Angiopoietin-1 Regulates Endothelial Cell Survival Through the Phosphatidylinositol 3′-Kinase/Akt Signal Transduction Pathway

Abstract

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.

Angiopoietin-2 at high concentration can enhance endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway

“…Ectopic expression of Ang2 in Ang2 nonexpressing cells promotes tumor angiogenesis when those cells are implanted in nude mice (Tanaka et al, 1999). In vitro experiments have shown that Ang1 has speci®c e ects on endothelial cells: it potently induces chemotactic response (Witzenbichler et al, 1998), network formation , sprouting (Koblizek et al, 1998;Kim et al, 1999) and survival in apoptosis Kim et al, 2000a). Although a recent report indicates that Ang2 (250 ng/ml) stimulates proliferation, migration, and release of nitric oxide in trophoblast cells that are expressing the Tie2 receptor (Dunk et al, 2000), the role of Ang2 on cultured endothelial cells is not known.…”

“…While Ang1 mRNA is mainly expressed in periendothelial cells, including vascular smooth muscle cells Suri et al, 1996;Kim et al, 2000a), Ang2 mRNA is selectively expressed in endothelial cells (Maisonpierre et al, 1997). To date, the regulation of Ang1 mRNA expression is an unknown, but we do have some information about the regulation of Ang2.…”

“…*P50.05 versus CB. Quanti®cation of apoptosis was performed as described previously (Kim et al, 2000a). Brie¯y,¯oating cells were collected with two PBS washes; adherent cells were collected by trypsinization.…”

“…The cell lysates were immunoprecipitated with anti-Tie2 antibody (Santa Cruz Biotechnology) or anti-p85 antibody (Upstate Biotechnology, Inc), and separated by SDS ± PAGE. After Western blotting with antiphosphotyrosine antibody (Upstate Biotechnology, Inc) (upper panels), the membranes were stripped and re-incubated with corresponding antibodies (lower panels) for detection of total Tie2 or p85 protein, as described previously (Kim et al, 2000a). The Akt at Ser473 phosphorylation assay was performed according to the manufacturer's protocol (New England BioLabs).…”

“…In lanes 800W and 200W, wortmannin (30 nM) was added to the cells 30 min before exposure to Ang2 (800 ng/ml) and Ang1 200 ng/ml). (c) Autoradiogram of thin-layer chromatography for PI 3'-kinase activity, performed as described previously (Kim et al, 2000a). Cells exposed to Ang2 for 10 min generated visible induction of phosphatidylinositol-3-monophosphate (PIP).…”