, 2005). Agonist activation induces conformational changes within VEGFR-2, followed by receptor dimerization and autophosphorylation of tyrosine residues in the intracellular kinase domains, which activates several intracellular pathways, displaying endothelial cell proliferation, migration, differentiation, tube formation, and vascular permeability increase and integrity (Hicklin and Ellis, 2005; Kerbel, 2008). Amblyomin-X is a Kunitz-type SPI recombinant protein of 15 kDa, obtained from

the cDNA library of Amblyomma cajennense salivary glands ( Batista et al., 2008), which shares similarities with TFPI ( Salemink et al., 1999) and inhibits Factor Xa (FXa) and consequently delays the time of blood coagulation BI2536 in vitro and ex vivo ( Batista et al., 2008, 2010). Recent evidence has extended our knowledge of the actions of Amblyomin-X, as Amblyomin-X treatment in C57BL6 mice reduced tumor mass and the number of metastatic events caused

by intravenous injection of murine melanoma B16F10 cells. Trichostatin A manufacturer In addition, in vitro Amblyomin-X treatment caused apoptosis in melanoma (SK-Mel-28) and pancreatic adenocarcinoma (Mia-PaCa-2) cells, and the proposed mechanisms are increased expression of the proteasome b2 catalytic subunit gene (PSBM2), decreased proteasomal activity and increased pool of poly-ubiquitinylated proteins ( Chudzinski-Tavassi et al., 2010). Considering the in vivo anti-tumor effects of Amblyomin-X and the role of SPI in neovascularization, Ribonucleotide reductase the present work investigated the effects of the Amblyomin-X on VGEF-A-induced in vivo angiogenesis and its actions on endothelial cell functions during the process. The findings highlight the effects of Amblyomin-X on endothelial cell proliferation and adhesion, mainly on VEGF-A-endothelial PECAM-1 expression, which may contribute to its modulatory effect on in vivo angiogenesis. Male Swiss mice (25–30 g) were fed on standard pellet diet and water ad libitum, and anesthetized

with a combination of ketamine (20 mg/kg) and xylazine solution (2 mg/kg, i.p) before each experimental procedure. All procedures were performed according to protocols approved by the Brazilian Society of Science of Laboratory Animals (SBCAL) for proper care and use of experimental animals. The Amblyomin-X protein (15 kDa) was obtained from a cDNA library of the salivary glands of the A. cajennense tick (GenBank accession AAT68575; Batista et al., 2008). Amblyomin-X was initially expressed in prokaryotic system (BL21(DE3) Escherichia coli) using the pAE vector. This kind of production inserts 6 histidin residues in the molecule ( Batista et al., 2010), becoming easier the protein purification process. However in the present study, it was used Amblyomin-X cloned and expressed in methylotrophic yeast system (Pichia pastoris) employing the pPIC9K vector (Faria et al., personal communication).