The vascular system is a complex, highly branched tubular network that is able to supply oxygen and nutrients to all body tissues and remove waste. During growth and tissue repair the vasculature undergoes coordinated expansion through a process known as angiogenesis, which involves the sprouting and branching of new blood vessels from pre-existing vasculature. Vascular endothelial growth factor A (VEGF-A) is the most potent inducer of angiogenesis. VEGF-A regulates blood vessel formation through the activation of three related tyrosine kinase receptors: VEGF receptors (VEGFRs)-l, -2, and -3. The main pro-angiogenic receptor expressed by endothelial cells is VEGFR-2. Upon ligand binding, VEGFR-2 activates multiple signalling pathways, which ultimately leads to the development of new blood vessels. The formation of VEGF-A concentration gradients plays an important role in the regulation of blood vessel growth. Endothelial cells in growing vascular sprouts modify their behaviour in response to their position within a VEGF-A gradient. I show that high doses of VEGF-A (40 ng/ml) remove 80% ofVEGFR-2 from the surface of human umbilical vein endothelial cells (HUVECs) over 2 h, leading to the rapid attenuation of VEGF-A-induced receptor signalling. In contrast, low doses of VEGF-A (2 ng/m!) trigger little receptor internalisation, with 10% of VEGFR-2 being lost over the same period. Although low doses preserve VEGFR-2 surface expression, receptor phosphorylation and p44/42 MAPK activation are down regulated to the same extent as high dose signals. Experiments show that surface-expressed VEGFR-2 remains active after prolonged exposure to 2 ng/ml ofVEGF-A; however, ELISA assays demonstrate that endothelial cells remove/sequester ~ 1 ng/ml VEGF-A from cell media. The termination of VEGF-A-induced signalling is therefore believed to result from either the inactivation or sequestration of extracellular VEGF-A. However, neither sequestration by soluble VEGFR-1 nor proteolysis by plasmin or matrix metalloproteinases can account for the loss of VEGF-A from cell media. Immunofluorescence microscopy shows that significant amounts of extracellular VEGF-A is localised to endothelial cell-cell junctions. However, junctional VEGF-A binding cannot be explained by key VEGF-A receptors or co-receptors, including VEGFR-1, VEGFR-2, neuropilin-1, vascular endothelial cadherin, {3 1 integrin, {3 3 integrin, glypicans-1 -6, and syndecans- 1-4. Proteomic analysis has identified the recently characterised VEGF-AIVEGFRbinding proteins CD146 and multimerin-2 as potential mediators of VEGF-A binding to the surface of endothelial cells. Future work will focus on elucidating their roles as regulators of extracellular VEGF-A distribution and/or concentration.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628991 |
| Date | January 2013 |
| Creators | Cowell, Christopher A. M. |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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