Effects of PI and PIII Snake Venom Haemorrhagic Metalloproteinases on the Microvasculature: A Confocal Microscopy Study on the Mouse Cremaster Muscle

Abstract

The precise mechanisms by which Snake Venom Metalloproteinases (SVMPs) disrupt the microvasculature and cause haemorrhage have not been completely elucidated, and novel in vivo models are needed. In the present study, we compared the effects induced by BaP1, a PI SVMP isolated from Bothrops asper venom, and CsH1, a PIII SVMP from Crotalus simus venom, on cremaster muscle microvasculature by topical application of the toxins on isolated tissue (i.e., ex vivo model), and by intra-scrotal administration of the toxins (i.e., in vivo model). The whole tissue was fixed and immunostained to visualize the three components of blood vessels by confocal microscopy. In the ex vivo model, BaP1 was able to degrade type IV collagen and laminin from the BM of microvessels. Moreover, both SVMPs degraded type IV collagen from the BM in capillaries to a higher extent than in PCV and arterioles. CsH1 had a stronger effect on type IV collagen than BaP1. In the in vivo model, the effect of BaP1 on type IV collagen was widespread to the BM of arterioles and PCV. On the other hand, BaP1 was able to disrupt the endothelial barrier in PCV and to increase vascular permeability. Moreover, this toxin increased the size of gaps between pericytes in PCV and created new gaps between smooth muscle cells in arterioles in ex vivo conditions. These effects were not observed in the case of CsH1. In conclusion, our findings demonstrate that both SVMPs degrade type IV collagen from the BM in capillaries in vivo. Moreover, while the action of CsH1 is more directed to the BM of microvessels, the effects of BaP1 are widespread to other microvascular components. This study provides new insights in the mechanism of haemorrhage and other pathological effects induced by these toxins.

Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function

“…Atr-III is a member of the high Mr metalloproteinase family, consistent with the effects of Ca 2+ , Mg 2+ and EDTA on its proteolytic activity [22,33,36,37,38]. Inhibition of proteolytic activity of Atr-III by adding Zn 2+ may be explained by the hypothesis that Atr-III might have other Zn 2+ -binding sites with different binding affinity.…”

“…It is particularly noteworthy that envenoming by Bothrops snakes are characterized by drastic hemostatic disturbances and inflammatory reactions with local tissue damage irradiating from the site of the bite [39]. Several studies demonstrate the ability of hemorrhagic metalloproteinases to degrade proteins of the basement membrane (BM) and other extracellular macromolecules using in vitro and in vivo strategies [38]. The broad substrate spectrum of Atr-III and other P-III SVMPs may be mediated not only by substrate recognition via the catalytic M domain but also by the non-enzymatic interaction of substrate with the non-proteinase domains (DC) in P-III SVMPs.…”

“…The latter contain exosites that direct the interaction of these P-III SVMPs to specific ECM targets, especially in microvessels or in the plasma membrane of cells and platelets [37]. Furthermore, the presence of these domains may prevent the inhibition of these large metalloproteinases by endogenous inhibitors mainly by α2M in plasma or body fluids [34,38]. Owing to their multiple functions, the P-III SVMPs containing MDC domains found mostly in viper venoms species may serve as potential tools to better understand how these enzymes affect the mechanisms underlying thrombosis, hemostasis, and inflammatory reactions [23].…”

Estudos de efeitos de uma metaloproteinase de veneno ofídico em células de músculo liso vascular: produção de fatores que modulam a migração e proliferação destas células e mecanismos e

“…A BaP1 não é tóxica para culturas de células endoteliais murinas e humanas (RUCAVADO et al, 1995), queratinócitos (RUCAVADO et al, 1998) e nem para macrófagos murinos (RUCAVADO et al, 2002). Em modelos experimentais in vivo, a BaP1 induziu hemorragia (GUTIÉRREZ et al, 1995, RUCAVADO et al, 1995PEREAÑES et al, 2013;CASTRO et al,2014, GUTIÉRREZ et al,2016), edema (PEREAÑES et al, 2013 e mionecrose moderada, com regeneração deficiente do músculo esquelético (RUCAVADO et al, 1995;HERNANDÉZ et al, 2011, HERRERA et al, 2015. Adicionalmente, esta metaloproteinase induziu inflamação local após sua injeção no coxim plantar e no músculo gastrocnêmio de camundongos (GUTIÉRREZ et al, 1995, RUCAVADO et al, 1995.…”

“…Ainda, estudos demostraram que a BaP1 induziu o influxo de neutrófilos para a cavidade peritoneal de camundongos (FERNANDES et al, 2006e CASTRO et al,2014, dependente da expressão de moléculas de adesão da família das integrinas (cadeia CD18) (FERNANDES et al, 2006). Na pele e tecido muscular de camundongos a BaP1 causou a liberação das gelatinases A (MMP-2) e B (MMP-9) (RUCAVADO et al, 2002), a ativação de MMP-2 em fibroblastos de humanos em cultura (SARAVIA-OTTEN et al, 2004) e a degradação de laminina e colágeno do tipo IV, no tecido auricular, na pele e no músculo cremaster de camundongos (JIMÉNEZ et al, 2008;HERRERA et al, 2015;HERRERA et al, 2016). A injeção intra-articular da BaP1, em ratos, desencadeou eventos inflamatórios, com a formação de edema e acúmulo de leucócitos na cavidade sinovial, acompanhada da liberação dos mediadores inflamatórios TNF- e prostaglandina E 2 (PGE 2 ) e nocicepção, dependente da liberação destes mediadores (FERNANDES et al, 2007) foi investigado e demanda estudos a respeito.…”