Regulação do processo de migração de células endoteliais (in vitro) por Jararagina-C, uma proteína tipo-disintegrina isolada do veneno de Bothrops jararaca

Abstract

Jararhagin is a PIII class SVMP with hemorrhagic activity, found in Bothrops jararaca venom. By the process of autolysis, Jararhagin gives rise to Jararhagin-C (JarC), a protein with disintegrin-like and cysteine-rich domains, containing the ECD motif in the disintegrin domain, which selectively recognizes the α2β1 integrin present in platelets and endothelial cells. Several studies have shown the ability of JarC to increase leukocyte adhesion and migration and to promote angiogenesis in experimental models in vivo. However, obtaining JarC from the venom is a limiting factor for the study of this toxin, as it represents about 1% of the crude venom, and expressing this protein in the recombinant form has solved this problem. Escherichia coli was successfully used to express the protein of interest in fusion with the SUMOUlp1 protein. In this work, we used native and recombinant JarC to investigate and compare, in vitro, its action on the migration of endothelial cells CRL-1730 in a Boyden chamber model using type I collagen as substrate and Fetal Bovine Serum (FBS) as cell migration promoting agent. We also verified the gene expression of the IL-8 chemokine by cells incubated with the proteins at 3h, 6h and 24h. Our results showed the effect of both JarC molecules in inhibiting FBS-stimulated cell migration. In the presence of FBS, JarC down regulated the expression of IL-8 at 3h and 6h, however, at 24h there was a slight increase in the expression of this chemokine. Regarding rJarC there was down regulation with 3h, stability with 6h and up regulation with 24h of treatment. However, the behavior of JarC (both in the native and recombinant form) in a culture of cells grown in the absence of FBS was completely different, being possible to observe an up regulation of IL-8 with 3 and 24 hours of stimulation, and a down regulation with 6 hours of stimulation. Our results clearly demonstrate a role for JarC, an ECD-disintegrin, in inhibiting endothelial cell migration. This effect was reproduced by the recombinant protein, confirming that its biological activity is preserved. A protein with an inhibitory action on endothelial cell migration may be a promising tool to treat pathologies that have angiogenesis as a central mechanism, such as age-related macular degeneration or tumor growth.