Journal: iScience

Article Title: Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

doi: 10.1016/j.isci.2023.108049

Figure Lengend Snippet: Augmented sVE-cadherin levels correlate with the loss of endothelial barrier function in patients with sepsis and in systemic inflammation in rats (A) Correlation analysis between sVE-cadherin levels and sequential organ failure assessement score (SOFA) of 20 patients is shown. (B) Correlation analysis between sVE-cadherin levels and volume resuscitation of cristalloids to stabilize the cardiovascular function of 20 patients is shown. (C) Representative H&E-staining of rat lungs are shown with and without the application of LPS. Scale bar is 100μm (n = 3). The panels below show, representative rat mesenteric microvessels following i.v. application of FITC-albumin to document microvascular loss of endothelial barrier function are shown. Scale bar is 50μm. (n = 5). (D) Quantitative analysis of alveolar septa thickness is shown in rats under control conditions and following LPS (∗p < 0.05, n = 3, unpaired t-te4st). (E) FITC-albumin extravasation in rat mesenteric microvessels was quantified by measuring the change of light intensity (ΔI) inside and outside the vessels as shown here for controls and 30 min after LPS application (∗∗∗∗p < 0.0001, n = 5, unpaired t-test). (F) Microcirculatory flow in rat mesenteric microvessels was investigated by the measurement of erythrocyte velocity [μm/s∗10 −3 ] under control conditions and after LPS application (∗∗∗∗p < 0.0001, n = 4 control, n = 5 LPS, unpaired t-test). (G) ELISA-based measurements of sVE-cadherin levels [ng/ml] are shown under control conditions and following the application of LPS (∗∗∗p < 0.001, n = 6 controls; n = 7 LPS, unpaired t-test). (H) Additional volume supply [ml] to maintain mean arterial pressure ≥60 mmHg under control conditions and following the application of LPS are shown (∗p < 0.05; n = 4 controls; n = 10 LPS, unpaired t-test). (I) Albumin levels of rats at the end of the experimental procedures under control conditions and after LPS application are shown (∗∗p < 0.01, n = 6 controls; n = 7 LPS, unpaired t-test). (J) Schematic overview of sVE-cad EC1-5 generation. CHO cells were transfected with the pcDNA-DEST47 plasmid containing sVE-cad EC1-5 and secrete sVE-cad EC1-5 into the supernatant. (K) Representative Western Blot of CHO cells and supernatants. In whole cell lysates of transfected CHO cells, a specific 90kDa protein band representing sVE-cadherin was detectable. CHO cells transfected with empty vector served as negative control. All pooled data in this figure are represented as mean ± SEM.

Article Snippet: Original Western Blot Data , https://doi.org/10.17632/ns3b3cd8jv.1 , Mendeley Data.

Techniques: Staining, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Western Blot, Negative Control

Journal: iScience

Article Title: Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

doi: 10.1016/j.isci.2023.108049

Figure Lengend Snippet: Generation and effects of sVE-cadherin EC1-5 (sVE-cad EC1-5 ) on endothelial cells (A) Application of sVE-cad EC1-5 on human endothelial cells (HDMEC) resulted in a dose-dependent decrease of transendothelial electrical resistance (TER); (∗∗p < 0.01, ∗∗∗∗p < 0.0001, n = 4 for each condition, one-Way ANOVA with Friedman and Dunn's Test.). (B) Permeability coefficient (P E ) measured as 70kDa FITC dextran flux across confluent (HDMEC). sVE-cad EC1-5 resulted in a significant increase in P E (∗p < 0.05, n = 12, one Way ANOVA with Sidak test). Lipopolysaccharide (LPS) served as a positive control (∗∗∗p < 0.001, n=12 (number dots are shown in the bargraph), one Way ANOVA with Sidak Test). (C) Representative Western Blot probed with cleaved caspase 3 antibody (used as marker of apoptosis). Only HDMEC treated with staurosporine showed a specific protein band for cleaved caspase 3. In lysates of sVE-cad EC1-5 treated cells, no cleaved caspase 3 was detectable; n = 4. (D) Cell viability assay showed that sVE-cad EC1-5 did not induce cell death (n = 18, 1-way ANOVA). Staurosporine was used as positive control (∗∗∗∗p < 0.0001, n = 18, 1-way ANOVA). (E) Representative immunofluorescence staining of HDMEC treated with sVE-cad EC1-5 for 24h. (VE-cadherin staining in red, F-actin in green; DAPI in blue; experiment shown is representative for n > 5, scale bar is 20μm). (F) Quantifications of VE-cadherin immunostaining of all experiments for the conditions shown in E). Application of HDMEC with sVE-cad EC1-5 resulted in a significant redistribution of VE-cadherin from the cell border to the cytoplasm as indicated by a flattening of the curve that measures the signal intensity at the cell border (∗p < 0.05). (G) Number of gaps/area counted in the immunostainings under control conditions and following the application of sVE-cad EC1-5 are shown. Gaps were counted in 20 randomly chosen areas from 5 independent experiments (∗∗∗∗p < 0.0001; unpaired two-tailed t-test). (H) Representative Western Blot of HDMEC cell lysate probed with VE-cadherin antibody directed against the extracellular domain of VE-cadherin. Beta actin is shown as a loading control. (I) The protein level is significantly reduced after the application of sVE-cad EC1-5 (∗p < 0.05, n = 5, unpaired two-tailed t-test); all pooled data in this figure are represented as mean ± SEM.

Article Snippet: Original Western Blot Data , https://doi.org/10.17632/ns3b3cd8jv.1 , Mendeley Data.

Techniques: Permeability, Positive Control, Western Blot, Marker, Viability Assay, Immunofluorescence, Staining, Immunostaining, Control, Two Tailed Test

Journal: iScience

Article Title: Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

doi: 10.1016/j.isci.2023.108049

Figure Lengend Snippet: Cell junction proteins associated with VE-cadherin and tight-junctions are reduced at the cell border in response to sVE-cad EC1-5 (A) Atomic force microscopy experiments demonstrated that the application of sVE-cad EC1-5 resulted in significantly reduced binding activity of VE-cadherin compared to control (∗p < 0.05, n = 4, paired two-tailed t-test). (B) Representative immunofluorescence staining for α-catenin, β-catenin, γ-catenin, ZO-1 and JAM-A of endothelial monolayer under control conditions and after 24h treatment with sVE-Cad EC1-5 ; insets highlight the changes at the level of the junctions; n > 5 for each of the conditions; scale bar is 20 μm. (C) Quantification of the immunostaining for the different junctional proteins α-catenin, β-catenin, γ-catenin, ZO-1 and JAM-A across 4–5 independent experiments are shown. Flattening of the curve that measures the signal intensity at the cell border indicates a loss/redistribution of the protein Quantification of F-actin staining as shown in Figure 2 E) is presented to document increased staining intensity by increased stress fiber formation (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, unpaired two-tailed t-test). (D) Representative Western Blot α-catenin, β-catenin, χ-catenin, ZO-1 and JAM-A under control conditions and following the application of sVE-cad EC1-5 . GAPDH served as loading control are shown. Quantifications of all Western blots performed are presented in Figure S2 C). (E) Representative Western blot of HDMEC lysates incubated with sVE-cad EC1-5 showed reduced VE-PTP protein levels compared to untreated cells. Reprobing of membranes for β-actin served as loading control. (F) Quantification of all Western blots performed for VE-PTP is shown (∗∗∗p < 0.001, n = 4, unpaired two-tailed t-test). (G) Co-immunostaining of VE-cadherin and VE-PTP showed a colocalization at the cell border under control conditions which was overall reduced following sVE-cad EC1-5 (n = 6; scale bar is 20 μm); all pooled data in this figure are represented as mean ± SEM.

Article Snippet: Original Western Blot Data , https://doi.org/10.17632/ns3b3cd8jv.1 , Mendeley Data.

Techniques: Microscopy, Binding Assay, Activity Assay, Control, Two Tailed Test, Immunofluorescence, Staining, Immunostaining, Western Blot, Incubation

Journal: iScience

Article Title: Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

doi: 10.1016/j.isci.2023.108049

Figure Lengend Snippet: VE-cad EC1-5 binds VE-PTP leading to loss of VE-cadherin/VE-PTP interaction (A–C) Molecular dynamic simulations revealed a direct interaction between sVE-cadherin specifically the 5 th EC-domain of sVE-cadherin with the 17th domain of VE-PTP protein. For details see text. (D) Representative pictures from proximity ligation assays (PLA) between VE-cadherin and VE-PTP are shown. Under control condition the interaction between both proteins is demonstrated as revealed by the red dots. Application of sVE-cad EC1-5 reduced the overall number of these spots confirming that sVE-cad EC1-5 interferes binding between VE-cadherin and VE-PTP. The lower pictures represent magnifications marked in the insets of the overview picture above; (n = 4; scale bar is 20 μm). (E) Western blot following for VE-PTP (left) and VE-cadherin (right) is shown following the incubation of supernatants of CHO-cells transfected with sVE-cad EC-1-5 and endothelial cell lysates which were then incubated with recombinant VE-PTP. Immunoprecipitation of VE-PTP from the supernatants and cell lysates was then performed (left) and membranes were reprobed with VE-cadherin to document a direct interaction between sVE-cad EC1-5 and VE-PTP. The lower bands with 70–90 kDa represent sVE-cadherin (n = 3); all pooled data in this figure are represented as mean ± SEM.

Article Snippet: Original Western Blot Data , https://doi.org/10.17632/ns3b3cd8jv.1 , Mendeley Data.

Techniques: Ligation, Control, Binding Assay, Western Blot, Incubation, Transfection, Recombinant, Immunoprecipitation

Journal: iScience

Article Title: Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

doi: 10.1016/j.isci.2023.108049

Figure Lengend Snippet: sVE-cad EC1-5 effects are mediated by the VE-PTP-dependent activation of RhoA/ROCK pathway (A) Measurements of transendothelial electrical resistance (TER) across endothelial monolayers was carried out. Application of sVE-cad EC1-5 reduced TER which was blocked by AKB9778 whereas AKB9778 alone augmented TER (∗p < 0.05; ∗∗∗∗p < 0.0001; n = 12, one-Way ANOVA with Friedman and Dunn's Test). (B) Representative immunofluorescence staining of VE-cadherin, VE-PTP and F-actin in endothelial monolayers (HDMEC) under control conditions, following incubation with sVE-cad EC1-5 alone or together with AKB9778 are show (n = 6, scale bar is 20 μm). (C) Representative Western Blot and quantitative analyses of HDMEC whole cell lysates displayed increased GEF-H1 protein levels after 24h incubation with sVE-cad EC1-5 compared to control conditions. GAPDH served as loading control. (D) Quantification of the Western blots performed as shown in C) is presented confirming an overall increase of GEF H1 (∗p < 0.05, n = 3, unpaired two-tailed t-test). (E) Western Blots following pulldown of activated GEF-H1 (RhoA G17) and incubation of the membranes with GEF-H1 antibodies, the total GEF-H1 levels of the input and β-actin as loading control are shown. (F) Quantification of the experiment shown in E) revealed an increased binding between RhoA and GEF-H1 after incubation with sVE-cad EC1-5 compared to untreated cells which was blocked by AKB9778 (∗p < 0.05, n = 6–7, ordinary 1-way ANOVA). (G) Representative Western blot after pulldown for active GTP-bound RhoA in endothelial cells under the different conditions are shown. Calpeptin and HIS-RhoA served as internal controls. Total RhoA from endothelial lysates are shown later in discussion. (H) Quantification of the experiment shown in (G) revealed overall augmented RhoA acitivity following the application of sVE-cad EC1-5 which was attenuated by co-incubation with AB9778 (∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; n = 5, ordinary 1-way ANOVA). (I) Representative immunostaining of endothelial monolayers (HDMECs) for VE-cadherin (red) and F-actin (green) are shown following incubation with sVE-cad EC1-5 or in combination with Rho-kinase inhibitor Y27632; n = 5; scale bar is 20 μm. (J) TER measurements across endothelial monolayers are shown after the application of sVE-cad EC1-5 , after Y27632 alone or in combination with sVE-cad EC1-5 (∗∗p < 0.01, n = 4, ordinary 1-way ANOVA); all pooled data in this figure are represented as mean ± SEM.

Article Snippet: Original Western Blot Data , https://doi.org/10.17632/ns3b3cd8jv.1 , Mendeley Data.

Techniques: Activation Assay, Immunofluorescence, Staining, Control, Incubation, Western Blot, Two Tailed Test, Binding Assay, Immunostaining

Journal: iScience

Article Title: Endothelial barrier dysfunction in systemic inflammation is mediated by soluble VE-cadherin interfering VE-PTP signaling

doi: 10.1016/j.isci.2023.108049

Figure Lengend Snippet:

Article Snippet: Original Western Blot Data , https://doi.org/10.17632/ns3b3cd8jv.1 , Mendeley Data.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Activation Assay, Proximity Ligation Assay, Western Blot, Software