A proteomic investigation into the mechanisms of VEGF‐adhesion crosstalk in endothelial cells.

UoM administered thesis: Phd

  • Authors:
  • Jenny James

Abstract

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 integrin‐ECM 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 pre‐existing vasculature. A better understanding of the factors and mechanisms involved in VEGF‐adhesion 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 VEGF‐adhesion 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 ≥two‐fold. A complementary phosphoproteomic strategy was adopted to analyse changes in protein phosphorylation within adhesion complexes following VEGF treatment. Phosphoproteomic analysis of VEGF‐induced enriched IAC and total cell lysate samples revealed that over 18% of HUVEC IAC phosphopeptides identified changed greater that ≥two‐fold. 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 protein‐protein interaction networks were constructed, statistical analyses employed, and detailed kinase prediction analysis performed. A range of proteins were highlighted as potentially important in VEGF‐adhesion crosstalk, including a role for Src kinase‐mediated protein phosphorylation. To investigate a role for Src kinase, VEGFinduced migration assays were performed; Src and FAK inhibition reduced VEGF‐induced migration of FN‐plated 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 VEGF‐adhesion 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 VEGF‐adhesion 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 follow‐up studies to be performed following on from the proteomic datasets generated here.

Details

Original languageEnglish
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Award date1 Aug 2018