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.
Subject(s)
Endothelial Cells/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neovascularization, Physiologic , Peptide Hormones/metabolism , Receptors, Calcitonin/metabolism , Vascular Endothelial Growth Factor A/metabolism , Analysis of Variance , Biomarkers/metabolism , Calcitonin Receptor-Like Protein , Cells, Cultured , Collagen , Drug Combinations , Gene Silencing , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Laminin , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Peptide Hormones/genetics , Phosphorylation , Proteoglycans , RNA, Messenger/analysis , RNA, Messenger/metabolism , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Protein 2 , Receptor Activity-Modifying Protein 3 , Receptor Activity-Modifying Proteins , Receptors, Calcitonin/antagonists & inhibitors , Receptors, Calcitonin/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-2/chemistry , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Human vascular endothelial cells express the urotensin-II (U-II) receptor and exhibit a strong in vitro angiogenic response to the peptide. Thus, in the present study an in vitro model, based on human umbilical vein endothelial cells (HUVEC) cultured on Matrigel, was used to characterize more in detail the signaling pathways that control the pro-angiogenic action of U-II. The activation of the U-II receptor (UT) was associated with an increase of intracellular calcium concentration. Both calcium rise and pro-angiogenic effect of the peptide can be blocked by U73122, a selective inhibitor of phospholipase-C, indicating that the signal transduction from UT mainly involves the phospholipase-C/IP(3) pathway. As far as the downstream signaling pathways are concerned, western blot analyses and experiments with specific inhibitors indicated that the U-II-induced self-organization of the cells into capillary-like structures was PKC dependent and involved the activation of the ERK1/2, but not p38-MAPK, transduction pathway. Interestingly, the pharmacological inhibition of PI3K (obtained with LY294002), hindered the capacity of U-II to induce a proangiogenic effect on HUVEC, suggesting that PI3K-dependent pathways also play a role in regulating the process.
Subject(s)
Endothelium, Vascular/cytology , Extracellular Signal-Regulated MAP Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Urotensins/physiology , Blotting, Western , Cells, Cultured , Endothelium, Vascular/enzymology , HumansABSTRACT
A recently proposed approach was used to model the self-organization into capillary-like structures of human vascular endothelial cells cultured on Matrigel. The model combines a Cellular Potts Model, considering cell adhesion, cytoskeletal rearrangement and chemotaxis, and a Partial Differential Equation model describing the release and the diffusion of a chemoattractant. The results were compared with the data from real in vitro experiments to establish the capability of the model to accurately reproduce both the spontaneous self-assembly of unstimulated cells and their self-organization in the presence of the pro-angiogenic factor adrenomedullin. The results showed that the model can accurately reproduce the self-assembly of unstimulated cells, but it failed in reproducing the adrenomedullin-induced self-organization of the cells. The extension of the model to include cell proliferation led to a good match between simulated and experimental patterns in both cases with predicted proliferation rates in agreement with the data of cell proliferation experiments.
Subject(s)
Adrenomedullin/pharmacology , Capillaries/drug effects , Capillaries/growth & development , Endothelial Cells/cytology , Endothelial Cells/drug effects , Models, Biological , Neovascularization, Physiologic/drug effects , Base Sequence , Capillaries/cytology , Capillaries/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Collagen , DNA Primers/genetics , Drug Combinations , Endothelial Cells/metabolism , Humans , In Vitro Techniques , Laminin , Neovascularization, Physiologic/genetics , Phenotype , Proteoglycans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Vascular Endothelial Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Urotensin-II (U-II), along its receptor UT, is widely expressed in the cardiovascular system, where it exerts regulatory actions under both physiological and pathological conditions. In the present study, human vascular endothelial cells (EC) from one arterious and three venous vascular beds were used to investigate in vitro their heterogeneity in terms of expression of U-II and UT and of angiogenic response to the peptide. Real-time PCR and immunocytochemistry demonstrated the expression of UT, as mRNA and protein, in all the EC populations investigated. U-II, on the contrary, was detectable only in EC from aorta and umbilical vein. U-II did not affect the proliferation rate of adult human EC, but induced a moderate proliferative effect on EC from human umbilical vein. When tested in the Matrigel assay, however, all EC exhibited a strong angiogenic response to the peptide, comparable to that of fibroblast growth factor-2 (FGF-2) and it was not associated to an increased expression of vascular endothelial growth factor (VEGF) and/or its receptors. The angiogenic effect of U-II was abolished by the UT antagonist palosuran. Overall, these data suggest that U-II, in addition to the well known role in the regulation of cardiovascular function, also exert a specific angiogenic activity.
Subject(s)
Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Neovascularization, Physiologic , Urotensins/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Endothelial Cells/cytology , Humans , Immunohistochemistry , Phenotype , Quinolines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Urea/analogs & derivatives , Urea/pharmacology , Urotensins/antagonists & inhibitors , Urotensins/biosynthesisABSTRACT
In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.