ATL-1, an analogue of aspirin-triggered lipoxin A4, is a potent inhibitor of several steps in angiogenesis induced by vascular endothelial growth factor.
OBJECTIVE
Vascular endothelial growth factor (VEGF) is the most important proangiogenic protein. We have demonstrated that ATL-1, a synthetic analogue of aspirin-triggered lipoxin A(4), inhibits VEGF-induced endothelial cell (EC) migration. In the present study, we investigated the effects of ATL-1 in several other actions stimulated by VEGF.


METHODS
Human umbilical vein ECs were treated with ATL-1 for 30 min before stimulation with VEGF. Cell proliferation was measured by thymidine incorporation. Adherent cells were determined by fluorescence intensity using a Multilabel counter. Expression and activity of matrix metalloproteinases (MMP) were analysed by western blot and zymography.


RESULTS
ATL-1 inhibited EC adhesion to fibronectin via interaction with its specific receptor. Furthermore, VEGF-induced MMP-9 activity and expression were reduced by pretreatment with ATL-1. Because the transcription factor NF-kappaB has been implicated in VEGF-mediated MMP expression and EC proliferation, we postulated that ATL-1 might modulate the NF-kappaB pathway and, indeed, ATL-1 inhibited NF-kappaB nuclear translocation. Pretreatment of EC with ATL-1 strongly decreased VEGF-dependent phosphorylation of phosphainositide 3-kinase (PI3-K) and extracellular signal-regulated kinase-2 (ERK-2), two signalling kinases involved in EC proliferation. Inhibition of VEGF-induced EC proliferation by ATL-1 was antagonized by sodium orthovanadate, suggesting that this inhibitory activity was mediated by a protein tyrosine phosphatase. This was confirmed by showing that ATL-1 inhibition of VEGF receptor-2 (VEGFR-2) phosphorylation correlates with SHP-1 association with VEGFR-2.


CONCLUSIONS
The synthetic 15-epi-lipoxin analogue, ATL-1, is a highly potent molecule exerting its effects on multiple steps of the VEGF-induced angiogenesis.
Cezar-de-Mello
P F T
PF
Departamento de Farmacologia e Psicobiologia, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil.
Vieira
A M
AM
Nascimento-Silva
V
V
Villela
C G
CG
Barja-Fidalgo
C
C
Fierro
I M
IM
D021581:Active Transport, Cell Nucleus; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D044045:Lipoxins / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000503:physiopathology / Q000517:prevention & control*; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000187:drug effects* / Q000378:metabolism; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
C447621:16-(4-fluorophenoxy)lipoxin A4; D044045:Lipoxins; D016328:NF-kappa B; D042461:Vascular Endothelial Growth Factor A; C040527:lipoxin A4; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6