Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization

Angiopoietin-1 (Ang1) has been recently identified as the major physiological ligand for the tyrosine kinase receptor Tie2 and assigned responsibility for recruiting and sustaining periendothelial support cells. Angiopoietin-2 (Ang2) was found to disrupt blood vessel formation in the developing embryo by antagonizing the effects of Ang1 and Tie2 and was thus considered to represent a natural Ang1/Tie2 inhibitor. In vivo effects of either angiopoietin on postnatal neovascularization, however, have not been previously described. Accordingly, we used the cornea micropocket assay of neovascularization to investigate the impact of angiopoietins on neovascularization in vivo. Neither Ang1 nor Ang2 alone promoted neovascularization. Pellets containing vascular endothelial growth factor (VEGF) alone induced corneal neovascularity extending from the limbus across the cornea. Addition of Ang1 to VEGF (Ang1+VEGF) produced an increase in macroscopically evident perfusion of the corneal neovasculature without affecting macroscopic measurements of length (0.58+/-0.03 mm) or circumferential neovascularity (136+/-10 degrees). In contrast, pellets containing Ang2+VEGF promoted significantly longer (0.67+/-0.05 mm) and more circumferential (160+/-15 degrees) neovascularity than VEGF alone or Ang1+VEGF (P<0.05). Excess soluble Tie2 receptor (sTie2-Fc) precluded modulation of VEGF-induced neovascularization by both Ang2 and Ang1. Fluorescent microscopic findings demonstrated enhanced capillary density (fluorescence intensity, 2.55+/-0.23 e(+9) versus 1.23+/-0.17 e+9, P<0.01) and increased luminal diameter of the basal limbus artery (39.0+/-2.8 versus 27.9+/-1.3 mu m, P<0.01) for Ang1+VEGF compared with VEGF alone. In contrast to Ang1+VEGF, Ang2+VEGF produced longer vessels and, at the tip of the developing capillaries, frequent isolated sprouting cells. In the case of Ang2+VEGF, however, luminal diameter of the basal limbus artery was not increased (26.7+/-1.9 versus 27.9+/-1.3, P=NS). These findings constitute what is to our knowledge the first direct demonstration of postnatal bioactivity associated with either angiopoietin. In particular, these results indicate that angiopoietins may potentiate the effects of other angiogenic cytokines. Moreover, these findings provide in vivo evidence that Ang1 promotes vascular network maturation, whereas Ang2 works to initiate neovascularization.