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Angiogenesis is the development of blood capillaries from pre-existing vessels. Vascular endothelial growth factor (VEGF) is a key regulator of vessel growth and regression, and acts as an endothelial survival factor by protecting endothelial cells from apoptosis. Many genes involved in cell proliferation and apoptosis are regulated by the nuclear factor kappa B (NFkappaB) transcription factor family. This study aimed to address the hypothesis that VEGF-mediated survival effects on endothelium involve NFkappaB. Using an NFkappaB-luciferase reporter adenovirus, we observed activation of NFkappaB following VEGF treatment of human umbilical vein endothelial cells. This was confirmed using electrophoretic mobility shift assay and found to involve nuclear translocation of NFkappaB sub-unit p65. However, NFkappaB activation occurred without degradation of inhibitory IkappaB proteins (IkappaBalpha, IkappaBbeta, and IkappaBepsilon). Instead, tyrosine phosphorylation of IkappaBalpha was observed following VEGF treatment, suggesting NFkappaB activation was mediated by degradation-independent dissociation of IkappaBalpha from NFkappaB. Adenovirus-mediated over-expression of either native IkappaBalpha, or of IkappaBalpha in which tyrosine residue 42 was mutated to phenylalanine, inhibited induction of NFkappaB-dependent luciferase activity in response to VEGF. Furthermore, VEGF-induced upregulation of mRNA for the anti-apoptotic protein Bcl-2 and cell survival following serum withdrawal was reduced following IkappaBalpha over-expression. This study highlights that different molecular mechanisms of NFkappaB activation may be involved downstream of stimuli which activate the endothelial lining of blood vessels.

Original publication

DOI

10.1016/j.bbrc.2005.12.095

Type

Journal article

Journal

Biochemical and biophysical research communications

Publication Date

02/2006

Volume

340

Pages

984 - 994

Addresses

Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College, London, UK. Jennifer.grosjean@imperial.ac.uk

Keywords

Endothelium, Vascular, Umbilical Veins, Cells, Cultured, Endothelial Cells, Humans, Adenoviridae, Neovascularization, Pathologic, Luciferases, Vascular Endothelial Growth Factor A, Tyrosine, NF-kappa B, Transcription Factors, RNA, Messenger, Blotting, Western, Signal Transduction, Apoptosis, Cell Survival, Transcription, Genetic, Up-Regulation, Phosphorylation, Time Factors, Transcription Factor RelA