Vascular Endothelial Integrity Affects the Severity of Enterovirus-Mediated Cardiomyopathy

Figures

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  Figure 1. Endothelial coxsackievirus and adenovirus receptor (CAR) deletion increases permeability of endothelial cell monolayer for coxsackievirus B3 (CVB3). (a). Trans-epithelial electrical resistance (TER) was measured in a trans-well chamber with cultured wild-type (WT) endothelial cells and endothelial cells in which CAR had been deleted (CAR-eKO (KO)). Trypan blue staining showed endothelial cells in the upper well. (b). WT and KO endothelial cells were treated with Green fluorescent protein (GFP)-CVB3, and GFP expression was observed under a fluorescent microscope. Blank, no endothelial cell cultured chamber. (c). Cell extracts were subjected to Western blot analysis using the indicated antibodies. GFP immunoblot bands indicate fold changes of GFP normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bands. All data are the mean ± s.e.m. from 3 independent experiments (scale bar, 100 µm). * p < 0.05 by two-tailed Student’s t-test.

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  Figure 2. CVB3 replication is delayed in endothelial cells. (a). WT and CAR-eKO (KO) endothelial cells were immunostained with antibodies against platelet endothelial cell adhesion molecule-1 (PECAM-1, green) and CAR (red) and analyzed by fluorescence microscopy (scale bar, 40 µm). Cell extracts were subjected to Western blot analysis using indicated antibodies. (b). WT and KO endothelial cells were treated with 1 m.o.i. CVB3 and then the cells were lysed at day 1, 3, 5, 7. Endothelial cell extract proteins were subjected to Western blot analysis using indicated antibodies. (c). Supernatants of CVB3-infected WT and KO cells were subjected to plaque forming unit (PFU) assay to measure release of virus into the supernatant.

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  Figure 3. Vascular permeability is increased in the heart of endothelial CAR knockout mice. (a). WT and KO mice were injected with 10% Evans blue dye (EBD) for 3 h. Heart was sectioned and subjected to fluorescence microscopy to determine the area of EBD uptake (white arrows; scale bar, 100 µm). (b). EBD concentration in the heart extract was measured by microplate reader (n = 5 per group). (c). WT and KO mice were treated with 70 kDa dextran-FITC, and heart sections were examined by fluorescence microscopy to determine the area of FITC uptake (left). Dextran-FITC concentration in the heart extract was measured at 488 nm in a microplate reader (right) (n = 8 per group; scale bar, 5 µm). All data are the mean ± s.e.m. * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-tailed Student’s t-test.

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  Figure 4. Endothelial CAR deletion increases inflammatory cell infiltration into the peritoneal cavity. (a,b). Ten-week-old male WT and KO mice were treated with PBS or 3% thioglycolate for 7 days, and peritoneal cells were isolated, stained with the indicated antibodies (monocytes = Gr-1 and macrophages = CD11b) and subjected to fluorescence-activated cell sorting (FACS) analysis. All data are the mean ± s.e.m. from 3 independent experiments. ** p < 0.01 and *** p < 0.001 by two-tailed Student’s t-test.

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  Figure 5. Effect of CAR deletion on CVB3-induced myocarditis. (a). Six-week-old male WT and KO mice were infected with 1 m.o.i. CVB3 for 5 days and then injected with EBD before being sacrificed. A heart section was examined by fluorescence microscopy. Red fluorescence represents EBD uptake by damaged myocardium (upper). The percent area of EBD was quantified with Scion Image (n = 4 per group). (b). Histological findings of sectioned heart show inflammatory cell infiltration and fibrosis using hematoxylin & eosin (H&E) and trichrome staining. (c). Virus titers in organs of WT and KO mice determined by PFU assay (n = 4 per group). All data are the mean ± s.e.m. from 3 independent experiments performed (scale bar, 100 µm). * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-tailed Student’s t-test.

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  Figure 6. CAR deletion disrupts endothelial cell VE-cadherin and PECAM-1 expression. (a). Endothelial cells were isolated from the livers of WT and KO (with deleted CAR) mice. Cell extracts were subjected to Western blot analysis using the indicated antibodies. (b). Immunofluorescence staining of endothelial cells to determine changes in cell morphology. All data are the mean ± s.e.m. from 3 independent experiments (scale bar, 60 µm). *** p < 0.001 by two-tailed Student’s t-test.

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  Figure 7. Disruption of the endothelial cell VE-cadherin and PECAM-1 expression by CAR ablation. (a). WT and KO mice liver were subjected to immunofluorescence staining using the indicated antibodies. Green, VE-cadherin and red, ZO-1. (b). Green, PECAM-1 and red, CAR in the vascular endothelial cells of liver, which are indicated by white arrows (scale bar, 40 µm and 15 µm). (c). WT and KO mouse hearts coronary vessel were subjected to immunofluorescence staining using the indicated antibodies. Green, VE-cadherin and red, CAR (scale bar, 15 µm).