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
]. 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
]. 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 6-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
genes that are responsible for mediating AM signaling. For example, TNF-α significantly reduced the expression of calcrl
(the gene encoding CLR), RAMP1
in cultured smooth muscle cells of human coronary artery in a time and dose-dependent manner [20
]. Moreover, Ono et al. have also shown that calcrl
expression was significantly decreased in lungs of LPS-induced septic mice, while RAMP3
expression levels were elevated nearly 40-fold [22
]. 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
]. 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
]. 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.