High Adrenomedullin Research Articles

Utility of Adrenomedullin in Predicting Outcomes in Patients with Acute Febrile Illness in the Tropics: A Prospective Observational Study.

Acute febrile illnesses (AFI) such as dengue, leptospirosis, and scrub typhus start with fever, but some patients can deteriorate rapidly. Identifying these patients at risk is necessary to ensure early referral. The study's objective was to assess the utility of serum adrenomedullin (ADM) as a biomarker in predicting outcomes in adult AFI patients presenting to the emergency department (ED). In this prospective observational study, all consecutive patients admitted with AFI through the ED between June 2023 and February 2024 were assessed for eligibility. Clinical and laboratory findings were noted. The serum samples sent routinely for biochemical tests were used to calculate ADM levels at admission and 48 hours later (whenever possible). Clinically relevant outcome variables were collected for all patients. The utility of ADM in predicting 7-day mortality was evaluated for primary outcome analysis using a stepwise binary logistic regression. A total of 181 patients were recruited, with dengue (n = 91, 50%) being the most common diagnosis. The median sequential organ failure assessment score at admission was 5 (IQR: 3-10). The 7-day mortality was 26 (14%, 95% CI: 9.3-19.5). The median admission ADM levels and difference in ADM levels at 48 hours were significantly higher in those who died by day 7. Adrenomedullin levels at admission were found to be an independent predictor of 7-day mortality (adjusted odds ratio: 1.002, 95% CI: 1.001-1.004, P = 0.005). High ADM levels can be used for early referral in patients with tropical AFI. There is a need to explore the utility of antibodies targeting ADM in clinical studies.

Adrenomedullin has a pivotal role in trophoblast differentiation: A promising nanotechnology-based therapeutic target for early-onset preeclampsia.

Early-onset preeclampsia (EOPE) is a severe pregnancy complication associated with defective trophoblast differentiation and functions at implantation, but manifestation of its phenotypes is in late pregnancy. There is no reliable method for early prediction and treatment of EOPE. Adrenomedullin (ADM) is an abundant placental peptide in early pregnancy. Integrated single-cell sequencing and spatial transcriptomics confirm a high ADM expression in the human villous cytotrophoblast and syncytiotrophoblast. The levels of ADM in chorionic villi and serum were lower in first-trimester pregnant women who later developed EOPE than those with normotensive pregnancy. ADM stimulates differentiation of trophoblast stem cells and trophoblast organoids in vitro. In pregnant mice, placenta-specific ADM suppression led to EOPE-like phenotypes. The EOPE-like phenotypes in a mouse PE model were reduced by a placenta-specific nanoparticle-based forced expression of ADM. Our study reveals the roles of trophoblastic ADM in placental development, EOPE pathogenesis, and its potential clinical uses.

Effects of enrichment strategies on outcome of adrecizumab treatment in septic shock: Post-hoc analyses of the phase II adrenomedullin and outcome in septic shock 2 trial.

Adrecizumab, a non-neutralizing antibody of adrenomedullin (ADM) was recently investigated regarding its potential to restore endothelial barrier function in septic shock patients with high plasma ADM levels. Circulating dipeptidyl peptidase 3 (cDPP3), a protease involved in the degradation of several cardiovascular mediators, represents another biological pathway strongly associated with outcome in septic shock, although unrelated to ADM. Therefore, the prognosis of patients with elevated cDPP3 may not be influenced by Adrecizumab. Also, time until initiation of treatment may influence efficacy. To evaluate effects of cDPP3-based enrichment on treatment efficacy of Adrecizumab. Post-hoc analysis of AdrenOSS-2, a phase-II, double-blind, randomized, placebo-controlled biomarker-guided trial of Adrecizumab. Compared to the total study cohort [HR for 28-day mortality of 0.84 (95% CI 0.53;1.31), p = 0.439], therapeutic benefit of Adrecizumab tended to be more pronounced in the subgroup of 249 patients with low cDPP3 (<50 ng/mL); [HR of 0.61 (95% CI 0.34;1.08), p = 0.085]. Median duration to study drug infusion was 8.5 h. In the subgroup of 129 patients with cDPP3 <50 ng/mL and an early start of treatment (<8.5 h after septic shock diagnosis) HR for 28-day mortality vs. placebo was 0.49 (95% CI 0.21-1.18), p = 0.105. In multivariate interaction analyses corrected for baseline disease severity, both cDPP3, as well as the cDPP3 * treatment interaction term were associated with a reduced HR for 28-day mortality in the Adrecizumab treated group; p = 0.015 for cDPP3 in univariate analysis, p = 0.025 for the interaction term between cDPP3 and treatment group. In contrast, treatment timing was not significantly associated with 28-day mortality in multivariate interaction analyses. In septic shock patients with high ADM levels, a further post-hoc enrichment strategy based on cDPP3 may indicate (with all the caveats to be considered for post-hoc subgroup analyses) that therapeutic efficacy is most pronounced in patients with lower cDPP3 levels.

Associations of Plasma Bioactive Adrenomedullin Levels with Cardiovascular Risk Factors in BRCA1/2 Mutation Carriers

Background Cardiovascular disease (CVD) is an important cause of morbidity and mortality in breast cancer survivors. Effective screening modalities to identify CVD risk are lacking in this population. Adrenomedullin (ADM) has been suggested as a biomarker for subclinical cardiac dysfunction in the general population. Levels of ADM have been proven to be responsive to lifestyle changes that lead to improved cardiovascular health. As BRCA1/2 mutation carriers are deemed to be at an increased risk for CVD, the aim of this study was to examine plasma ADM levels in a cohort of BRCA mutation carriers and to assess their association with cardiovascular risk factors. Methods Plasma ADM concentrations were measured in 292 female BRCA1/2 mutation carriers with and without a history of breast cancer. Subjects were classified into high versus low ADM levels based on the median ADM level in the entire cohort (13.8 pg/mL). Logistic regression models were used to estimate the odds ratios (OR) of having elevated ADM levels by several cardiovascular risk factors. Results Of all women (median age: 43 years), 57.5% had a previous diagnosis of breast cancer. The median time between diagnosis and study entry was three years (range: 0 – 32 years). Women presenting with metabolic syndrome had 22-fold increased odds of having elevated ADM levels (p < 0.001). Elevated ADM levels were associated with lower cardiorespiratory fitness (OR = 0.88, p < 0.001) and several parameters of obesity (p < 0.001). ADM levels were higher in women who have ever smoked (OR = 1.72, p = 0.02). ADM levels were not associated with a previous diagnosis of breast cancer (p = 0.28). Conclusions This is the first study in BRCA mutation carriers that has linked circulating ADM levels to traditional cardiovascular risk factors. The long-term clinical implications of these findings are yet to be determined.

Abstract P6-10-16: High circulating levels of adrenomedullin are associated with metabolic syndrome and low cardiorespiratory fitness in BRCA1 and BRCA2 mutation carriers

Abstract BACKGROUND: Risk factors for cardiovascular disease (CVD) and breast cancer overlap substantially. Identification of the underlying mechanisms associated with this co-occurrence is of great public health importance. Adrenomedullin (AM) is an almost ubiquitously expressed peptide with vasodilator and natriuretic properties. Previous studies have observed a link between high AM levels and worse prognosis in patients with myocardial infarction and heart failure, indicating a crucial role in the pathophysiology of CVD. Moreover, AM is expressed in 80% of sporadic breast cancers, and the degree of expression is associated with tumour growth, local tumour progression and bone metastases. High plasma AM levels are linked to arterial hypertension, diabetes, obesity and smoking. Preliminary evidence suggests that AM influences the osteoclast differentiation mediated by Receptor Activator of NF-κB Ligand (RANKL), an important signalling pathway in BRCA1-associated breast tumorigenesis. OBJECTIVE: Besides an elevated risk of breast cancer, recent studies revealed that BRCA mutation carriers are potentially at higher cardiovascular risk. The value of AM in BRCA mutation carriers is unknown. The aim of this study was to examine circulating plasma AM levels in BRCA mutation carriers and assess their association with modifiable risk factors. METHODS: AM concentrations were measured in 290 BRCA1/2 mutation carriers without overt CVD, participating in the randomized controlled LIBRE study (NCT numbers: NCT02087592, NCT02516540), by an immunoassay (sphingotest® bio-ADM®). Subjects were classified into high versus low AM levels based on the median plasma AM level in the entire cohort (13.9 pg/mL). Univariate logistic regression models were used to estimate the odds ratio (OR) of having high circulating AM levels by metabolic syndrome (MetS), cardiorespiratory fitness (CRF), body mass index (BMI), circulating insulin, smoking, current age, BRCA mutation status (BRCA1 or BRCA2) and previous diagnosis of breast cancer. MetS was defined in accordance with the criteria of the Third Adult Treatment Panel (ATP III). CRF was measured by peak oxygen uptake (VO2peak) assessed on a cycle ergometer via standardized cardiopulmonary exercise testing. RESULTS: Of all women (median age: 43 years), 57.9% had a previous diagnosis of breast cancer. The median time between diagnosis and study entry was 3 years (range: 0-32 years). Women fulfilling the criteria of metabolic syndrome had over 17 times higher odds of having increased AM levels compared to those who did not meet the criteria (OR = 17.49, p &amp;lt; 0.001). Moreover, high AM levels were associated with lower VO2peak (OR = 0.91, p &amp;lt; 0.001), higher BMI (OR = 1.24, p &amp;lt; 0.001) and higher circulating insulin levels (OR = 1.1, p &amp;lt; 0.001). AM levels were higher in women who have ever smoked (OR = 1.73, p = 0.022), and AM levels increased with the number of pack-years smoked (OR = 1.03, p = 0.06). AM levels were not associated with age (p = 0.143), BRCA mutation status (p = 0.51) or previous diagnosis of breast cancer (p = 0.48). After adjustment for confounding variables, we observed that MetS (OR = 10.78, p = 0.002) and VO2peak (OR = 0.92, p = 0.001) were independent determinants of circulating AM. CONCLUSIONS: This is the first study in BRCA mutation carriers that has linked circulating AM levels to MetS and CRF. The long-term clinical implications of these findings are yet to be determined. Conflict of interest: The study is funded by the German Cancer Aid (Deutsche Krebshilfe) within the Priority Program “Primary Prevention of Cancer” (Grant no. 110013). JS and OH are employed by Sphingotec GmbH, a company having patent rights in and commercializing the bio-ADM assay. SG and MK received grants from Sphingotec GmbH. JL, MYD, MB and MH have nothing to disclose. Citation Format: Jacqueline Lammert, Sabine Grill, Maryam Yahiaoui-Doktor, Maryam Basrai, Joachim Struck, Oliver Hartmann, Martin Halle, Marion Kiechle. High circulating levels of adrenomedullin are associated with metabolic syndrome and low cardiorespiratory fitness in BRCA1 and BRCA2 mutation carriers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-16.

A double-blind, placebo-controlled, randomised, multicentre, proof-of-concept and dose-finding phase II clinical trial to investigate the safety, tolerability and efficacy of adrecizumab in patients with septic shock and elevated adrenomedullin concentration (AdrenOSS-2)

IntroductionSepsis remains a major health problem with an increasing incidence, high morbidity and high mortality. Apart from treatment with antibiotics and organ support, no approved specific adjunct therapies currently exist....

Adrenomedullin and tumour microenvironment.

Adrenomedullin (AM) is a regulatory peptide whose involvement in tumour progression is becoming more relevant with recent studies. AM is produced and secreted by the tumour cells but also by numerous stromal cells including macrophages, mast cells, endothelial cells, and vascular smooth muscle cells. Most cancer patients present high levels of circulating AM and in some cases these higher levels correlate with a worst prognosis. In some cases it has been shown that the high AM levels return to normal following surgical removal of the tumour, thus indicating the tumour as the source of this excessive production of AM. Expression of this peptide is a good investment for the tumour cell since AM acts as an autocrine/paracrine growth factor, prevents apoptosis-mediated cell death, increases tumour cell motility and metastasis, induces angiogenesis, and blocks immunosurveillance by inhibiting the immune system. In addition, AM expression gets rapidly activated by hypoxia through a HIF-1α mediated mechanism, thus characterizing AM as a major survival factor for tumour cells. Accordingly, a number of studies have shown that inhibition of this peptide or its receptors results in a significant reduction in tumour progression. In conclusion, AM is a great target for drug development and new drugs interfering with this system are being developed.

The role of glutamic acid 73 in adrenomedullin interactions with rodent AM2 receptors

Adrenomedullin (AM) is a peptide, which is important for vascular development. There is much interest in the clinical potential of its receptors. The mode of AM binding to its receptors is poorly understood. Previous studies have identified amino acid Glu74, which is found in the receptor activity-modifying protein (RAMP3) subunit of the AM2 receptor as important for high affinity AM interactions with this receptor. Its reciprocal residue in RAMP1 (Trp) impedes AM interactions in the closely related human calcitonin gene-related peptide (CGRP) receptor. The Glu is conserved in RAMP3 across species, supporting its role in contributing to AM binding. We mutated this residue in rat and mouse RAMP3 to Ala, Lys and Trp to determine its function in rodent AM2 receptors. Only the Trp substitution in mouse RAMP3 produced a substantial reduction in AM potency. However, mutation of the Lys found in rat RAMP1 to Glu enhanced AM potency. Although Glu is highly conserved in RAMP3, this work suggests that it may only make a small or indirect contribution to AM interactions. Nevertheless, the equivalent amino acid in RAMP1 may serve to impair high affinity AM interactions.

Adrenomedullin expression in epithelial ovarian cancers and promotes HO8910 cell migration associated with upregulating integrin α5β1 and phosphorylating FAK and paxillin

BackgroundEpithelial ovarian cancer (EOC) is one of the leading causes of cancer deaths in women worldwide. Adrenomedullin (AM) is a multifunctional peptide which presents in various kinds of tumors.MethodsIn this study, we characterized the expression and function of AM in epithelial ovarian cancer using immunohistochemistry staining. Exogenous AM and small interfering RNA (siRNA) specific for AM receptor CRLR were treated to EOC cell line HO8910. Wound healing assay and flow cytometry were used to measure the migration ability and expression of integrin α5 of HO8910 cells after above treatments. Western blot was used to examine the phosphorylation of FAK and paxillin.ResultsWe found that patients with high AM expression showed a higher incidence of metastasis, larger residual size of tumors after cytoreduction and shorter disease-free and overall survival time. Exogenous AM induced ovarian cancer cell migration in time- and dose- dependent manners. AM upregulated the expression of integrin α5 and phosphorylation of FAK, paxillin as well.ConclusionsOur results suggested that AM contributed to the progression of EOC and had additional roles in EOC cell migration by activating the integrin α5β1 signaling pathway. Therefore, we presumed that AM could be a potential molecular therapeutic target for ovarian carcinoma.

The relationship of adrenomedullin and left ventricular remodeling in older patients with essential hypertension

ObjectiveTo investigate the relationship of adrenomedullin (ADM) and left ventricular remodeling in older patients with essential hypertension.Methods77 essential hypertension patients were divided into normal left ventricular group (normal group), left...

Fetal Loss of Adrenomedullin Causes Maternal Vascular Pathology Typical of Preeclampsia.

Adrenomedullin (AM) is a 52-amino acid peptide vasodilator that is elevated 3 to 5-fold in normal human pregnancies but is often blunted in pregnancy complications such as fetal growth restriction, gestational diabetes, and preeclampsia. Our previous studies have shown that genetic reduction of maternal AM leads to poor pregnancy outcomes, but the role of fetal AM remains unknown. Therefore, we examined placentas of Adm-/- and Adm+/+ pups at midgestation for structural, functional, and developmental phenotypes. Histologic and morphometric analysis revealed normal differentiation and layer formation in the Adm-/- placentas. Doppler ultrasound of umbilical cord blood flow showed that placental impedance was unaffected by fetal loss of Adm. Although histology and lectin staining showed no difference in the total cross-sectional area of fetal and maternal vessels, the fetal vessels of the Adm-/- placentas were large and underbranched compared to those of wildtype littermates. Electron microscopy of methyl-methacrylate casts confirmed that Adm-/- placentas had reduced fetal vessel branching; a hallmark feature of preeclampsia. Strikingly, the Adm-/- placentas also had reduced numbers of decidual natural killer (NK) cells and reduced remodeling of maternal spiral arteries; other known features of preeclampsia. To further investigate the effects of AM signaling on NK cells, isolated primary NK cells were treated with AM, which resulted in significantly altered cytokine expression compared to untreated controls. Primary smooth muscle cell cultures exposed to supernatant from AM-treated NK cells also showed a significant increase in smooth muscle cell apoptosis, indicating that the AM-mediated change in NK cell cytokine expression may be required for normal spiral artery remodeling. To confirm that this phenotype is specific to lack of fetal AM, ovarian transplantation was performed from Adm+/- to Adm+/+ females. The placentas of Adm-/- pups from wildtype mothers receiving heterozygote ovary transplants recapitulated the reduced NK cell invasion and failed spiral artery remodeling originally seen in heterozygous mothers, demonstrating that lack of AM specifically from the fetal compartment is responsible for the observed preeclampsia-like pathology. Furthermore, we were able to rescue this phenotype by generating a transgenic mouse model with increased fetal AM expression, termed Admhi/hi. High fetal AM led to recruitment of increased decidual NK cells and normal spiral artery remodeling. RT-PCR screening revealed that numerous cytokines known to play important roles in the placenta had opposite regulation in Admhi/hi versus Adm-/- placentas. Taken together, these data indicate that loss of fetal AM causes reduced fetal vascularization and maternal spiral artery remodeling characteristic of preeclampsia, and that these phenotypes can be rescued by overexpressing fetal AM. These findings demonstrate that the dosage of fetal AM is a critical factor for adapting the maternal vasculature to pregnancy and may be important in preventing the development of preeclampsia. This work is supported by The Burroughs Wellcome Fund, NIH/NICHD RO1 HD46970, HD060860, NIH/NHLBI F30 HL104778, and the March of Dimes Foundation. (poster)

Expression and role of adrenomedullin in renal tumors and value of its mRNA levels as prognostic factor in clear‐cell renal carcinoma

Antiangiogenic therapies are used for advanced clear-cell renal carcinomas (cRCC), but without curative possibilities, underlining the need for new therapeutic targets. Adrenomedullin (AM), a multifunctional peptide, is highly expressed in several tumors and 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). In this study, real-time quantitative reverse-transcription-PCR showed AM mRNA levels were higher in cRCC and in chromophobe renal carcinomas (chRCC) than in normal renal tissue. Interestingly, AM mRNA expression in cRCC correlated strongly with VEGF-A mRNA expression. Immunohistochemically, AM, CLR and RAMP2 were localized in the carcinomatous epithelial compartment of cRCC. Interestingly, RAMP3 immunostaining was found only in the inflammatory cells that infiltrated tumors, suggesting a cross talk between tumor cells and the microenvironment. We also observed that cRCC cells BIZ and 786-O expressed and secreted AM into the culture medium. In vitro, exogenous AM treatment stimulated cell proliferation, migration and invasion, indicating the cell can respond to AM. The action of AM was specific and was mediated by the CLR/RAMP2 and CLR/RAMP3 receptors. Clinical data showed the prognostic value of AM. High AM mRNA levels were associated with an increased risk of relapse after curative nephrectomy for cRCC. These findings highlight the implication of the AM pathway in the metastatic process and the prognostic relevance of AM in cRCC and point to a potential new therapeutic target.

Plasma adrenomedullin as an independent predictor of future cardiovascular events in high-risk patients: Comparison with C-reactive protein and adiponectin

This study investigated the predictive power of plasma adrenomedullin (AM) for future cardiovascular (CV) events. In 121 patients with multiple CV risk factors and/or disease, plasma concentrations of AM, high sensitive C-reactive protein (hs-CRP), and adiponectin were measured. During follow-up periods (mean, 3.5 years) after the baseline assessment, 28 patients newly experienced CV events such as stroke/transient ischemic attack, acute coronary syndrome, and congestive heart failure. The plasma level of AM, but not hs-CRP or adiponectin, was significantly higher in patients who had CV events than in event-free subjects. When the patients were divided into three groups by tertiles of basal levels of AM (<10.1, 10.1–13.1, and ≥13.1 fmol/mL), cumulative event-free rates by the Kaplan–Meier method were decreased according to the increase in basal AM levels (83.2%, 68.6%, and 52.8% in the lowest, middle, and highest tertiles of AM, respectively; log-rank test, P = 0.033). By univariate Cox regression analysis, previous coronary artery disease, creatinine clearance, and plasma AM and hs-CRP levels were significantly associated with CV events during follow-up. Among these possible predictors, high plasma AM ( P = 0.004) and low creatinine clearance ( P = 0.043) were independent determinants for morbidity in multivariate analysis. These findings indicate that plasma AM is a powerful independent predictor of future CV events in high-risk patients, suggesting its predictive value is superior to that of hs-CRP or adiponectin.

Hypoxic Induction of Receptor Activity-Modifying Protein 2 Alters Regulation of Pulmonary Endothelin-1 by Adrenomedullin: Induction under Normoxia Versus Inhibition under Hypoxia

The vasodilator adrenomedullin (AM) is up-regulated in pulmonary hypertension, and inhaled AM is beneficial in patients. Therefore, we investigated the effects of AM on pulmonary endothelin-1 (ET-1). In normoxic isolated rat lungs (IRL) and rat pulmonary artery endothelial cells (RPAEC), the calcitonin gene-related peptide type-1 receptor (CGRP1R) antagonist human (h)CGRP(8-37) decreased ET-1 secretion, and the AM receptor antagonist hAM(22-52) had no effect. Exogenous AM (1 and 10 pM) increased ET-1 levels, which was abolished by hCGRP(8-37) and protein kinase A (PKA) inhibition. At 50 and 100 pM, AM decreased ET-1, an effect sensitive to hAM(22-52), NO inhibition, and protein kinase G (PKG) inhibition. In RPAEC, these results were attributed to altered ET-1 gene expression; low exogenous AM also promoted activity of endothelin-converting enzyme, and high AM increased the number of endothelin type-B (ETB) receptor sites. Hypoxia significantly elevated AM and ET-1 levels in IRL and RPAEC, and hAM(22-52), NO inhibition, or PKG inhibitors caused a further ET-1 rise. These interventions also prevented the hypoxia-related increase in ETB sites in RPAEC. In RPAEC, both high AM and hypoxia down-regulated receptor activity-modifying protein (RAMP)1, but they up-regulated RAMP2 protein and AM receptor sites, and RAMP2 silencing by small interference RNA proved its pivotal role for signal switching. In conclusion, endogenous pulmonary AM up-regulates ET-1 and endothelin-converting enzyme activity under physiological conditions, via CGRP1R and PKA. In contrast, hypoxia-induced high AM levels, via AM1 receptor and NO/PKG, down-regulate ET-1 gene expression and promote expression of ETB receptors. This hypoxia-related switch of AM signaling can be attributed to up-regulation of the RAMP2/AM1 receptor system.

Plasma adrenomedullin level and development of hypertension

The hypotensive peptide adrenomedullin (AM) is assumed to act as a factor inhibitory on elevation of blood pressure and on progression of the hypertensive organ damage, and plasma AM levels are elevated in patients with hypertension. The aim of the present study is to explore whether or not a rise in plasma AM levels precedes the development of hypertension. Normotensive local residents without apparent cardiovascular or renal disease (n = 177) were divided into low and high AM groups based on the median concentration of AM in plasma (11.9 fmol/ml), and followed up for 3 years for development of hypertension. The incidence of hypertension was higher in the residents with high AM than low AM levels (27.8 vs 11.5%, P < 0.01), whereas a similar analysis of plasma levels of atrial or brain natriuretic peptides revealed no such difference. The plasma AM level was found to be a significant parameter for the development of hypertension in a univariate analysis (P < 0.01), but not in a multivariate analysis. Meanwhile, the plasma AM level was significantly (P < 0.01) correlated with age and body mass index (BMI), two variables independently significant for the development of hypertension. The present findings suggest that an elevation of the plasma AM level associated with aging and increased BMI precedes the development of hypertension in the normotensive subjects.

Increased plasma levels of adrenomedullin, a vasoactive peptide, in patients with end-stage pulmonary disease

Aim To study adrenomedullin (AM) plasma levels in patients with severe lung disease and to analyze the relationship between AM and heart changes, hemodynamics and blood gases. Methods Case control study of 56 patients (36 men, 20 women) with severe lung disease and 9 control subjects (7 men, 2 women). Patients with end-stage pulmonary disease, including chronic obstructive pulmonary disease (COPD, n=11), cystic fibrosis (CF, 26), idiopatic pulmonary fibrosis (ILD, n=9), and idiopatic pulmonary arterial hypertension (PAH, n=10), who were evaluated for lung trasplantation between January 1997 and September 2000, and nine patients who underwent lung surgery for a solitary benign nodule. AM plasma levels in pulmonary artery (mixed venous blood, vein) and aorta or femoral artery (arterial, art), art and vein blood gases, pulmonary hemodynamics, systemic hemodynamics, two-dimensional transthoracic echocardiography and echo-Doppler study. Results Plasma AM (art and ven) levels were higher among patients' group compared to the controls (AMart p<0.02 and AMven p<0.04) for CF, ILD, PAH (AMart, pg ml −1 Controls 13.7±3.6, COPD 22.8±6.2, CF 28.1±11.4, ILD 34.1±14.3, PAH 35.1±18.9; AMven, pg ml −1 Controls 14.2±4.8, COPD 28.1±12.6, CF 31.7±14.1, ILD 38.7±16.5, PAH 40.1±4.4). We found with a trend towards higher concentration in ILD and PAH patients compared to COPD and CF but no statistical significant differences. Mixed-venous AM was higher than arterial AM in all groups resulting in AM uptake (AMPulmUp pg min −1 Controls 4.8±22.6, COPD 21.1±44.9, CF 20.6±45.1, ILD 23.7±38.5, PAH 29.9±49.7). The univariate analysis showed a weak but significant correlation between AMart and mean systemic arterial pressure, heart rate, mean pulmonary arterial pressure and systemic vascular resistance. In the multivariate analysis, four variables emerged as independent factors of AMart including mean pulmonary arterial pressure, heart rate, mean systemic arterial pressure and left ventricular diastolic diameter ( F=8.6, p<0.00001, r=0.60, r 2=0.32). A similar weak correlation was apparent between AMven, systemic vascular resistance, and mean pulmonary arterial pressure. The results of multivariate analysis identify right atrial enlargement, mean right atrial pressure, heart rate and left ventricular dimensions as the only independent variables related to AMven ( F=4.3, p<0.0004 r=0.56, r 2=0.26). AM pulmonary uptake was significantly correlated with AMven ( r=0.65), but not with hemodynamic, blood gas and echocardiographic variables. Conclusions AM plasma levels are elevated in patients with severe lung disease in face of a preserved pulmonary uptake. These results suggest that the high AM plasma levels in patients with severe lung disease are not caused by a reduced pulmonary clearance, instead suggesting a systemic production.

Selective Inactivation of Adrenomedullin over Calcitonin Gene-related Peptide Receptor Function by the Deletion of Amino Acids 14-20 of the Mouse Calcitonin-like Receptor

The receptors for the neuropeptide calcitonin (CT) gene-related peptide (CGRP) and the multifunctional peptide hormone adrenomedullin (AM) are calcitonin-like receptor (CLR)/receptor-activity-modifying protein (RAMP) 1 and CLR/RAMP2 heterodimers, respectively. Here, the amino acid sequence TRNKIMT, corresponding to the residues 14-20 of the N terminus of the mouse (m) CLR, was found to be required for a functional mCLR/RAMP2 AM receptor. The deletion of amino acids 14-20 (Delta14-20) or their substitution by alanine (14-20A) did not affect the heterodimerization of the mCLR with mRAMP1 or mRAMP2, and the levels of expression at the surface of transiently transfected COS-7 cells were not altered. In mRAMP1/mCLR- or mRAMP1/mCLR-(Delta14-20)-expressing cells CGRP stimulated cAMP formation with EC(50) values of 0.12 +/- 0.01 and 1.5 +/- 0.4 nm, respectively. In mRAMP2/mCLR-expressing cells the EC(50) of AM was 0.8 +/- 0.2 nm. However, in cells expressing mRAMP2/mCLR-(Delta14-20) up to 10(-6) m AM failed to stimulate cAMP production. In mRAMP2/mCLR-(14-20A) expressing cells the cAMP response to AM was minimally restored, and the EC(50) was >100 nm. In conclusion, the deletion of the amino acid sequence TRNKIMT of the extreme N terminus of the mCLR maintained CGRP receptor function of mRAMP1/receptor heterodimers, but AM no longer activated the mutant mCLR-(Delta14-20) in the presence of mRAMP2. The TRNKIMT sequence is required for normal mCLR/mRAMP2 association, and as a consequence, high affinity AM binding signaling the activation of adenylyl cyclase.

Cloning, characterization and central nervous system distribution of receptor activity modifying proteins in the rat.

Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin and calcitonin (CT) are structurally and functionally related neuropeptides. It has recently been shown that the molecular pharmacology of CGRP and ADM is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Furthermore, RAMP proteins have also been shown to govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. In this study, we have cloned the rat RAMP family and characterized the pharmacology of rat CGRP and ADM receptors. Rat RAMP1, RAMP2 and RAMP3 shared 72%, 69% and 85% homology with their respective human homologues. As expected CRLR-RAMP1 coexpression conferred sensitivity to CGRP, whilst association of RAMP2 or RAMP3 with CRLR conferred high affinity ADM binding. Using specific oligonucleotides we have determined the expression of RAMP1, RAMP2 and RAMP3 mRNAs in the rat central nervous system by in situ hybridization. The localization of RAMP mRNAs was heterogeneous. RAMP1 mRNA was predominantly expressed in cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most abundant in hypothalamus; and RAMP3 was restrictively expressed in thalamic nuclei. Interestingly, in specific brain areas only a single RAMP mRNA was often detected, suggesting mutual exclusivity in expression. These data allow predictions to be made of where each RAMP protein may heterodimerize with its partner G-protein-coupled receptor(s) at the cellular level and consequently advance current understanding of cellular sites of action of CGRP, ADM, amylin and CT. Furthermore, these localization data suggest that the RAMP family may associate and modify the behaviour of other, as yet unidentified neurotransmitter receptors.

Effect of adrenomedullin on the production of endothelin-1 and on its vasoconstrictor action in resistance arteries: evidence for a receptor-specific functional interaction in patients with heart failure

Endothelin-1 (ET-1) and adrenomedullin (ADM) are both produced in the arterial wall, but have opposing biological actions. Evidence from experimental animals suggests a functional interaction between ET-1 and ADM. We have tested this in humans. Small resistance arteries were obtained from gluteal biopsies taken from patients with chronic heart failure (CHF) due to coronary heart disease (CHD), or with CHD and preserved ventricular function. The contractile responses to big ET-1 and to ET-1 in both sets of vessels were studied in the absence (control) and presence of ADM at 20 pmol/l (low ADM) or 200 pmol/l (high ADM), using wire myography. ADM did not affect the conversion of big ET-1 into ET-1 in vessels from patients with either CHD or CHF. Low ADM did not alter the contractile response to ET-1 in vessels from patients with CHF. Low ADM was not tested in vessels from patients with CHD, but high ADM did not affect this response in arteries from these patients. High ADM did, however, significantly reduce the vasoconstrictor effect of ET-1 in vessels from patients with CHF. The maximum response, as a percentage of the response to high potassium, was 199% (S.E.M. 25%) in the control experiments (n = 14), 205% (27%) in the low-ADM (n = 7) studies and 150% (17%) in the high-ADM (n = 6) experiments (P &lt; 0.001). Furthermore, the Hill coefficient increased from 0.57±0.05 in the absence of ADM to 1.16±0.15 in the high-ADM experiments, indicating that ADM at 200 pmol/l specifically antagonized one receptor type in vessels from patients with CHF. We conclude that there is a one-site receptor interaction between ADM and ET-1 that is specific for vessels from patients with CHF. This functional interaction between ADM and ET-1 in resistance arteries may be of pathophysiological importance in CHF.

Effect of adrenomedullin on the production of endothelin-1 and on its vasoconstrictor action in resistance arteries: evidence for a receptor-specific functional interaction in patients with heart failure

Endothelin-1 (ET-1) and adrenomedullin (ADM) are both produced in the arterial wall, but have opposing biological actions. Evidence from experimental animals suggests a functional interaction between ET-1 and ADM. We have tested this in humans. Small resistance arteries were obtained from gluteal biopsies taken from patients with chronic heart failure (CHF) due to coronary heart disease (CHD), or with CHD and preserved ventricular function. The contractile responses to big ET-1 and to ET-1 in both sets of vessels were studied in the absence (control) and presence of ADM at 20 pmol/l (low ADM) or 200 pmol/l (high ADM), using wire myography. ADM did not affect the conversion of big ET-1 into ET-1 in vessels from patients with either CHD or CHF. Low ADM did not alter the contractile response to ET-1 in vessels from patients with CHF. Low ADM was not tested in vessels from patients with CHD, but high ADM did not affect this response in arteries from these patients. High ADM did, however, significantly reduce the vasoconstrictor effect of ET-1 in vessels from patients with CHF. The maximum response, as a percentage of the response to high potassium, was 199% (S.E.M. 25%) in the control experiments (n=14), 205% (27%) in the low-ADM (n=7) studies and 150% (17%) in the high-ADM (n=6) experiments (P<0.001). Furthermore, the Hill coefficient increased from 0.57+/-0.05 in the absence of ADM to 1.16+/-0.15 in the high-ADM experiments, indicating that ADM at 200 pmol/l specifically antagonized one receptor type in vessels from patients with CHF. We conclude that there is a one-site receptor interaction between ADM and ET-1 that is specific for vessels from patients with CHF. This functional interaction between ADM and ET-1 in resistance arteries may be of pathophysiological importance in CHF.