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.
Serogroup B outer membrane vesicles (OMV) with iron regulated proteins (IRP) from Neisseria meningitidis constitute the antigen for the vaccine against the disease caused by this bacterium. Aiming to enhance final OMV concentration, seven batch experiments were carried out under four different conditions: (i) with original Catlin medium; (ii) with original Catlin medium and lactate and amino acids pulse at the 6th cultivation hour; (iii) with Catlin medium with double initial concentrations of lactate and amino acids and (iv) Catlin medium without glycerol and with double initial concentrations of lactate and amino acids. The cultivation experiments were carried out in a 7-L bioreactor under the following conditions: 36°C, 0.5atm, overlay air 1L/min, agitation: 250-850 rpm, and O(2) control at 10%, 20 h. After lactate and amino acids exhaustion, cell growth reached stationary phase and a significant release increase of OMV was observed. According to the Luedeking & Piret model, OMV liberation is non-growth associated. Glycerol was not consumed during cultivation. The maximum OMV concentration value attained was 162 mg/L with correspondent productivity of 8.1mg/(Lh) employing Catlin medium with double initial concentrations of lactate and amino acids. The obtained OMV satisfied constitution and protein pattern criteria and were suitable for vaccine production.
Specific blood coagulation inhibitors from hematophagous organisms, with different structures and novel mechanism of action, have been described and they represent promising agents for the treatment of a variety of human diseases related to coagulation and cancer. In our lab, the salivary glands transcriptome of the adult Amblyomma cajennense tick was previously characterized by expressed sequence tags (EST). A transcript that codes for a tissue factor pathway inhibitor (TFPI)-like protein with unique structure was found, and the recombinant form of this protein was named Amblyomin-X. This protein was able to inhibit the factor Xa amidolytic activity and the activation of factor X by the extrinsic tenase complex (FVIIa/TF). Herein, it was performed functional and structural evaluation of Amblyomin-X. The CD assay and molecular dynamics simulations revealed that Amblyomin-X is structurally stable and the naturally unfolded regions as well as the presence of three disulfide bridges in its Kunitz-type domain seem to sustain its inhibitory activity. Regarding the electrostatic potential mapping on the Kunitz-type region, the pattern of charged residues was not quite the same in comparison to human TFPI-1 and TFPI-2, pointing out there might be distinct functional and structural features, which are going to be experimentally exploited.
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