Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3.
Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells. (+info)
Early induction of angiogenetic signals in gliomas of GFAP-v-src transgenic mice.
Angiogenesis is a prerequisite for solid tumor growth. Glioblastoma multiforme, the most common malignant brain tumor, is characterized by extensive vascular proliferation. We previously showed that transgenic mice expressing a GFAP-v-src fusion gene in astrocytes develop low-grade astrocytomas that progressively evolve into hypervascularized glioblastomas. Here, we examined whether tumor progression triggers angiogenetic signals. We found abundant transcription of vascular endothelial growth factor (VEGF) in neoplastic astrocytes at surprisingly early stages of tumorigenesis. VEGF and v-src expression patterns were not identical, suggesting that VEGF activation was not only dependent on v-src. Late-stage gliomas showed perinecrotic VEGF up-regulation similarly to human glioblastoma. Expression patterns of the endothelial angiogenic receptors flt-1, flk-1, tie-1, and tie-2 were similar to those described in human gliomas, but flt-1 was expressed also in neoplastic astrocytes, suggesting an autocrine role in tumor growth. In crossbreeding experiments, hemizygous ablation of the tumor suppressor genes Rb and p53 had no significant effect on the expression of VEGF, flt-1, flk-1, tie-1, and tie-2. Therefore, expression of angiogenic signals is an early event during progression of GFAP-v-src tumors and precedes hypervascularization. Given the close similarities in the progression pattern between GFAP-v-src and human gliomas, the present results suggest that these mice may provide a useful tool for antiangiogenic therapy research. (+info)
In vitro hematopoietic and endothelial cell development from cells expressing TEK receptor in murine aorta-gonad-mesonephros region.
Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+ cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34(+) and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1(+) endothelial cells on the OP9 stromal layer, while TEK- cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells. (+info)
Angiopoietins 3 and 4: diverging gene counterparts in mice and humans.
The angiopoietins have recently joined the members of the vascular endothelial growth factor family as the only known growth factors largely specific for vascular endothelium. The angiopoietins include a naturally occurring agonist, angiopoietin-1, as well as a naturally occurring antagonist, angiopoietin-2, both of which act by means of the Tie2 receptor. We now report our attempts to use homology-based cloning approaches to identify new members of the angiopoietin family. These efforts have led to the identification of two new angiopoietins, angiopoietin-3 in mouse and angiopoietin-4 in human; we have also identified several more distantly related sequences that do not seem to be true angiopoietins, in that they do not bind to the Tie receptors. Although angiopoietin-3 and angiopoietin-4 are strikingly more structurally diverged from each other than are the mouse and human versions of angiopoietin-1 and angiopoietin-2, they appear to represent the mouse and human counterparts of the same gene locus, as revealed in our chromosomal localization studies of all of the angiopoietins in mouse and human. The structural divergence of angiopoietin-3 and angiopoietin-4 appears to underlie diverging functions of these counterparts. Angiopoietin-3 and angiopoietin-4 have very different distributions in their respective species, and angiopoietin-3 appears to act as an antagonist, whereas angiopoietin-4 appears to function as an agonist. (+info)
Role of the Ets transcription factors in the regulation of the vascular-specific Tie2 gene.
The Tie2 gene encodes a vascular endothelium-specific receptor tyrosine kinase that is required for normal vascular development and is also upregulated during angiogenesis. The regulatory regions of the Tie2 gene that are required for endothelium-specific gene expression in vivo have been identified. However, the transcription factors required for Tie2 gene expression remain largely unknown. We have identified highly conserved binding sites for Ets transcription factors in the Tie2 promoter. Mutations in 2 particular binding sites lead to a 50% reduction in the endothelium-specific activity of the promoter. We have compared the ability of several members of the Ets family to transactivate the Tie2 promoter. Our results demonstrate that 1 of 3 distinct isoforms of the novel Ets transcription factor NERF, NERF2, is expressed in endothelial cells and can strongly transactivate the regulatory regions of the Tie2 gene in comparison to other Ets factors, which have little or no effect. NERF2 can bind to the Tie2 promoter Ets sites in electrophoretic mobility shift assays. These studies support a role for Ets factors in the regulation of vascular-specific gene expression and suggest that the novel Ets factor NERF2 may be a critical transcription factor in specifying the expression of the Tie2 gene in vascular endothelial cells. (+info)
Two independent mechanisms essential for tumor angiogenesis: inhibition of human melanoma xenograft growth by interfering with either the vascular endothelial growth factor receptor pathway or the Tie-2 pathway.
Protein ligands and receptor tyrosine kinases that specifically regulate endothelial cell function are mainly involved in physiological as well as in disease-related angiogenesis. These ligand/receptor systems include the vascular endothelial growth factor (VEGF) and the angiopoietin (Ang) families, and their receptors, the VEGF receptor family and the tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (Tie) family. In the present study, the contribution of these endothelium-specific ligand/receptor systems to tumor angiogenesis was evaluated. A375v human melanoma cells, which express at least the angiogenic growth factors VEGF, VEGF-C, and Ang-1, were stably transfected to overexpress the extracellular ligand-binding domains of the endothelium-specific receptor tyrosine kinases fms-like tyrosine kinase-1 (Flt-1), Flt-4, Tie-1, and Tie-2, respectively. In vitro proliferation and colony formation assays confirmed that expression of the extracellular receptor domains inhibited neither tumor cell mitogenesis nor the ability to produce anchorage-independent growth. Nude mouse xenografts revealed that interference with either the VEGF receptor pathway or the Tie-2 pathway resulted in a significant inhibition of tumor growth and tumor angiogenesis. In contrast, interference with the Flt-4 pathway or the Tie-1 pathway was without significant effect. Our results show that both the VEGF receptor pathway and the Tie-2 pathway are essential for A375v melanoma xenograft growth. The inhibition of the VEGF receptor pathway cannot be compensated by the Tie-2 pathway, nor vice versa. These findings suggest that the VEGF receptor pathway and the Tie-2 pathway have to be considered as two independent mediators essential for the process of in vivo angiogenesis. (+info)
Octamer-dependent in vivo expression of the endothelial cell-specific TIE2 gene.
The TIE2 gene, also known as TEK, encodes a tyrosine kinase receptor that is required for the normal development of the vascular system during embryogenesis. TIE2 is specifically expressed in endothelial cells; however, the transcriptional mechanisms that regulate this highly restricted pattern of expression remain unknown. Here we demonstrate that a consensus octamer element located in the 5'-flanking region of TIE2 is required for normal expression in embryonic endothelial cells. Transgenic embryos carrying a TIE2/LacZ construct spanning 2.1 kilobases of upstream regulatory sequences exhibit expression of the reporter transgene specifically in endothelial cells. Site-directed mutagenesis of a consensus octamer element located in this region results in the loss of enhancer activity and significantly impairs the endothelial expression of the reporter transgene. Consistent with the in vivo data, in vitro DNA-protein binding studies show that the consensus octamer element displays an endothelial cell-specific pattern of binding, suggesting an interaction with a protein complex consisting of Oct1 and an endothelial cell-restricted cofactor. These data identify a novel role for the octamer element as an essential regulator of TIE2 expression, define the first known transcriptional pathway that mediates the expression of a developmental endothelial cell gene, and provide insights into the transcriptional mechanisms that regulate development of the vasculature during embryogenesis. (+info)
Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes.
PURPOSE: To elucidate the potential role of angiopoietins and the Tie2 system in choroidal neovascularization. METHODS: Surgically excised choroidal neovascular membranes (CNVMs) were obtained at vitrectomy from five eyes with age-related macular degeneration, three eyes with idiopathic neovascular maculopathy, and two eyes had degenerative myopia and two eyes had angioid streaks. Light microscopic immunohistochemistry was performed to detect cytokines such as vascular endothelial growth factor (VEGF), Ang1, and Ang2 and cellular components such as retinal pigment epithelial (RPE) cells, macrophages, and endothelial cells. Immunofluorescent double staining using confocal microscopy was performed to identify the cell types that secrete specific cytokines. RESULTS: Ang1 and Ang2 were positive in all surgically excised CNVMs, regardless of the primary disease. Double staining revealed that many of the cytokeratin, CD68 and factor VIII positive cells also had Ang1 and Ang2 immunoreactivities. In contrast to Ang1, Ang2 immunoreactivity tends to be higher in the highly vascularized regions of many CNVMs, and the localization was very similar to that of VEGF staining. Almost all vascular structures had prominent immunoreactivity for Tie2, which was confirmed by double staining for Tie2 and factor VIII. Tie2 immunoreactivity was also observed in the RPE monolayer and in pigmented, polygonal, and fibroblast-like cells in the stroma. CONCLUSIONS: Present findings that Ang2 and VEGF are co-upregulated and that Tie2 is expressed in a variety of cell types in CNVMs further support a crucial role of the interaction between VEGF and Ang2 in pathologic angiogenesis of CNVM formation. (+info)