiple functions in protection against hypertension, renal injury, cardiac infarction, cardiac hypertrophy and restenosis (Fig. 1).

Section snippets

Preparation of adenovirus carrying human AM cDNA

Human adrenomedullin (hAM) cDNA was cloned into adenoviral shuttle vector pAdTrack-CMV. The adenoviral shuttle vector (pAdTrack-CMV) or adenoviral shuttle vector carrying hAM cDNA under the control of cytomegalovirus (CMV) promoter was co-transformed into E. coli BJ5183 cells along with an adenoviral backbone vector pAdEasy-1 containing all Ad5 sequences except for E1 and E3 genes [18]. The resultant adenoviral plasmid (pAd.CMV-hAM) generated by homologous recombination was linearized with Pac

Concluding remarks and future directions

Gene transfer is a novel means of providing sustained and localized delivery of the required therapeutic protein. Currently, viral vectors are the vehicles of choice for gene delivery because of their high transfer efficiency. The most commonly used viral vectors are adenovirus, adeno-associated virus (AAV) and retrovirus. Retroviral vector has limitations as it can only infect dividing cells. Recombination-deficient adenovirus is an excellent vector for gene delivery because of its high

Acknowledgments

This work was supported by National Institutes of Health grants HL 29397 and HL 52196.

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