title: Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.
abstract: Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.
authors.affiliation: Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil.
authors.forename: C
authors.initials: C
authors.lastname: Baldo
authors.affiliation: Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil.
authors.forename: D S
authors.initials: DS
authors.lastname: Lopes
authors.affiliation: Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil.
authors.forename: E L
authors.initials: EL
authors.lastname: Faquim-Mauro
authors.affiliation: LIM62, Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: J F
authors.initials: JF
authors.lastname: Jacysyn
authors.affiliation: Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 48149, Münster, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Niland
authors.affiliation: Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 48149, Münster, Germany.
authors.forename: J A
authors.initials: JA
authors.lastname: Eble
authors.affiliation: Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil.
authors.forename: P B
authors.initials: PB
authors.lastname: Clissa
authors.affiliation: Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil. Electronic address: ana.moura@butantan.gov.br.
authors.forename: A M
authors.initials: AM
authors.lastname: Moura-da-Silva
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001485:Basement Membrane / Q000187:drug effects; D018929:Cell Culture Techniques; D021982:Cell-Matrix Junctions / Q000187:drug effects; D003094:Collagen / Q000187:drug effects*; D003435:Crotalid Venoms / Q000032:analysis / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004338:Drug Combinations; D042783:Endothelial Cells / Q000187:drug effects; D005353:Fibronectins / Q000187:drug effects*; D005434:Flow Cytometry; D022001:Focal Adhesions / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells; D007797:Laminin; D008666:Metalloendopeptidases / Q000032:analysis / Q000494:pharmacology*; D008954:Models, Biological; D011509:Proteoglycans; D000097146:Bothrops jararaca Venom
chemical_list: D003094:Collagen; D003435:Crotalid Venoms; D004338:Drug Combinations; D005353:Fibronectins; D007797:Laminin; D008666:Metalloendopeptidases; D011509:Proteoglycans; D000097146:Bothrops jararaca Venom; C059485:matrigel
keywords: Collagen; Endothelial cell; Extracellular matrix; Hemorrhage; Metalloproteinase; Snake venom