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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 isolated toxins on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or different toxins isolated from B. asper venom, 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 toxins 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 toxins: (A) 100 µg of hemorrhagic metalloproteinase BaP1; (B) 10 µg of coagulant thrombin-like serine proteinase; (C) 40 µg of the phospholipase A2 homologue myotoxin II; (D) 80 µg of myotoxin II. *p<0.05 when compared with control preparations treated with PBS alone.
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pntd-0000318-g004: Effect of isolated toxins on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or different toxins isolated from B. asper venom, 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 toxins 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 toxins: (A) 100 µg of hemorrhagic metalloproteinase BaP1; (B) 10 µg of coagulant thrombin-like serine proteinase; (C) 40 µg of the phospholipase A2 homologue myotoxin II; (D) 80 µg of myotoxin II. *p<0.05 when compared with control preparations treated with PBS alone.

Mentions: To determine which component of the venom is responsible for the activity on the lymphatic vessels, single isolated toxins were tested. Application of BaP1, a hemorrhagic metalloproteinase, and of a thrombin-like coagulant serine proteinase isolated from B. asper venom did not induce any effect in the lumen of lymphatics (Fig 4 A,B), at doses inducing widespread hemorrhage, in the case of BaP1, and defibrination, in the case of the thrombin-like enzyme. In contrast, the PLA2 homologue myotoxin II reproduced the effects induced by crude venom in lymphatics, since it induced a dose-dependent reduction in the lumen of these vessels (Fig 4 C, D) and a concomitant halting in the lymph flow (Table 1).


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 isolated toxins on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or different toxins isolated from B. asper venom, 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 toxins 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 toxins: (A) 100 µg of hemorrhagic metalloproteinase BaP1; (B) 10 µg of coagulant thrombin-like serine proteinase; (C) 40 µg of the phospholipase A2 homologue myotoxin II; (D) 80 µg of myotoxin II. *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-g004: Effect of isolated toxins on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or different toxins isolated from B. asper venom, 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 toxins 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 toxins: (A) 100 µg of hemorrhagic metalloproteinase BaP1; (B) 10 µg of coagulant thrombin-like serine proteinase; (C) 40 µg of the phospholipase A2 homologue myotoxin II; (D) 80 µg of myotoxin II. *p<0.05 when compared with control preparations treated with PBS alone.
Mentions: To determine which component of the venom is responsible for the activity on the lymphatic vessels, single isolated toxins were tested. Application of BaP1, a hemorrhagic metalloproteinase, and of a thrombin-like coagulant serine proteinase isolated from B. asper venom did not induce any effect in the lumen of lymphatics (Fig 4 A,B), at doses inducing widespread hemorrhage, in the case of BaP1, and defibrination, in the case of the thrombin-like enzyme. In contrast, the PLA2 homologue myotoxin II reproduced the effects induced by crude venom in lymphatics, since it induced a dose-dependent reduction in the lumen of these vessels (Fig 4 C, D) and a concomitant halting in the lymph flow (Table 1).

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