Gliki, Georgia; (2001) Signalling mechanisms mediating biological functions of vascular endothelial growth factor in endothelial cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

It was previously reported that VEGF stimulates prostacyclin (PGI2) production in human umbilical vein endothelial cells (HUVECs) via activation of the extracellular signal-regulated kinase (ERK) cascade. For this thesis, the contribution of other signalling pathways in VEGF-induced PGI2 production was investigated. Work carried out also intended to elucidate the signalling mechanisms that mediate VEGF-induced activation of ERK in HUVECs. VEGF- induced cytosolic phospholipase A2 (cPLA2) phosphorylation and PGI2 production were blocked by GF109203X and Calphostin C, two structurally unrelated, specific PKC inhibitors. These inhibitors also blocked VEGF-induced ERK and MEK (mitogen-activated protein kinase kinase) phosphorylation. VEGF increased PKC activity and immunoreactivity of PKCδ, α and ԑ in the particulate fraction of HUVECs while the [symbol] isoform was unaffected. Association of Raf 1 (the upstream activator of the MEK-ERK pathway) with PKCδ was increased by VEGF, as determined by co-immunoprecipitation of Raf 1 with PKCδ. A PKCδ-selective inhibitor, rottlerin, abrogated VEGF-induced ERK activation and PGI2 production while a PKCα-selective inhibitor, Go6976, had no effect on ERK activation but partially inhibited PGI2 production in response to VEGF. Antisense oligonucleotides against PKCδ, but not against PKCα, reduced VEGF-induced ERK activation. Inhibition of Ca2+ fluxes by BAPTA/AM inhibited PGI2 production but had no effect on VEGF-induced ERK activation. Neither inhibition nor activation of the NO/cGMP pathway had any effect on ERK activation and PGI2 synthesis. These findings indicate that activation of PKC plays a crucial role in VEGF signalling via the ERK cascade and implicate PKCδ as a key mediator of this pathway. The role of PKC in angiogenesis was also addressed. Inhibitors of PKC markedly decreased VEGF-induced angiogenesis, as determined in an in vitro model of tube formation. PKC inhibitors and PKC downregulation, induced by prolonged phorbol ester treatment, inhibited VEGF-induced phosphorylation of Akt, the PI3K effector. These findings suggest that PKC mediates VEGF signalling via the PI3K/Akt pathway. In conclusion, the results from this thesis indicate that PKC is a crucial mediator of VEGF signal transduction pathways and of two biological functions of VEGF, namely PGI2 production and angiogenesis. These results also suggest that distinct PKC isoforms are utilized by VEGF in eliciting distinct biological effects.

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