Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein-coupled receptor (CLR), which heterodimerizes with three receptor activity-modifying proteins (RAMP1-3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein-tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses.

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina.

PURPOSE: Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. METHODS: Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. RESULTS: Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. CONCLUSION: Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties.

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

Fluorescently labeled adrenomedullin allows real-time monitoring of adrenomedullin receptor trafficking in living cells.

The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM1), a heterodimer consisting of the calcitonin receptor-like receptor (CLR), a G protein-coupled receptor, associated with the receptor activity-modifying protein 2 (RAMP2). Full length and N-terminally shortened ADM peptides were synthesized using Fmoc/tBu solid phase peptide synthesis and site-specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)-catalyzed azide alkyne cycloaddition. For the first time, Tam-labeled ligands allowed the observation of co-internalization of the whole ligand-receptor complex in living cells co-transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2-complex is routed to the degradative pathway. Moreover, we found that the N-terminus of ADM is not a crucial component of the peptide sequence in terms of AM1 internalization behavior.

Receptor activity-modifying proteins of adrenomedullin (RAMP2/3): Roles in the pathogenesis of ARDS.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung condition characterized by widespread inflammation and pulmonary edema. Adrenomedullin (AM), a bioactive peptide with various functions, is expected to be applied in treating ARDS. Its functions are regulated primarily by two receptor activity-modifying proteins, RAMP2 and RAMP3, which bind to the AM receptor calcitonin receptor-like receptor (CLR). However, the roles of RAMP2 and RAMP3 in ARDS remain unclear. We generated a mouse model of ARDS via intratracheal administration of lipopolysaccharide (LPS), and analyzed the pathophysiological significance of RAMP2 and RAMP3. RAMP2 expression declined with LPS administration, whereas RAMP3 expression increased at low doses and decreased at high doses of LPS. After LPS administration, drug-inducible vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2-/-) showed reduced survival, increased lung weight, and had more apoptotic cells in the lungs. DI-E-RAMP2-/- mice exhibited reduced expression of Epac1 (which regulates vascular endothelial cell barrier function), while RAMP3 was upregulated in compensation. In contrast, after LPS administration, RAMP3-/- mice showed no significant changes in survival, lung weight, or lung pathology, although they exhibited significant downregulation of iNOS, TNF-α, and NLRP3 during the later stages of inflammation. Based on transcriptomic analysis, RAMP2 contributed more to the circulation-regulating effects of AM, whereas RAMP3 contributed more to its inflammation-regulating effects. These findings indicate that, while both RAMP2 and RAMP3 participate in ARDS pathogenesis, their functions differ distinctly. Further elucidation of the pathophysiological significance and functional differences between RAMP2 and RAMP3 is critical for the future therapeutic application of AM in ARDS.

Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling.

Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor.

Receptor activity modifying protein-3 mediates the protumorigenic activity of lysyl oxidase-like protein-2.

Lysyl oxidase-like protein-2 (LOXL2) induces epithelial to mesenchymal transition and promotes invasiveness. To understand the mechanisms involved, we examined the effect of LOXL2 overexpression in MCF-7 cells on gene expression. We found that LOXL2 up-regulated the expression of receptor activity modifying protein-3 (RAMP3). Expression of RAMP3 in MDA-MB-231 cells in which LOXL2 expression was inhibited restored vimentin expression, invasiveness, and tumor development. Inhibition of RAMP3 expression in MDA-MB-231 cells mimicked the effects produced by inhibition of LOXL2 expression and was accompanied by inhibition of p38 phosphorylation. LOXL2 overexpression in these cells did not restore invasiveness, suggesting that RAMP3 functions downstream to LOXL2. LOXL2 and RAMP3 are strongly coexpressed in human colon, breast, and gastric carcinomas but not in normal colon or gastric epithelial cells. RAMP3 associates with several G-protein-coupled receptors forming receptors for peptides, such as adrenomedullin and amylin. We hypothesized that RAMP3 could function as a transducer of autocrine signals induced by such peptides. However, the proinvasive effects of RAMP3 could not be abrogated following inhibition of the expression or activity of these peptides. Our experiments suggest that the protumorigenic effects of LOXL2 are partially mediated by RAMP3 and that RAMP3 inhibitors may function as antitumorigenic agents. -

Adrenomedullin: a possible regulator of germinal vesicle breakdown.

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.

The antifibrosis effect of adrenomedullin in human lung fibroblasts.

Adrenomedullin (AM) is a regulatory peptide involved in cellular proliferation and protein synthesis. The authors investigated AM and the AM receptor system in the human fetal lung fibroblasts (HFLFs), and assessed whether AM can inhibit proliferation and collagen synthesis in HFLFs under hypoxia. Fibroblasts were exposed to hypoxia (2% O(2)) after the addition of AM. The effects of AM and transforming growth factor ß1 (TGF-ß1) on the proliferation of fibroblasts were determined by the methanethiosulfonate (MTS) assay. Total collagen synthesis was determined by [(3)H]proline incorporation. TGF-ß1 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The concentration of intracellular calciumion ([Ca(2+)](i)) in fibroblasts was detected with a laser scanning confocal microscope. AM, adrenomedullin receptor (ADMR), calcitonin receptor-like receptor (CRLR), AM receptor chaperone receptor activity-modifying protein-1 (RAMP1),RAMP2, and RAMP3 were detected in the HFLFs. The hypoxia-induced increases in cell proliferation, collagen synthesis, and TGF-ß1 production were inhibited by AM. AM also inhibited proliferation and collagen synthesis in fibroblasts induced by TGF-ß1. AM caused a decrease of the hypoxia-induced [Ca(2+)](i) in fibroblasts. This study suggests that AM is produced by HFLFs and AM may function as an antifibrosis factor that protects cells from hypoxic pulmonary damage through its receptors.

Altered Expression of Adrenomedullin 2 and its Receptor in the Adipose Tissue of Obese Patients.

CONTEXT: Adrenomedullin 2 (AM2) plays protective roles in the renal and cardiovascular systems. Recent studies in experimental animals demonstrated that AM2 is an adipokine with beneficial effects on energy metabolism. However, there is little information regarding AM2 expression in human adipose tissue. OBJECTIVE: To investigate the pattern and regulation of the expression of AM2 and its receptor component in human adipose tissue, in the context of obesity and type 2 diabetes. METHODS: We measured metabolic parameters, serum AM2, and expression of ADM2 and its receptor component genes in abdominal subcutaneous and visceral adipose tissue in obese (with or without type 2 diabetes) and normal-weight women. Serum AM2 was assessed before and 6 to 9 months after bariatric surgery. Expression/secretion of AM2 and its receptor were assessed in human adipocytes. RESULTS: ADM2 mRNA in both fat depots was higher in obese patients, whether diabetic or not. Although serum AM2 was significantly lower in obese patients, it was not changed after bariatric surgery. AM2 and its receptor complex were predominantly expressed by adipocytes, and the expression of CALCRL, encoding a component of the AM2 receptor complex, was lower in both fat depots of obese patients. Incubating adipocytes with substances mimicking the microenvironment of obese adipose tissue increased ADM2 mRNA but reduced both AM2 secretion into culture media and CALCRL mRNA expression. CONCLUSIONS: Our data indicate that AM2 signaling is suppressed in adipose tissue in obesity, involving lower receptor expression and ligand availability, likely contributing to insulin resistance and other aspects of the pathophysiology associated with obesity.

Adrenomedullin insufficiency alters macrophage activities in fallopian tube: a pathophysiologic explanation of tubal ectopic pregnancy.

Ectopic pregnancy is the major cause of maternal morbidity and mortality in the first trimester of pregnancy. Tubal ectopic pregnancy (TEP) accounts for nearly 98% of all ectopic pregnancies. TEP is usually associated with salpingitis but the underlying mechanism in salpingitis leading to TEP remains unclear. Adrenomedullin (ADM) is a peptide hormone abundantly expressed in the fallopian tube with potent anti-inflammatory activities. Its expression peaks at the early luteal phase when the developing embryo is being transported through the fallopian tube. In the present study, we demonstrated reduced expression of ADM in fallopian tubes of patients with salpingitis and TEP. Using macrophages isolated from the fallopian tubes of these women, our data revealed that the salpingistis-associated ADM reduction contributed to aggravated pro-inflammatory responses of the tubal macrophages resulting in production of pro-inflammatory and pro-implantation cytokines IL-6 and IL-8. These cytokines activated the expression of implantation-associated molecules and Wnt signaling pathway predisposing the tubal epithelium to an adhesive and receptive state for embryo implantation. In conclusion, this study provided evidence for the role of ADM in the pathogenesis of TEP through regulating the functions of tubal macrophages.

PulmoBind Imaging Measures Reduction of Vascular Adrenomedullin Receptor Activity with Lack of effect of Sildenafil in Pulmonary Hypertension.

Endothelial dysfunction is a core pathophysiologic process in pulmonary arterial hypertension (PAH). We developed PulmoBind (PB), a novel imaging biomarker of the pulmonary vascular endothelium. 99mTechnetium (99mTc)-labelled PB binds to adrenomedullin receptors (AM1) densely expressed in the endothelium of alveolar capillaries. We evaluated the effect of sildenafil on AM1 receptors activity using 99mTc-PB. PAH was induced in rats using the Sugen/hypoxia model and after 3 weeks, animals were allocated to sildenafil (25 or 100 mg/kg/day) for 4 weeks. 99mTc-PB uptake kinetics was assessed by single-photon emission computed tomography. PAH caused right ventricular (RV) hypertrophy that was decreased by low and high sildenafil doses. Sildenafil low and high dose also improved RV function measured from the tricuspid annulus plane systolic excursion. Mean integrated pulmonary uptake of 99mTc-PB was reduced in PAH (508% · min ± 37, p < 0.05) compared to controls (630% · min ± 30), but unchanged by sildenafil at low and high doses. Lung tissue expressions of the AM1 receptor components were reduced in PAH and also unaffected by sildenafil. In experimental angio-proliferative PAH, sildenafil improves RV dysfunction and remodeling, but does not modify pulmonary vascular endothelium dysfunction assessed by the adrenomedullin receptor ligand 99mTc-PB.

Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs.

Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.

Stromal fibroblasts present in breast carcinomas promote tumor growth and angiogenesis through adrenomedullin secretion.

Tumor- or cancer-associated fibroblasts (TAFs or CAFs) are active players in tumorigenesis and exhibit distinct angiogenic and tumorigenic properties. Adrenomedullin (AM), a multifunctional peptide plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). We show that AM and AM receptors mRNAs are highly expressed in CAFs prepared from invasive breast carcinoma when compared to normal fibroblasts. Immunostaining demonstrates the presence of immunoreactive AM and AM receptors in the CAFs (n = 9). The proliferation of CAFs is decreased by anti-AM antibody (αAM) and anti-AM receptors antibody (αAMR) treatment, suggesting that AM may function as a potent autocrine/paracrine growth factor. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing CAFs as demonstrated by reduced numbers of the vessel structures, suggesting that AM is one of the CAFs-derived factors responsible for endothelial cell-like and pericytes recruitment to built a neovascularization. We show that MCF-7 admixed with CAFs generated tumors of greater volume significantly different from the MCF-7 xenografts in nude mice due in part to the induced angiogenesis. αAMR and AM22-52 therapies significantly suppressed the growth of CAFs/MCF-7 tumors. Histological examination of tumors treated with AM22-52 and aAMR showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells, induced apoptosis and decrease of tumor cell proliferation. Our findings highlight the importance of CAFs-derived AM pathway in growth of breast carcinoma and in neovascularization by supplying and amplifying signals that are essential for pathologic angiogenesis.

Endothelial Restoration of Receptor Activity-Modifying Protein 2 Is Sufficient to Rescue Lethality, but Survivors Develop Dilated Cardiomyopathy.

RAMPs (receptor activity-modifying proteins) serve as oligomeric modulators for numerous G-protein-coupled receptors, yet elucidating the physiological relevance of these interactions remains complex. Ramp2 null mice are embryonic lethal, with cardiovascular developmental defects similar to those observed in mice null for canonical adrenomedullin/calcitonin receptor-like receptor signaling. We aimed to genetically rescue the Ramp2(-/-) lethality in order to further delineate the spatiotemporal requirements for RAMP2 function during development and thereby enable the elucidation of an expanded repertoire of RAMP2 functions with family B G-protein-coupled receptors in adult homeostasis. Endothelial-specific expression of Ramp2 under the VE-cadherin promoter resulted in the partial rescue of Ramp2(-/-) mice, demonstrating that endothelial expression of Ramp2 is necessary and sufficient for survival. The surviving Ramp2(-/-) Tg animals lived to adulthood and developed spontaneous hypotension and dilated cardiomyopathy, which was not observed in adult mice lacking calcitonin receptor-like receptor. Yet, the hearts of Ramp2(-/-) Tg animals displayed dysregulation of family B G-protein-coupled receptors, including parathyroid hormone and glucagon receptors, as well as their downstream signaling pathways. These data suggest a functional requirement for RAMP2 in the modulation of additional G-protein-coupled receptor pathways in vivo, which is critical for sustained cardiovascular homeostasis. The cardiovascular importance of RAMP2 extends beyond the endothelium and canonical adrenomedullin/calcitonin receptor-like receptor signaling, in which future studies could elucidate novel and pharmacologically tractable pathways for treating cardiovascular diseases.

Functional Analysis of the Adrenomedullin Pathway in Malignant Pleural Mesothelioma.

INTRODUCTION: Malignant pleural mesothelioma (MPM) grows aggressively within the thoracic cavity and has a very low cure rate, thus highlighting the need for identification of new therapeutic targets. Adrenomedullin (AM) is a multifunctional peptide that is highly expressed in several tumors and plays an important role in angiogenesis and tumor growth after binding to its receptors, calcitonin receptor-like receptor/receptor activity-modifying protein 2 (CLR/RAMP2) and calcitonin receptor-like receptor/receptor activity-modifying protein 3 (CLR/RAMP3). METHODS: Real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the steady-state levels of AM, CLR, RAMP2 and RAMP3 messenger RNA (mRNA) transcripts in normal pleural tissue (n=5) and MPM (n=24). The expression of these candidates at protein level was revealed by immunohistochemistry. We also characterized the expression and regulation by hypoxia of AM system in MPM cell lines and MeT-5A cells. In vitro and in vivo studies were performed to determine the functional role of AM system in MPM. RESULTS: In this study, real-time quantitative reverse transcriptase polymerase chain reaction showed twofold to 10-fold higher levels of AM messenger RNA in MPM tissue than in normal pleural tissue. The MPM cell lines H2452, H2052, and human mesothelioma cell line MSTO-211H showed a significant increase in expression of AM messenger RNA under hypoxic conditions. Our results also show that AM stimulates cell proliferation in vitro through the Raf1 proto-oncogene, serine/threonine kinase (CRAF)/ Mitogen-activated protein kinase kinase 1 (MEK)/Extracellular regulated MAPKinase (ERK) pathway. Furthermore, the proliferation, migration, and invasion of MPM cells were decreased after treatment with anti-AM (αAM) and anti-AM receptor antibodies, thus indicating that MPM cells are regulated by AM. The action of AM was specific and mediated by CLR/RAMP2 and CLR/RAMP3 receptors. In vivo, αAM and AM22-52 antagonist therapies blocked angiogenesis and induced apoptosis in MSTO-211H xenografts, thereby resulting in tumor regression. Histologic examination of tumors treated with AM22-52 and αAM antibody showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. CONCLUSIONS: Our findings highlight the importance of the AM pathway in growth of MPM and in neovascularization by supplying and amplifying signals that are essential for pathologic neoangiogenesis and lymphangiogenesis.

Pathophysiological roles of adrenomedullin-RAMP2 system in acute and chronic cerebral ischemia.

The accessory protein RAMP2 is a component of the CLR/RAMP2 dimeric adrenomedullin (AM) receptor and is the primary determinant of the vascular functionality of AM. RAMP2 is highly expressed in the brain; however, its function there remains unclear. We therefore used heterozygous RAMP2 knockout (RAMP2+/-) mice, in which RAMP2 expression was reduced by half, to examine the actions of the endogenous AM-RAMP2 system in cerebral ischemia. To induce acute or chronic ischemia, mice were subjected to middle cerebral artery occlusion (MCAO) or bilateral common carotid artery stenosis (BCAS), respectively. In RAMP2+/- mice subjected to MCAO, recovery of cerebral blood flow (CBF) was slower than in WT mice. AM gene expression was upregulated after infarction in both genotypes, but the increase was greater in RAMP2+/- mice. Pathological analysis revealed severe nerve cell death and demyelination, and a higher level of oxidative stress in RAMP2+/- mice. In RAMP2+/- mice subjected to BCAS, recovery of cerebral perfusion was slower and less complete than in WT mice. In an 8-arm radial maze test, RAMP2+/- mice required more time to solve the maze and showed poorer reference memory. They also showed greater reductions in nerve cells and less compensatory capillary growth than WT mice. These results indicate the AM-RAMP2 system works to protect nerve cells from both acute and chronic cerebral ischemia by maintaining CBF, suppressing oxidative stress, and in the case of chronic ischemia, enhancing capillary growth.