Abstract
Intermedin (IMD) is a recently discovered peptide closely related to adrenomedullin. Its principal physiological activity is its role in the regulation of the cardiovascular system, where it exerts a potent hypotensive effect. In addition, data were recently provided showing that this peptide is able to exert a clearcut pro-angiogenic effect both in vitro and in vivo. IMD acts through the non-selective interaction with receptor complexes formed by the dimerization of calcitonin-like receptor (CLR) with the receptor activity-modifying proteins RAMP1, 2 or 3. Thus, in the present study, the role of CLR/RAMP complexes in mediating the pro-angiogenic effect induced by IMD on human umbilical vein endothelial cells (HUVECs) cultured on Matrigel was examined. Real-time PCR demonstrated the expression of IMD, CLR/RAMP1 and CLR/RAMP2 (but not CLR/RAMP3) mRNA in HUVECs. IMD exerted a significant in vitro angiogenic action, specifically triggered by the binding of the peptide to CLR/RAMP complexes. Both CLR/RAMP1 and CLR/RAMP2 appeared to mediate the pro-angiogenic effect, which was associated with a significant increase of vascular endothelial growth factor (VEGF) mRNA expression 18 h following IMD administration, indicating that the observed pro-angiogenic effects are related, at least in part, to an increased synthesis of this growth factor promoted by the peptide. Western blot analysis, however, showed a significant increase of VEGF receptor-2 phosphorylation as early as 5 min following IMD administration, indicating that IMD induces a pro-angiogenic response in human vascular endothelial cells not only via CLR/RAMP-induced release of VEGF, but also during signal initiation and propagation by transactivating the VEGF receptor-2 machinery.
MeSH terms
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Analysis of Variance
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Biomarkers / metabolism
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Calcitonin Receptor-Like Protein
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Cells, Cultured
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Collagen
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Drug Combinations
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Endothelial Cells / metabolism*
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Gene Silencing
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Humans
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Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Laminin
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Membrane Proteins / antagonists & inhibitors
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Neovascularization, Physiologic*
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Peptide Hormones / genetics
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Peptide Hormones / metabolism*
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Phosphorylation
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Proteoglycans
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RNA, Messenger / analysis
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RNA, Messenger / metabolism
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Receptor Activity-Modifying Protein 1
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Receptor Activity-Modifying Protein 2
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Receptor Activity-Modifying Protein 3
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Receptor Activity-Modifying Proteins
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Receptors, Calcitonin / antagonists & inhibitors
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Receptors, Calcitonin / genetics
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Receptors, Calcitonin / metabolism*
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Reverse Transcriptase Polymerase Chain Reaction
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Signal Transduction
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Vascular Endothelial Growth Factor A / genetics
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Vascular Endothelial Growth Factor A / metabolism*
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Vascular Endothelial Growth Factor Receptor-2 / chemistry
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Vascular Endothelial Growth Factor Receptor-2 / genetics
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Vascular Endothelial Growth Factor Receptor-2 / metabolism
Substances
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ADM2 protein, human
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Biomarkers
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CALCRL protein, human
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Calcitonin Receptor-Like Protein
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Drug Combinations
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Intracellular Signaling Peptides and Proteins
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Laminin
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Membrane Proteins
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Peptide Hormones
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Proteoglycans
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RAMP1 protein, human
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RAMP2 protein, human
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RAMP3 protein, human
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RNA, Messenger
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Receptor Activity-Modifying Protein 1
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Receptor Activity-Modifying Protein 2
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Receptor Activity-Modifying Protein 3
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Receptor Activity-Modifying Proteins
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Receptors, Calcitonin
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Vascular Endothelial Growth Factor A
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matrigel
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Collagen
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Vascular Endothelial Growth Factor Receptor-2