No abstract

-Lipoxins (LX) and aspirintriggered LX (ATL) are eicosanoids generated during inflammation via transcellular biosynthetic routes that elicit distinct anti-inflammatory and proresolution bioactions, including inhibition of leukocytemediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, and inhibition of cell proliferation and migration. Recently, it was reported that aspirin induces heme oxygenase-1 (HO-1) expression on endothelial cells (EC) in a COX-independent manner, what confers protection against prooxidant insults. However, the underlying mechanisms remain unclear. In this study, we investigated whether an aspirin-triggered lipoxin A4 stable analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1) was able to induce endothelial HO-1. Western blot analysis showed that ATL-1 increased HO-1 protein expression associated with increased mRNA levels on EC in a timeand concentration-dependent fashion. This phenomenon appears to be mediated by the activation of the G protein-coupled LXA4 receptor because pertussis toxin and Boc-2, a receptor antagonist, significantly inhibited ATL-1-induced HO-1 expression. We demonstrate that treatment of EC with ATL-1 inhibited VCAM and E-selectin expression induced by TNF-␣ or IL-1␤. This inhibitory effect of the analog is modulated by HO-1 because it was blocked by SnPPIX, a competitive inhibitor that blocks HO-1 activity. Our results establish that ATL-1 induces HO-1 in human EC, revealing an undescribed mechanism for the anti-inflammatory activity of these lipid mediators. signaling transduction; resolution of inflammation LIPOXINS (LX) are endogenous lipid mediators that dampen the host response and orchestrate resolution of inflammation. In humans, three main biosynthetic pathways are described for LX formation, each involves transcellular biosynthetic use of intermediates between distinct cell types that are in close proximity during inflammatory responses (43). Monocytes, eosinophils, and airway-epithelial cells can convert arachidonate into 15S-hydroxyeicosatetraenoic acid (15S-HETE) by a 15-lipoxygenase (LO) catalyzed reaction. 15S-HETE is rapidly taken up by neutrophils and converted to lipoxin A 4 by a 5-LO-catalyzed reaction (43). The second pathway for lipoxin biosynthesis was determined for interactions that occur predominantly within the vasculature between 5-LO, present in myeloid cells, and 12-LO, which is present in platelets. The 5-LO product leukotriene A 4 is converted in a transcellular manner by platelet 12-LO to lipoxins (43). More recently, a third major pathway for lipoxin generation was discovered that involves aspirin and the action of cyclooxygenase (COX)-2 and 5-LO (11). Endothelial and epithelial cells express COX-2 in response to diverse stimuli such as cytokines, hypoxia, and bacterial infections. Aspirin acetylates COX-2 and switches its catalytic activity for conversion of arachidonic acid to 15R-HETE in lieu of prostanoid biosynthesis. 15R-HETE is released from endot...

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.

An antivenom protein has been identified in the blood of the snake Crotulus durissus terriJicus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotulus serum (CICS), and to analyze its mechanism of action.CICS has been purified from blood serum of the Crotulus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23 -25-kDa subunits. Two different subunit peptides were identified by SDSPAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues.The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex.The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.

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.