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Effects of Bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation.

Mora J, Mora R, Lomonte B, Gutiérrez JM - PLoS Negl Trop Dis (2008)

Bottom Line: B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow.In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat.Moreover, fucoidan significantly reduced venom-induced footpad edema.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitología, Universidad de Costa Rica, San José, Costa Rica.

ABSTRACT

Background: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied.

Methodology/principal findings: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A(2) (PLA(2)) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA(2) homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells.

Conclusions/significance: Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA(2)s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.

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Effect of metalloproteinase inhibitor Batimastat and myotoxin inhibitor fucoidan on the activity of B. asper venom on mouse mesentery collecting lymphatics.Before application, venom solutions were incubated with either Batimastat or fucoidan, as described in Methods. The mesentery was exposed and 50 µL of either PBS or the mixtures of 100 µg venom and inhibitors, dissolved in 50 µL PBS, were applied onto the preparation. Observations were performed as described in the legend of Fig 2. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom incubated with Batimastat (A) or fucoidan (B). *p<0.05 when compared with control preparations treated with PBS alone.
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pntd-0000318-g005: Effect of metalloproteinase inhibitor Batimastat and myotoxin inhibitor fucoidan on the activity of B. asper venom on mouse mesentery collecting lymphatics.Before application, venom solutions were incubated with either Batimastat or fucoidan, as described in Methods. The mesentery was exposed and 50 µL of either PBS or the mixtures of 100 µg venom and inhibitors, dissolved in 50 µL PBS, were applied onto the preparation. Observations were performed as described in the legend of Fig 2. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom incubated with Batimastat (A) or fucoidan (B). *p<0.05 when compared with control preparations treated with PBS alone.

Mentions: To confirm that involvement of specific venom components in the effect of crude venom on lymphatics, the ability of several inhibitors to block the effect of B. asper venom was assessed. Incubation of B. asper venom with the metalloproteinase inhibitor Batimastat resulted in complete abrogation of hemorrhagic activity, without preventing the lymphatic alterations induced by the venom (Fig 5A). In contrast, incubation of venom with fucoidan, a known inhibitor of myotoxic PLA2s, completely abolished the effect of venom on the lymphatics (Fig 5B), without affecting its hemorrhagic activity. Control preparations in which Batimastat or fucoidan alone were applied to mesentery did not show any observable effect (not shown).


Effects of Bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation.

Mora J, Mora R, Lomonte B, Gutiérrez JM - PLoS Negl Trop Dis (2008)

Effect of metalloproteinase inhibitor Batimastat and myotoxin inhibitor fucoidan on the activity of B. asper venom on mouse mesentery collecting lymphatics.Before application, venom solutions were incubated with either Batimastat or fucoidan, as described in Methods. The mesentery was exposed and 50 µL of either PBS or the mixtures of 100 µg venom and inhibitors, dissolved in 50 µL PBS, were applied onto the preparation. Observations were performed as described in the legend of Fig 2. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom incubated with Batimastat (A) or fucoidan (B). *p<0.05 when compared with control preparations treated with PBS alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563035&req=5

pntd-0000318-g005: Effect of metalloproteinase inhibitor Batimastat and myotoxin inhibitor fucoidan on the activity of B. asper venom on mouse mesentery collecting lymphatics.Before application, venom solutions were incubated with either Batimastat or fucoidan, as described in Methods. The mesentery was exposed and 50 µL of either PBS or the mixtures of 100 µg venom and inhibitors, dissolved in 50 µL PBS, were applied onto the preparation. Observations were performed as described in the legend of Fig 2. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom incubated with Batimastat (A) or fucoidan (B). *p<0.05 when compared with control preparations treated with PBS alone.
Mentions: To confirm that involvement of specific venom components in the effect of crude venom on lymphatics, the ability of several inhibitors to block the effect of B. asper venom was assessed. Incubation of B. asper venom with the metalloproteinase inhibitor Batimastat resulted in complete abrogation of hemorrhagic activity, without preventing the lymphatic alterations induced by the venom (Fig 5A). In contrast, incubation of venom with fucoidan, a known inhibitor of myotoxic PLA2s, completely abolished the effect of venom on the lymphatics (Fig 5B), without affecting its hemorrhagic activity. Control preparations in which Batimastat or fucoidan alone were applied to mesentery did not show any observable effect (not shown).

Bottom Line: B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow.In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat.Moreover, fucoidan significantly reduced venom-induced footpad edema.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitología, Universidad de Costa Rica, San José, Costa Rica.

ABSTRACT

Background: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied.

Methodology/principal findings: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A(2) (PLA(2)) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA(2) homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells.

Conclusions/significance: Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA(2)s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.

Show MeSH
Related in: MedlinePlus