Novel pathways in microvascular signalling

The regulation of microvascular survival impacts both developmental remodelling of the vasculature, and various pathologies. The aim of this thesis was to examine the role of a recently identified A20 binding inhibitor of NF-kB, a zinc finger protein termed ABIN-2 in endothelial protection. More specifically, looking at its effects on inflammation and apoptosis in endothelia both at the cellular level and in vivo. The involvement of ABIN-2 in the Tie2 receptor pathway was also examined. Tie2 is an endothelial receptor essential for blood vessel formation and promotes endothelial survival. A transfection protocol was established allowing expression ABIN-2 in up to 90% of endothelial cells. ABIN-2 was shown to reduce apoptosis in endothelial cells as well as improve cell survival following growth factor deprivation. This effect was inhibited by Wortmannin and LY294002 which are known inhibitors of phatidylinositol-3 kinase. Expression of the truncated form of ABIN-2 lacking the carboxy terminal of ABIN-2 did not protect cells from apoptosis. In addition, expression of the truncated form prevented cell rescue by angiopoeitin-1 from apoptosis. The chick chorioallantoic membrane was used as an in vivo model for testing the role of ABIN-2 in vessel inflammation. It was possible to use this model to transfect plasmids into live microvessels using electroporation resulting in high yield expression of target protein. This model was adapted to look at microvessel inflammation and apoptosis. Expression of ABIN-2 in microvessels reduced leukocyte rolling following tumour necrosis factor-a and lipopolysaccharide induced inflammation. The truncated form of ABIN-2 lacking the carboxy terminal did not reduce microvessel inflammation. The NF-kB inhibitor PTDC was found to suppress leukocyte rolling. ABIN-2 expression also appeared to give limited protection against apoptosis in vivo.