Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2

The endothelial cell (EC) specific tyrosine kinase receptor, Tie2,, interac ts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang 2). Ang1 stimulates Tie2 receptor autophosphorylation. while Ang? has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We stud ied the effects of Ang1 and Ang2, in an in vitro model of angiogenesis. Hum an ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditio ned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length o ver residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293 T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary like tubes within 48 h (DI: 24.58+/-5.91 and 19.13+/ -7.86, respectively) vs. control (DI: 2.73+/-1.68 and 2.15+/-1.45, respecti vely, both P<0.001). Interestingly, CM from two independent cell lines over expressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22+/-3.00 and 9.72+/-4.84, both P<0.005 vs. control) although the de gree of angiogenesis was significantly less then that seen with Ang1. Addit ion of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocke d by an excess of soluble Tie2. receptor (20 mug/ml). Both Ang1* and Ang2 i nduced modest increases in [H-3]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-expos ure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a di rect role in stimulating Tie2 receptor signaling and inducing in vitro angi ogenesis. Our findings suggest that the physiological role of Ang? is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions . (C) 2001 Elsevier Science B.V. All rights reserved.