Angiogenesis, the growth of new capillaries from preexisting ones, occurs through dynamic functions of the endothelial cells (EC), including migration, which is essential to achieve an organized formation of the vessel sprout. We demonstrated previously that an aspirintriggered lipoxin analog, 15-epi-16-(para-fluoro)-phenoxylipoxin A 4 (ATL-1), inhibits vascular endothelial growth factor (VEGF)-induced EC migration. In the present study, we investigated the effects of ATL-1 in the actin cytoskeleton reorganization of EC stimulated with VEGF. Pretreatment of EC with ATL-1 caused a reduction in VEGF-induced stress fibers and therefore reduced the intracellular content of filamentous actin. A concomitant impairment in stress-activated protein kinase (SAPK2/ p38) phosphorylation suggests that ATL inhibition of VEGF-stimulated actin polymerization involves the SAPK2/p38 pathway. Moreover, ATL-1 treatment inhibited focal adhesion clustering due to inhibition of focal adhesion kinase (FAK) phosphorylation and the subsequent association of FAK with the actin cytoskeleton. This final event, which ultimately allows cell migration, was reverted by an LX receptor antagonist, but not by a cys-LT1R antagonist, indicating an effect via the Gprotein-linked LXA 4 receptor. Together our results provide evidence that ATL-1 inhibits EC migration via the concerted inhibition of actin polymerization and proper assembly of focal adhesions, supporting a role for these novel lipid mediators as angiogenesis modulators.

Background and purpose: 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. Key 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-kB has been implicated in VEGF-mediated MMP expression and EC proliferation, we postulated that ATL-1 might modulate the NF-kB pathway and, indeed, ATL-1 inhibited NF-kB nuclear translocation. Pretreatment of EC with ATL-1 strongly decreased VEGFdependent 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 and implications:The synthetic 15-epi-lipoxin analogue, ATL-1, is a highly potent molecule exerting its effects on multiple steps of the VEGF-induced angiogenesis.