Increased angiogenesis parallels cardiac tissue remodelling in experimental acute Trypanosoma cruzi infection

Figures

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  Fig. 1:. acute Chagas disease (CD) in Swiss Webster mice infected with the Y strain of T. cruzi. Mice were infected with 104 blood trypomastigote forms and the following parameters were evaluated in a kinetic study: parasitemia (A), survival rate (B), tissue parasitism (C) and inflammatory infiltrates (D). Mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) test, p < 0.01** and p < 0.001***. N = 20. dpi: days post-infection; NI: noninfected.

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  Fig. 2:. western blotting of proangiogenic proteins in the heart. Representative immunoblot analysis of hypoxia-inducible factor-1α (HIF-1α) (132 kDa) (A), vascular endothelial growth factor (VEGF) (42 kDa) (B), Flk-1 (150 kDa) (C) and phosphorylated extracellular-signal-regulated protein kinase (pERK) 1/2 (44 kDa) (D) levels in cardiac tissue. Hearts of noninfected (NI) mice and Trypanosoma cruzi infected at 8, 15 and 22 days post-infection (dpi) were harvested, and protein lysates were probed with anti-HIF-1α, anti-VEGF, anti-Flk-1 and anti-pERK1/2 antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (36 kDa) was used as a loading control. One-way analysis of variance (ANOVA) test, p < 0.05*, p < 0.01** and p < 0.001***. N = 5 for each group. IV: index of variation of the mean ± standard error of the mean (SEM).

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  Fig. 3:. immunofluorescence of vascular endothelial growth factor (VEGF) in cardiac tissue. VEGF distribution (green) in noninfected (NI) (A) and Trypanosoma cruzi infected (B). NI mice displaying VEGF in blood vessels among cardiac fibres (thin arrows) and arterioles (&) (A). In T. cruzi infected tissues, VEGF can be observed in blood vessels (thin arrows), in areas of connective tissue (thick arrows) and diffuse in the cytoplasm of cardiomyocytes (B). Notice the VEGF striated staining pattern in myofibrils (#). Bar graph shows the quantification of fluorescence intensity (C). Mean ± standard error of the mean (SEM). Unpaired t test, p < 0.001***. Bars = 20 µm. N = 3 for each group. dpi: days post-infection.

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  Fig. 4:. immunohistochemistry of cardiac Flk-1. Flk-1 content (green) in noninfected (NI) (A) and Trypanosoma cruzi infected (B) heart. NI mice heart reveals weak labelling for Flk-1 in the blood vessel (thin arrows) located between cardiac fibres (A), whereas 15 days post-infection (dpi) infected animals exhibit intense Flk-1 staining in the blood vessel (thin arrows) (B). Bar graph shows the percentage of stained area quantification (C). Mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) test, p < 0.05*. Bars = 20 µm. N = 3 for each group.

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  Fig. 5:. evaluation of cardiac angiogenesis. Hematoxylin and eosin (H&E) staining of noninfected (NI) (A) and Trypanosoma cruzi infected at 8 (B), 15 (C) and 22 days post-infection (dpi) (D), showing blood vessels (arrow). By immunofluorescence of NI (left panel) and T. cruzi infected cardiac tissues (right panel) (E), we can observe an increase in CD31 (F, G and J) and α-smooth muscle actin (α-SMA) positive blood vessel (arrow) in infected tissue (H, I and K), indicating angiogenesis and vascular maturation. Mean ± standard error of the mean (SEM). Unpaired t test, p < 0.001***. N = 5 for each group. Bar = 10 µm.

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  Fig. 6:. confocal microscopy analysis of α-smooth muscle actin (α-SMA) positive cells. Immunostaining (green) of α-SMA shows the mural cells in arterioles of cardiac tissue (A-C). A continuous staining pattern can be observed in the controls (A), while in infected hearts the α-SMA staining is clearly delimited around individuals myofibroblasts near blood vessels 15 and 22 days post-infection (dpi) (arrow) (B and C). Bar = 10 µm. N = 5 for each group. NI: noninfected.

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  Fig. 7:. analysis of fibrosis by histochemistry. Masson’s trichrome staining shows the collagen distribution in noninfected (NI) (A) and in T. cruzi infected hearts at 15 (B) and 22 (C) days post-infection (dpi). Notice the strong collagen staining around blood vessels infiltrating through the cardiac tissues in infected animals. Bar = 10 µm. N = 5 for each group.