ABSTRACT
OBJECTIVE: Angiopoietin-1 (Ang-1) is an important regulator of angiogenesis in endothelial cells. It promotes migration, proliferation, and differentiation of cells, although the regulating factors involved in these processes remain unclear. In this study, we evaluated the contribution of the transcription factor early growth response-1 (Egr-1) to Ang-1-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression of Egr-1 was evaluated with real-time PCR and immunoblotting, whereas Egr-1 DNA binding activity was monitored with electrophoretic mobility shift assays. Cell migration was measured with wound healing and Boyden chamber assays, whereas cell proliferation and differentiation of cells into capillary-like tube structures were monitored with cell counting, BrdU incorporation and Matrigels. To selectively inhibit Egr-1 expression, we used both siRNA oligonucleotides and specific DNAzymes. Egr-1 mRNA expression rose approximately 9-fold within 2 hours of Ang-1 exposure and declined thereafter. Upregulation of Egr-1 expression was accompanied by an increase in nuclear mobilization and augmented DNA binding. These processes were mediated through the Erk1/2, PI-3 kinase/AKT, and mTOR pathways. Knockdown of Egr-1 expression completely abrogated Ang-1-induced endothelial migration and significantly reduced proliferation and capillary-like tube formation of HUVECs that overexpress Ang-1. CONCLUSIONS: Ang-1 triggers significant and transient induction of Egr-1, and Egr-1 contributes to Ang-1-induced endothelial cell migration and proliferation.
Subject(s)
Angiopoietin-1/metabolism , Cell Differentiation , Cell Movement , Cell Proliferation , Early Growth Response Protein 1/metabolism , Endothelial Cells/metabolism , Neovascularization, Physiologic , Cells, Cultured , DNA/metabolism , DNA, Single-Stranded/metabolism , Early Growth Response Protein 1/genetics , Endothelial Cells/enzymology , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction , TOR Serine-Threonine Kinases , Time Factors , Transfection , Up-Regulation , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Angiopoietin-1 (Ang-1), ligand for the endothelial cell-specific Tie-2 receptors, promotes migration and proliferation of endothelial cells, however, whether these effects are promoted through the release of a secondary mediator remains unclear. In this study, we assessed whether Ang-1 promotes endothelial cell migration and proliferation through the release of interleukin-8 (IL-8). Ang-1 elicited in human umbilical vein endothelial cells (HUVECs) a dose- and time-dependent increase in IL-8 production as a result of induction of mRNA and enhanced mRNA stability of IL-8 transcripts. IL-8 production is also elevated in HUVECs transduced with retroviruses expressing Ang-1. Neutralization of IL-8 in these cells with a specific antibody significantly attenuated proliferation and migration and induced caspase-3 activation. Exposure to Ang-1 triggered a significant increase in DNA binding of activator protein-1 (AP-1) to a relatively short fragment of IL-8 promoter. Upstream from the AP-1 complex, up-regulation of IL-8 transcription by Ang-1 was mediated through the Erk1/2, SAPK/JNK, and PI-3 kinase pathways, which triggered c-Jun phosphorylation on Ser63 and Ser73. These results suggest that promotion of endothelial migration and proliferation by Ang-1 is mediated, in part, through the production of IL-8, which acts in an autocrine fashion to suppress apoptosis and facilitate cell proliferation and migration.
Subject(s)
Angiopoietin-1/pharmacology , Autocrine Communication/drug effects , Cell Movement/drug effects , Endothelial Cells/cytology , Endothelial Cells/drug effects , Interleukin-8/biosynthesis , Transcription Factor AP-1/metabolism , Animals , Cell Proliferation/drug effects , Cells, Cultured , Endothelial Cells/enzymology , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Humans , Interleukin-8/genetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , RNA Stability/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retroviridae , Transduction, Genetic , Umbilical Veins/cytology , Umbilical Veins/drug effects , Umbilical Veins/enzymologyABSTRACT
The aims of the present study are to investigate the presence and distribution of angiotensin II (Ang II), as well as AT1 and AT2 receptors, in endocardial endothelial cells (EECs) and to determine if the effect of Ang II on intracellular calcium in these cells is mediated via the AT1 or the AT2 receptor. Immunofluorescence and 3D confocal microscopy techniques were used on 20-week-old fetal human EECs. Our results showed that Ang II and its receptors, the AT1 and the AT2 types, are present and exhibit a different distribution in human EECs. Ang II labelling is found throughout the cell with a fluorescence signal higher in the cytosol when compared with the nucleus. Like Ang II, the AT1 receptor fluorescence signal is also homogeneously distributed in human EECs but with a preferential labelling at the level of the nucleus, while the AT2 receptor labelling is solely present in the nucleus. Using fluo-3 and 3D confocal microscopy technique, superfusion of human EECs with increasing concentration of Ang II induced a dose-dependent sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of Ang II on human EEC's intracellular Ca2+ ([Ca2+]) was completely prevented by losartan, an AT1 receptor antagonist. Our results suggest that Ang II, as well as AT1 and AT2 receptors, is present but differentially distributed in EECs of 20-week-old fetal human hearts, and that the AT1 receptor mediates the effects of Ang II on [Ca2+]i in these cells.
