Abstract
Decay-accelerating factor (DAF), a membrane-bound complement regulatory protein, is up-regulated on endothelial cells (ECs) following treatment with vascular endothelial growth factor (VEGF), providing enhanced protection from complement-mediated injury. We explored the signaling pathways involved in this response. Incubation of human umbilical vein ECs with VEGF induced a 3-fold increase in DAF expression. Inhibition by flk-1 kinase inhibitor SU1498 and failure of placental growth factor (PlGF) to up-regulate DAF confirmed the role of VEGF-R2. The response was also blocked by pretreatment with phospholipase C-gamma (PLCgamma) inhibitor U71322 and protein kinase C (PKC) antagonist GF109203X. In contrast, no effect was seen with nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA). Use of PKC agonists and isozyme-specific pseudosubstrate peptide antagonists suggested a role for PKCalpha and -epsilon in VEGF-mediated DAF up-regulation. This was confirmed by transfection of ECs with PKCalpha and -epsilon dominant-negative constructs, which in combination completely abrogated induction of DAF by VEGF. In contrast, LY290042, a phosphoinositide 3-kinase (PI3K) inhibitor, significantly augmented DAF expression, suggesting a negative regulatory role for phosphoinositide 3-kinase. The widely used immunosuppressive drug cyclosporin A (CsA) inhibited DAF induction by VEGF in a dose-dependent manner. The VEGF-induced DAF expression was functionally effective, significantly reducing complement-mediated EC lysis, and this cytoprotective effect was reversed by CsA. These data provide evidence for a VEGF-R2-, phospholipase C-gamma-, and PKCalpha/epsilon-mediated cytoprotective pathway in ECs. This may represent an important mechanism for the maintenance of vascular integrity during chronic inflammation involving complement activation. Moreover, inhibition of this pathway by CsA may play a role in CsA-mediated vascular injury.
Highlights
§ To whom correspondence should be addressed: Cardiovascular Medicine Unit, Eric Bywaters Center, Imperial College London, Hammersmith Hospital, Du Cane Rd., London W12 0NN, United Kingdom
Vascular endothelial growth factor (VEGF)-induced Up-regulation of Decay-accelerating factor (DAF) Is Mediated via VEGF-R2 and PLC␥—DAF is expressed at a low level on resting endothelial cells (ECs) in culture
We have shown previously that DAF expression on human vascular ECs may be regulated via distinct agonist-specific pathways, leading us to propose that DAF induction may be important in the maintenance of vascular integrity during inflammation, thrombosis, and angiogenesis [16, 36, 37]
Summary
§ To whom correspondence should be addressed: Cardiovascular Medicine Unit, Eric Bywaters Center, Imperial College London, Hammersmith Hospital, Du Cane Rd., London W12 0NN, United Kingdom. We describe in detail the VEGF-R2- and PKC-dependent signaling pathway by which VEGF induces DAF expression on the EC surface, so enhancing cytoprotection against complement-mediated injury. This resulted in a dose-dependent inhibition of VEGF-induced DAF up-regulation (not shown), which was maximal following pretreatment with 10 M U73122 (Fig. 1C).
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