Effect of BaP1 and CsH1 on size and density of gaps between adjacent smooth muscle and pericytes on mouse cremaster muscle vasculature.

<p>Isolated cremaster muscles were incubated with either 30 μg of BaP1 or 15 μg of CsH1 (model without blood flow). In another experiment, anesthetized mice were injected by intrascrotal route with either 60 μg of BaP1 or 30 μg of CsH1 (model with blood flow). Controls were incubated or injected with PBS. After 15 min of exposition to toxin in each model, whole cremaster muscles were fixed and immunostained for observation by confocal microscopy and analysis of the gaps between adjacent smooth muscle and pericytes. Results are expressed as the mean ± SEM of the (A, B) gap size and (C, D) gap density (number of gaps per vessel area) of at least five images of arterioles and PCV per cremaster (n = 4). (E) Representative three-dimensional images of each vessel type immunostained for actin α smooth muscle are shown for BaP1 30 μg and control in the model without blood flow. Notice the increase in the gap size (arrows) in arterioles and PCV of treated tissues as compared to control. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; PCV: post-capillary venules.</p

Effect of BaP1 and CsH1 on type IV collagen from vascular BM on mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with either (A) 30 μg of BaP1 or (C) 15 μg of CsH1 (model without blood flow). In another experiment, anesthetized mice were injected by intrascrotal route with either (B) 60 μg of BaP1 or (D) 30 μg of CsH1 (model with blood flow). Controls were incubated or injected with PBS. After 15 min of exposition to toxin in each model, whole cremaster muscles were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for type IV collagen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type per animal (n = 4). (E) Representative three-dimensional images of each vessel type immunostained for type IV collagen are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions) for CsH1 30 μg and control applied in anesthetized mice (i.e. model with blood flow). The images show a decrease in fluorescence intensity for type IV collagen in BM of arterioles, capillaries, and PCV of treated tissues as compared to control. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. Col IV: type IV collagen; PCV: post-capillary venules.</p

Dose and time dependency of BaP1 effects on type IV collagen from vascular BM on isolated mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with different amounts of BaP1 (10, 30 and 100 μg) for either 5 or 15 min (model without blood flow). Control tissues were incubated with PBS. Whole tissues were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for type IV collagen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type: (A) arterioles, (B) capillaries, and (C) PCV per cremaster (n = 4). Below each graph, representative three-dimensional images of each vessel type immunostained for type IV collagen are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions) for BaP1 (30 μg) and control at 15 min. The images show a decrease in fluorescence intensity for type IV collagen in BM of capillaries of treated tissues as compared to control, whereas no significant reduction was observed in arterioles and PCV. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; Col IV: type IV collagen; PCV: post-capillary venules.</p

Effect of BaP1 and CsH1 on vascular permeability after intradermical application.

<p>Mice received an intravenous injection of 200 μl of Evans Blue dye (6 mg/ml). After 5 min, mice were injected by intradermal route, in the ventral abdominal region, with either 2 μg of BaP1 or 1 μg of CsH1. PBS was injected as control in another group of animals. After 15 min mice were sacrificed by cervical dislocation, their skin was removed and the area of extravasation was measured. (A) Results are expressed as the mean ± SEM (n = 5). **p < 0.001 as compared to CsH1. (B) Figure shows representative images from five animals analyzed.</p

Effect of BaP1 on laminin and nidogen from vascular BM on isolated mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with 30 μg of BaP1 for 15 min (model without blood flow). Control tissues were incubated with PBS. Whole tissues were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for (A) laminin and (B) nidogen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type per cremaster (n = 4). (C) Representative three-dimensional images of each vessel type immunostained for laminin are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions). The images show a decrease in fluorescence intensity for laminin in BM of capillaries and PCV of treated tissues as compared to control, whereas no reduction in the fluorescence intensity was observed for nidogen. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. Lam: laminin; Nid: nidogen; PCV: post-capillary venules.</p

Western blot analysis of basement membrane components in skin homogenates.

<p>Groups of five mice were injected by intradermal route in the ventral abdominal region with either BaP1 (PI, 75 μg), BlatH1 (PII, 1.5 μg), CsH1 (PIII, 35 μg) SVMPs or PBS (lane C). After 15 min, mice were sacrificed, their skin was removed, and an area of 12 mm diameter was dissected out. Tissues of the same group were homogenized and centrifuged, and the supernatant collected. Then, 10–20 μL of each skin homogenate sample were separated under reducing conditions on 4–15% Tris–HCl SDS-PAGE gradient gels, and transferred to nitrocellulose membranes. Immunodetection was performed with (A) anti-collagen type IV, (B) anti-collagen type VI, (C) anti-laminin, and (D) anti-nidogen 1. The anti-GAPDH antibody was used as loading control. The reaction was detected using an anti-rabbit peroxidase antibody and a chemiluminescent substrate. Images were obtained with the ChemiDoc XRS+ System (BioRad).</p

Immunolocalization of SVMPs with vascular basement membrane on cremaster muscle <i>ex vivo</i>.

<p>Isolated cremaster muscles were incubated for 15 min with equi-hemorrhagic amounts of either BaP1 (PI, 30 μg), BlatH1 (PII, 3.5 μg) or CsH1 (PIII, 15 μg) SVMPs labeled with Alexa Fluor 647 (blue). Control tissues were incubated with the SVMPs without labeling and no fluorescence was detected. Whole tissues were fixed with 4% paraformaldehyde and immunostained with anti-collagen IV following the secondary antibody labeled with Alexa Fluor 488 (green). Tissues were visualized in a Zeiss LSM 5 Pascal laser-scanning confocal microscope. Three-dimensional reconstitution of the images and analysis of co-localization were carried out with the IMARIS x64 7.4.2 software as described in Methods. (A) Distribution of the SVMPs in the cremaster muscle tissue. Scale bar represents 150 μm. (B) White areas represent co-localization of the SVMPs (blue) with collagen IV (green) of vascular basement membrane in PCV, arterioles, and capillaries. Scale bar represents 20 μm. Results are expressed as the mean ± SEM of (C) percentage of material of SMVPs co-localized with collagen IV of vascular basement membrane, and (D) Pearson´s correlation coefficient of at least four vessels type per tissue (n = 3). *p<0.001 when compared with BaP1 (PI) SVMP for post-capillary venules (PCV), arterioles, and capillaries.</p

Degradation of extracellular matrix proteins identified in wound exudates (see Methods section for details).

<p>^a Not detected.</p><p>Degradation of extracellular matrix proteins identified in wound exudates (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003731#sec002" target="_blank">Methods</a> section for details).</p

(A), (B) SDS-PAGE of <i>Crotalus simus</i> PIII SVMP and crude venom.

<p>Samples were run on a 12% gel, and stained with (A) Coomassie blue or (B) Pro-Q Emerald 300 glycoprotein stain (Molecular probes). Lane 1: SVMP, reducing conditions; lane 2: SVMP, non-reducing conditions; lane 3: <i>Crotalus simus</i> venom, reducing conditions; lane 4: <i>Crotalus simus</i> venom, non-reducing conditions. The P-III metalloproteinase is a major component of the venom; it is glycosylated and has a molecular mass of 55 kDa. (C) and (D) Light micrographs of sections of lung tissue from mice injected intravenously with either saline solution (C) or 100 μg of <i>C. simus</i> PIII SVMP. Mice were sacrificed one h after injection and tissue samples were obtained and routinely processed for embedding in paraffin and further staining with hematoxylin-eosin. Notice prominent hemorrhage in the pulmonary tissue in (D) (arrow). 125 X.</p

Western blot analysis of basement membrane components in exudates collected from the gastrocnemius.

<p>Groups of five mice were injected in the right gastrocnemius with either BaP1 (PI, 75 μg), BlatH1 (PII, 3 μg), or CsH1 (PIII, 50 μg) SVMPs. After 15 min, mice were sacrificed, a 5 mm incision was made in the skin overlying the injected muscle, and a heparinized capillary tube was introduced under the skin to collect the wound exudate fluid; exudate samples from a single treatment were then pooled. Afterwards, 100 μg of protein of each sample was separated under reducing conditions on 4–15% Tris–HCl SDS-PAGE, and transferred to nitrocellulose membranes. Immunodetection was performed with (A) anti-collagen type IV, (B) anti-collagen type VI, (C) anti-laminin, and (D) anti-nidogen 1. The reaction was detected using an anti-rabbit peroxidase antibody and a chemiluminescent substrate. Images were obtained with the ChemiDoc XRS+ System (BioRad).</p