Vascular endothelial growth factor A isoform-specific regulation of endothelial cell function

• Main Author: Fearnley, Gareth William
• Other Authors: Ponnambalam, Sreenivasan ; Wheatcroft, Stephen B.
• Published: University of Leeds 2015
• Subjects: 570
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669619

Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase (RTK) vascular endothelial growth factor 2 (VEGFR2) triggers an array of downstream signal transduction pathways which modulate a multitude of endothelial cell responses, such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Multiple splice isoforms of VEGF-A exist, yet it is unclear how different VEGF-A isoforms bind to the same RTK to program distinct cellular responses. The work presented in this PhD thesis evaluated VEGF-A isoforms for their ability to program VEGFR2 endocytosis, post-translational modification, proteolysis and terminal degradation. Such changes in VEGFR2 status were linked to downstream signal transduction and gene expression, with relevance to cell function and vascular physiology. VEGF-A isoforms differentially promoted VEGFR2 tyrosine transautophosphorylation and endocytosis. Different VEGFR2-VEGF-A complexes exhibit altered ubiquitination, a hallmark of trafficking through the endosome-lysosome system for subsequent terminal degradation and proteolysis. VEGF-A isoform-specific VEGFR2 phosphorylation coupled with endocytosis and delivery to early endosomes is required for isoform-specific activation of the MEK1-ERK1/2 signal transduction pathway and endothelial cell proliferation. VEGF-A isoforms also exhibited differences in their ability to stimulate arterial regeneration in a mouse hind limb ischaemia model. VEGF-A isoform-specific ERK1/2 activation was essential for the phosphorylation of activating transcription factor 2 (ATF-2) at residue T71. Differential activation of ATF-2 regulated VEGF-A isoform-specific gene transcription (e.g. VCAM-1) and endothelial cell responses, such as leukocyte recruitment. Additionally, basal ATF-2-pT71 levels are required to maintain endothelial cell cycle commitment, via repressing p53-dependent gene transcription. Furthermore, VEGF-A isoforms promoted differential PLC1 phosphorylation and a subsequent isoform-specific increase in cytosolic calcium ions. A functional consequence of this VEGF-A isoform-specific calcium ion flux, was differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2 (NFAT1) in order to regulate endothelial cell migration. Thus, this study provides a mechanistic framework for understanding how different ligand isoforms differentially program RTK functionality in health and disease.