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.

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 β3 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.

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In Brazil and in most of the Latin America countries, the control of the cattle rabies relies upon the indiscriminate culling of the Desmodus rotundus inside a radius of 12 km around an outbreak. The culling is performed by applying warfarin paste in the back of the bats, in the hope that when it returns to its roost after foraging, its conspecifics would ingest the paste during social grooming and die of hemorrhage. This control approach is performed in absentia with the surveillance effort, since if bats die indiscriminately, the effects in the social structure of the colonies are unknown. It is believed that the indiscriminate culling helps the rabies virus spread among bats and consequently, the spillover to livestock. For this reason, any sampling effort in bats and even the identification of cattle rabies outbreaks transmitted by bats is useless to predict future outbreaks or even mitigate current outbreaks. Even though the tendency of the cattle rabies outbreaks is decreasing in Brazil, this should be carefully considered, since rabies surveillance efforts are heterogeneous and decreasing. Even so, the nationwide space-time cattle rabies outbreak shows a wave pattern, increasing the urge of the revision of the current surveillance and control actions. The present work proposes a new approach aiming at decreasing the spillover to livestock by incorporating ecological features of the D. rotundus, most importantly turning the surveillance-control rationale which is currently reactive to livestock rabies cases to a surveillance system reactive to changes in the incidence of vampire bat attacks on the livestock and selective bat population control. For example, estimating the roosts' carrying capacity and contact networks allows the interruption of rabies virus spread by reducing the bat population of target individuals in selected roosts or roost communities. Moreover, the terrain slope dependency of the bat foraging behavior allows to find more efficiently the roost from which bats are attacking a farm or conversely, to find attacked farms if an occupied bat roost is known. These practices could increase the efficiency of the surveillance and control and the cost-effectiveness of the current cattle rabies control program.