Objective-Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. Methods and Results-sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). 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 dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. Conclusions-These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling. (Arterioscler Thromb Vasc

STRUCTURED ABSTRACT Objectives Our purpose was to determine whether factors that regulate angiogenesis are altered in peripheral arterial disease (PAD) and whether these factors are associated with the severity of PAD. Background Alterations in angiogenic growth factors occur in cardiovascular disease (CVD), but whether these factors are altered in PAD or correlate with disease severity is unknown. Methods Plasma was collected from patients with PAD (n=46) and healthy control subjects (n=23). PAD patients included those with intermittent claudication (IC, n=23) and critical limb ischemia (CLI, n=23). Plasma angiopoietin-2 (Ang2), soluble Tie2 (sTie2), vascular endothelial growth factor (VEGF), soluble VEGF receptor-1 (sVEGFR-1), and placenta growth factor (PlGF) were measured by ELISA. In vitro, endothelial cells (ECs) were treated with recombinant VEGF to investigate effects on sTie2 production. Results Plasma concentrations of sTie2 (P<0.01), Ang2 (P<0.001), and VEGF (P<0.01), but not PlGF or sVEGFR-1, were significantly greater in PAD patients compared to controls. Plasma Ang2 was significantly increased in both IC and CLI compared to controls (p<0.0001), but there was no difference between IC and CLI. Plasma VEGF and sTie2 were similar in controls and IC but were significantly increased in CLI (P<0.001 vs. control or IC). Increased sTie2 and VEGF were independent of CVD risk factors or the ankle-brachial index, and VEGF treatment of ECs in vitro significantly increased sTie2 shedding. Conclusions VEGF and sTie2 are significantly increased in CLI, and sTie2 production is induced by VEGF. These proteins may provide novel biomarkers for CLI, and sTie2 may be both a marker and a cause of CLI.