Contrasting Actions of Selective Inhibitors of Angiopoietin-1 and Angiopoietin-2 on the Normalization of Tumor Blood Vessels

Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have complex actions in angiogenesis and vascular remodeling due to their effects on Tie2 receptor signaling. Ang2 blocks Ang1-mediated activation of Tie2 in endothelial cells under certain conditions but is a Tie2 receptor agonist in others. We examined the effects of selective inhibitors of Ang1 (mL4-3) or Ang2 (L1-7[N]), alone or in combination, on the vasculature of human Colo205 tumors in mice. The Ang2 inhibitor decreased the overall abundance of tumor blood vessels by reducing tumor growth and keeping vascular density constant. After inhibition of Ang2, tumor vessels had many features of normal blood vessels (normalization), as evidenced by junctional accumulation of vascular endothelial-cadherin, junctional adhesion molecule-A, and platelet/endothelial cell adhesion molecule-1 in endothelial cells, increased pericyte coverage, reduced endothelial sprouting, and remodeling into smaller, more uniform vessels. The Ang1 inhibitor by itself had little noticeable effect on the tumor vasculature. However, when administered with the Ang2 inhibitor, the Ang1 inhibitor prevented tumor vessel normalization, but not the reduction in tumor vascularity produced by the Ang2 inhibitor. These findings are consistent with a model whereby inhibition of Ang2 leads to normalization of tumor blood vessels by permitting the unopposed action of Ang1, but decreases tumor vascularity primarily by blocking Ang2 actions.Solid tumors require angiogenesis—the formation of new blood vessels from existing vessels—for survival, growth, and metastasis.¹ Tumor vessels are structurally and functionally abnormal.^1,2 They exist in a constantly dynamic state of sprout formation, proliferation, remodeling, or regression. Structurally, tumor vessels tend to be leaky and tortuous, lacking the hierarchical arrangement of arterioles, capillaries, and venules.² Pericytes that attach to and help stabilize normal vessels are loosely associated with the endothelium of tumor vessels.^1,2 These vascular abnormalities result in impaired and heterogeneous blood flow. In tumors, angiogenesis inhibitors not only cause vessel regression or retardation of vessel growth, but they can also induce vascular normalization.^1,2,3The complicated regulation of angiogenesis and vascular maturation involves multiple signaling cascades driven by endothelial-cell specific growth factors and their receptors. One of these, vascular endothelial growth factor (VEGF) has been extensively studied,⁴ but angiopoietins and other growth factors are also involved.^5,6 The angiopoietin ligands (Ang1 and Ang2) and their receptor (Tie2) have essential roles in vascular development.^7,8 Ang1 is produced by vascular mural cells, pericytes, and certain other cells, whereas Ang2 and Tie2 are expressed primarily by endothelial cells.Angiogenesis and vascular remodeling involve a complex coordination of Ang1 and Ang2 signaling through Tie2.⁵ The traditional view of Ang1 and Ang2 signaling is that the growth factors have opposing effects on Tie2 receptor activation: Ang1 binds to Tie2 to promote vascular maturation and integrity, whereas Ang2 acts as a naturally occurring antagonist of Ang1.^7,8,9,10,11 Although a number of studies indicate an antagonistic role of Ang2, recent studies have shown that Ang2 can have an agonistic role depending on the experimental environment.^12,13,14,15 If expressed at high concentrations or for long durations in cultured endothelial cells, Ang2—like Ang1—can induce Tie2 receptor phosphorylation.^13,16 Ang2 can also promote chemotaxis, tube formation, migration, and sprouting of endothelial cells in the absence of Ang1,¹⁴ which support the view that Ang2 actions are context- dependent.Normalization of tumor vascular morphology and function has been demonstrated with numerous angiogenesis inhibitors.^1,17,18 Ang1 and Ang2 regulate vascular maturation and integrity during development; however, their effects on normalization of tumor vessels are not known. Tumors grown in mice lacking Ang2 have a more mature vascular phenotype, but it is not known whether Ang1 plays a role.¹⁹ The effects of individual angiopoietins on the tumor vasculature have not been studied extensively in loss-of-function experiments, due largely to the limited availability of selective angiopoietin inhibitors. Some clues to the effects of Ang1 and Ang2 on tumor vessels have been garnered through overexpression of the ligands in tumor cell xenografts.^{20,21,22,23,24,25,26} These studies, however, have yielded conflicting data,^{20,21,22,23,24,25,26} the ligands were administered at nonphysiological levels, and the results were restricted to prevention studies. Studies blocking the Tie2 receptor have shown reduced tumor angiogenesis,^27,28,29,30 but the specific roles of each ligand cannot be differentiated. Pharmacological angiopoietin inhibitors using antisense, aptamer, and peptide-Fc fusion protein (peptibody) technologies are currently being developed, but published studies have been restricted to inhibition of Ang1 or Ang2 alone.^31,32,33 Studies using aptamers or peptibodies that potently neutralize Ang2 activity showed that Ang2 antagonism resulted in inhibition of angiogenesis and tumor growth.^31,32 Inhibition of Ang1 in a cell line stably transfected with antisense RNA resulted in reduced tumor growth and angiogenesis.³³To gain a better understanding of the effects of Ang1 and Ang2 on blood vessels in tumors, we used selective inhibitors (peptibodies) of Ang1 and Ang2, alone or in combination, in Colo205 tumors. These studies focused on Colo205 tumors, as this model is sensitive to angiopoietin inhibitors.³¹ We found that inhibition of Ang1 alone had little effect on the tumor vasculature, whereas inhibition of Ang2 resulted in fewer tumor vessels and normalization of the surviving tumor vessels. When the Ang2 inhibitor was administered with the Ang1 inhibitor, tumor vessel normalization did not occur, but the Ang2 inhibitor-mediated reduction in vascularity was unaffected. These findings suggest that inhibition of Ang2 leads to unopposed Ang1 activity and results in normalization of tumor vessels. In contrast, the Ang2 inhibitor-mediated reduction in tumor vascularity was Ang1-independent.