Controlling angiogenesis : functional studies of angiomotin

Sammanfattning: The expansion of a blood circulatory network by the process of angiogenesis is essential for embryonic as well as postnatal growth. Deregulated blood vessel formation may also negatively contribute to the pathogenesis of diseases, e.g. cancer and diabetic retinopathy. Furthermore, compounds that interfere with angiogenesis signalling pathways have shown great promise when used in clinical trials for patients suffering from cancer or macular degeneration. The formation of a functional blood vessel involves several distinct steps. Cells of the vessel wall, i.e. endothelial cells, extend filopodia that sense pro-angiogenic signals and direct migration and the formation of the vessel network. Circulation is established when two endothelial sprouts fuse and form a continuous lumen. Angiomotin has previously been implicated to play a role in endothelial migration and to be a possible target for anti-angiogenic therapy. The aim of this thesis was to investigate the biological role of angiomotin during vascular development and to elucidate the signalling pathways involved. In this thesis it is demonstrated that the angiomotin gene is expressed as two isoforms with distinct functions. The shorter isoform, p80-angiomotin, is expressed during the migratory phase of retinal angiogenesis, whereas the other isoform, p130-angiomotin, is expressed during and after vessel maturation. In vitro, these isoforms exhibit opposite functions. For instance, p80-angiomotin promotes cell migration whereas p130-angiomotin promotes actin fibre formation and cell contacts. It is further shown that p80-angiomotin expression removes p130-angiomotin from cell junctions resulting in a migratory switch. The angiomotin signalling pathway was analysed by identifying binding proteins by two different approaches, peptide pull down and yeast two-hybrid screening. Data are presented demonstrating that the PDZ-binding domain of angiomotin binds to a polarity protein complex as well as to a Rho-GEF, which has previously been shown to mediate endothelial cell migration. These findings argue that angiomotin acts as a scaffold for proteins regulating cell polarity and GTPase activity. The biological role of angiomotin during mouse and zebrafish embryogenesis was also studied. Genetic ablation of angiomotin in the mouse results in vascular defects in the intersomitic region as well as dilated vessels in the brain and embryonic lethality after E11. Furthermore, knockdown of angiomotin in zebrafish impaired the migration of intersegmental vessels and caused dilation of vessels in the brain, confirming the phenotype found in the mouse. It is further shown that angiomotin deficient endothelial cells have an intact proliferative response to VEGF but exhibit defects in migration. Taken together, these data indicate that angiomotin is essential for normal embryonic vessel formation and that it may affect directional cell migration by controlling cell polarity and GTPase activity.