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)
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)
Angiopoietin-1 is an apoptosis survival factor for endothelial cells.
We examined the effect of angiopoietin-1 (Ang1) on apoptosis in human umbilical vein endothelial cells (HUVECs). Ang1 (5-1000 ng/ml) dose-dependently inhibited apoptosis under a serum-deprived state. A significant apoptotic inhibition occurred with as low as 50 ng/ml. Two hundred ng/ml of Ang1 inhibited to approximately 50% of the control apoptotic rates for 96 h. Furthermore, an augmented antiapoptotic effect of Ang1 by the addition of 20 ng/ml vascular endothelial growth factor was observed. This Ang1-induced strong antiapoptotic effect in endothelial cells is a novel and intriguing finding and could be an additional description of Ang1-induced direct biological function. (+info)
Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF.
In contrast with the prevailing view that most tumors and metastases begin as avascular masses, evidence is presented here that a subset of tumors instead initially grows by coopting existing host vessels. This coopted host vasculature does not immediately undergo angiogenesis to support the tumor but instead regresses, leading to a secondarily avascular tumor and massive tumor cell loss. Ultimately, however, the remaining tumor is rescued by robust angiogenesis at the tumor margin. The expression patterns of the angiogenic antagonist angiopoietin-2 and of pro-angiogenic vascular endothelial growth factor (VEGF) suggest that these proteins may be critical regulators of this balance between vascular regression and growth. (+info)
The transcription factor MEF2C-null mouse exhibits complex vascular malformations and reduced cardiac expression of angiopoietin 1 and VEGF.
The MEF2 family of transcription factors has been implicated in transcriptional regulation in a number of different cell types. Targeted deletion of the MEF2C gene, in particular, revealed its importance for early cardiogenesis (Q. Lin et al., 1997, Science 276, 1404-1407). We report here that this deletion also resulted in vascular anomalies characterized by extreme variability in lumen size and defects in remodeling. While primary vascular networks formed in the yolk sac of the mutants, they failed to remodel into more complex vascular structures. Likewise, although the primordia of the dorsal aortae formed normally, anomalies were observed in these vessels later in development. Dorsal and anterior to the heart, the aortae exhibited abnormally small lumens, as did the anterior cardinal veins and intersegmental arteries. In contrast, the dorsal aortae and intersegmental arteries caudal to the heart were grossly enlarged. Cranial vessels were also enlarged and less branched than normal. Endocardiogenesis in the mutant was abnormal with the endothelial cells exhibiting a number of aberrant phenotypes. These endocardial defects were accompanied by a notable reduction in angiopoietin 1 and VEGF mRNA production by the myocardium, indicating that MEF2C is required for myocardial expression of these important endothelial-directed cytokines and thus for correct endocardial morphogenesis. (+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)
Expression of angiopoietin-1, angiopoietin-2, and the Tie-2 receptor tyrosine kinase during mouse kidney maturation.
The Tie-2 receptor tyrosine kinase transduces embryonic endothelial differentiation, with Angiopoietin-1 (Ang-1) acting as a stimulatory ligand and Ang-2 postulated to be a naturally occurring inhibitor. Expression of these genes was sought during mouse kidney maturation from the onset of glomerulogenesis (embryonic day 14 [E14]) to the end of nephron formation (2 wk postnatal [P2]), and during medullary maturation into adulthood (P8). Using Northern and slot blotting of RNA extracted from whole organs, these three genes were expressed throughout the experimental period with peak levels at P2 to P3. By in situ hybridization analysis at E18, P1, and P3, Ang-1 mRNA was found to localize to condensing renal mesenchymal cells, proximal tubules, and glomeruli in addition to maturing tubules of the outer medulla. In contrast, Ang-2 transcripts were more spatially restricted, being detected only in differentiating outer medullary tubules and the vasa recta bundle area. Using in situ hybridization and immunohistochemistry, Tie-2 was detected in capillaries of the nephrogenic cortex, glomerular tufts, cortical interstitium, and medulla including vessels in the vasa recta. Using Western blotting of protein extracted from whole organs, Tie-2 protein was detected between E14 and P8 with tyrosine phosphorylated Tie-2 evident from E18. These data are consistent with the hypothesis that Tie-2 has roles in maturation of both glomeruli and vasa rectae. (+info)
New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF.
Our analyses in several different tumor settings challenge the prevailing view that malignancies and metastases generally initiate as avascular masses that only belatedly induce vascular support. Instead, we find that malignant cells rapidly co-opt existing host vessels to form an initially well-vascularized tumor mass. Paradoxically, the co-opted vasculature does not undergo angiogenesis to support the growing tumor, but instead regresses (perhaps as part of a normal host defense mechanism) via a process that involves disruption of endothelial cell/smooth muscle cell interactions and endothelial cell apoptosis. This vessel regression in turn results in necrosis within the central part of the tumor. However, robust angiogenesis is initiated at the tumor margin, rescuing the surviving tumor and supporting further growth. The expression patterns of Angiopoietin-2 (the natural antagonist for the angiogenic Tie2 receptor) and vascular endothelial growth factor (VEGF) strongly implicate these factors in the above processes. Angiopoietin-2 is highly induced in co-opted vessels, prior to VEGF induction in the adjacent tumor cells, providing perhaps the earliest marker of tumor vasculature and apparently marking the co-opted vessels for regression. Subsequently, VEGF upregulation coincident with Angiopoietin-2 expression at the tumor periphery is associated with robust angiogenesis. Thus, in tumors, Angiopoietin-2 and VEGF seem to reprise the roles they play during vascular remodeling in normal tissues, acting to regulate the previously underappreciated balance between vascular regression and growth. (+info)