Mechanisms regulating phenotypic change and survival of vascular cells: Studies of the expression, subcellular localisation and regulation of focal adhesion kinase

Focal adhesion kinase (FAK) has been implicated in cellular processes linked to adhesion, migration and survival of animal cells. Evidence that FAK is involved in cellular locomotion suggested that it may also play a role in phenotypic transition of VSMC from a quiescent to a migratory state as seen in the development of neointimal hyperplasia. The expression of FAK and two other focal adhesion components, paxillin and p130Cas, were upregulated in early aortic medial explant culture, leading to outgrowth and phenotypic modulation of VSMC. Explant treatment with PDGF-BB, IGF-I and bFGF also led to explant outgrowth and increased protein expression of FAK, paxillin and p130Cas. These results indicate that increased expression of FAK may play a role in the early changes leading to phenotypic plasticity of VSMC. Investigation of the subcellular localisation of FAK demonstrated nuclear expression of full length FAK (p125FAK) in VSMC and HUVECs, in addition to its localisation to focal adhesions. A p50 NH2-terminal domain FAK fragment (p50N-FAK) was exclusively localised in the nucleus in both cell types, whilst a p55 COOH- terminal FAK fragment (p55C-FAK) was found only in the cytosol. The findings of nuclear localisation of piand intracellular segregation of p50N-FAK and p55C- FAK suggest novel regulatory roles for FAK and FAK-related species in vascular cells. Another major aim of this thesis was to examine the role of FAK in the endothelial cell apoptotic response. Apoptosis of HUVECs resulted in caspase-dependent p125FAK proteolysis and increased nuclear accumulation of p50N-FAK, which were preceded by rapid FAK tyrosine dephosphorylation at residues Y861, Y407 and Y397. Y861 phosphorylation was shown to be maintained via a PKC-dependent mechanism and regulated very early in the HUVEC apoptotic response. These findings provide novel insights into the apoptotic regulation of FAK in HUVECs and the signalling mechanisms important for maintaining FAK phosphorylation, which may have relevance for VSMC and other cell types