Fig 1.
(A), (B) SDS-PAGE of Crotalus simus PIII SVMP and crude venom.
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: Crotalus simus venom, reducing conditions; lane 4: Crotalus simus 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 C. simus 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.
Table 1.
Tryptic peptide ion sequences obtained by de novo CID-MS/MS sequencing of Crotalus simus PIII SVMP.
Fig 2.
Immunolocalization of SVMPs with vascular basement membrane on cremaster muscle ex vivo.
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.
Fig 3.
Western blot analysis of basement membrane components in skin homogenates.
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).
Fig 4.
Western blot analysis of basement membrane components in exudates collected from the gastrocnemius.
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).
Table 2.
Extracellular matrix proteins identified in wound exudate collected from mice injected with PI, PII or PIII SVMPs.
Table 3.
Degradation of extracellular matrix proteins identified in wound exudates (see Methods section for details).