Inhibition of angiopoietin 2 attenuates lumen formation of tumour-associated vessels in vivo

Anti-angiogenic therapy, inhibition of a co-operative process with vascular endothelial cells and pericytes could be an effective strategy to treat malignant tumours. Apart from vascular endothelial growth factor (VEGF), angiopoietin 2 (Ang2) is a promising target of anti-angiogenic therapy. Although inhibition of Ang2 has been shown to decrease tumour size in preclinical and phase I trials, its mechanisms of action remain largely unknown. To elucidate the mechanisms of Ang2 inhibition, we have focused on differentiation of the vessels as well as on growth of the vessels, especially in vivo. L1-10, a selective Ang2 inhibitor was used. The in vitro effects of Ang2 inhibition or addition of Ang2 using HUVECs were also examined. Growth and differentiation of tumour-associated vessels were investigated in xenografts derived from a colon cancer treated by L1-10. Effects of VEGF inhibition were also examined to discriminate Ang2-specific action on the tumour-associated vessels. In vitro studies showed that VEGF enhanced proliferation and tube formation of HUVECs, and caused a significant increase in Rac1 and CDC42 expression when cultured in the collagen matrix gel, whereas neither Ang2 nor L1-10 affected in vitro behaviour of HUVECs or levels of the proteins. In vivo, on the other hand, we found that Ang2 inhibition with treatment of L1-10 dose-dependently decreased tumour growth. Furthermore, we found that L1-10 treatment extends the tumour-associated vessels whilst it suppressed a sound lumen formation. Histological analysis on xenografts suggests that Ang2 inhibition could have disturbed in vivo vascular differentiation. Our data provide a novel aspect that Ang2 may play an essential role in in vivo vascular differentiation, thus supporting a rationale for Ang2-targeted therapy against colon cancer.