Human adrenomedullin (AM) functions as a circulating hormone and as a local paracrine mediator with multiple biological activities. We investigated the metabolism of AM by examining its fragmentation in human serum. Adrenomedullin was rapidly cleaved in human serum, but was relatively stable in plasma. We showed that AM was rapidly digested by thrombin in serum, with AM(13-44) as the main product. On the basis of these data, we prepared AM analogs in which Arg-44 was replaced by Ala, Lys, and D-Arg, respectively. These analogs were resistant to thrombin and showed comparable biological activity to native AM. Furthermore, the bioavailabilities of these peptides were improved after subcutaneous administration in rats. These AM analogs may be promising drug candidates for clinical applications.

Sepsis remains a major medical challenge, for which, apart from improvements in supportive care, treatment has not relevantly changed over the last few decades. Vasodilation and vascular leakage play a pivotal role in the development of septic shock, with vascular leakage being caused by disrupted endothelial integrity. Adrenomedullin (ADM), a free circulating peptide involved in regulation of endothelial barrier function and vascular tone, is implicated in the pathophysiology of sepsis. ADM levels are increased during sepsis, and correlate with extent of vasodilation, as well as with disease severity and mortality. In vitro and preclinical in vivo data show that administration of ADM exerts anti-inflammatory, antimicrobial, and protective effects on endothelial barrier function during sepsis, but other work suggests that it may also decrease blood pressure, which could be detrimental for patients with septic shock. Work has been carried out to negate ADMs putative negative effects, while preserving or even potentiating its beneficial actions. Preclinical studies have demonstrated that the use of antibodies that bind to the N-terminus of ADM results in an overall increase of circulating ADM levels and improves sepsis outcome. Similar beneficial effects were obtained using coadministration of ADM and ADM-binding protein-1. It is hypothesized that the mechanism behind the beneficial effects of ADM binding involves prolongation of its half-life and a shift of ADM from the interstitium to the circulation. This in turn results in increased ADM activity in the blood compartment, where it exerts beneficial endothelial barrier-stabilizing effects, whereas its detrimental vasodilatory effects in the interstitium are reduced. Up till now, in vivo data on ADM-targeted treatments in humans are lacking; however, the first study in septic patients with an N-terminus antibody (Adrecizumab) is currently being conducted.

Adrenomedullin (AM) is a multifunctional evolutionarily highly conserved peptide. Although its genomic and amino acid (aa) sequences are known in several mammalian species and in fish, the structure of the AM gene remains unknown in intermediate phyla, including birds. Here, we report the structure and aa sequence of the chicken (c) AM ortholog. The cAM gene is located at the short arm of chromosome 5, which shows high synteny with the short arm of human (h) chromosome 11, where hAM is located. Key sequences in the third intron have been conserved which allow for an alternative splicing mechanism, similar to the one found in mammals. The preprohormone contains two peptides with high homology to human proadrenomedullin N-terminal 20 peptide (PAMP) and hAM. We found through real-time PCR and immunocytochemistry cAM mRNA and peptide expression in a variety of chicken tissues, which parallel patterns observed for mammals, with the exception that cAM levels are almost non-detectable in brain. Similarly to mammals, cAM expression is upregulated under hypoxic conditions and following dexamethasone treatment. These data demonstrate a high degree of homology between the cAM gene and its mammalian ortholog and evolutionary conservation of the regulatory mechanisms controlling its expression.

Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can "re-route" the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.

Intermedin (IMD) is a newly identified member of the calcitonin family of peptides that shares structural and functional homology with adrenomedullin (AM). In vivo cardiovascular effects of AM have been described, but relatively little is known of the in vivo actions of IMD. The purpose of this study was to compare the regional haemodynamic effects of IMD with those of AM in conscious rats, and investigate possible underlying mechanisms.

Secondary lymphedema is a debilitating condition, and genetic factors predisposing to its development remain largely unknown. Adrenomedullin (AM) is peptide encoded, together with proadrenomedullin N-terminal peptide (PAMP), by the Adm gene (adrenomedullin gene). AM and its putative receptor calcitonin receptor-like receptor (CLR) are implicated in angiogenesis and lymphangiogenesis during embryogenesis and wound healing, suggesting their possible involvement in secondary lymphedema. To investigate whether AM deficiency predisposes to secondary lymphedema, we used heterozygous adult mice with Adm gene-knockin stop mutation, which selectively abrogated AM, but preserved PAMP, expression (Adm(AM+/Δ) animals). After hind limb skin incision, Adm messenger RNA expression was upregulated in wounded tissue of both Adm(AM+/+) and Adm(AM+/Δ) mice. However, only Adm(AM+/Δ) animals developed limb swelling and histopathological lymphedematous changes, including epidermal thickening, elevated collagen fiber density, and increased microvessel diameter. Secondary lymphedema was prevented when circulating AM levels in Adm(AM+/Δ) mice were restored by systemic peptide delivery. In human skin, CLR was expressed in tissue components affected by lymphedema, including epidermis, lymphatics, and blood vessels. Our study identified a previously unrecognized role for endogenous AM as a key factor in secondary lymphedema pathogenesis and provided experimental in vivo evidence of an underlying germ-line genetic predisposition to developing this disorder.

Adrenomedullin (AM) is a 52 amino acid peptide involved in the pathophysiology of several human diseases. Here we show the gene structure, organ distribution, and regulated expression of AM in monkey. The monkey AM (mAM) gene is located on the short arm of chromosome 9 and it codes for a 185 amino acid preprohormone, which contains two amidated peptides identical to the human AM and proadrenomedullin N-terminal 20 peptide. The promoter region of the mAM gene contains a variety of transcription factor binding motifs. mAM is widely expressed throughout many organs as shown by real-time PCR and immunohistochemical techniques, and we have found similar levels of circulating plasma AM in monkeys and humans. A significant upregulation of the mAM mRNA was observed in monkey cells exposed to low oxygen tension conditions, TGF-beta1, all-trans-retinoic acid, and dexamethasone. Our collective data show a high degree of homology between mAM and hAM, which renders the monkey an attractive animal model for future pharmacological and pre-clinical studies targeting AM.

The signaling peptides adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and CGRP have overlapping and distinct functions in the cardiovascular, lymphatic, and nervous systems by activating three shared receptors comprised of the class B GPCR CLR in complex with a RAMP1, -2, or -3 modulatory subunit. Here, we report that AM2/IMD, which is thought to be a non-selective agonist, is kinetically selective for CLR-RAMP3, known as the AM 2 R. AM2/IMD-AM 2 R elicited substantially longer duration cAMP signaling than the eight other peptide-receptor combinations due to AM2/IMD slow off-rate binding kinetics. The regions responsible for the slow off-rate were mapped to the AM2/IMD mid-region and the RAMP3 extracellular domain. MD simulations revealed how these bestow enhanced stability to the complex. Our results uncover AM2/IMD-AM 2 R as a cognate pair with unique temporal features, define the mechanism of kinetic selectivity, and explain how AM2/IMD and RAMP3 collaborate to shape the signaling output of a clinically important GPCR.

Adrenomedullin (ADM) has been shown to take part in physiological and pathological angiogenesis. The purpose of this study was to investigate whether ADM signaling is involved in choroidal neovascularization (CNV) using a mouse model.

Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases-a major site of treatment-refractory tumor growth in patients with advanced disease.

Adrenomedullin (ADM) is a peptide hormone first discovered in 1993 in pheochromocytoma. It is synthesized by endothelial and vascular smooth muscle cells and diffuses freely between blood and interstitium. Excretion of ADM is stimulated by volume overload to maintain endothelial barrier function. Disruption of the ADM system therefore results in vascular leakage and systemic and pulmonary oedema. In addition, ADM inhibits the renin-angiotensin-aldosterone system. ADM is strongly elevated in patients with sepsis and in patients with acute heart failure. Since hallmarks of both conditions are vascular leakage and tissue oedema, we hypothesize that ADM plays a compensatory role and may exert protective properties against fluid overload and tissue congestion. Recently, a new immunoassay that specifically measures the biologically active ADM (bio-ADM) has been developed, and might become a biomarker for tissue congestion. As a consequence, measurement of bio-ADM might potentially be used to guide diuretic therapy in patients with heart failure. In addition, ADM might be used to guide treatment of (pulmonary) oedema or even become a target for therapy. Adrecizumab is a humanized, monoclonal, non-neutralizing ADM-binding antibody with a half-life of 15 days. Adrecizumab binds at the N-terminal epitope of ADM, leaving the C-terminal side intact to bind to its receptor. Due to its high molecular weight, the antibody adrecizumab cannot cross the endothelial barrier and consequently remains in the circulation. The observation that adrecizumab increases plasma concentrations of ADM indicates that ADM-binding by adrecizumab is able to drain ADM from the interstitium into the circulation. We therefore hypothesize that administration of adrecizumab improves vascular integrity, leading to improvement of tissue congestion and thereby may improve clinical outcomes in patients with acute decompensated heart failure. A phase II study with adrecizumab in patients with sepsis is ongoing and a phase II study on the effects of adrecizumab in patients with acute decompensated heart failure with elevated ADM is currently in preparation.

Adrenomedullin is a neuropeptide known for its cardiovascular activities and anti-inflammatory effects. Here, we investigated the effect of adrenomedullin in a model of experimental autoimmune encephalomyelitis (EAE) that mirrors chronic progressive multiple sclerosis. A short-term systemic treatment with adrenomedullin reduced clinical severity and incidence of EAE, the appearance of inflammatory infiltrates in spinal cord and the subsequent demyelination and axonal damage. This effect was exerted at multiple levels affecting both early and late events of the disease. Adrenomedullin decreased the presence/activation of encephalitogenic Th1 and Th17 cells and down-regulated several inflammatory mediators in peripheral lymphoid organs and central nervous system. Noteworthy, adrenomedullin inhibited the production by encephalitogenic cells of osteopontin and of Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), two critical cytokines in the development of EAE. At the same time, adrenomedullin increased the number of IL-10-producing regulatory T cells with suppressive effects on the progression of EAE. Furthermore, adrenomedullin generated dendritic cells with a semi-mature phenotype that impaired encephalitogenic responses in vitro and in vivo. Finally, adrenomedullin regulated glial activity and favored an active program of neuroprotection/regeneration. Therefore, the use of adrenomedullin emerges as a novel multimodal therapeutic approach to treat chronic progressive multiple sclerosis.

Adrenomedullin (ADM) is a multifunctional hormone that regulates processes as diverse as blood pressure and cell growth. Although expressed in the ovary, the role of ADM in this organ is not clear. In the present study, we found the expression of ADM receptor and receptor activity-modifying proteins in mouse cumulus cells but not in the oocytes. We report that germinal vesicle breakdown (GVBD), which is required for oocyte maturation, is not inhibited by ADM alone. However, ADM in the presence of the nitric oxide donor sodium nitroprusside (SNP) significantly inhibited GVBD. Furthermore, the ADM- and SNP-dependent inhibition of GVBD was abrogated by Akt blockade. Additionally, Akt expression and phosphorylation was exhibited by ADM, suggesting that Akt signaling upstream in cumulus cells is responsible. Additionally, immunohistochemical analysis revealed that ADM was localized in the granulosa cells of developed follicles, implying the possibility that ADM physiologically affects oocyte maturation in vivo. Our results provide the evidence that ADM can act as a GVBD regulator.

Chondrocyte apoptosis may have a pivotal role in the development of osteoarthritis. Interest has increased in the use of anti-apoptotic compounds to protect against osteoarthritis development. In this work, we investigated the effect of adrenomedullin (AM), a 52 amino-acid hormone peptide, and a 31 amino-acid truncated form, AM(22-52), on chondrocyte apoptosis. Bovine articular chondrocytes (BACs) were cultured under hypoxic conditions to mimic cartilage environment and then treated with Fas ligand (Fas-L) to induce apoptosis. The expression of AM and its calcitonin receptor-like receptor (CLR)/receptor activity-modifying protein (RAMP) (receptor/co-receptor) was assessed by immunostaining. We evaluated the effect of AM and AM(22-52) on Fas-L-induced chondrocyte apoptosis. FAS expression was appreciated by RT-qPCR and immunostainings. The expression of hypoxia-inducible factor 1α (HIF-1α), CLR and one co-receptor (RAMP2) was evidenced. With BACs under hypoxia, cyclic adenosine monophosphate production increased dose-dependently with AM stimulation. AM significantly decreased caspase-3 activity (mean 35% decrease; p = 0.03) as a marker of Fas-L-induced apoptosis. Articular chondrocytes treated with AM showed significantly reduced cell death, along with downregulated Fas expression and production, as compared with AM(22-52). AM decreased articular chondrocyte apoptosis by downregulating a Fas receptor. These findings may pave the way for novel therapeutic approaches in osteoarthritis.

The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13-52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13-52) binds to an apparent single population of sites with similar affinities (K(D) of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (B(max) of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1-52) and human adrenomedullin-(13-52) competed against specific [125I]human adrenomedullin-(13-52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide alpha (hCGRPalpha), [Cys(Et)(2,7)]hCGRPalpha and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13-52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPgammaS (100 microM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13-52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1-52) and [Cys(Et)(2,7)]hCGRPalpha against specific [125I]human adrenomedullin-(13-52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13-52) binding sites that are resistant to human adrenomedullin-(22-52) and human CGRP-(8-37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.

Adrenomedullin has been shown to be overexpressed in many tumors, including gastric cancer tumors; however, its mechanism of action remains unclear. In this study, we examined the role of adrenomedullin in the pathogenesis of gastric cancer. Using clinical specimens and immunohistochemistry, we found that the expression levels of adrenomedullin and its receptors are inordinately elevated as compared to the adjacent non-tumor gastric tissues. We used siRNA gene silencing, in BGC-823 gastric cancer cell lines, to target adrenomedullin genes, and found that increased adrenomedullin expression results in the proliferation of tumor cells, tumor invasion, and metastasis. Furthermore, we found that under hypoxic conditions, gastric cancer BGC-823 cells exhibit higher expression levels of adrenomedullin and various other related proteins. Our results indicate the involvement of adrenomedullin in microvessel proliferation and partially in the release of hypoxia in solid tumors. Knockdown of adrenomedullin expression, at the protein level, reduced the levels of phosphoprotein kinase B and B-cell lymphoma 2 but increased the levels of cleaved-caspase3 and Bcl 2 associated x protein (Bax). Therefore, we hypothesized siRNA targeting of adrenomedullin genes inhibits various serine/threonine kinases via a signaling pathway that induces cell apoptosis. SiRNA targeting of adrenomedullin genes and green fluorescent control vectors were used to transfect BGC-823 cells, and western blot analyses were used to detect changes in the rates of autophagy in related proteins using confocal laser scanning microscopy. No significant changes were detected. Therefore, the knockdown of adrenomedullin and its receptors may represent a novel treatment strategy for gastric cancer.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

Within the tumor microenvironment, tumor cells and endothelial cells regulate each other. While tumor cells induce angiogenic responses in endothelial cells, endothelial cells release angiocrine factors, which act on tumor cells and other stromal cells. We report that tumor cell-derived adrenomedullin has a pro-angiogenic as well as a direct tumor-promoting effect, and that endothelium-derived CC chemokine ligand 2 (CCL2) suppresses adrenomedullin-induced tumor cell proliferation. Loss of the endothelial adrenomedullin receptor CALCRL or of the G-protein Gs reduced endothelial proliferation. Surprisingly, tumor cell proliferation was also reduced after endothelial deletion of CALCRL or Gs. We identified CCL2 as a critical angiocrine factor whose formation is inhibited by adrenomedullin. Furthermore, CCL2 inhibited adrenomedullin formation in tumor cells through its receptor CCR2. Consistently, loss of endothelial CCL2 or tumor cell CCR2 normalized the reduced tumor growth seen in mice lacking endothelial CALCRL or Gs. Our findings show tumor-promoting roles of adrenomedullin and identify CCL2 as an angiocrine factor controlling adrenomedullin formation by tumor cells.

Adrenomedullin (AM) is postulated to exert organ-protective effects. It is expressed in the renal glomeruli, but its roles in the glomerular podocytes have been poorly elucidated. In the present study, we investigated the expression and regulation of AM in recently established conditionally immortalized mouse podocyte cell line in vitro and podocyte injury model in vivo. The cultured differentiated podocytes expressed AM mRNA and secreted measurable amount of AM. AM secretion from the podocytes was increased by H(2)O(2), hypoxia, puromycin aminonucleoside (PAN), albumin overload, and TNF-alpha. Real-time RT-PCR analysis revealed that AM mRNA expression in the podocytes was enhanced by PAN and TNF-alpha, both of which were suppressed by mitochondrial antioxidants. Furthermore, AM expression was upregulated in the glomerular podocytes of PAN nephrosis rats. These results indicated that AM expression in the podocytes was upregulated by stimuli or condition relevant to podocyte injury, suggesting its potential role in podocyte pathophysiology.

Adrenomedullin (AM), a 52-amino acid ringed-structure peptide with C-terminal amidation, was originally isolated from human pheochromocytoma. AM are widely distributed in various tissues and acts as a local vasoactive hormone in various conditions.