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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. Key Words: endothelial cells Ⅲ angiopoietins Ⅲ angiogenesis Ⅲ transcription factors Ⅲ cell migration T he receptor tyrosine kinase (Tie-2) and its associated ligands, the angiopoietins, have emerged as important regulators of angiogenesis both in adults and in embryos. In adult mice, Ang-1 stimulates in vivo vascular remodeling, vascular enlargement, wound healing, and lymphangiogenesis. 1 In addition, Ang-1 inhibits endothelial cell (EC) apoptosis and stimulates migration, proliferation, and differentiation of these cells. 1 Despite the importance of the Ang-1/Tie-2 receptor pathway to vascular homeostasis and angiogenesis, little is known about transcription factors that are activated downstream from it. Daly et al 2 have reported that Ang-1 inhibits the transcriptional activity of FoxO-1 downstream of the PI-3 kinase/AKT pathway, thereby reducing the expression of several proapoptotic proteins. Elk-1 is activated by Ang-1 downstream from the Erk1/2 pathway and forms a complex by binding serum response factor and serum response element in various promoters. 3 However, neither of these studies addressed the importance of these transcription factors in mediating the biological functions of the Ang-1/Tie-2 pathway. Conclusion-Early growth response-1 (Egr-1) has recently been identified as a transcription factor that is significantly induced downstream from Tie-2 receptors. 4 Egr-1 is an immediateearly gene that is rapidly and transiently induced by many stimuli, including hypoxia, shear stress, and injury. On activation it binds promoter regions of several growth factors, cytokines, recepto...

Angiopoietin 1 (Ang-1) is the main ligand for endothelial cell-specific tyrosine kinase (Tie-2) receptors and it promotes migration and proliferation and inhibits apoptosis and vascular leakage. The exact mechanisms through which the Ang-1 exerts these effects remain unclear. The authors exposed human umbilical vein endothelial cells (HUVECs) to Ang-1 (300 ng/mL) for 4 h and conducted gene expression profiling using oligonucleotide microarrays. Real-time polymerase chain reaction (PCR) was also conducted to verify several of the genes that were regulated by Ang-1. Exposure to Ang-1 resulted in induction of 86 genes that are involved in endothelial cell (EC) proliferation, differentiation, migration, and survival. Thirty-six of these genes, including stanniocalcin, cyclin D1, vascular endothelial growth factor C, fms-related tyrosine kinase 1, interleukin 8, and CXCR4 have previously been shown to be induced by vascular endothelial growth factor (VEGF), suggesting significant similarities between VEGF and Ang-1 pathways. Ang-1 exposure also inhibited mRNA expressions of 49 genes, most of which are involved in cell cycle arrest, apoptosis, and suppression of transcription. These results indicate that Ang-1 triggers coordinated responses in endothelial cells designed to inhibit the expression of proapoptotic and antiproliferative genes and up-regulate proproliferative, proangiogenic, and antiapoptotic pathways. Moreover, we also found that the Erk1/2, phosphatidylinositol (PI) 3-kinase, and the mTOR pathways are involved in Ang-1-induced gene expression in HUVECs.