Post-transcriptional control of Amblyomin-X on secretion of vascular endothelial growth factor and expression of adhesion molecules in endothelial cells.

Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000 ng/10 µL; each 48 h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and ß3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process.

Actions of the Kunitz-type serine protease inhibitor Amblyomin-X on VEGF-A-induced angiogenesis.

Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 µl; each 48 h) inhibited VEGF-A-induced (10 ng/10 µl; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules.

Antiallodynic effect and side effects of Phα1ß, a neurotoxin from the spider Phoneutria nigriventer: comparison with ω-conotoxin MVIIA and morphine.

Phα1ß is a potent toxin obtained from the spider Phoneutria nigriventer that blocks neuronal voltage-sensitive Ca(2+) channels. This study compared the antiallodynic effects of Phα1ß, ω-conotoxin MVIIA and morphine in mice and their side effects in rats. Mechanical allodynia was measured in mice receiving single intrathecal administration of Phα1ß, ω-conotoxin MVIIA or morphine before or after the incisional plantar procedure. The effect of the treatments on cardiovascular profile and global neurological were evaluated in rats. The expression of pro or anti-inflammatory cytokines of human polymorph mononuclear cells was also evaluated. Preemptive use of ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site) induced shorter antiallodynic effect than Phα1ß (100 pmol/site) in mice. Post-incision administration of Phα1ß (200 pmol/site) induced longer mechanical antiallodynic effect than ω-conotoxin MVIIA (1.0 or 10 pmol/site) or morphine (1000 pmol/site). Intrathecal injection of Phα1ß (200 pmol/site) and morphine (433 pmol/site) did not change while ω-conotoxin MVIIA (100 pmol/site) increased the heart rate in rats 3 h after its administration. Phα1ß (200 pmol/site), ω-conotoxin MVIIA (100 pmol/site) and morphine (433 pmol/site) did not change mean arterial pressure 0.5 and 3 h after their administration. The treatments did not alter neurological performance assessed by global neurological evaluation and open-field test. The tested drugs did not induced expression of pro or anti-inflammatory cytokines in CD4 monocytes. In conclusion, preemptive administration Phα1ß in mice induced longer antiallodynic effect than ω-conotoxin MVIIA and morphine. Phα1ß also induced a longer mechanical antiallodynic effect than ω-conotoxin MVIIA and morphine when used after the surgical incision. The present results suggest that Phα1ß has a potential application in the management of postoperative pain with low side effects.