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• W2946341736 abstract "Article14 May 2019free access Transparent process VE-PTP inhibition stabilizes endothelial junctions by activating FGD5 Laura J Braun Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Maren Zinnhardt Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Matthias Vockel Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Hannes C Drexler Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Kevin Peters Aerpio Pharmaceuticals, Cincinnati, OH, USA Search for more papers by this author Dietmar Vestweber Corresponding Author [email protected] orcid.org/0000-0002-3517-732X Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Laura J Braun Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Maren Zinnhardt Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Matthias Vockel Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Hannes C Drexler Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Kevin Peters Aerpio Pharmaceuticals, Cincinnati, OH, USA Search for more papers by this author Dietmar Vestweber Corresponding Author [email protected] orcid.org/0000-0002-3517-732X Max Planck Institute of Molecular Biomedicine, Münster, Germany Search for more papers by this author Author Information Laura J Braun1, Maren Zinnhardt1, Matthias Vockel1,†, Hannes C Drexler1, Kevin Peters2 and Dietmar Vestweber *,1 1Max Planck Institute of Molecular Biomedicine, Münster, Germany 2Aerpio Pharmaceuticals, Cincinnati, OH, USA †Present address: Institute for Human Genetics, University of Münster, Münster, Germany *Corresponding author. Tel: +49 251 70365 210; Fax: +49 251 70365 299; E-mail: [email protected] EMBO Rep (2019)20:e47046https://doi.org/10.15252/embr.201847046 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Inhibition of VE-PTP, an endothelial receptor-type tyrosine phosphatase, triggers phosphorylation of the tyrosine kinase receptor Tie-2, which leads to the suppression of inflammation-induced vascular permeability. Analyzing the underlying mechanism, we show here that inhibition of VE-PTP and activation of Tie-2 induce tyrosine phosphorylation of FGD5, a GTPase exchange factor (GEF) for Cdc42, and stimulate its translocation to cell contacts. Interfering with the expression of FGD5 blocks the junction-stabilizing effect of VE-PTP inhibition in vitro and in vivo. Likewise, FGD5 is required for strengthening cortical actin bundles and inhibiting radial stress fiber formation, which are each stimulated by VE-PTP inhibition. We identify Y820 of FGD5 as the direct substrate for VE-PTP. The phosphorylation of FGD5-Y820 is required for the stabilization of endothelial junctions and for the activation of Cdc42 by VE-PTP inhibition but is dispensable for the recruitment of FGD5 to endothelial cell contacts. Thus, activation of FGD5 is a two-step process that comprises membrane recruitment and phosphorylation of Y820. These steps are necessary for the junction-stabilizing effect stimulated by VE-PTP inhibition and Tie-2 activation. Synopsis Inflammation-induced vascular permeability is suppressed by inhibiting the endothelial tyrosine phosphatase VE-PTP. FGD5, a GEF for Cdc42, is essential for this effect and acts as a direct substrate of VE-PTP, which stabilizes endothelial junctions. FGD5 tyrosine phosphorylation is essential for endothelial junction stabilization. VE-PTP inhibition activates FGD5 in two steps: by Tie-2, Rap-1 dependent junction recruitment and by phosphorylation of Y820. FGD5 supports cortical actin and inhibits radial stress fibers by activating Cdc42 and Rac1. Introduction Endothelial cells determine the permeability of the blood vessel wall and thereby control extravasation of solutes, macromolecules, and leukocytes. During inflammation, vascular permeability is locally increased in the microcirculation, which enables plasma proteins and leukocytes to fight infections. However, hyperpermeability causes severe consequences in diseases such as diabetes and can be catastrophic in acute settings such as stroke, myocardial infarction, or acute respiratory distress syndrome. Molecular mechanisms that enhance vascular permeability in inflammatory settings target specifically junctions between endothelial cells. Endothelial junction integrity relies on several adhesion molecules among which vascular endothelial (VE)-cadherin is of major importance 1-3. It is essential for the formation of these junctions and the entire vascular system during embryonic development 4, 5. Interference with the function of VE-cadherin by adhesion-blocking antibodies is sufficient to enhance leukocyte extravasation 6 and vascular permeability 7 in vivo, and enhancing the interaction of VE-cadherin with the actin cytoskeleton blocked both of these processes 8. However, when different organs were compared it was found that adhesion-blocking antibodies 7 and the conditional inactivation of the VE-cadherin gene 9 were sufficient to enhance vascular permeability in some, but not in all, organs of the mouse. Even the structural integrity of endothelial junctions as judged by electron microscopy was not affected by ablating the VE-cadherin gene in adult mice 9. The VE-protein tyrosine phosphatase (PTP) associates with VE-cadherin and supports its function 10, 11. Inflammatory cytokines as well as leukocytes docking to endothelium dissociate VE-PTP from VE-cadherin 11, 12, a process which is required in vivo for the induction of enhanced vascular permeability 13. The association of VE-PTP with VEGFR-2 is also potentially relevant for the regulation of VEGF-mediated effects on endothelial junctions 14, 15. VE-PTP also associates with Tie-2, an endothelial tyrosine kinase receptor involved in angiogenesis and vascular remodeling 16, 17. Gene inactivation of VE-PTP causes embryonic lethality at midgestation due to overactivation of Tie-2 and enhanced endothelial proliferation and vessel enlargement 17-19. Besides vascular remodeling, Tie-2 is well known to protect the vasculature against plasma leakage induced by various pro-inflammatory mediators 20-22. The importance of VE-PTP for this junction-protective effect of Tie-2 was first suggested by the beneficial effect of the VE-PTP inhibitor AKB-9778 in mouse models of retinopathy. The inhibitor suppressed ocular neovascularization and blocked VEGF-induced vascular leakage and retinal detachment 23. Directly comparing the effects of VE-PTP on VE-cadherin and on Tie-2 in the adult mouse, we found recently that the inhibitor AKB-9778 as well as conditional gene inactivation of VE-PTP could counteract histamine, VEGF, and LPS-induced vascular permeability in various organs. This effect was mediated by Tie-2, since it was strongly reduced upon silencing the expression of Tie-2 in these mice 9. Of note, baseline vascular permeability was enhanced by the VE-PTP inhibitor in vivo in the absence of Tie-2, revealing the supportive effect of VE-PTP on VE-cadherin under these conditions. Thus, inhibition of VE-PTP in vivo has opposing effects on endothelial junctions due to its different effects on VE-cadherin and on Tie-2. However, activation of Tie-2 via inhibition of VE-PTP protects endothelial junctions against inflammation-induced destabilization and even overrides the negative effect of VE-PTP inhibition on the junction-stabilizing function of VE-cadherin 9. Mechanistically, Tie-2 signaling was suggested to counteract VEGF-induced permeability across endothelial monolayers by blocking VE-cadherin endocytosis 24. In addition, it was shown that the Tie-2 ligand angiopoietin-1 (Ang1) activated the GTPase Rac1 which in turn blocked the activation of RhoA and thereby relieved the pulling forces of actomyosin radial stress fibers (RSF) on endothelial junctions 22, 25. We could confirm this and found that the activation of Rap1 is an additional essential signaling step downstream of Tie-2 and upstream of Rac1 activation in this pathway 9. Rap1 is a well-characterized GTPase known to enhance the stability of endothelial junctions 26-29. Stimulation of the GEF Epac1 by pharmacologically enhancing intracellular cAMP levels or by a cAMP analogue (007) triggers the activation of Rap1, enhances the formation and tension of junction-stabilizing circumferential actin bundles (CAB), and reduces pulling forces on junctions by counteracting RSF formation 30-33. FGD (FYVE, RhoGEF, and PH domain containing) 5 is a GEF of Cdc42 and belongs to the FGD GEF family that contains FGD1 to FGD6 as well as the FGD1-related Cdc42-GEF (FRG). They all share a Dbl homology, a FYVE, and two pleckstrin homology (PH) domains. FGD5 is by far the largest member in this family containing 30 tyrosine residues in the mouse and a large N-terminus comprising about half of the molecule which is potentially unstructured. This GEF was reported to be specifically expressed in hematopoietic stem cells 34 and in endothelial cells based on in situ hybridization and was found to be involved in vascular pruning, endothelial cell network formation, and directional movement in the mouse embryo 35, 36. In cultured endothelial cells, it has been implicated in the stabilization of endothelial junctions, where it was acting downstream of the cAMP-Epac1-Rap1 pathway, stimulating Cdc42-dependent activation of the kinase MRCK which phosphorylated S19 of the regulatory light chain of non-muscle myosin II, stimulating junctional CAB 30. This was reproduced by Pannekoek et al 33, although in this study silencing of FGD5 had only a limited effect on cAMP-Epac1-Rap1-mediated junction stabilization. Here, we have analyzed the mechanism whereby the inhibition of VE-PTP stabilizes endothelial junctions. Searching for targets of VE-PTP, we identified FGD5 as a direct substrate. Determining which tyrosine residue is targeted by VE-PTP enabled us to show that tyrosine phosphorylation of FGD5 is required for the full activation of FGD5. We found that VE-PTP inhibition triggers enhanced FGD5 phosphorylation by blocking its dephosphorylation as well as by activating the kinase activity of Tie-2. FGD5 was required for the junction-stabilizing effect of a VE-PTP inhibitor in vitro and in vivo. These effects were due to enhancing circumferential actin bundles as well as blocking the tension of radial stress fibers on junctions. Results FGD5 is a substrate of VE-PTP In order to understand more about the mechanism of how VE-PTP modulates endothelial junction integrity, we searched for novel substrates of this tyrosine phosphatase. To this end, changes of protein phosphorylation were analyzed by comparing untreated mouse bEnd.5 cells with cells treated for 30 min with the VE-PTP inhibitor AKB-9778. Tryptic digests of cell lysates were subjected to affinity isolation on TiO2 microspheres, and isolated phospho-peptides from each cell population were analyzed and compared by mass spectrometry. In total, 6,569 class I phosphosites could be identified across both samples measured by two methods (HCD and MSA, see Materials and Methods). Of these, 341 phosphosites were specifically detected in the AKB-9778-treated samples with a localization probability > 0.75 in both the HCD- and the MSA-based measurements, among them a considerable fraction of phospho-tyrosine sites (n = 56, 16.4%; Dataset EV1). Several of the identified phosphosites were found in proteins that are potentially relevant for the regulation of junctions, such as FGD5, Tie-2, Tie-1, ZO-1, claudin-5, p120, VEGFR-3, JAM-C, neuropilin-1, afadin, PAR-3, ArhGAP12, and SHP-2. Since another proteomics approach showed that the Cdc42 GEF FGD5 was one of the major proteins isolated with an anti-phospho-tyrosine antibody from bEnd.5 cells after AKB-9778 treatment (Drexler et al, unpublished), we decided to analyze this protein further. To verify the substrate character of FGD5, bEnd.5 mouse endothelioma cells were briefly pre-treated with peroxyvanadate, and cell lysates were subjected to pull-down assays with a GST-VE-PTP fusion protein containing the cytoplasmic tail of VE-PTP (fused to glutathione S-transferase) and an analogous protein carrying an inactivating CS/DA point mutation in the active center of the phosphatase domain. The mutation leaves substrate recognition intact, yet the phosphatase activity is lost, which strongly stabilizes the enzyme–substrate interaction. For the detection of mouse FGD5, polyclonal antibodies were raised against and purified on a recombinant form of the first 248 amino acids of FGD5 (Fig EV1). As shown in Fig 1A, the phosphatase-dead trapping mutant of VE-PTP bound strongly to FGD5, whereas binding to the WT protein was hardly stronger than to the GST-negative control. In agreement with this result, FGD5 could only be efficiently immunoprecipitated from HUVEC lysates with an anti-phospho-tyrosine antibody when these cells were pre-treated for 30 min with the VE-PTP inhibitor AKB-9785 (Fig 1B). Corresponding to this finding, knocking down VE-PTP in HUVEC by siRNA enhanced tyrosine phosphorylation of FGD5 as shown in immunoblots (Fig 1C). Subcutaneous administration of AKB-9785 in mice for 1 h followed by immunoprecipitation of FGD5 from lung lysates and subsequent anti-phospho-tyrosine immunoblotting also revealed a 3- to 3.5-fold increase in pY levels of FGD5 in vivo (Fig 1D). We conclude that FGD5 is a substrate of VE-PTP. Click here to expand this figure. Figure EV1. FGD5 N-term antibody efficiently detects murine FGD5 WT MDMVEC were transfected with control or FGD5 siRNA, lysed, and immunoblotted for FGD5 (FGD5 N-term), pre-serum, and α-tubulin. FGD5 N-term was used to precipitate FGD5 from WT MDMVEC lysates. As an isotype control, lysates were incubated with rabbit pre-serum (Iso). Precipitates and lysates were immunoblotted for FGD5 (FGD5 N-term) and α-tubulin. Confluent WT MDMVEC monolayers were stained for FGD5 (FGD5 N-term) and PECAM-1. As a control for specific FGD5 staining by FGD5 N-term, cells were incubated with pre-serum. Scale bars 30 μm. Data information: Results are representative of at least two independent experiments. Download figure Download PowerPoint Figure 1. FGD5 is a substrate of VE-PTP bEnd.5 cells were stimulated with peroxyvanadate for 7 min, lysed, pre-cleared by incubation with GST, and submitted to pulldowns with GST, GST-VE-PTP-WT, or GST-VE-PTP-CS/DA (as indicated). Pulldowns and total cell lysates were analyzed by immunoblot for FGD5, GST, and α-tubulin. Lysates of HUVEC treated with vehicle (−) or 5 μM AKB-9785 (+) for 30 min were submitted to immunoprecipitation (IP) with 4G10. Precipitates were immunoblotted for FGD5 and cell lysates for FGD5 and α-tubulin. Lysates of HUVEC transfected with VE-PTP-targeting or control siRNA were submitted to immunoprecipitation of FGD5. Precipitates and lysates were immunoblotted for phospho-tyrosines (4G10), FGD5, VE-PTP and α-tubulin. FGD5 was precipitated from total lung lysates of C57BL/6 mice that received subcutaneous injections of vehicle or AKB-9785 for 1 h. Precipitates and lysates were immunoblotted for phospho-tyrosines (4G10), FGD5, PECAM-1 and α-tubulin. Data information: Results are representative of two (D) or three (A–C) independent experiments. Download figure Download PowerPoint FGD5 translocates to cell contacts upon VE-PTP inhibition Activation of GEFs is usually accompanied by recruitment to membranes at their site of action. For FGD5, it had been reported that it is translocated to endothelial cell contacts upon activation of the Rap1 GEF Epac1 with the cAMP analogue 8-pCPT-2′-O-Me-cAMP (007) 30. Therefore, we tested whether inhibition of VE-PTP would have a similar effect. As shown in Fig 2A and B, AKB-9785 indeed enhanced the recruitment of FGD5 to junctions of HUVEC monolayers in a similar way as the reagent 007. At the same time, we observed a straightening of endothelial junctions (judged by staining for VE-cadherin) and reduced radial stress fibers. VE-cadherin staining levels were unchanged at cell contacts (Fig 2C). Due to the similarities of the effects, we tested whether 007 would also stimulate the phosphorylation of FGD5. However, immunoblot analysis revealed that only AKB-9785 but not 007 treatment of HUVEC triggered tyrosine phosphorylation of FGD5 (Fig 2D). This strongly suggests that tyrosine phosphorylation of FGD5 is not required for its recruitment to junctions. Figure 2. Rap1 mediates translocation to cell contacts but not phosphorylation of FGD5 upon VE-PTP inhibition Confluent HUVEC monolayers were stimulated with vehicle, 200 μM 007, or 5 μM AKB-9785 for 20 min. Cells were fixed, permeabilized, and stained for FGD5, VE-cadherin, and F-actin. Scale bars 30 μm. Quantification of FGD5 signal intensities at cell contacts relative to FGD5 signal intensities in adjacent areas of the cytoplasm of 20–25 cells as shown in (A). Quantification of VE-cadherin signals at cell–cell junctions relative to cytoplasmic signals of 20–25 cells as displayed in (A). FGD5 was immunoprecipitated from HUVEC lysates after stimulation with 200 μM 007 for 20 min or 5 μM AKB-9785 for 30 min. Precipitates and lysates were immunoblotted for phospho-tyrosines (4G10), FGD5, and α-tubulin. HUVEC were transfected with Rap1a/b-targeting or control siRNA and treated with 5 μM AKB-9785 or vehicle. Fixed and permeabilized monolayers were stained for FGD5 and F-actin. Scale bars 30 μm. FGD5 signal intensities at cell contacts relative to FGD5 signal intensities in the cytoplasm of 20–25 cells as shown in (E). FGD5 immunoprecipitates from HUVEC transfected with Rap1a/b-targeting or control siRNA and treated with 5 μM AKB-9785 or vehicle for 30 min were immunoblotted for 4G10 and FGD5, and cell lysates for Rap1 and α-tubulin. Quantification of 4G10 signals as shown in (G). 4G10 signal intensities are displayed relative to the amount of precipitated FGD5 in the respective sample and normalized to control siRNA- and vehicle-treated cells. Data information: Graphs represent mean ± SEM. Results are representative of three (A, D, G) or four (E) independent experiments or pooled from three (B, C, H) or four (F) independent experiments. Statistical significance was tested using one-way (B) or two-way ANOVA (F, H) ***P < 0.001, n.s., not significant. Download figure Download PowerPoint We have shown recently that Tie-2 activation, induced by VE-PTP inhibition, leads to the activation of Rap1. Furthermore, Rap1 was needed for the junction-stabilizing effect of the VE-PTP inhibitor 9. Therefore, we tested whether Rap1, although not sufficient to trigger tyrosine phosphorylation of FGD5, would be involved in this process. To this end, we inhibited the expression of Rap1a/b in HUVEC by siRNA and re-analyzed the effects of AKB-9785 on junction recruitment and tyrosine phosphorylation of FGD5. As shown in Fig 2G, Rap1a/b expression was largely repressed. The consequence was a strong inhibition of AKB-9785-induced junction recruitment of FGD5 (Fig 2E and F), whereas the induction of tyrosine phosphorylation was unaffected (Fig 2G and H). We conclude that Rap1 is needed for the recruitment of FGD5 to junctions upon VE-PTP inhibition, but is not involved in the tyrosine phosphorylation of FGD5 downstream of VE-PTP. Stimulation of Tie-2 activates FGD5 Since VE-PTP inhibition stabilizes endothelial junctions by activating Tie-2 9, we tested whether Tie-2 would be relevant for the induction of tyrosine phosphorylation of FGD5. To this end, we transfected HUVEC with siRNA targeting Tie-2 and stimulated with AKB-9785 48 h later. Western blots of immunoprecipitated FGD5 revealed a 44% reduction of phospho-tyrosine levels upon Tie-2 siRNA treatment (Fig 3A and B). Thus, either Tie-2 itself or a kinase downstream of Tie-2 is responsible for the phosphorylation of FGD5. Since inhibition of Tie-2 expression was highly efficient, it is unlikely that the remaining phosphorylation of FGD5 was due to residual Tie-2. Therefore, it cannot be excluded that another kinase is also involved in the phosphorylation of FGD5. Figure 3. Tie-2 activation induces FGD5 phosphorylation, translocation, and activation of Cdc42 FGD5 was immunoprecipitated from HUVEC stimulated with vehicle or 5 μM AKB-9785 for 30 min after transfection with Tie-2-targeting or control siRNA. Precipitates were probed for phospho-tyrosines (4G10) and FGD5, and lysates for Tie-2 and α-tubulin. 4G10 signal intensities from three similar experiments as the one displayed in (A) were normalized to the amount of precipitated FGD5. HUVEC were starved in serum-free medium, treated with 1 μg/ml COMP-Ang1 (CA1) or 5 μM AKB-9785, followed by FGD5 immunoprecipitation and immunoblotting for phospho-tyrosines (4G10), FGD5, Tie-2, and α-tubulin. Confluent HUVEC monolayers were treated with 1 μg/ml COMP-Ang1 or vehicle for 20 min, and fixed, permeabilized cells were stained for FGD5, VE-cadherin, and F-actin. Scale bars 30 μm. Quantification of FGD5 signal intensities at cell contacts relative to FGD5 signal intensities in the cytoplasm of the same cell upon vehicle or Ang1 stimulation as in (D) with 20 cells analyzed. Serum-starved HUVEC were treated with 200 μM 007, 5 μM AKB-9785, 600 ng/ml Ang1, or vehicle for 20 min, and Cdc42 activation levels were compared using a colorimetric Cdc42 G-LISA activity assay. Data information: Results are representative of three independent experiments (A, C, D) or pooled from three (B, E) or five independent experiments (F). Statistical significance was tested using one-way ANOVA. *P < 0.05, ***P < 0.001. Results are displayed as mean ± SEM. Download figure Download PowerPoint Next, we tested whether direct stimulation of Tie-2 with the agonist Ang1, without inhibiting VE-PTP, would also stimulate FGD5. We found that the treatment of confluent HUVEC monolayers with recombinant COMP-Ang1 was sufficient to stimulate FGD5 tyrosine phosphorylation and translocation of FGD5 to cellular junctions (Fig 3C–E). Likewise, Ang1 was able to stimulate Cdc42 activation, as was analyzed by a colorimetric Cdc42 G-LISA assay (Fig 3F). Thus, FGD5 acts downstream of Tie-2 and could be involved in the junction-stabilizing effect of this receptor. FGD5 is required in vitro for the endothelial junction-stabilizing effect of VE-PTP inhibition To analyze the functional relevance of FGD5, we tested whether inhibition of FGD5 expression would interfere with the junction-stabilizing effect induced by VE-PTP inhibition or Tie-2 activation. To this end, we transfected HUVEC with FGD5 siRNA, seeded them on Transwell filters, and tested the effect of AKB-9785 on paracellular permeability induced by thrombin. Under control siRNA conditions, thrombin-induced permeability for FITC-dextran was prevented by AKB-9785 (Fig 4A). In contrast, upon depletion of FGD5, AKB-9785 could no longer significantly compensate the permeability-enhancing effect of thrombin (Fig 4A). FGD5 expression was inhibited by siRNA by 83% as determined by immunoblot analysis (Fig 4B). Since endothelial cells also express FGD1 and FGD6, we analyzed whether FGD5 siRNA or control siRNA would interfere with the mRNA levels of FGD1, FGD5, and FGD6. We found reduction of the mRNA level solely for FGD5 (Fig 4C). Figure 4. FGD5 is necessary to counteract thrombin-induced vascular leak induction by inhibition of VE-PTP Paracellular permeability for 250 kD FITC-dextran was determined for HUVEC transfected with FGD5-targeting or control siRNA. Cells were treated with vehicle, thrombin, or a combination of thrombin and 5 μM AKB-9785 (as indicated). Total cell lysates of HUVEC transfected with control or FGD5-targeting siRNA were immunoblotted for expression of FGD5 and α-tubulin. mRNA levels of FGD1, FGD5, and FGD6 in HUVEC transfected with FGD5 siRNA, represented as expression relative to control siRNA-treated cells. Impedance measurements of control or FGD5 siRNA-treated HUVEC at 4,000 Hz using the ECIS ΖΘ system. After pre-treatment with vehicle, 5 μM AKB-9785, or 1 μg/ml COMP-Ang1 (black arrowheads), cells were stimulated with thrombin (open arrowheads). Resistance values were normalized to the average resistance over 30 min prior to pre-treatment. Paracellular permeability for 250 kD FITC-dextran of control or FGD5 siRNA-treated HUVEC under basal conditions. Data information: Results are pooled from three (C), four (D, E), or five independent experiments with three filters per assay (A) or representative of five independent experiments (B). Statistical significance was tested using two-way ANOVA (A) or Student's t-test (E). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Results are shown as mean ± SEM. Download figure Download PowerPoint To analyze endothelial junction integrity by an alternative method, we applied the electric cell impedance sensing (ECIS) technique. Control or FGD5 siRNA-treated HUVEC were cultured on ECIS slides; treated for 30 min with COMP-Ang1, AKB-9785, or vehicle; and then exposed to thrombin. As shown in Fig 4D (direct resistance measurements in Fig EV2), COMP-Ang1 and AKB-9785 partially compensated the loss of electrical resistance caused by thrombin, whereas this compensating effect was lost when FGD5 expression was inhibited by siRNA. Thus, FGD5 is essential for the junction-stabilizing effect caused by VE-PTP inhibition or Tie-2 activation. Interestingly, FGD5 siRNA had no effect on basal permeability for FITC-dextran in Transwell filter assays (Fig 4E). Click here to expand this figure. Figure EV2. FGD5 is necessary to counteract the thrombin-induced drop in electrical resistanceImpedance measurements of control or FGD5 siRNA-treated HUVEC at 4,000 Hz using the ECIS ΖΘ system. After pre-treatment with vehicle, 5 μM AKB-9785, or 1 μg/ml COMP-Ang1 (black arrowheads), cells were stimulated with thrombin (open arrowheads). Download figure Download PowerPoint VE-PTP inhibition requires FGD5 to counteract histamine-induced vascular permeability in vivo We have shown previously that VE-PTP inhibition protects in vivo against histamine-induced vascular permeability in the skin and that this effect was mediated by the activation of Tie-2 9. To analyze whether FGD5 is required for this protection against the induction of vascular permeability, we used an in vivo siRNA approach for FGD5. Stabilized siRNA targeting FGD5 and control siRNA were intravenously injected in combination with a polyethyleneimine-based transfection reagent, and knockdown efficiency was determined 48 h later by immunoblot analysis of lung lysates. Administration of in vivo FGD5 siRNA efficiently inhibited FGD5 expression by 80% (±5%) as determined for 16 mice (Fig 5A and B). The expression of endothelial markers such as VE-cadherin, Tie-2, VE-PTP, and PECAM-1 was not compromised. This siRNA approach was combined with a Miles assay for the skin. Based on i.v. injected Evans blue and intradermal challenge with histamine, we found that permeability induction was clearly reduced by AKB-9785 in mice treated with control siRNA, whereas this protective effect of AKB-9785 was largely lost in mice with blocked expression of FGD5 (Fig 5C). Silencing efficiency is documented for some of these mice in Fig 5D. These results suggest that FGD5 is required for the vascular barrier-protective effect of VE-PTP in vivo. Figure 5. VE-PTP inhibition requires FGD5 to counteract histamine-induced vascular permeability in the skin Total lung lysates of C57BL/6 mice i.v. injected with control or FGD5-targeting siRNA for 48 h were immunoblotted for the indicated antigens. Percentages indicate the FGD5 protein levels relative to VE-cadherin levels, with the ratio of the FGD5 and VE-cadherin signal of the first lane arbitrarily set as 100. The average FGD5 knockdown efficiency in murine lungs was determined by quantification of immunoblot signals for FGD5 relative to VE-cadherin of 16 control or FGD5-targeting siRNA-treated animals each. Mice were treated with FGD5 or control siRNA as in (A). After 48 h, they were injected subcutaneously with 0.6 mg AKB-9785 or vehicle followed by intravenous injection of Evans Blue dye 30 min later and intradermal injections of PBS or histamine another 15 min later. After 30 min, mice were sacrificed, and the dye was extracted from skin samples and quantified by measuring absorbance at 620 nm. Total lung lysates of mice in (C) were immunoblotted for the indicated antigens. Data information: Data are representative of three independent experiments (A, D) or pooled from three independent experiments with 5–6 mice (B) or 3 mice per group (C). Results are displayed as mean ± SEM. Statistical significance was tested using Student's t-test (B) or two-way ANOVA (C). **P < 0.01, ***P < 0.001, n.s., not significant. Download figure Download PowerPoint FGD5 is required for the induction of cortical actin bundles and for the suppression of radial stress fibers Thrombin is known to interfere with endothelial junction integrity by stimulating the formation of radial stress fibers, which" @default.
• W2946341736 created "2019-05-29" @default.
• W2946341736 creator A5009450784 @default.
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• W2946341736 date "2019-05-14" @default.
• W2946341736 modified "2023-10-16" @default.
• W2946341736 title "<scp>VE</scp>‐<scp>PTP</scp>inhibition stabilizes endothelial junctions by activating<scp>FGD</scp>5" @default.
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