The Potential Pathogenic Contributions of Endothelial Barrier and Arterial Contractile Dysfunction to Shock Due to B. anthracis Lethal and Edema Toxins

Figures

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  Figure 1. This figure shows key components of an adherens junction (AJ). VE-cadherin homophilic ligation (brown structures) mediates calcium-dependent adhesion of adjacent endothelial cells (blue structures). The cytoplasmic domain of VE-cadherin binds ß-catenin (ß-cat), which in turn recruits IQGAP1 (IQ) and α-catenin to AJs. IQGAP1 binds and stabilizes GTP-bound (Cdc42) and Rac1, whereas α-catenin (α-cat) directly interacts with actin and mediates formation of cortical actin bundles. P120-catenin (p120-cat) associates with the juxtamembrane region of VE-cadherin and prevents VE-cadherin internalization. p120-cat also recruits p190RhoGAP (p190) to AJs and provides the mechanism for RhoA inhibition at the level of AJs. γ-catenin (γ-cat) interacts with VE-cadherin and recruits desmoplakin (Des) and vimentin to AJs. (Reproduced from [38]. Copyright 2010, Annual Reviews).

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  Figure 2. Concentration dependent effects of medium only, lethal factor alone (LF), LFE687c (an inactive LF) protective antigen alone (PA) or lethal toxin (LT) on albumin permeability. (A) Human lung microvascular endothelial cell monolayers grown on porous membranes of two compartment (upper and lower) inserts were incubated with medium alone or medium containing 1 μg/mL LF, 1 μg/mL LFE687c and PA, or varying amounts of LF in the presence of 1 μg/mL PA. After 72 h, fluorescein isothiocyanate labeled human serum albumin (FITC-HSA) was added to the upper compartment of the inserts. After 2 h the amount of FITC-HSA in the bottom compartment was measured using a fluorescent microplate reader. Values were calculated as the micrograms of FITC-HSA per hour per square centimeter and reported as the means ± SE for a minimum of three independent experiments. (B) Monolayers were grown and treated and data was collected as in (A). * p < 0.001, † p < 0.01 versus medium alone. (Reproduced from [44]. Copyright 2005, Elsevier.)

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  Figure 3. Phase contrast morphology and immunofluorescence visualization of F-actin, VE-cadherin, and nuclei. Cells were incubated with medium alone (A–C), or medium containing 1 μg/mL of lethal factor (LF) (D–F), 1 μg/mL protective antigen (PA) (G–I) or both (i.e., lethal toxin (J–L). After 72 h, monolayers were washed, fixed and stained with Hochst 33342 (blue), F-actin (green), VE-cadherin (red) as described [44]. Phase contrast images and corresponding immunofluorescence images were visualized using an inverted microscope (40× objective). Lethal toxin (LT) induced cellular elongation and small interendothelial gaps (black arrows) compared with medium or LF or PA alone. In addition, LT increased central F-actin stress fibers and decreased VE-cadherin immunofluorescence. Images were representative of five separate experiments. (Reproduced from [44]. Copyright 2005, Elsevier.)

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  Figure 4. Lethal toxin (LeTx) induces permeability and gap formation in wild-type rat pulmonary microvascular endothelial cells (RPMEC). (A) LeTx induces permeability in RPMEC after 45 min of treatment. Monolayers were grown on filter inserts and exposed to LeTx (2 μg/mL). The transfer of Alex Fluor-dextran (3 kDa) through the monolayer was measured. The bar represents change in fluorescence intensity in the lower chamber compared with control groups. Data are represented as mean ± SD (n = 3). (B) LT promotes gap formation between endothelial cells. RPMEC monolayers were grown on cover slips and then treated with LT (2 μg/mL) for 30 min. The borders between endothelial cells were visualized by staining plasma membranes and nuclei. Arrows point to regions where gaps formed between cells. Gaps were evident in LeTx exposed cells but not controls. This figure is representative of three different experiments. (Reproduced from [52]. Copyright 2012, John Wiley and Sons.)

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  Figure 5. Panels (A–C) compare the effects of pretreatment of aortic rings for 60 min with diluent, protective antigen (PA) alone or four increasing concentrations of edema toxin (ET, 100, 200, 400 or 800 ng/mL) on the mean (±SEM) contractile force the rings subsequently generated during stimulation with increasing phenylephrine (PE) concentrations (Panel (A)), the mean (±SEM) maximal contractile force (MCF) they developed during PE stimulation (Panel (B)), and the mean (±SEM) estimated concentration of PE producing 50% of the MCF (Panel (C)). The contractile force shown in Panel (A) and the MCF shown in Panel (B) were calculated as a percentage of peak contractile force rings generated when exposed initially to 60 mM KCL. The number of rings used in each experiment (n) is shown in the key. (Adapted from Li et al. [77]. 2013, American Physiological Society.)

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  Figure 6. Panels (A–C) show the serial mean (±SEM) contractile force rings pre-contracted for 10 min with 1, 10 or 100 μM phenylephrine (PE) generated during 90 min of subsequent treatment with protective antigen (PA, 1600 ng/mL) or edema toxin (ET, 800 ng/mL). Panels (D–F) compare the mean (±SEM) relaxation (calculated based on the data from Panels (A–C)) recorded at 90 min in rings treated with PA alone versus ET. The contractile forces shown in Panels (A–C) were calculated as a percentage of peak contractile force rings generated when exposed initially to 60 mM KCL. The number of rings used in each experiment (n) is shown in the key in Panels (A–C). Adapted from Li et al. [77]. 2013, American Physiological Society.)

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  Figure 7. Panels (A–C) compare the effects of pretreatment of aortic rings for 60 min with diluent, protective antigen (PA) alone or four increasing concentrations of lethal toxin (LT, 40, 80, 160, 320 or 640 ng/mL) on the mean (±SEM) contractile force the rings subsequently generated during stimulation with increasing phenylephrine (PE) concentrations (Panel (A)), the mean (±SEM) maximal contractile force (MCF) they developed during PE stimulation (Panel (B)), and the mean (±SEM) estimated concentration of PE producing 50% of the MCF, EC50 (Panel (C)). The contractile force shown in Panel (A) and the MCF shown in Panel (B) were calculated as a percentage of peak contractile force rings generated when exposed initially to 60 mM KCL. The number of rings used in each experiment (n) is shown in the key. (Adapted from Li et al. [77]. 2013, American Physiological Society.)

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  Figure 8. Comparison of the contractile response to phenylephrine treatment of aortic rings isolated from animals 4, 15, 24 or 48 h following the initiation of 24 h in vivo challenges with normal saline (control), edema toxin (ETx) or lethal toxin (LTx). Panels (A–D) show the mean (±SEM) maximal contractile force (MCF) and Panels (E–H) show the mean (±SEM) estimated concentration of phenylephrine producing 50% of the MCF (EC50) for aortic rings isolated from control (open bar), ETx (black bar) and LT (grey bar) challenged animals. Only control and LTx animals were studied at 15 h. Numbers of animals providing rings for study for each challenge and time point are shown in Panels (A–D). Levels of significance for the comparisons between toxin and control rings are provided in the figure. (Adapted from [78]. 2017, American Physiological Society.)

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  Figure 9. Comparison of the serial mean (±SEM) percent changes in mean arterial blood pressure (MAP) (y-axis) with graded in vivo phenylephrine treatment (x-axis) in animals following the initiation 24 h challenges of normal saline (control, open circles), edema toxin (ETx, black diamonds) or lethal toxin (LTx, grey triangles). Comparison between ETx and control animals are shown with measures at 24 and 48 h (Panels (A,B) respectively), and the comparisons between LTx and control are shown with measures at15 and 48 h (Panels (C,D) respectively). Numbers of animals studied for each challenge and time point are shown in the figure. * Level of significance for the change in the difference between groups across the phenylephrine doses (i.e., the dose interaction). (Adapted from [78]. 2017, American Physiological Society.)

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  Figure 10. Comparison of proportional survival (Panel (A)), and serial mean (±SEM) changes in mean arterial blood pressure (MAP, Panel (B)) over 48h in animals following initiation of a 24 h challenge (grey shaded area) of normal saline (control, Con) or edema toxin (ETx) and 24 h concurrent treatment (open labeled bar) with adefovir (ADF) or diluent (not labeled). Animals challenged with normal saline and treated with diluent or adefovir are designated with open circles and open inverted triangles respectively and animals challenged with ETx and treated with diluent or adefovir are designated with black or grey diamonds respectively. Initial numbers of animals studied are shown in the figure. * Level of significance for the overall difference between groups across the time period. ** Level of significance for the change in the difference between groups across the time period (i.e., the time interaction). (Adapted from Suffredini et al. [78]. 2017, American Physiological Society.)

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  Figure 11. This figure compares the mean (±SEM) contractile force aortic rings generated during stimulation with increasing phenylephrine concentrations (Panel (A)), the mean (±SEM) maximal contractile force (MCF) developed during PE stimulation (Panel (B)), and the mean (±SEM) estimated concentration of PE producing 50% of the MCF (EC50, Panel (C)) after pretreatment with protective antigen (PA) (1600 ng/mL) combined with placebo or with edema toxin (ET) (800 ng/mL of edema factor and 1600 ng/mL of PA) combined with either placebo or l-nitro-arginine methyl-ester (l-NAME) in increasing concentrations of 0.03, 0.3, 3, or 30 μM. The brackets and p-values demonstrate the levels of significance for comparisons of respective groups of rings. (Adapted from Li et al. [79]. 2016, American Physiological Society.)