Tie2 is an endothelial receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis. Tie2 regulates the transition from vascular quiescence to angiogenesis. Consequently, Tie2 plays a critical role in early stages of tumor angiogenesis. The ligands for Tie2-Angiopoietin-1 (Ang1) and Ang2-each exhibit opposite effects on the vasculature. Stimulation of Tie2 by Ang1 promotes vascular maturation and stabilization, whereas stimulation by Ang2 destabilizes blood vessels to facilitate angiogenesis. Remarkably, both Ang1 and Ang2 bind Tie2 with high affinity (-3 nM), and both ligands have been shown to induce Tie2 autophosphorylation in various experimental systems. These and other findings suggest that different types of Tie2 receptor complexes can be assembled on the endothelial cell surface in response to Ang1 versus Ang2. The goal of this study is to develop molecular probes that recognize the components of the Tie2 receptor complex that are responsible for differential effects of Ang1 and Ang2 signaling. This exploratory grant proposes to use cell-based, Tie2-proximity-directed panning of specialized peptide and antibody phage display libraries to isolate phage that recognize proteins associated with the Tie2 receptor. In addition, phage probes will be sought that recognize determinants of the Tie2 receptor that respond uniquely to either Ang1 or Ang2. All phage peptides and antibodies will first be validated by cell-based binding assays and endothelial cell functional assays and then tested using in vivo angiogenesis assays to determine the effects of these compounds on vascularization and vascular permeability. Validated ligands have strong potential for development as biomarker-specific probes with applications in basic science, therapeutics, diagnostics, and imaging of early events in tumor angiogenesis.
