Jararhagin Disruption of Endothelial Cell Anchorage Is Enhanced in Collagen Enriched Matrices
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Toxicon 108 (2015) 240e248 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices C. Baldo a, D.S. Lopes b, E.L. Faquim-Mauro a, J.F. Jacysyn c, S. Niland d, J.A. Eble d, * P.B. Clissa a, A.M. Moura-da-Silva a, a Laboratorio de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900, Sao~ Paulo, SP, Brazil b Instituto de Genetica e Bioquímica, Universidade Federal de Uberlandia,^ Uberlandia,^ MG, Brazil c LIM62, Faculdade de Medicina da Universidade de Sao~ Paulo, Sao~ Paulo, SP, Brazil d Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 48149, Münster, Germany article info abstract Article history: Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and Received 21 August 2015 ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic Received in revised form activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the 19 October 2015 study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accu- Accepted 27 October 2015 mulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen Available online 31 October 2015 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- Keywords: Snake venom dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo Metalloproteinase experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells Endothelial cell in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibro- Extracellular matrix nectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen Hemorrhage and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin Collagen 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 a2b1 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. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction hemorrhage that involves direct damage of microvessels (Baldo et al., 2010). Extracellular matrix (ECM) consists of a complex macromolec- Venom induced hemorrhage is a relevant symptom of enven- ular network responsible for tissue structural integrity and modu- omation with viper snakes and causes the massive local damage by lation of cellular activity. Collagens are the most abundant proteins disruption of capillary vessels. Consequently, blood leaks into tis- in the ECM. Collagen remodeling by matrix metalloproteinases sues and distal tissues become ischemic due to the lack of blood (MMPs) is commonly observed during normal tissue homeostasis. supply. Venom-induced hemorrhage has been attributed to the A rapid and extensive degradation of collagens is also detected in ability of snake venom metalloproteinases (SVMPs) to degrade some pathological situations, such as cancer, autoimmune diseases basement membrane (BM) components (Baramova et al., 1989; (Rosenblum et al., 2010), and on pathogenesis of venom-induced Escalante et al., 2006). However, different SVMPs are described and they vary considerably in hemorrhagic activity. PIII-class SVMPs are generally potent hemorrhagins while PI-class SVMPs present weak or undetectable hemorrhagic activity. Since both PI * Corresponding author. and PIII-SVMPs are equally able to degrade ECM components, the E-mail address: [email protected] (A.M. Moura-da-Silva). http://dx.doi.org/10.1016/j.toxicon.2015.10.016 0041-0101/© 2015 Elsevier Ltd. All rights reserved. C. Baldo et al. / Toxicon 108 (2015) 240e248 241 structural difference that could account for the hemorrhagic ac- previously described (Paine et al., 1992) with modifications tivity is the presence of additional adhesive domains, present only (Moura-da-Silva et al., 2003). BnP1 was purified from B. neuwiedi in PIII SVMPs, which could potentiate the catalytic action of the venom according to Baldo et al. (2008). The purity of toxins was toxins by binding to ECM or endothelial cells (Fox and Serrano, evaluated by SDS-PAGE 12.5%, under reducing conditions. Toxins ® 2005; Kamiguti et al., 1996; Tanjoni et al., 2010). This issue was were used in the native form or labeled with Alexa Fluor 488 investigated by our group comparing the mechanisms related to (Molecular Probes, USA) according to the manufacturer in- hemorrhagic activity of jararhagin, a well-known hemorrhagic PIII structions. BSA (Bovine serum albumin e Sigma Aldrich, USA) was SVMP isolated from Bothrops jararaca venom (Paine et al., 1992) and used as control of non-toxic protein in different sets of BnP1, a non-hemorrhagic PI SVMP isolated from Bothrops neuwiedi experiments. venom (Baldo et al., 2008). The results demonstrated that hemor- rhagic activity of jararhagin is correlated to its ability to bind to 2.2. Cell culture collagen (Baldo et al., 2010; Moura-da-Silva et al., 2008). In vivo, this toxin is able to selectively degrade the fibrillar collagen and base- HUVECs were harvested by type IV collagenase (Worthington, ment membrane collagen IV, co-localizing with the basement USA) digestion as described by Jaffe et al. (1973) and cultured on membrane of capillaries and venules on mouse skin (Baldo et al., gelatin-coated plastic dishes in RPMI 1640 medium supplemented 2010). On the other hand, BnP1 hydrolyzed ECM components with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 UI/mL in vitro (Baldo et al., 2008), but in vivo the toxin did not accumulate penicillin, 100 mg/mL streptomycin, 50 uM 2-mercaptoethanol, 5 U/ nearby blood vessels nor hydrolyzed BM collagens. These evidences mL heparin and 20 ng/mL bFGF. For bidimensional cultures (2D), 96 have been recently confirmed by Herrera et al., 2015 observing or 24 wells microplates were coated with type I collagen (BD tissue localization of two hemorrhagic SVMPs, CsH1 (PIII-SVMP Bioscience, USA), type IV collagen (BD Bioscience, USA), fibronectin from Crotalus simus) and BlatH1 (PII-SVMP from Bothriechis later- (Invitrogen, USA) or matrigel (Sigma Aldrich, USA) in a final con- alis), but not of BaP1, a weakly-hemorrhagic PI-SVMP from Bothrops centration of 50 mg/mL. Cells cultured on gelatin 1% or uncoated asper. In this sense, these observations strongly suggest the relevant plates were used as a control. For three-dimensional cultures (3D), role of interactions between adhesive domains present in the 30 mL of matrigel were added to a 24 well microplates containing toxins and BM components enhancing the proteolytic effect of glass coverslips (13 mm), following incubation of 30 min, 37 C. these enzymes, allowing disruption of microvessel walls. After that, 0.5 Â 105 cells were added on the top of gelled matrigel Endothelial cells are also essential for the integrity of micro- and cultured in the HUVECs medium for approximately 18 h, 37 C. vessels and the role of endothelial cells damage on hemorrhagic After that, the capillary-like structures could be observed. activity by PIII-SVMPs is another still open question. In vitro,both hemorrhagic and non-hemorrhagic SVMPs induce apoptosis of 2.3. Immunofluorescence assays endothelial cells at comparable levels (Diaz et al., 2005; Tanjoni et al., 2005). Additionally, hemorrhage induced by jararhagin Immunofluorescence experiments were carried out using in vivo occurs much faster than the apoptosis of endothelial cells markers for actin cytoskeleton (rhodamine-phalloidin), focal ad- in vitro, and apoptotic cells were not detected on hemorrhagic le- hesions (vinculin), and basement membrane (collagen IV and sions induced by SVMP from B. asper venom (Jimenez et al., 2008), laminin) as described below. In order to investigate the interaction ruling out that apoptosis of these cells is the prevalent mechanism of toxins with HUVECs, cells were treated with 800 nM Alexa Fluor for hemorrhages observed in vivo. However, apparently discrepant 488-labeled jararhagin (Alexa488-Jar), Alexa Fluor 488-labeled results obtained in vivo and in vitro may be due to the absence of BnP1 (Alexa488-BnP1) or Alexa Fluor 488-labeled BSA (Alexa488- experimental conditions that accurately model a complex micro- BSA) for 30 min. The samples were fixed in 3.7% formaldehyde environment observed in vivo. Most of these studies were per- for 10 min at room temperature. Nonspecific staining was blocked formed using endothelial cells grown on conventional culture by incubating the coverslips for 1 h at room temperature with a bottles, which are static and rigid surfaces. Therefore, they are very solution of Phosphate Buffer Saline (PBS) containing 1% triton X- distinct from the matrix microenvironment