Materials and Methods: Confluent human umbilical vein endothelial cells (HUVECs) at 4–5th passage were cultured in M199 without supplements or serum for 24 hrs, and then treated with recombinant Ang1 or Ang2 for 24hrs. TNF-alpha (100ng/ml) and phosphatydilinositol (PI) 3-K inhibitor LY-294002 (100nM) were added to the medium 3 hrs prior to Ang treatment.

Results: Effects of Ang1 and Ang2 on Tie2: In non-stimulated ECs, Ang1 decreased Tie2 expression in a dose-dependent manner, while Ang2 treatment did not change Tie2 expression. Neither Ang1 nor Ang2 affected cell viability or proliferation. To test whether Ang1-induced Tie2 downregulation was mediated by activation of PI3 kinase, ECs were treated with PI3 kinase inhibitor, LY294002. LY294002 treatment alone did not change Tie2 expression. LY294002 did not affect Ang1-induced Tie2 downregulation. Effects of Ang1 and Ang2 on Tie2 expression of TNF-alpha-stimulated ECs: Both Ang1 and Ang2 were able to attenuate TNF-alpha-induced Tie2 upregulation. Effects of Ang on TNFalpha-induced Tie2 upregulation were not blocked by LY294002. Neither Ang1 nor Ang2 affected viability or proliferation rates of TNF-alpha-stimulated ECs.

Discussion: In this study, we found that angiopoietins can regulate expression of their receptor, Tie2. Ang1, but not Ang2, down-regulated Tie2 expression on ECs without TNF-alpha stimulation. Both Ang1 and Ang2 attenuated TNF-alphainduced upregulation of Tie2. Regulation of Tie2 expression by Ang was not dependent on PI3 kinase, a pathway common for various Tie2 signaling. The Ang-Tie2 system appears to have a feedback system that may be regulating the overall activity of the Tie2 system. Dysregulation of the feedback system in the Ang-Tie2 system may be associated with pathological angiogenesis.