Elsevier

Peptides

Volume 28, Issue 11, November 2007, Pages 2164-2170
Peptides

Mice heterozygous for adrenomedullin exhibit a more extreme inflammatory response to endotoxin-induced septic shock

https://doi.org/10.1016/j.peptides.2007.08.012Get rights and content

Abstract

Adrenomedullin (AM) is a highly conserved peptide that can act as a potent vasodilator, anti-microbial factor and anti-inflammatory factor. Several studies have implicated diverse roles for AM in regulating the inflammatory and hemodynamic responses to septic shock. Moreover, during sepsis the receptors that mediate AM signaling [calcitonin receptor-like receptor (calcrl) and receptor activity modifying proteins (RAMP) 2 and 3] undergo dynamic and robust changes in their expression. Although numerous studies have used animal models to study the role of administered or increased AM in septic animals, genetic studies to determine the consequences of reduced AM during septic shock have not yet been performed. Here, we used a murine model of lipopolysaccharide (LPS)-induced septic shock to assess the inflammatory response in mice heterozygous for the AM gene. Following LPS challenge, AM+/− mice had higher expression of TNF-α and IL-1β than LPS-treated wild-type (WT) controls. Consequently, serum TNF-α was also significantly elevated in LPS-treated AM+/− mice compared to WT LPS-treated mice. We also observed higher serum levels of liver enzymes, suggesting more advanced end-organ damage in mice with genetically reduced AM. Finally, we found that RAMP2 and calcrl expression levels were markedly reduced in LPS-treated mice, whereas RAMP3 expression was significantly elevated. Importantly, these changes in receptor gene expression were conserved in AM+/− mice, demonstrating that AM peptide itself does not impact directly on the expression of the genes encoding its receptors. We, therefore, conclude that during septic shock the dynamic modulation of AM and its receptors primarily functions to dampen the inflammatory response.

Introduction

Adrenomedullin (AM) is a peptide encoded by a highly conserved gene that may have evolved from an antimicrobial peptide in early eukaryotic organisms into a potent vasodilator in higher mammalian species [37]. AM causes relaxation of vascular smooth muscle cells (VSMCs) [7], reduces endothelial cell permeability [10] and is a biologically relevant antimicrobial peptide involved in the innate immune response [1]. The 52-amino acid peptide is produced and secreted by many mammalian tissues and is most highly expressed by VSMCs [30] and endothelial cells [29]. Stimuli for AM synthesis and secretion include angiotensin II, endothelin-1, hypoxia, oxidative stress and inflammatory cytokines such as TNF-α and IL-1β [6]. Thus, the biological functions of AM in mammals are numerous, diverse and likely inter-related.

Plasma levels of AM are significantly elevated in humans with a wide variety of physiological and pathological conditions, including cardiovascular disease, normal pregnancy and septic shock [8]. In patients with septic shock, AM peptide levels are 25 to 30 folds higher than in normal individuals [11], [21]. Since AM is a potent vasodilator [15], it is reasonable to assume that increased plasma AM contributes to the extreme hypotension observed in the early stages of septic shock. However, our recent studies using genetically engineered mice that lack one copy of the AM gene demonstrate that reduction of endogenous AM to 50% of wild-type (WT) levels has no effect on the acute hypotension that occurs in an LPS-induced murine model of septic shock [2]. These results suggest that AM may play other primary roles during septic shock.

AM possesses anti-inflammatory [9], bactericidal [1], and positive inotropic [12] properties, which are all beneficial responses to sepsis. When treated with endotoxin, mice over-expressing AM in their vasculature experience less severe hemodynamic and inflammatory responses, less liver damage and lower mortality rates compared to WT endotoxin-treated controls [28]. AM has also been shown to reduce TNF-α expression and release in macrophage cell lines and rat Kupffer cells [33]. More recently, administration of AM to rats with α-toxin-induced sepsis reduced vascular hyperpermeability and resulted in dramatically improved survival rates [31]. Finally, dynamic and robust changes in the expression of AM and its receptors occur in the lungs in response to septic shock [4], [22]. Taken together, these results suggest that the beneficial roles of AM during septic shock may primarily be to minimize organ damage by influencing the immune response and/or vascular permeability, rather than by regulating blood pressure. Yet, experiments to genetically confirm the primary function of AM during septic shock have not yet been performed.

The AM peptide contains a six-residue ring structure and amidated C-terminus which, due to conserved sequence homology and structural motifs, places it in the calcitonin family of peptides, including calcitonin, calcitonin gene related peptide (CGRP), amylin and intermedin [37]. Peptides of this family also share a unique mechanism of G-protein coupled receptor signaling by a novel class of single transmembrane proteins called receptor activity modifying proteins (RAMPs). RAMPs were first identified through their association with the calcitonin receptor-like receptor (CLR) and can interact with many other class II GPCRs to determine receptor ligand binding specificity [27]. In the case of CLR, association with RAMP1 produces a CGRP receptor, while association with RAMP2 or RAMP3 produces a receptor specific for AM. In this way, the spatial and temporal expression of RAMP proteins determines the tissue responsiveness to either CGRP or AM.

During inflammation and septic shock, there are robust and dynamic changes in the expression of the RAMP and calcrl genes that are responsible for mediating AM signaling. For example, TNF-α significantly reduced the expression of calcrl (the gene encoding CLR), RAMP1 and RAMP2 in cultured smooth muscle cells of human coronary artery in a time and dose-dependent manner [20]. Moreover, Ono et al. [22] have also shown that calcrl and RAMP2 expression was significantly decreased in lungs of LPS-induced septic mice, while RAMP3 expression levels were elevated nearly 40-fold. In a related fashion, the amount of AM binding protein (AMBP) is significantly reduced during the hypodynamic phase of sepsis, which may account for the reduced responsiveness to elevated plasma AM during the late phase of sepsis [32], [34], [36], [37]. These results suggest that the modulation of AM signaling during septic shock is complex (involving both receptor modulation and active peptide bioavailability) and finely tuned in order to maintain homeostatic balance in response to severe physiological insults. However, whether AM signaling itself is involved in these dynamic receptor responses remains unclear.

Our previous studies with genetically engineered mouse models have shown that mice lacking both copies of the AM gene or the calcrl gene die at mid-gestation from extreme hydrops fetalis and cardiovascular defects [3], [5]. Adult female mice heterozygous for AM display profound reproductive defects [16] and are protected from hypertension-induced cardiovascular end-organ damage [2]. Otherwise, adult male and female AM heterozygous mice are born at the expected Mendelian ratios, survive to adulthood and have normal blood pressures under basal and stressed conditions with no obvious phenotypic defects.

To determine if genetic reduction of endogenous AM affects the septic response in mice, we challenged AM+/− mice in an LPS-induced model of septic shock. Since AM is consistently reported as an anti-inflammatory peptide, we were particularly interested in determining whether genetic reduction of endogenous AM in vivo could alter the inflammatory response in septic animals. We also used our genetic model to determine if the dynamic gene expression changes observed in the AM receptor signaling genes during septic shock are dependent on the expression levels of AM peptide.

Section snippets

Experimental animals and LPS treatment

The generation and phenotype of mice with a targeted deletion of the AM gene have been previously described [3]. Experiments were carried out with 8–10-week-old male mice bred under a controlled environment and were approved by the Institutional Animal Care and Use Committee of the University of North Carolina, Chapel Hill. Animals used in these experiments were produced by AM+/− intercrosses and were maintained on an isogenic 129S6/SvEv genetic background. To induce septic shock in WT and AM+/−

AM gene expression is induced significantly less in AM+/− mice treated with LPS

Using quantitative RT-PCR, AM gene expression was measured in lungs of WT and AM+/− mice treated with or without LPS for 2 h. In untreated mice, the basal level of AM gene expression in AM+/− mice was 53% of WT mice, consistent with our previous demonstration that the gene targeting strategy effectively reduced AM gene expression by approximately half wild-type levels [3]. AM gene expression was dramatically elevated in both WT and AM+/− mice following LPS administration. However, the level of AM

Discussion

Many in vitro studies have suggested an important regulatory role for AM in sepsis and other inflammatory diseases [13], [18], [19], [30], [33], [35]. Moreover, genetic over-expression of AM in the mouse vasculature or therapeutic administration of AM peptide in rats or mice had beneficial effects on reducing the inflammatory and hemodynamic insults elicited by septic shock [28]. However, whether genetic reduction of AM can cause a more severe response to sepsis has not yet been determined. The

Acknowledgements

The authors would like to thank current and past members of the Caron Laboratory for helpful advice and discussions. This work was supported in part with funds from the Burroughs Wellcome Fund and a National Institutes of Health grant HD046970 to KMC.

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