Adhesion of cells to the surrounding extra cellular matrix (ECM) is primarily mediated by integrin cell surface receptors, which mechanically link the ECM and actin cytoskeleton, enabling the positioning of cells within tissues. Upon integrinâECM binding, signalling and adaptor proteins are recruited, leading to the formation of intracellular integrin adhesion complexes (IACs). These complexes and resulting integrin signalling pathways coordinate their activity with growth factor receptors and associated signalling pathways, regulating downstream processes such as adhesion, migration and proliferation. One example of crosstalk between growth factor and integrin mediated adhesion involves vascular endothelial growth factor (VEGF) in endothelial cells (ECs), and contributes to angiogenesis, the formation of new blood vessels from preâexisting vasculature. A better understanding of the factors and mechanisms involved in VEGFâadhesion crosstalk would enhance understanding of vascular development, and highlight ways that therapeutics can be used to treat angiogenic associated disorders. The aim of this project was to identify mediators of VEGFâadhesion crosstalk in ECs. Initially, a protocol for human umbilical vein endothelial cell (HUVEC) IAC enrichment was optimised. Briefly, HUVECs were plated on fibronectin for two hours to allow IAC formation, followed by crosslinking to stabilise IACs, cell body removal, and collection of remaining IACs. Mass spectrometric analyses of enriched IACs defined, for the first time, the HUVEC IAC composition, a network of 297 proteins. This dataset was comparable to previously reported IAC composition datasets, confirming successful identification of IAC components. HUVEC IACs were then enriched following VEGF treatment, and proteomic analysis revealed that the abundance of only 1% of proteins changed â¥twoâfold. A complementary phosphoproteomic strategy was adopted to analyse changes in protein phosphorylation within adhesion complexes following VEGF treatment. Phosphoproteomic analysis of VEGFâinduced enriched IAC and total cell lysate samples revealed that over 18% of HUVEC IAC phosphopeptides identified changed greater that â¥twoâfold. Together, these data suggested that, while adhesion complex composition remains largely unchanged during VEGFadhesion crosstalk, changes and signalling events occur mainly through phosphorylation. From these proteomic and phosphoproteomic datasets, a range of proteinâprotein interaction networks were constructed, statistical analyses employed, and detailed kinase prediction analysis performed. A range of proteins were highlighted as potentially important in VEGFâadhesion crosstalk, including a role for Src kinaseâmediated protein phosphorylation. To investigate a role for Src kinase, VEGFinduced migration assays were performed; Src and FAK inhibition reduced VEGFâinduced migration of FNâplated HUVECs, with combined kinase inhibition showing greater effects. This study presents one of the first to (1) perform a global proteomic analysis of EC IACs, and (2) investigate VEGFâadhesion crosstalk mechanisms using proteomic and phosphoproteomic workflows. The datasets derived in this study contain a great deal of information, which has been used here to define the HUVEC IAC protein composition and identify candidates that may contribute to VEGFâadhesion crosstalk. For example, Src kinase has been highlighted as a potential mediator of crosstalk, and future studies leading from this data may aid discovery of associated therapeutic targets. This study has the potential to provide a base for many future studies; for example there is unlimited potential for candidate selection and followâup studies to be performed following on from the proteomic datasets generated here.
|Date of Award||1 Aug 2018|
- The University of Manchester
|Supervisor||Ann Canfield (Supervisor) & Martin Humphries (Supervisor)|