Iolanda M. Fierro

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 A₄, 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‐κB has been implicated in VEGF‐mediated MMP expression and EC proliferation, we postulated that ATL‐1 might modulate the NF‐κB pathway and, indeed, ATL‐1 inhibited NF‐κB 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 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.British Journal of Pharmacology (2008) 153, 956–965; doi:10.1038/sj.bjp.0707650; published online 14 January 2008

Lipoxins and their aspirin-triggered carbon-15 epimers have emerged as mediators of key events in endogenous anti-inflammation and resolution. However, the implication of these novel lipid mediators on cardiovascular diseases such as hypertension, atherosclerosis, and heart failure has not been investigated. One of the major features shared by these pathological conditions is the increased production of reactive oxygen species (ROS) generated by vascular NAD(P)H oxidase activation. In this study, we have examined whether an aspirin-triggered lipoxin A₄ analog (ATL-1) modulates ROS generation in endothelial cells (EC). Pre-treatment of EC with ATL-1 (1–100 nM) completely blocked ROS production triggered by different agents, as assessed by dihydrorhodamine 123 and hydroethidine. Furthermore, ATL-1 inhibited the phosphorylation and translocation of the cytosplamic NAD(P)H oxidase subunit p47^phox to the cell membrane as well as NAD(P)H oxidase activity. Western blot and immunofluorescence microscopy analyses showed that ATL-1 (100 nM) impaired the redox-sensitive activation of the transcriptional factor NF-κB, a critical step in several events associated to vascular pathologies. These results demonstrate that ATL-1 suppresses NAD(P)H oxidase-mediated ROS generation in EC, strongly indicating that lipoxins may play a protective role against the development and progression of cardiovascular diseases.

Angiogenesis, the growth of new capillaries from pre-existing 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 aspirin-triggered lipoxin analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄ (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 G-protein-linked LXA₄ 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.

Lipoxins (LX) are bioactive eicosanoids that can be formed during cell to cell interactions in human tissues to self limit key responses in host defense and promote resolution. Aspirin treatment initiates biosynthesis of carbon 15 epimeric LXs, and both series of epimers (LX and aspirin-triggered 15-epi-LX) display counter-regulatory actions with neutrophils. In this study, we report that synthetic lipoxin A₄ (LXA₄) and 15-epi-LXA₄ (i.e., 15(R)-LXA₄ or aspirin-triggered LXA₄) are essentially equipotent in inhibiting human polymorphonuclear leukocytes (PMN) in vitro chemotaxis in response to leukotriene B₄, with the maximum inhibition (∼50% reduction) obtained at 1 nM LXA₄. At higher concentrations, 15-epi-LXA₄ proved more potent than LXA₄ as its corresponding carboxyl methyl ester. Also, exposure of PMN to LXA₄ and 15-epi-LXA₄ markedly decreased PMN transmigration across both human microvessel endothelial and epithelial cells, where 15-epi-LXA₄ was more active than LXA₄ at “stopping” migration across epithelial cells. Differences in potency existed between LXA₄ and 15-epi-LXA₄ as their carboxyl methyl esters appear to arise from cell type-specific conversion of their respective carboxyl methyl esters to their corresponding carboxylates as monitored by liquid chromatography tandem mass spectrometry. Both synthetic LXA₄ and 15-epi-LXA₄ as free acids activate recombinant human LXA₄ receptor (ALXR) to regulate gene expression, whereas the corresponding methyl ester of LXA₄ proved to be a partial ALXR antagonist and did not effectively regulate gene expression. These results demonstrate the potent stereospecific actions shared by LXA₄ and 15-epi-LXA₄ for activating human ALXR-regulated gene expression and their ability to inhibit human PMN migration during PMN vascular as well as mucosal cell to cell interactions.

Proliferative states such as chronic inflammation, ischemic diseases, and cancer are often accompanied by intense angiogenesis, a highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, and maturation. Aspirin-triggered lipoxins (ATLs), the 15R enantiomeric counterparts of lipoxins (LXs), are endogenous mediators generated during multicellular responses that display potent immunomodulatory actions. Herein, we report a novel action for the ATL stable analog 15-epi-16-(para-fluoro)-phenoxy-lipoxin A₄(denoted ATL-1), which proved to be a potent inhibitor of angiogenesis. This ATL inhibited endothelial cell proliferation in the 1 to 10 nM range by ∼50% in cells stimulated with either vascular endothelial growth factor (VEGF) at 3 ng/ml or leukotriene D₄ at 10 nM. In addition, ATL-1 (in a 10–100 nM range) inhibited VEGF (3 ng/ml)-induced endothelial cell chemotaxis. In a granuloma in vivo model of inflammatory angiogenesis, ATL-1 treatment (10 μg/mouse) reduced by ∼50% the angiogenic phenotype, as assessed by both vascular casting and fluorescence. Together, these results identify a novel and potent previously unappreciated action of aspirin-triggered 15-epi-LX.

Multicellular host responses to infection, injury or inflammatory stimuli lead to the formation of a broad range of chemical mediators by the host. The integrated response of the host is essential to health and disease; thus it is important to achieve a more complete understanding of the molecular and cellular events governing the formation and actions of endogenous mediators of resolution that appear to control the duration of inflammation. Lipoxins are trihydroxytetraene-containing lipid mediators that can be formed during cell-cell interactions and are predominantly counterregulators of some well-known mediators of inflammation. Since this circuit of lipoxin formation and action appears to be of physiological relevance for the resolution of inflammation, therapeutic modalities targeted at this system are likely to have fewer unwanted side effects than other candidates and current anti-inflammatory therapies. Here, we present an overview of the recent knowledge about the biosynthesis and bioactions of these anti-inflammatory lipid mediators.