Jararhagin Induce a Specialized Form of Apoptosis - Anoikis - in Endothelial Cells

¹Tanjoni, I.; ²Weinlich,  R.; ¹Della-Casa, M.S.; ¹Clissa, P.B.; ³De Freitas, M.S.; ³Barja-Fidalgo, C.; ²Amarante-Mendes, G.P.; ¹Moura-da-Silva, A. M.

1 Laboratório de Imunopatologia, Instituto Butantan, 2 Laboratório de Biologia Celular e Molecular, Instituto de Ciências Biomédicas/ USP, 3 Departamento de Farmacologia, Instituto de Biologia, UERJ

Jararhagin is a hemorrhagic snake venom metalloproteinase (SVMP) involved in systemic and local effects of Bothrops jararaca venom. In this study, we evaluated the effect of jararhagin on cultures of endothelial cells (tEnd), fibroblasts (A31) and murine peritoneal adherent cells (MPACs). Growth, viability and adhesion were evaluated by MTT assays and induction of apoptosis by flow cytometric analysis of DNA content. The exposure of tEnd to jararhagin (20 and 40 mg/mL) resulted in inhibition of proliferation and decrease of cell viability following the loss of cell adhesion and the increase in the number of apoptotic cells and activation of caspase 3. When observed with a phase-contrast microscope, exposure of tEnd to jararhagin led cells to retract, round up and detach. Cytoskeleton organization of jararhagin-treated tEnd cultures was then analysed by immunoflorescence microscopy using phalloidin staining, showing changes in cell shape with decrease of the cell spreading. In addition, we verified by western blotting that jararhagin was able to induce rearrangement of the actin network with a slight decrease in the F-actin content followed by the increase in the G-actin content. Using Triton X-100 insoluble fraction, we verified a decrease of the amount of FAK associated with actin cytoskeleton and a moderate decrease in tyrosyne phosphorylation of proteins with molecular weight about 125 kDa. Both morphological alterations and apoptosis of endothelial cells induced by jararhagin were abolished when the toxin was pre-incubated with EDTA showing the importance of catalytic activity in these effects. Moreover, the number of apoptotic cells under non-adherent conditions was not increased after treatment with jararhagin, showing that jararhagin exert indirect cytotoxic activity on the tEnd. Treatment of MPACs and fibroblast cultures with similar concentrations of jararhagin did not result in any of the effects observed for endothelial cells. Concluding, jararhagin-induced apoptosis is selective to endothelial cells and occurs by interfering in the adhesion mechanisms. Therefore, jararhagin is a useful tool for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomena named anoikis. In addition, studies using jararhagin may elucidate the role of MMPs and ADAMs in anoikis and rearrangement of the actin network.

Supported by FAPESP.

Correspondence to: belleclues@yahoo.com