Angiopoietin-1 (Ang1) is a strong apoptosis survival factor for endothelial cells. In this study, the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells was examined. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of Tie2 and the p85 subunit of phosphatidylinositol 3'-kinase (PI 3'-kinase) and increased PI 3'-kinase activity in a dose-dependent manner. The PI 3'-kinase-specific inhibitors wortmannin and LY294002 blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3'-kinase-dependent manner. Expression of a dominant-negative form of Akt reversed the Ang1-induced antiapoptotic effect. Ang1 mRNA and protein were present in vascular smooth muscle cells but not in endothelial cells. Cultured vascular smooth muscle cells, but not human umbilical vein endothelial cells, secreted Ang1. These findings indicate that the Tie2 receptor, PI 3'-kinase, and Akt are crucial elements in the signal transduction pathway leading to endothelial cell survival induced by the paracrine activity of Ang1.

Abstract-Angiopoietin-1 (Ang1) is a strong inducer of endothelial cell sprouting, which is a first step in both angiogenesis and neovascularization. We examined the mechanisms underlying Ang1-induced cell sprouting using porcine pulmonary artery endothelial cells. Ang1 induced the nondirectional and directional migration of endothelial cells mediated through the Tie2 but not the Tie1 receptor. Ang1 induced tyrosine phosphorylation of p125 FAK , and this phosphorylation was dependent on phosphatidylinositol (PI) 3Ј-kinase activity. Ang1 induced the secretion of plasmin and matrix metalloproteinase-2 (MMP-2), which is inhibited by PI 3Ј-kinase inhibitors. Ang1 also induced the secretion of small amounts of proMMP-3 and proMMP-9 but not proMMP-1. Ang1 suppressed the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), but not of TIMP-1. Addition of ␣ 2 -antiplasmin, a combination of TIMP-1 and TIMP-2, or PI 3Ј-kinase inhibitors inhibited Ang1-induced sprouting activity. Therefore, Ang1-induced sprouting activity in endothelial cells may be accomplished by cytoskeletal changes and secretion of proteinases and may be largely mediated through intracellular PI 3Ј-kinase activation. (Circ Res. 2000;86:952-959.)

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.z 1999 Federation of European Biochemical Societies.

Normally, tissue factor (TF) is not expressed on the surface of endothelial cells, but its expression can be induced by vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-a. However, the signaling pathway(s) affecting this induction is unknown. Using human umbilical vein endothelial cells, we found that inhibitors of guanine-cytosine-rich DNA binding protein and nuclear factor (NF)-kB suppressed VEGF- and TNF-a-induced expression and activity of TF. However, unexpectedly, phosphatidylinositol (PI) 3'-kinase inhibitor enhanced the VEGF- and TNF-a-induced expression and activity of TF. Angiopoietin-1 (Ang1), a strong activator of intracellular PI 3'-kinase/Akt, inhibited the induction of TF by VEGF and TNF-a, whereas Ang1 itself did not produce any significant effect on TF. Selective activation (or inactivation) of PI 3'-kinase/Akt by using adenoviral transfer reduced (or enhanced) TNF-a-induced expression of TF mRNA and protein, regardless of Ang1 treatment. From these results, we conclude that Ang1 inhibits the up-regulation of TF expression, possibly through activation of PI 3'-kinase/Akt in endothelial cells. Ang1 may be useful as an inhibitor of VEGF- and TNF-a-induced coagulation, inflammation, and cancer progression.

Adrenomedullin (AM) is a multifunctional peptide in human pheochromocytoma. To evaluate whether AM could be an angiogenic factor, we examined its effect on kinases and angiogenic processes. AM induced tyrosine phosphorylation of Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase1/2 (ERK1/2) by using distinct signaling pathways in human umbilical vein endothelial cells (HUVECs). AM also phosphorylated focal adhesion kinase, and phosphatidylinositol 3'-kinase inhibitor inhibited AM-induced focal adhesion kinase phosphorylation. Pretreatment with high concentrations of AM22-52, a putative AM receptor antagonist, partially suppressed AM-induced phosphorylation of Akt, ERK1/2, and focal adhesion kinase. AM and vascular endothelial growth factor produced increases in DNA synthesis and migration in HUVECs. AM induced tube formation in HUVECs, and its effect was inhibited by pretreatment with phosphatidylinositol 3'-kinase inhibitor or ERK1/2 inhibitor. AM induced sprouting in porcine pulmonary arterial endothelial cells and promoted neovessel formation in a mouse Matrigel plug assay. Inhibitors of phosphatidylinositol 3'-kinase and ERK1/2 inhibited AM-induced endothelial sprouting in vitro and angiogenesis in vivo. AM exerts angiogenic activity through activation of Akt, MAPK, and focal adhesion kinase in endothelial cells.

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 NH P -terminal and COOHterminal 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 Nterminus (V21 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.z 1999 Federation of European Biochemical Societies.

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