Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2.
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The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly induced by COMP-Ang1 also in transfected cells that do not express Tie2. When cotransfected, Tie2 formed heteromeric complexes with Tie1, enhanced Tie1 activation, and induced phosphorylation of a kinase-inactive Tie1 in a ligand-dependent manner. Tie1 phosphorylation was also induced by native Ang1 and Ang4, although less efficiently than with COMP-Ang1. In conclusion, we show that Tie1 phosphorylation is induced by multiple angiopoietin proteins and that the activation is amplified via Tie2. These results should be important in dissecting the signal transduction pathways and biological functions of Tie1. (+info)
Differential regulation and properties of angiopoietin-like proteins 3 and 4.
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Angiopoietin-like protein 3 and 4 (Angptl3 and Angptl4) are two members of the angiopoietin-like family of proteins. These two closely related proteins have been reported to similarly affect lipid metabolism through their capacity to inhibit lipoprotein lipase. We undertook a series of studies to compare the structure, function, and regulation of Angptl3 and Angptl4. Previously, we reported that Angptl4 exists as variable-sized oligomers that contain intermolecular disulfide bonds. We now have evidence that although there are no intermolecular disulfide bonds evident in Angptl3, higher molecular weight forms do exist. In addition, Angptl4 exhibits a widespread distribution of tissue expression, while Angptl3 is exclusively expressed in the liver. Treatments with various ligands of nuclear receptors reveal that Angptl3 is a target gene of liver X receptor, while Angptl4 expression is activated by ligands of all peroxisome proliferator-activated receptors. Expression of Angptl4 in adipose tissue and liver is induced by fasting, while Angptl3 expression is not appreciably affected by nutritional status. We suggest that the differential regulation of Angptl3 and Angptl4 by sites of expression, nutritional status, and ligands of nuclear receptors may confer unique roles of each in lipoprotein metabolism. (+info)
Recombinant angioarrestin secreted from mouse melanoma cells inhibits growth of primary tumours.
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Angioarrestin is a recently described anti-angiogenic protein whose expression is down-regulated in solid tumours of various origins. It has a sequence identical to angiopoietin related protein-1. In this study we investigated anti-tumour properties of angioarrestin in B16 (F10) melanoma tumour model. We constructed an expression vector encoding human angioarrestin under the control of EF-1alpha promoter. This vector was transferred to B16 (F10) cells and recombinant angioarrestin secreted from the transfected cells was tested for anti-angiogenic activity using endothelial cell proliferation assay. Finally, mice were injected subcutaneously with cells that had been transfected with either angioarrestin-encoding vector or empty vector and tumor growth was compared. The obtained recombinant angioarrestin inhibited proliferation of bovine aortic endothelial cells. Tumours derived from an angioarrestin-secreting B16 (F10) cell clone grew in vivo more slowly than tumours derived from a cell clone transfected with empty vector. These data show, to our knowledge for the first time, that angioarrestin can inhibit primary melanoma tumour growth. (+info)
The angiopoietin/Tie-2 system in proliferative sickle retinopathy: relation to vascular endothelial growth factor, its soluble receptor Flt-1 and von Willebrand factor, and to the effects of laser treatment.
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AIM: To determine plasma levels of angiopoietin-1 and angiopoietin-2 (Ang-1, Ang-2), their soluble receptor Tie-2, vascular endothelial growth factor (VEGF), its soluble receptor Flt-1 (as indices of angiogenesis), and von Willebrand factor (vWf, marking endothelial damage/dysfunction) in sickle cell disease (SCD) patients with proliferative sickle retinopathy (PSR), with non-proliferative retinopathy (NPR), or no retinopathy (NR) and in control subjects with normal haemoglobin (AA subjects). In addition, to determine changes with panretinal laser photocoagulation (PRP) therapy. METHODS: Research indices were measured (ELISA) in 24 SCD patients who had PSR, 16 with NPR, 16 with NR, and from 23 AA subjects. Eight patients received PRP therapy and plasma was obtained before laser treatment and at 6 months after the last PRP session. RESULTS: Ang-1, Ang-2, VEGF, and vWf (but not Tie-2 or sFlt-1) were raised in SCD patients compared to AA subjects (p<0.01) but there were no differences among the three SCD subgroups. Significant correlations were between Ang-1 and VEGF, Ang-1 and Tie-2, and VEGF and sFlt-1 in patients with SCD (r = 0.67-0.88). Plasma Ang-2, VEGF, sFlt-1, and vWf levels did not change, but Ang-1 fell and Tie-2 rose significantly following PRP therapy. CONCLUSIONS: SCD patients have raised plasma angiopoietins (Ang-1, Ang-2), VEGF, and vWf compared to AA subjects. These indices did not differ according to severity of retinopathy and only limited changes occurred following PRP. The elevated growth factor levels in SCD may have obscured any association with retinopathy. (+info)
Roles of reactive oxygen species in angiopoietin-1/tie-2 receptor signaling.
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In this study we identified the involvement of reactive oxygen species (ROS) in signaling and biological effects of the angiopoietin-1 (Ang-1)/tie-2 receptor pathway. Exposure of human umbilical vein endothelial cells to Ang-1 (50 ng/ml) induced rapid and transient production of ROS, particularly superoxide anions. ROS production was attenuated by preincubation with a peptide (gp91ds-tat) that inhibits the association of the gp91(phox) subunit with the p47(phox) subunit of NADPH oxidase and by the expression of a dominant-negative form of Rac-1 (Rac1N17). These results suggest that ROS production in response to Ang-1 exposure originates mainly from a Rac-1-dependent NADPH oxidase. Overexpression of antioxidants (superoxide dismutase and catalase) and Rac1N17, as well as preincubation with selective inhibitors of NADPH oxidase augmented basal p38 phosphorylation, inhibited Ang-1-induced PAK-1 phosphorylation and potentiated Ang-1-induced Erk1/2 phosphorylation but had no influence on AKT and SAPK/JNK phosphorylation by Ang-1. Exposure to Ang-1 (100 ng/ml) for 5 h induced a threefold increase in endothelial cell migration, a response that was strongly inhibited by overexpression of antioxidants, Rac1N17, and selective NADPH oxidase inhibitors. We conclude that activation of tie-2 receptors by Ang-1 triggers the production of ROS through activation of NADPH oxidase and that ROS generation by Ang-1 promotes endothelial cell migration while negatively regulating Erk1/2 phosphorylation. (+info)
Microbial regulation of intestinal radiosensitivity.
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We describe a method for treating germ-free (GF) mice with gamma-irradiation and transplanting them with normal or genetically manipulated bone marrow while maintaining their GF status. This approach revealed that GF mice are markedly resistant to lethal radiation enteritis. Furthermore, administering lethal doses of total body irradiation to GF mice produces markedly fewer apoptotic endothelial cells and lymphocytes in the mesenchymal cores of their small intestinal villi, compared with conventionally raised animals that have acquired a microbiota from birth. Analysis of GF and conventionally raised Rag1-/- mice disclosed that mature lymphocytes are not required for the development of lethal radiation enteritis or the microbiota-associated enhancement of endothelial radiosensitivity. Studies of gnotobiotic knockout mice that lack fasting-induced adipose factor (Fiaf), a fibrinogen/angiopoietin-like protein normally secreted from the small intestinal villus epithelium and suppressed by the microbiota, showed that Fiaf deficiency results in loss of resistance of villus endothelial and lymphocyte populations to radiation-induced apoptosis. Together, these findings provide insights about the cellular and molecular targets involved in microbial regulation of intestinal radiosensitivity. (+info)
Cooperative interaction of Angiopoietin-like proteins 1 and 2 in zebrafish vascular development.
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Angiopoietin-like protein (Angptl) 1 and Angptl2, which are considered orphan ligands, are highly homologous, particularly in the fibrinogen-like domain containing the putative receptor binding site. This similarity suggests potentially cooperative functions between the two proteins. In this report, the function of Angptl1 and Angptl2 is analyzed by using morpholino antisense technology in zebrafish. Knockdown of both Angptl1 and Angptl2 produced severe vascular defects due to increased apoptosis of endothelial cells at the sprouting stage. In vitro studies showed that Angptl1 and Angptl2 have antiapoptotic activities through the phosphatidylinositol 3-kinase/Akt pathway, and coinjection of constitutively active Akt/protein kinase B mRNA rescued impaired vascular development seen in double knockdown embryos. These results provide a physiological demonstration of the cooperative interaction of Angptl1 and Angptl2 in endothelial cells through phosphatidylinositol 3-kinase/Akt mediated antiapoptotic activities. (+info)
The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.
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Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia. (+info)