Adrenomedullin Research Articles

Identification of Adrenomedullin-Induced S-Nitrosylated Proteins in JEG-3 Placental Cells

Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is important during placentation. Dysregulation of the process leads to pregnancy complications. S-nitrosylation of proteins is associated with cell invasion in many cell types. Adrenomedullin (ADM), a polypeptide expressed abundantly in the first-trimester placentas, induces EVCT invasion by upregulation of protein S-nitrosylation. This study aimed to identify the S-nitrosylated proteins induced by ADM in the JEG-3 placental cells. By using affinity chromatography followed by mass spectrometric analysis, tubulin, enolase, eukaryotic translation initiation factor 4A1, actin, annexin II (ANX II), and glyceraldehyde 3-phosphate dehydrogenaseprotein-1 were found to be S-nitrosylated by ADM. In vitro treatment with ADM or S-Nitrosoglutathione (GSNO) significantly increased the ANX II surface expression, but not its total expression in the JEG-3 cells. Translocation of ANX II to cell surface has been reported to act as a cell surface receptor to plasmin, plasminogen, and tissue plasminogen activator (tPA), thereby stimulating cell invasion and migration. However, in this study, ADM-induced surface expression of ANX II in the JEG-3 cells was not associated with changes in the secretory and membrane-bound tPA activities. Future studies are required to understand the roles of surface expression of S-nitrosylated ANX II on trophoblast functions. To conclude, this study provided evidences that ADM regulated the nitric oxide signaling pathway and modulated trophoblast invasion.

Adrenomedullin: A Novel Therapeutic for the Treatment of Inflammatory Bowel Disease.

Adrenomedullin (AM) is a bioactive peptide with various physiological functions, including vasodilation, angiogenesis, anti-inflammation, organ protection, and tissue repair. AM suppresses inflammatory cytokine production in the intestinal mucosa, improves vascular and lymphatic regeneration and function, mucosal epithelial repair, and immune function in the intestinal bacteria of animal models with intestinal inflammation. We have been promoting translational research to develop novel therapeutic agents for inflammatory bowel disease (IBD) using AM and have started clinical research for IBD patients since 2010. A multicenter clinical trial is currently underway in Japan for patients with refractory ulcerative colitis and Crohn’s disease. Moreover, since current AM administration is limited to continuous intravenous infusion, the development of a subcutaneous formulation using long-acting AM is underway for outpatient treatment.

Human adrenomedullin and its binding protein attenuate tissue injury and inflammation following hepatic ischemia reperfusion in rabbits

BackgroundLiver injury caused by ischemia reperfusion (I/R) during surgical procedures, such as liver resection or liver transplantation, is a major cause of liver damage and graft failure. The current method of treatment is mostly preventative (i.e., ischemic preconditioning). While a number of pharmacological modalities have been studied to reduce hepatic I/R injury, none have been entirely successful. It has been demonstrated that the administration of adrenomedullin (AM) in combination with AM-binding protein (AM/AMBP-1) exerts significant protective effects in various pathological conditions. In an effort to develop AM/AMBP-1 as a novel therapeutic for hepatic I/R injury, the present study examined the effect of a low dose of human AM, which does not induce hypotension, in combination with human AMBP-1 in a rabbit model of hepatic I/R (i.e., non-rodent species). MethodsIschemia of 70% of the liver was induced by placing a microvascular clip across the hilum of the left and median lobes for 60 min. The clip was then removed to commence reperfusion. At 15 min following clip removal (i.e., reperfusion), human AM/AMBP-1 was administered intravenously via the ear marginal vein continuously for 30 min. At 20 h, blood and tissue samples were collected for various measurements. ResultsThe serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase) and lactate dehydrogenase, were elevated following hepatic I/R. The administration of AM/AMBP-1 significantly decreased these levels by 58, 44, 41%, respectively. Hepatic I/R increased the direct and total bilirubin levels, whereas treatment with human AM/AMBP-1 decreased these levels by 60% and 69%, respectively. Treatment with AM/AMBP-1 also inhibited interleukin-6 gene expression by 95%. There were no changes in tumor necrosis factor-α (TNF-α) gene expression and myeloperoxidase activity (MPO), lactate and Suzuki scores after treatment. The treatment, however, reduced apoptosis post-hepatic I/R in the ischemic portion of the liver. ConclusionAdditional experiments with AM and AMBP-1 alone are needed to completely interpret the experimental results in this non-rodent species of hepatic I/R injury. The present study suggests that human AM/AMBP-1 may be developed as a novel therapeutic to attenuate hepatic I/R associated inflammation and liver injury.

Proteomics to improve phenotyping in obese patients with heart failure with preserved ejection fraction.

Recent evidence points towards a distinct obese phenotype among patients with heart failure with preserved ejection fraction (HFpEF). We aimed to identify differentially expressed circulating biomarkers in obese HFpEF patients and link them to disease severity and outcomes. From the LIFE-Heart study, 999 patients with HFpEF and 999 patients without heart failure (no-HF) were selected and 92 circulating serum biomarkers were measured using a proximity extension assay. Elevation of identified biomarkers was validated in 220 patients from the Aldo-DHF trial with diagnosed HFpEF. HFpEF patients were older and had more comorbidities including coronary artery disease and type 2 diabetes as compared to no-HF patients (P < 0.05 for all). After adjusting for covariates, adrenomedullin (ADM), galectin-9 (Gal-9), thrombospondin-2 (THBS-2), CD4, and tumour necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) were significantly higher in obese HFpEF patients [body mass index (BMI) ≥30 kg/m2 , n=464] as compared to lean HFpEF (BMI <30 kg/m2 , n=535) and obese no-HF patients (BMI ≥30 kg/m2 , n=387) (P < 0.001 for both); these findings were verified in the Aldo-DHF validation cohort (P < 0.001). Except for CD4 these proteins were associated with increased estimates of left atrial pressure in a linear fashion. Importantly, ADM and CD4 were associated with increased mortality in obese HFpEF patients after adjusting for covariates. Obese HFpEF patients exhibit higher circulating biomarkers of volume expansion (ADM), myocardial fibrosis (THBS-2) and systemic inflammation (Gal-9, CD4) compared to obese non-HFpEF or lean HFpEF patients. These findings support the clinical definition of a distinct obese HFpEF phenotype and might merit further investigation.

Soluble Angiotensin Converting Enzyme 2 (ACE2) Is Upregulated and Soluble Endothelial Nitric Oxide Synthase (eNOS) Is Downregulated in COVID-19-induced Acute Respiratory Distress Syndrome (ARDS).

A damaged endothelium is an underlying condition of the many complications of COVID-19 patients. The increased mortality risk associated with diseases that have underlying endothelial dysfunction, such as acute respiratory distress syndrome (ARDS), suggests that endothelial (e) nitric oxide synthase (NOS)-derived nitric oxide could be an important defense mechanism. Additionally, intravenous recombinant angiotensin converting enzyme 2 (ACE2) was recently reported as an effective therapy in severe COVID-19, by blocking viral entry, and thus reducing lung injury. Very few studies exist on the prognostic value of endothelium-related protective molecules in severe COVID-19 disease. To this end, serum levels of eNOS, inducible (i) NOS, adrenomedullin (ADM), soluble (s) ACE2 levels, and serum (s) ACE activity were measured on hospital admission in 89 COVID-19 patients, hospitalized either in a ward or ICU, of whom 68 had ARDS, while 21 did not. In our cohort, the COVID-19-ARDS patients had considerably lower eNOS levels compared to the COVID-19 non-ARDS patients. On the other hand, sACE2 was significantly higher in the ARDS patients. iNOS, ADM and sACE activity did not differ. Our results might support the notion of two distinct defense mechanisms in COVID-19-derived ARDS; eNOS-derived nitric oxide could be one of them, while the dramatic rise in sACE2 may also represent an endogenous mechanism involved in severe COVID-19 complications, such as ARDS. These results could provide insight to therapeutical applications in COVID-19.

Upregulated expression of hypoxia reactive genes in peripheral blood mononuclear cells from chronic liver disease patients

Liver fibrosis induces intrahepatic microcirculation disorder and hypoxic stress. Hypoxic stress has the potential for an increase in the possibility of more liver fibrosis and carcinogenesis. Liver biopsy is a standard method that evaluates of intrahepatic hypoxia, however, is invasive and has a risk of bleeding as a complication. Here, we investigated the hypoxia reactive gene expressions in peripheral blood mononuclear cells (PBMC) from chronic liver disease patients to evaluate intrahepatic hypoxia in a non-invasive manner. The subjects enrolled for this study were composed of 20 healthy volunteers (HV) and 48 patients with chronic liver disease (CLD). CLD patients contained 24 patients with chronic hepatitis（CH）and 24 patients with liver cirrhosis (LC). PBMC were isolated from heparinized peripheral blood samples. We measured the transcriptional expression of hypoxia reactive genes and inflammatory cytokines by quantitative RT-PCR. mRNA expression of adrenomedullin (AM), vascular endothelial growth factor A (VEGFA) superoxide dismutase (SOD), glutathione peroxidase (GPx) (p < 0.05), Interleukin-6 (IL-6), transforming growth factor-beta (TGF-β) and heme oxygenase-1 (HO-1) in CLD group were significantly higher than HV. AM mRNA expression is correlated with serum lactate dehydrogenase (LDH), serum albumin (Alb), IL6, and SOD mRNA expression. The hypoxia reactive gene expression in PBMCs from CLD patients was more upregulated than HV. Especially, angiogenic genes were notably upregulated and correlated with liver fibrosis. Here, we suggest that mRNA expression of AM in PBMCs could be the biomarker of intrahepatic hypoxia.

Combined evaluation of plasma B-type natriuretic peptide and urinary liver-type fatty acid-binding protein/creatinine ratio is related to worsening renal function in patients undergoing elective percutaneous coronary intervention.

There are few reports on the significance for the combined evaluation of blood humoral factors and urinary biomarkers in terms of worsening renal function (WRF) after coronary angiography (CAG)/percutaneous coronary arterial intervention (PCI). Urinary liver type-fatty acid-binding protein (L-FABP), neutrophil gelatinase associated lipocalin (NGAL), and adrenomedullin (AM) were measured less than 24h before and 3h, 6h, 1day, and 2days after CAG/PCI. WRF was defined as a > 20% decrease in the estimated GFR. WRF occurred in seven of 100 patients and the increase in L-FABP/creatinine (Cr) at 1day after CAG/PCI was significantly higher in the WRF group than in the non-WRF group. Plasma B-type natriuretic peptide (BNP) before CAG/PCI and L-FABP/Cr at 1day after CAG/PCI were independent predictors for WRF. The areas under the receiver-operating characteristic curves were as follows: 0.760 for BNP before CAG/PCI, 0.731 for L-FABP/Cr at 1day after CAG/PCI, and 0.892 for BNP and L-FABP/Cr. Urinary AM levels after PCI/CAG were negatively correlated only to serum potassium levels. Gene expressions of AM and AM-receptor were detectable in renal tubule epithelial cells. AM increased intracellular second messenger levels in a dose-dependent manner. Our results suggest that combined evaluation of plasma BNP and urinary L-FABP/Cr is useful as a predictor of renal dysfunction in CAG/PCI patients.

Comparison of nitric oxide and adrenomedullin levels of children with attention deficit hyperactivity disorder and anxiety disorder

Abstract Objectives Many studies show that adrenomedullin (ADM) is associated with nitric oxide (NO) and various mechanisms and is involved in the etiopathogenesis of schizophrenia, bipolar disorder and autism by oxidative stress and HPA axis dsyregulation. The aim of this study comparison of nitric oxide and adrenomedullin levels in children with ADHD, AD and healthy control included in our study, especially due to their effect mechanisms as they may predict anxiety symptom, was to investigate the relationship between nitric oxide and adrenomedullin levels and anxiety symptoms in children with ADHD, AD and healthy control. Methods The study included 27 ADHD, 27 AD and 23 healthy children without any previous drug use, without comorbid disease. The semi-structured interview was conducted by the researcher in all the children attending the study. Sociodemographic information form, Conner’s Parent and Teacher Rating scale and State-Trait Anxiety Inventory (STAI) were evaluated. NO level measured by spectrophotometer, ADM levels were measured by ELISA. Results There was no statistically significant difference in the serum NO and ADM levels of the children included in the sampling group according to age and sex. There was no statistically significant difference between NO and ADM levels between ADHD, AD and control groups. There was no statistically significant relationship between serum NO and ADM levels and ADHD, AD and control group children of state-trait anxiety scores. Conclusions These findings may suggest that NO and ADM levels in children with ADHD, AD do not show these diseases and that these parameters are not associated with anxiety symptoms.

Adrenomedullin increases cAMP accumulation and BDNF expression in rat DRG and spinal motor neurons.

Objective(s):Adrenomedullin (AM) has high expression in the spinal cord. In this study, we investigated the expression of AM and its receptor components, including calcitonin receptor-like receptor (CLR) and receptor activity modifying proteins (RAMPs) in dorsal root ganglion (DRG) and spinal motor (SM) neurons. Furthermore, the effects of AM on cAMP/cAMP response element-binding protein (CREB), AKT/glycogen synthase kinase-3 beta (GSK-3β) signaling pathways, and expressions of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were evaluated. Materials and Methods:Rat embryonic DRG and SM neurons were isolated, purified, and cultured. Real-time PCR was used to assess expressions of AM, CLR, and RAMPs. cAMP levels, p-CREB, BDNF, and NT-3 were determined using an enzyme-linked immunosorbent assay. p-AKT and p-GSK-3β levels were determined by western blotting. Real-time PCR showed expressions of AM, CLR, RAMP2, and RAMP3 in both DRG and SM neurons. Results:AM increased cAMP accumulation and p-CREB levels in DRG and SM neurons. AM increased p-AKT and p-GSK-3β in DRG, but not SM neurons. AM significantly increased BDNF expression in both DRG and SM neurons. There was also an increase in NT-3 level in both DRG and SM neurons, which is statistically significant in SM neurons. Conclusion:These results showed both DRG and SM neurons are targets of AM actions in the spinal cord. An increase in BDNF expression by AM in both DRG and SM neurons suggests the possible beneficial role of AM in protecting, survival, and regeneration of sensory and motor neurons.

Calcitonin receptors in GtoPdb v.2021.2

This receptor family comprises a group of receptors for the calcitonin/CGRP family of peptides. The calcitonin (CT), amylin (AMY), calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CGRP, AM, AMY, and CT receptors [131, 74, 71]) are generated by the genes CALCR (which codes for the CT receptor) and CALCRL (which codes for the calcitonin receptor-like receptor, CLR, previously known as CRLR). Their function and pharmacology are altered in the presence of RAMPs (receptor activity-modifying proteins), which are single TM domain proteins of ca. 150 amino acids, identified as a family of three members; RAMP1, RAMP2 and RAMP3. There are splice variants of the CT receptor; these in turn produce variants of the AMY receptor [131], some of which can be potently activated by CGRP. The endogenous agonists are the peptides calcitonin, α-CGRP (formerly known as CGRP-I), β-CGRP (formerly known as CGRP-II), amylin (occasionally called islet-amyloid polypeptide, diabetes-associated polypeptide), adrenomedullin and adrenomedullin 2/intermedin. There are species differences in peptide sequences, particularly for the CTs. CTR-stimulating peptide (CRSP) is another member of the family with selectivity for the CT receptor but it is not expressed in humans [94]. CLR (calcitonin receptor-like receptor) by itself binds no known endogenous ligand, but in the presence of RAMPs it gives receptors for CGRP, adrenomedullin and adrenomedullin 2/intermedin. There are several approved drugs that target this receptor family, such as pramlintide, erenumab, and the "gepant" class of CGRP receptor antagonists.

CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

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.

Identification of Diagnostic Biomarkers and Their Correlation with Immune Infiltration in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease of the central retina, with no suitable biomarkers for early diagnosis and treatment. This study aimed to find potential diagnostic biomarker candidates for AMD and investigate their immune-related roles in this pathology. Weight gene correlation analysis was first performed based on data from the Gene Expression Omnibus database and 20 hub genes were identified. The functional enrichment analyses showed that the innate immune response, inflammatory response, and complement activation were key pathways associated with AMD. Complement C1s (C1S), adrenomedullin (ADM), and immediate early response 5 like (IER5L) were identified as the crucial genes with favorable diagnostic values for AMD by using LASSO analysis and multiple logistic regression. Furthermore, a 3-gene model was constructed and proved to be of good diagnostic and predictive performance for AMD (AUC = 0.785, 0.840, and 0.810 in training, test, and validation set, respectively). Finally, CIBERSORT was used to evaluate the infiltration of immune cells in AMD tissues. The results showed that the NK cells, CD4 memory T cell activation, and macrophage polarization may be involved in the AMD process. C1S, ADM, and IER5L were correlated with the infiltration of the above immune cells. In conclusion, our study suggests that C1S, ADM, and IER5L are promising diagnostic biomarker candidates for AMD and may regulate the infiltration of immune cells in the occurrence and progression of AMD.

Adrenomedullin Is a Diagnostic and Prognostic Biomarker for Acute Intracerebral Hemorrhage.

Hemorrhagic stroke remains an important health challenge. Adrenomedullin (AM) is a vasoactive peptide with an important role in cardiovascular diseases, including stroke. Serum AM and nitrate–nitrite and S-nitroso compounds (NOx) levels were measured and compared between healthy volunteers (n = 50) and acute hemorrhagic stroke patients (n = 64). Blood samples were taken at admission (d0), 24 h later (d1), and after 7 days or at the time of hospital discharge (d7). Neurological severity (NIHSS) and functional prognosis (mRankin) were measured as clinical outcomes. AM levels were higher in stroke patients at all times when compared with healthy controls (p < 0.0001). A receiving operating characteristic curve analysis identified that AM levels at admission > 69.0 pg/mL had a great value as a diagnostic biomarker (area under the curve = 0.89, sensitivity = 80.0%, specificity = 100%). Furthermore, patients with a favorable outcome (NIHSS ≤ 3; mRankin ≤ 2) experienced an increase in AM levels from d0 to d1, and a decrease from d1 to d7, whereas patients with unfavorable outcome had no significant changes over time. NOx levels were lower in patients at d0 (p = 0.04) and d1 (p < 0.001) than in healthy controls. In conclusion, AM levels may constitute a new diagnostic and prognostic biomarker for this disease, and identify AM as a positive mediator for hemorrhagic stroke resolution.

Novel Chemically Modified Curcumin (CMC) Analogs Exhibit Anti-Melanogenic Activity in Primary Human Melanocytes.

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.

Adrenomedullin Expression Characterizes Leukemia Stem Cells and Associates With an Inflammatory Signature in Acute Myeloid Leukemia.

Adrenomedullin (ADM) is a hypotensive and vasodilator peptide belonging to the calcitonin gene-related peptide family. It is secreted in vitro by endothelial cells and vascular smooth muscle cells, and is significantly upregulated by a number of stimuli. Moreover, ADM participates in the regulation of hematopoietic compartment, solid tumors and leukemias, such as acute myeloid leukemia (AML). To better characterize ADM involvement in AML pathogenesis, we investigated its expression during human hematopoiesis and in leukemic subsets, based on a morphological, cytogenetic and molecular characterization and in T cells from AML patients. In hematopoietic stem/progenitor cells and T lymphocytes from healthy subjects, ADM transcript was barely detectable. It was expressed at low levels by megakaryocytes and erythroblasts, while higher levels were measured in neutrophils, monocytes and plasma cells. Moreover, cells populating the hematopoietic niche, including mesenchymal stem cells, showed to express ADM. ADM was overexpressed in AML cells versus normal CD34+ cells and in the subset of leukemia compared with hematopoietic stem cells. In parallel, we detected a significant variation of ADM expression among cytogenetic subgroups, measuring the highest levels in inv(16)/t(16;16) or complex karyotype AML. According to the mutational status of AML-related genes, the analysis showed a lower expression of ADM in FLT3-ITD, NPM1-mutated AML and FLT3-ITD/NPM1-mutated cases compared with wild-type ones. Moreover, ADM expression had a negative impact on overall survival within the favorable risk class, while showing a potential positive impact within the subgroup receiving a not-intensive treatment. The expression of 135 genes involved in leukemogenesis, regulation of cell proliferation, ferroptosis, protection from apoptosis, HIF-1α signaling, JAK-STAT pathway, immune and inflammatory responses was correlated with ADM levels in the bone marrow cells of at least two AML cohorts. Moreover, ADM was upregulated in CD4+ T and CD8+ T cells from AML patients compared with healthy controls and some ADM co-expressed genes participate in a signature of immune tolerance that characterizes CD4+ T cells from leukemic patients. Overall, our study shows that ADM expression in AML associates with a stem cell phenotype, inflammatory signatures and genes related to immunosuppression, all factors that contribute to therapy resistance and disease relapse.

Adrenomedullin Triggers Migraine Attacks

Adrenomedullin is a peptide from the family of calcitonin/calcitonin gene-related peptide (CGRP) that may be involved in the regulation of the

Adrenomedullin Ameliorates Pulmonary Fibrosis by Regulating TGF-ß-Smads Signaling and Myofibroblast Differentiation.

Pulmonary fibrosis is an irreversible, potentially fatal disease. Adrenomedullin (AM) is a multifunctional peptide whose activity is regulated by receptor activity-modifying protein 2 (RAMP2). In the present study, we used the bleomycin (BLM)-induced mouse pulmonary fibrosis model to investigate the pathophysiological significance of the AM-RAMP2 system in the lung. In heterozygous AM knockout mice (AM+/-), hydroxyproline content and Ashcroft scores reflecting the fibrosis severity were significantly higher than in wild-type mice (WT). During the acute phase after BLM administration, FACS analysis showed significant increases in eosinophil, monocyte, and neutrophil infiltration into the lungs of AM+/-. During the chronic phase, fibrosis-related molecules were upregulated in AM+/-. Notably, nearly identical changes were observed in RAMP2+/-. AM administration reduced fibrosis severity. In the lungs of BLM-administered AM+/-, the activation level of Smad3, a receptor-activated Smad, was higher than in WT. In addition, Smad7, an antagonistic Smad, was downregulated and microRNA-21, which targets Smad7, was upregulated compared to WT. Isolated AM+/- lung fibroblasts showed less proliferation and migration capacity than WT fibroblasts. Stimulation with TGF-β increased the numbers of α-SMA-positive myofibroblasts, which were more prominent among AM+/- cells. TGF-β-stimulated AM+/- myofibroblasts were larger and exhibited greater contractility and extracellular matrix production than WT cells. These cells were α-SMA (+), F-actin (+), and Ki-67(-) and appeared to be nonproliferating myofibroblasts (non-p-MyoFbs), which contribute to the severity of fibrosis. Our findings suggest that in addition to suppressing inflammation, the AM-RAMP2 system ameliorates pulmonary fibrosis by suppressing TGF-β-Smad3 signaling, microRNA-21 activity and differentiation into non-p-MyoFbs.

Lymphatic endothelial-cell expressed ACKR3 is dispensable for postnatal lymphangiogenesis and lymphatic drainage function in mice.

Atypical chemokine receptor ACKR3 (formerly CXCR7) is a scavenging receptor that has recently been implicated in murine lymphatic development. Specifically, ACKR3-deficiency was shown to result in lymphatic hyperplasia and lymphedema, in addition to cardiac hyperplasia and cardiac valve defects leading to embryonic lethality. The lymphatic phenotype was attributed to a lymphatic endothelial cell (LEC)-intrinsic scavenging function of ACKR3 for the vascular peptide hormone adrenomedullin (AM), which is also important during postnatal lymphangiogenesis. In this study, we investigated the expression of ACKR3 in the lymphatic vasculature of adult mice and its function in postnatal lymphatic development and function. We show that ACKR3 is widely expressed in mature lymphatics and that it exerts chemokine-scavenging activity in cultured murine skin-derived LECs. To investigate the role of LEC-expressed ACKR3 in postnatal lymphangiogenesis and function during adulthood, we generated and validated a lymphatic-specific, inducible ACKR3 knockout mouse. Surprisingly, in contrast to the reported involvement of ACKR3 in lymphatic development, our analyses revealed no contribution of LEC-expressed ACKR3 to postnatal lymphangiogenesis, lymphatic morphology and drainage function.

Study Protocol for a Randomized, Double-Blind, Placebo-Controlled, Phase-II Trial: AdrenoMedullin for Ischemic Stroke Study

Adrenomedullin (AM), a vasoactive peptide, has strong anti-inflammatory and angiogenic properties, which have been reported to ameliorate the consequences of ischemic stroke in several animal models. After a phase I study in healthy volunteers, two phase II trials of AM for inflammatory bowel diseases have been recently completed. The current AdrenoMedullin For Ischemic Stroke (AMFIS) study aims to assess the safety and efficacy of AM in patients with acute ischemic stroke. The AMFIS study is an investigator-initiated, randomized, double-blind, phase-II trial. AM or placebo will be administered to patients with non-cardioembolic ischemic stroke within 24h after stroke onset. In the first cohort of the AMFIS study, patients will be randomly allocated to the investigation treatment A (30μg/kg of AM in total for 7 days, n=20) or placebo group (n=10). In the second cohort, patients will be assigned to the investigation treatment B (56μg/kg of AM in total for 7 days, n=20) or placebo group (n=10). Serious adverse events related to the protocol treatment will be evaluated as the primary outcome. All adverse events will be analyzed as the secondary outcome. Regarding efficacy endpoints, the change in National Institutes of Health Stroke Scale and modified Rankin Scale scores will be compared between investigation treatment and placebo groups. AM is expected to be a safe and effective treatment for ischemic stroke.

PROGNOSTIC VALUE OF MID-REGIONAL PROADRENOMEDULLIN (MR-PROADM) IN PATIENTS WITH CHRONIC KIDNEY DISEASE AND HYPERTENSION

Objective: Adrenomedullin (ADM) is a peptide hormone with an antihypertensive effect. ADM plasma concentration is elevated in hypertension, heart failure and chronic kidney disease (CKD). Unfortunately, ADM is an unstable peptide, therefore the stable MR-proADM protein, produced in the course of ADM synthesis in a 1:1 ratio, maybe used as a biomarker in diagnostic procedures. The aim of the study was to assess the MR-proADM plasma concentration in conservatively treated patients with CKD and hypertension comparing to healthy volunteers in regard to kidney function, blood pressure control and cardiovascular parameters. Design and method: The plasma concentration of MR-proADM was evaluated in 80 patients with chronic kidney disease conservatively treated and hypertension (median age 76 years) and in 27 healthy volunteers using ELISA test. The blood pressure control and echocardiography were assessed. The statistical analysis included the stages of CKD, the degree of hypertension and the presence of cardiovascular complications. Results: The plasma MR-proADM concentration was higher in patients with CKD comparing to the control group (247.32 pg / ml vs 65.20 pg / ml; p &lt; 0.001). There was a significant positive correlation between MR-proADM and the dimensions of the left ventricle (p = 0.0039, r = 0.33) and left atrium (p = 0.026, r = 0.26), and a negative correlation between MR-proADM and the ejection fraction rate (p = 0.043 r = -0.23). There were no significant differences in the plasma concentration of MR-proADM depending on the degree of hypertension and CKD stages. Conclusions: Chronic kidney disease is associated with an increased level of plasma MR-proADM similarly to abnormal volume of heart cavities and reduced left ventricular ejection. However, the usefulness of MR-proADM protein as a biomarker of CKD and increased cardiovascular risk requires further research.