Intermedin (adrenomedullin2) stabilizes the endothelial barrier and antagonizes thrombin‐induced barrier failure in endothelial cell monolayers

• Published in: British journal of pharmacology 2012-01, Vol.165 (1), p.208-222
• Main Authors: Aslam, M, Pfeil, U, Gündüz, D, Rafiq, A, Kummer, W, Piper, HM, Noll, T
• Format: Article
• Language: English
• Subjects: Adherens Junctions - drug effects; Adherens Junctions - physiology; Biological and medical sciences; Calcitonin Gene-Related Peptide - genetics; Calcitonin Gene-Related Peptide - metabolism; CGRP8–37; Cyclic AMP - genetics; Cyclic AMP - metabolism; Endothelial Cells - drug effects; Endothelial Cells - physiology; Endothelium, Vascular - physiology; Gene Expression Regulation - drug effects; Humans; intermedin; Medical sciences; Myosin Light Chains - genetics; Myosin Light Chains - metabolism; Peptide Hormones - pharmacology; Pharmacology. Drug treatments; Rac1; rac1 GTP-Binding Protein - genetics; rac1 GTP-Binding Protein - metabolism; RAMP2; Real-Time Polymerase Chain Reaction; Receptors, Adrenomedullin - genetics; Receptors, Adrenomedullin - metabolism; Research Papers; Reverse Transcriptase Polymerase Chain Reaction; rhoA GTP-Binding Protein - genetics; rhoA GTP-Binding Protein - metabolism; RNA - genetics; RNA - metabolism; Signal Transduction; thrombin; Thrombin - pharmacology
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/j.1476-5381.2011.01540.x

BACKGROUND AND PURPOSE Intermedin is a member of the calcitonin gene‐related‐peptide (CGRP) family expressed in endothelial cells and acts via calcitonin receptor‐like receptors (CLRs). Here we have analysed the receptors for intermedin and its effect on the endothelial barrier in monolayers of human umbilical vein endothelial cells (HUVECs). EXPERIMENTAL APPROACH We analysed the effect of intermedin on albumin permeability, contractile machinery, actin cytoskeleton and VE‐cadherin in cultured HUVECs. KEY RESULTS Intermedin concentration‐dependently reduced basal endothelial permeability to albumin and antagonized thrombin‐induced hyperpermeability. Intermedin was less potent (EC50 1.29 ± 0.12 nM) than adrenomedullin (EC50 0.24 ± 0.07 nM) in reducing endothelial permeability. These intermedin effects were inhibited by AM22–52 and higher concentrations of αCGRP8–37, with pA2 values of αCGRP8–37 of 6.4 for both intermedin and adrenomedullin. PCR data showed that HUVEC expressed only the CLR/RAMP2 receptor complex. Intermedin activated cAMP/PKA and cAMP/Epac signalling pathways. Intermedin's effect on permeability was blocked by inhibition of PKA but not of eNOS. Intermedin antagonized thrombin‐induced contractile activation, RhoA activation and stress fibre formation. It also induced Rac1 activation, enhanced cell–cell adhesion and antagonized thrombin‐induced loss of cell–cell adhesion. Treatment with a specific inhibitor of Rac1 prevented intermedin‐mediated barrier stabilization. CONCLUSION AND IMPLICATIONS Intermedin stabilized endothelial barriers in HUVEC monolayers via CLR/RAMP2 receptors. These effects were mediated via cAMP‐mediated inactivation of contractility and strengthening of cell–cell adhesion. These findings identify intermedin as a barrier stabilizing agent and suggest intermedin as a potential treatment for vascular leakage in inflammatory conditions.