Angiopoitein-1 (Ang1) is a relatively recently discovered vascular ligand which has been shown to have substantial potential therapeutic applications in treating a range of pathologies including: transplant atherosclerosis, stroke, diabetic retinopathies and sepsis. However, this large glycoprotein shows variable solubility and biological activity as a recombinant protein and is difficult to produce. This project aims to develop small, stable Ang1 mimetic proteins for use as potential therapeutic lead molecules.

Based on the mechanism by which the native ligand activates its receptor, a small synthetic ligand was designed and expressed in E.Coli. The synthetic ligand was isolated and purified and its ability to bind the angiopoietin receptor analysed by in vitro ELISA. Cell surface binding was examined by immunoflouresence staining and the ability of the ligand to activate cellular signalling was tested by phospho-specific immunoblotting. Functionally, the influence of the ligand on endothelial cells migration was studied using Boyden chamber chemo tactic assay.

A synthetic ligand was produced of molecular mass 12kDa compared with the 70kDa native ligand. This ligand binds angiopoietin receptor in vitro. In cellular assays the ligand interacts with the endothelial cell surface and activates the angiopoietin receptor. In addition it stimulates downstream signalling pathways including the phosphatidylinositol 3-kinase/Akt and Erk1/2 pathways. The ligand activates endothelial cell migration.

The novel synthetic ligands are easy to produce, can be expressed in E Coli, are highly soluble and stable, activate the angiopoietin receptor. The properties of these synthetic ligands suggest they may be lead molecules for generating potential therapeutic Ang1 mimetics.