Abstract 482: VEGF Induces MMP-dependent Tie2 Cleavage via PI3K-AKT
OBJECTIVES: Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved from endothelial cells and produces a 75 kDa soluble receptor fragment (sTie2), however, the signaling mechanisms and effector molecules responsible for the shedding phenomenon are unknown. We investigated mechanisms responsible for Tie2 shedding.
MATERIALS: Human umbilical vein endothelial cells (HUVECs), NIH-3T3, or HEK293 cells exogenously expressing full-length Tie2 were serum starved for varying timepoints. The conditioned media (CM) were then analyzed for the presence of Tie2 by ELISA or westernblot using a Tie2-specific antibody recognizing the extracellular domain. VEGF-A165 (R&D systems) was used for experimentation. Pharmacologic inhibitors of p38 MAPK, Akt, and PI3K were also used. Adenovirus encoding myristylated Akt, wildtype PTEN, dominant negative PTEN and catalytically inactive PTEN were used to infect HUVECs to discern signaling mechanisms. Additionally, the functional effects of sTie2 were examined using concentrated CM in response to Ang1/Ang2-mediated Tie2 stimulation in HUVECs.
RESULTS: In HUVECs, Tie2 shedding was both constitutive and VEGF-inducible. Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either constitutively active, myristoylated Akt or dominant negative PTEN, which increased Akt phosphorylation. Additionally, sTie2 inhibited both Ang1 and Ang2 mediated Tie2 activation, signaling, and cellular responses.
CONCLUSIONS: VEGF regulates Tie2-Angiopoietin signaling by inducing Tie2 shedding via the PI3K/Akt pathway. Physiologically, sTie2 likely functions to maintain vascular homeostasis by modulating Ang1/Ang2- mediated Tie2 activation. These results suggest a previously unrecognized mechanism by which VEGF directly acts to regulate the Tie2-Angiopoietin pathway.