Artículos de revistas: "Phosphatase, PTPRG, Endothelial cells, permeability"

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Essler, Markus, Karin Hermann, Mutsuki Amano, Kozo Kaibuchi, Jürgen Heesemann, Peter C. Weber y Martin Aepfelbacher. "Pasteurella multocida Toxin Increases Endothelial Permeability via Rho Kinase and Myosin Light Chain Phosphatase". Journal of Immunology 161, n.º 10 (15 de noviembre de 1998): 5640–46. http://dx.doi.org/10.4049/jimmunol.161.10.5640.

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Abstract Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho. Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells. Stimulation of endothelial layers with PMT enhanced transendothelial permeability >10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation. PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase. Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase. The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.

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Vestweber, Dietmar. "Vascular Endothelial Protein Tyrosine Phosphatase Regulates Endothelial Function". Physiology 36, n.º 2 (1 de marzo de 2021): 84–93. http://dx.doi.org/10.1152/physiol.00026.2020.

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Vascular endothelial protein tyrosine phosphatase (VE-PTP) is a receptor-type PTP (RPTP), predominantly expressed in vascular endothelial cells. It regulates embryonic and tumor angiogenesis and controls vascular permeability and homeostasis in inflammation. Major substrates are the tyrosine kinase receptor Tie-2 and the adhesion molecule VE-cadherin. This review describes how VE-PTP controls vascular functions by its various substrates and the therapeutic potential of VE-PTP in various pathophysiological settings.

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Wachtel, M., K. Frei, E. Ehler, A. Fontana, K. Winterhalter y S. M. Gloor. "Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition". Journal of Cell Science 112, n.º 23 (1 de diciembre de 1999): 4347–56. http://dx.doi.org/10.1242/jcs.112.23.4347.

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Regulation of epithelial and endothelial permeability is essential for proper function of compartmentalized organisms, and tyrosine phosphorylation plays an important role in this process. We analyzed the impact of protein tyrosine phosphatase (PTP) inhibition on the structure of endothelial junctional proteins. In human umbilical vein endothelial cells (HUVECs) the PTP inhibitors phenylarsine oxide (PAO) and pervanadate induced proteolysis of the tight junction protein occludin. Occludin proteolysis was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline (PHEN), but not by inhibitors against other types of proteases. The junctional proteins ZO-1, cadherin and beta-catenin were not cleaved. Under conditions of occludin proteolysis, PAO treatment elevated permeability for FITC-dextran. Simultaneous incubation of HUVECs with PAO and PHEN inhibited the rise in permeability by more than 60%. PAO treatment lead to progressive disappearance of occludin from the cell periphery. In contrast, ZO-1, cadherin and beta-catenin retained their positions at the sites of intercellular contact. Simultaneous administration of PAO and PHEN greatly prevented the redistribution of occludin. These results demonstrate a selective cleavage of occludin by a metalloproteinase and suggest that this process can contribute to the control of paracellular permeability in endothelial cells.

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Kaestner, Charlotte L., Amin Sobh, Jianping Li, Alberto Riva, Richard Lynn Bennett y Jonathan D. Licht. "Functional CRISPR Screening Identifies Ptprg As a Driver of Migration and Adhesion in NSD2-E1099K ALL". Blood 138, Supplement 1 (5 de noviembre de 2021): 1149. http://dx.doi.org/10.1182/blood-2021-154009.

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Abstract Background: Acute Lymphoblastic Leukemia (ALL) is the most common childhood cancer and frequently infiltrates the central nervous system (CNS). CNS-directed therapy is currently limited to intrathecal and systemic high-dose methotrexate, or less commonly craniospinal irradiation, both of which are associated with substantial neurotoxicity. A lack of mechanistic understanding of the mechanisms of CNS infiltration presents an obstacle for the development of more specific and less toxic therapeutic approaches. We previously showed that ALL cells with a specific mutation (E1099K) in the histone methyltransferase NSD2 have aggressive CNS tropism by not only infiltrating the leptomeninges but also the brain parenchyma in murine xenografts models. Analysis of cBioPortal data shows that NSD2-E1099K is associated with a higher rate of testicular involvement in ALL also suggesting more aggressive infiltration behavior of the tumor. Accordingly, using gene editing to revert mutant NSD2 back to wild-type, we also showed that NSD2-E1099K cells have an enhanced ability to migrate and adhere in vitro. RNA-seq data on four NSD2-E1099K cell lines revealed genes that may play a role in ALL brain infiltration. However, it remains unknown which of those upregulated genes could be potential therapeutic targets against CNS leukemia. Aim: This study aims to Identify therapeutically targetable genes that are important for migration of NSD2-E1099K ALL cells Methods: Using a focused CRISPR-gene-knockout library of 5600 sgRNAs directed against 500 genes upregulated in NSD2-E1099K cells, we ascertained the necessity of the selected genes for migration in the RCH-ACV cell line. Candidate genes were evaluated for cellular dependency using a CRISPR-loss of function screen and the cancer dependency map portal. Overexpression of the candidate genes in NSD2-E1099K cell lines was confirmed with qPCR analysis. Candidate genes were validated by individual shRNA knockdown followed by migration and adhesion assays. Results: Our study identified genes whose knockout led to enhancement of migration and others whose knockout resulted in inhibition of migration. Protein Tyrosine Phosphatase Receptor Type G (PTPRG) was one of the top candidate genes whose knockout resulted in inhibition of migration. Dependency map analysis showed that PTPRG is not a commonly essential gene and a CRISPR-based-loss-of function screen performed in parallel to the migration screen confirmed that ALL cell survival is not dependent on PTPRG. We also found that PTPRG is overexpressed in multiple NSD2-E1099K ALL cell lines. Individual Knockdown of PTPRG in NSD2-E1099K ALL cell lines not only inhibited migration, but also led to a loss of adhesion ability to endothelial cells of the Blood Brain Barrier. Conclusions: Our findings implicate PTPRG as an important modulator of migration and adhesion in ALL cells and a potential therapeutic target for preventing ALL brain infiltration, especially in NSD2-E1099K ALL. Disclosures Licht: Epizyme: Research Funding.

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Kevil, Christopher G., Naotsuka Okayama y J. Steven Alexander. "H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity". American Journal of Physiology-Cell Physiology 281, n.º 6 (1 de diciembre de 2001): C1940—C1947. http://dx.doi.org/10.1152/ajpcell.2001.281.6.c1940.

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We previously reported that exposure of endothelial cells to H2O2results in a loss of cell-cell apposition and increased endothelial solute permeability. The purpose of this study was to determine how tyrosine phosphorylation and tyrosine phosphatases contribute to oxidant-mediated disorganization of endothelial cell junctions. We found that H2O2caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues. H2O2exposure also results in increased endothelial monolayer permeability, which was attenuated by pp60, an inhibitor of src kinase. Inhibition of protein tyrosine phosphatase activity by phenylarsine oxide (PAO) demonstrated a similar permeability profile compared with H2O2, suggesting that tyrosine phosphatase activity is important in maintaining a normal endothelial solute barrier. Immunofluorescence shows that H2O2exposure caused a loss of pan-reactive cadherin and β-catenin from cell junctions that was not blocked by the src kinase inhibitor PP1. H2O2also caused β-catenin to dissociate from the endothelial cytoskeleton, which was not prevented by PP1. Finally, we determined that PP1 did not prevent cadherin internalization. These data suggest that oxidants like H2O2produce biological effects through protein phosphotyrosine modifications by decreasing total cellular phosphatase activity combined with increased src kinase activity, resulting in increased endothelial solute permeability.

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Gloor, Sergio M., Adrien Weber, Naoto Adachi y Karl Frei. "Interleukin-1 Modulates Protein Tyrosine Phosphatase Activity and Permeability of Brain Endothelial Cells". Biochemical and Biophysical Research Communications 239, n.º 3 (octubre de 1997): 804–9. http://dx.doi.org/10.1006/bbrc.1997.7557.

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Kelly, J. J., T. M. Moore, P. Babal, A. H. Diwan, T. Stevens y W. J. Thompson. "Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+and permeability". American Journal of Physiology-Lung Cellular and Molecular Physiology 274, n.º 5 (1 de mayo de 1998): L810—L819. http://dx.doi.org/10.1152/ajplung.1998.274.5.l810.

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Cytosolic Ca2+concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 μM) produced higher [Ca2+]ilevels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]iresponse in RPMVECs, we investigated whether reduced activation of store-operated Ca2+entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+followed by readdition media with 2 mM extracellular Ca2+increased RPMVEC [Ca2+]ito a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]iin RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 μM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]ipromotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]isignaling pathways or dominant Ca2+-independent mechanisms from controlling cellular gap formation and permeability.

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Kim, Soo Hyeon, Young-Rak Cho, Hyeon-Ju Kim, Joa Sub Oh, Eun-Kyung Ahn, Hye-Jin Ko, Byung Joon Hwang et al. "Antagonism of VEGF-A–induced increase in vascular permeability by an integrin α3β1-Shp-1-cAMP/PKA pathway". Blood 120, n.º 24 (6 de diciembre de 2012): 4892–902. http://dx.doi.org/10.1182/blood-2012-05-428243.

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Abstract In cancer, VEGF-induced increase in vascular permeability results in increased interstitial pressure, reducing perfusion and increasing hypoxia, which reduce delivery of chemotherapeutic agents and increase resistance to ionizing radiation. Here, we show that both TIMP-2 and Ala + TIMP-2, a TIMP-2 mutant without matrix metalloproteinase inhibitory activity, antagonize the VEGF-A–induced increase in vascular permeability, both in vitro and in vivo. Like other agents known to preserve endothelial barrier function, TIMP-2 elevates cytosolic levels of cAMP and increases cytoskeletal-associated vascular endothelial cadherin in human microvascular endothelial cells. All of these effects are completely ablated by selective knockdown of integrin α3β1 expression, expression of a dominant negative protein tyrosine phosphatase Shp-1 mutant, administration of the protein tyrosine phosphatase inhibitor orthovanadate, or the adenylate cyclase inhibitor SQ22536. This TIMP-2–mediated inhibition of vascular permeability involves an integrin α3β1-Shp-1-cAMP/protein kinase A-dependent vascular endothelial cadherin cytoskeletal association, as evidenced by using siRNAs to integrin α3β1 and Shp-1, or treatment with Shp-1 inhibitor NSC87877 and protein kinase A inhibitor H89. Our results demonstrate the potential utility for TIMP-2 in cancer therapy through “normalization” of vascular permeability in addition to previously described antiangiogenic effects.

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Staddon, J. M., K. Herrenknecht, C. Smales y L. L. Rubin. "Evidence that tyrosine phosphorylation may increase tight junction permeability". Journal of Cell Science 108, n.º 2 (1 de febrero de 1995): 609–19. http://dx.doi.org/10.1242/jcs.108.2.609.

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Tight junction permeability control is important in a variety of physiological and pathological processes. We have investigated the role of tyrosine phosphorylation in the regulation of tight junction permeability. MDCK epithelial cells and brain endothelial cells were grown on filters and tight junction permeability was determined by transcellular electrical resistance (TER). The tyrosine phosphatase inhibitor pervanadate caused a concentration- and time-dependent decrease in TER in both MDCK and brain endothelial cells. However, as expected, pervanadate resulted in the tyrosine phosphorylation of many proteins; hence interpretation of its effects are extremely difficult. Phenylarsine oxide, a more selective tyrosine phosphatase inhibitor, caused the tyrosine phosphorylation of relatively few proteins as analyzed by immunoblotting of whole cell lysates. This inhibitor, like pervanadate, also elicited a decrease in TER in the two cell types. In the MDCK cells, the action of phenylarsine oxide could be reversed by the subsequent addition of the reducing agent 2,3-dimercaptopropanol. Immunocytochemistry revealed that phenylarsine oxide rapidly stimulated the tyrosine phosphorylation of proteins associated with intercellular junctions. Because of the known influence of the adherens junction on tight junctions, we analyzed immunoprecipitates of the E-cadherin/catenin complex from MDCK cells treated with phenylarsine oxide. This revealed an increase in the tyrosine phosphorylation of beta-catenin, but not of alpha-catenin. However, the tight junction associated protein ZO-1 was also tyrosine phosphorylated after PAO treatment. These data indicate that tight junction permeability may be regulated via mechanisms involving tyrosine phosphorylation of adherens junction and tight junction proteins.

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Juettner, Vanessa V., Kevin Kruse, Arkaprava Dan, Vinh H. Vu, Yousaf Khan, Jonathan Le, Deborah Leckband, Yulia Komarova y Asrar B. Malik. "VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism". Journal of Cell Biology 218, n.º 5 (4 de abril de 2019): 1725–42. http://dx.doi.org/10.1083/jcb.201807210.

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Vascular endothelial (VE) protein tyrosine phosphatase (PTP) is an endothelial-specific phosphatase that stabilizes VE-cadherin junctions. Although studies have focused on the role of VE-PTP in dephosphorylating VE-cadherin in the activated endothelium, little is known of VE-PTP’s role in the quiescent endothelial monolayer. Here, we used the photoconvertible fluorescent protein VE-cadherin-Dendra2 to monitor VE-cadherin dynamics at adherens junctions (AJs) in confluent endothelial monolayers. We discovered that VE-PTP stabilizes VE-cadherin junctions by reducing the rate of VE-cadherin internalization independently of its phosphatase activity. VE-PTP serves as an adaptor protein that through binding and inhibiting the RhoGEF GEF-H1 modulates RhoA activity and tension across VE-cadherin junctions. Overexpression of the VE-PTP cytosolic domain mutant interacting with GEF-H1 in VE-PTP–depleted endothelial cells reduced GEF-H1 activity and restored VE-cadherin dynamics at AJs. Thus, VE-PTP stabilizes VE-cadherin junctions and restricts endothelial permeability by inhibiting GEF-H1, thereby limiting RhoA signaling at AJs and reducing the VE-cadherin internalization rate.

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Yuan, Yuan, F. Y. Meng, Q. Huang, James Hawker y H. Mac Wu. "Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function". American Journal of Physiology-Heart and Circulatory Physiology 275, n.º 1 (1 de julio de 1998): H84—H93. http://dx.doi.org/10.1152/ajpheart.1998.275.1.h84.

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The transendothelial movement of solutes is a dynamic process controlled by a complex interaction between the cytoskeleton and adhesion proteins. The aim of this study was to examine whether protein tyrosine phosphorylation is involved in the regulation of endothelial barrier function. The apparent permeability coefficient of albumin ( P a) was measured in isolated and perfused coronary venules. Tyrosine phosphatase inhibitors, including phenylarsine oxide and sodium orthovanadate, dose and time dependently increased basal P a. Western blot analysis of cultured coronary venular endothelial cells revealed that inhibition of tyrosine phosphatase induced an increase in phosphotyrosine content in a number of proteins, including bands at 65–70 and 120–130 kDa, which were identified as paxillin and focal adhesion kinase (pp125FAK), respectively. The time course and dose responsiveness of protein tyrosine phosphorylation were tightly correlated with those of increases in P a. Furthermore, stimulation of endothelial cells with histamine or phorbol myristate acetate (PMA) enhanced tyrosine phosphorylation of paxillin and pp125FAK, which was blocked by the tyrosine kinase inhibitor damnacanthal. Correspondingly, the increases in venular permeability elicited by histamine and PMA were abolished in damnacanthal-treated venules. Taken together, the data suggest a possible involvement of protein tyrosine phosphorylation in the control of endothelial barrier function. Paxillin and its associated focal adhesion proteins may play a specific role in agonist-induced hyperpermeability responses in the endothelium of exchange vessels.

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Bindewald, K., D. Gündüz, F. Härtel, S. C. Peters, C. Rodewald, S. Nau, M. Schäfer, J. Neumann, H. M. Piper y T. Noll. "Opposite effect of cAMP signaling in endothelial barriers of different origin". American Journal of Physiology-Cell Physiology 287, n.º 5 (noviembre de 2004): C1246—C1255. http://dx.doi.org/10.1152/ajpcell.00132.2004.

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cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5′- N-(ethylcarboxamido)-adenosine (NECA), the β-adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures.

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Soni, Dheeraj, Sushil C. Regmi, Dong-Mei Wang, Auditi DebRoy, You-Yang Zhao, Stephen M. Vogel, Asrar B. Malik y Chinnaswamy Tiruppathi. "Pyk2 phosphorylation of VE-PTP downstream of STIM1-induced Ca2+ entry regulates disassembly of adherens junctions". American Journal of Physiology-Lung Cellular and Molecular Physiology 312, n.º 6 (1 de junio de 2017): L1003—L1017. http://dx.doi.org/10.1152/ajplung.00008.2017.

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Vascular endothelial protein tyrosine phosphatase (VE-PTP) stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of stromal interaction molecule 1 (STIM1) activation of store-operated Ca2+ entry (SOCE) in regulating AJ assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted Stim1 knockout mice, indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated permeability response. Thus Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP, in turn, binds to and activates Src, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca2+ signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.

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Grinnell, K. L., B. Casserly y E. O. Harrington. "Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium". American Journal of Physiology-Lung Cellular and Molecular Physiology 298, n.º 3 (marzo de 2010): L361—L370. http://dx.doi.org/10.1152/ajplung.00374.2009.

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Pulmonary edema is mediated in part by disruption of interendothelial cell contacts. Protein tyrosine phosphatases (PTP) have been shown to affect both cell-extracellular matrix and cell-cell junctions. The SH2 domain-containing nonreceptor PTP, SHP2, is involved in intercellular signaling through direct interaction with adherens junction proteins. In this study, we examined the role of SHP2 in pulmonary endothelial barrier function. Inhibition of SHP2 promoted edema formation in rat lungs and increased monolayer permeability in cultured lung endothelial cells. In addition, pulmonary endothelial cells demonstrated a decreased level of p190RhoGAP activity following inhibition of SHP2, events that were accompanied by a concomitant increase in RhoA activity. Furthermore, immunofluorescence microscopy confirmed enhanced actin stress fiber formation and diminished interendothelial staining of adherens junction complex-associated proteins upon SHP2 inhibition. Finally, immunoprecipitation and immunoblot analyses demonstrated increased tyrosine phosphorylation of VE-cadherin, β-catenin, and p190RhoGAP proteins, as well as decreased association between p120-catenin and VE-cadherin proteins. Our findings suggest that SHP2 supports basal pulmonary endothelial barrier function by coordinating the tyrosine phosphorylation profile of VE-cadherin, β-catenin, and p190RhoGAP and the activity of RhoA, signaling molecules important in adherens junction complex integrity.

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Timmerman, Ilse, Mark Hoogenboezem, Anton M. Bennett, Dirk Geerts, Peter L. Hordijk y Jaap D. van Buul. "The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation". Molecular Biology of the Cell 23, n.º 21 (noviembre de 2012): 4212–25. http://dx.doi.org/10.1091/mbc.e12-01-0038.

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Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated β-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin–mediated cell–cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin–controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate β-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin–associated β-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell–cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell–cell junctions by dephosphorylating VE-cadherin–associated β-catenin and promoting the mobility of VE-cadherin at the plasma membrane.

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Fu, Panfeng, Ramaswamy Ramchandran, Mark Shaaya, Longshuang Huang, David L. Ebenezer, Ying Jiang, Yulia Komarova et al. "Phospholipase D2 restores endothelial barrier function by promoting PTPN14-mediated VE-cadherin dephosphorylation". Journal of Biological Chemistry 295, n.º 22 (23 de abril de 2020): 7669–85. http://dx.doi.org/10.1074/jbc.ra119.011801.

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Increased permeability of vascular lung tissues is a hallmark of acute lung injury and is often caused by edemagenic insults resulting in inflammation. Vascular endothelial (VE)-cadherin undergoes internalization in response to inflammatory stimuli and is recycled at cell adhesion junctions during endothelial barrier re-establishment. Here, we hypothesized that phospholipase D (PLD)-generated phosphatidic acid (PA) signaling regulates VE-cadherin recycling and promotes endothelial barrier recovery by dephosphorylating VE-cadherin. Genetic deletion of PLD2 impaired recovery from protease-activated receptor-1–activating peptide (PAR-1–AP)-induced lung vascular permeability and potentiated inflammation in vivo. In human lung microvascular endothelial cells (HLMVECs), inhibition or deletion of PLD2, but not of PLD1, delayed endothelial barrier recovery after thrombin stimulation. Thrombin stimulation of HLMVECs increased co-localization of PLD2-generated PA and VE-cadherin at cell-cell adhesion junctions. Inhibition of PLD2 activity resulted in prolonged phosphorylation of Tyr-658 in VE-cadherin during the recovery phase 3 h post-thrombin challenge. Immunoprecipitation experiments revealed that after HLMVECs are thrombin stimulated, PLD2, VE-cadherin, and protein-tyrosine phosphatase nonreceptor type 14 (PTPN14), a PLD2-dependent protein-tyrosine phosphatase, strongly associate with each other. PTPN14 depletion delayed VE-cadherin dephosphorylation, reannealing of adherens junctions, and barrier function recovery. PLD2 inhibition attenuated PTPN14 activity and reversed PTPN14-dependent VE-cadherin dephosphorylation after thrombin stimulation. Our findings indicate that PLD2 promotes PTPN14-mediated dephosphorylation of VE-cadherin and that redistribution of VE-cadherin at adherens junctions is essential for recovery of endothelial barrier function after an edemagenic insult.

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Panchatcharam, Manikandan, Abdel K. Salous, Jason Brandon, Sumitra Miriyala, Jessica Wheeler, Pooja Patil, Manjula Sunkara, Andrew J. Morris, Diana Escalante-Alcalde y Susan S. Smyth. "Mice With Targeted Inactivation of Ppap2b in Endothelial and Hematopoietic Cells Display Enhanced Vascular Inflammation and Permeability". Arteriosclerosis, Thrombosis, and Vascular Biology 34, n.º 4 (abril de 2014): 837–45. http://dx.doi.org/10.1161/atvbaha.113.302335.

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Objective— Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein–coupled receptor–mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate. LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in the development and human disease. Approach and Results— We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek promoter–mediated inactivation of Ppap2b resulted in embryonic lethality because of vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tyrosine kinase Tek promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3 deficiency led to significant increases in vascular permeability at baseline and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein–coupled receptor–dependent LPA signaling. Conclusions— Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.

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Broermann, Andre, Mark Winderlich, Helena Block, Maike Frye, Jan Rossaint, Alexander Zarbock, Giuseppe Cagna et al. "Dissociation of VE-PTP from VE-cadherin is required for leukocyte extravasation and for VEGF-induced vascular permeability in vivo". Journal of Experimental Medicine 208, n.º 12 (24 de octubre de 2011): 2393–401. http://dx.doi.org/10.1084/jem.20110525.

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We have recently shown that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial membrane protein, associates with VE-cadherin and is required for optimal VE-cadherin function and endothelial cell contact integrity. The dissociation of VE-PTP from VE-cadherin is triggered by vascular endothelial growth factor (VEGF) and by the binding of leukocytes to endothelial cells in vitro, suggesting that this dissociation is a prerequisite for the destabilization of endothelial cell contacts. Here, we show that VE-cadherin/VE-PTP dissociation also occurs in vivo in response to LPS stimulation of the lung or systemic VEGF stimulation. To show that this dissociation is indeed necessary in vivo for leukocyte extravasation and VEGF-induced vascular permeability, we generated knock-in mice expressing the fusion proteins VE-cadherin-FK 506 binding protein and VE-PTP-FRB* under the control of the endogenous VE-cadherin promoter, thus replacing endogenous VE-cadherin. The additional domains in both fusion proteins allow the heterodimeric complex to be stabilized by a chemical compound (rapalog). We found that intravenous application of the rapalog strongly inhibited VEGF-induced (skin) and LPS-induced (lung) vascular permeability and inhibited neutrophil extravasation in the IL-1β inflamed cremaster and the LPS-inflamed lung. We conclude that the dissociation of VE-PTP from VE-cadherin is indeed required in vivo for the opening of endothelial cell contacts during induction of vascular permeability and leukocyte extravasation.

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Spring, Kathleen, Catherine Chabot, Simon Langlois, Line Lapointe, Nguyen Thu Ngan Trinh, Christine Caron, Jagoda K. Hebda, Julie Gavard, Mounib Elchebly y Isabelle Royal. "Tyrosine phosphorylation of DEP-1/CD148 as a mechanism controlling Src kinase activation, endothelial cell permeability, invasion, and capillary formation". Blood 120, n.º 13 (27 de septiembre de 2012): 2745–56. http://dx.doi.org/10.1182/blood-2011-12-398040.

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Abstract DEP-1/CD148 is a receptor-like protein tyrosine phosphatase with antiproliferative and tumor-suppressive functions. Interestingly, it also positively regulates Src family kinases in hematopoietic and endothelial cells, where we showed it promotes VE-cadherin–associated Src activation and endothelial cell survival upon VEGF stimulation. However, the molecular mechanism involved and its biologic functions in endothelial cells remain ill-defined. We demonstrate here that DEP-1 is phosphorylated in a Src- and Fyn-dependent manner on Y1311 and Y1320, which bind the Src SH2 domain. This allows DEP-1–catalyzed dephosphorylation of Src inhibitory Y529 and favors the VEGF-induced phosphorylation of Src substrates VE-cadherin and Cortactin. Accordingly, RNA interference (RNAi)–mediated knockdown of DEP-1 or expression of DEP-1 Y1311F/Y1320F impairs Src-dependent biologic responses mediated by VEGF including permeability, invasion, and branching capillary formation. In addition, our work further reveals that above a threshold expression level, DEP-1 can also dephosphorylate Src Y418 and attenuate downstream signaling and biologic responses, consistent with the quiescent behavior of confluent endothelial cells that express the highest levels of endogenous DEP-1. Collectively, our findings identify the VEGF-dependent phosphorylation of DEP-1 as a novel mechanism controlling Src activation, and show this is essential for the proper regulation of permeability and the promotion of the angiogenic response.

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Ramos-Perez, Willy, Diana Escalante-Alcalde, Kevin Lynch y Susan Schwab. "The role of lipid phosphate phosphatase 1 in lymphocyte egress from lymphoid organs (173.39)". Journal of Immunology 188, n.º 1_Supplement (1 de mayo de 2012): 173.39. http://dx.doi.org/10.4049/jimmunol.188.supp.173.39.

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Abstract The signaling lipid sphingosine 1-phosphate (S1P) regulates vascular permeability, lymphocyte trafficking, and inflammation. The concentration of S1P is high in blood and lymph compared to lymphoid organs, and this difference guides lymphocyte exit from lymphoid organs into circulation. However, little is known about how S1P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. Lipid phosphate phosphatase 3 (LPP3) is essential to maintain low S1P in the thymus, and we asked whether its homolog LPP1 cooperates with LPP3 to regulate lymphoid S1P. Measurements of Lpp1 messenger RNA indicate that, like Lpp3, Lpp1 is highly expressed by thymic endothelial and epithelial cells, and lymph node (LN) lymphatic endothelial, blood endothelial, and fibroblastic reticular cells. Using mice deficient in LPP1, we have found that LPP1 is not required to maintain low S1P in thymus or LN or to enable efficient lymphocyte egress. Mice doubly deficient in LPP1 and LPP3 preliminarily have a small accumulation of mature thymocytes and minor reduction in the number of T cells in lymph compared to mice deficient in LPP3 alone, suggestive of an egress defect. Further work will test the hypothesis that LPP1 and LPP3 work together to maintain low lymphoid organ S1P and efficient lymphocyte circulation.

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21

Kolosova, Irina A., Shwu-Fan Ma, Djanybek M. Adyshev, Peyi Wang, Motoi Ohba, Viswanathan Natarajan, Joe G. N. Garcia y Alexander D. Verin. "Role of CPI-17 in the regulation of endothelial cytoskeleton". American Journal of Physiology-Lung Cellular and Molecular Physiology 287, n.º 5 (noviembre de 2004): L970—L980. http://dx.doi.org/10.1152/ajplung.00398.2003.

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We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99–L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-α abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-α isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.

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22

Ushakumari, Chinchu Jagadan, Qiong L. Zhou, Yu-Hua Wang, Sijia Na, Michael C. Rigor, Cindy Y. Zhou, Max K. Kroll, Benjamin D. Lin y Zhen Y. Jiang. "Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice". Cells 11, n.º 15 (25 de julio de 2022): 2288. http://dx.doi.org/10.3390/cells11152288.

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Neutrophil elastase (NE) plays a pivotal role in inflammation. However, the mechanism underlying NE-mediated inflammation in obesity remains unclear. Here, we report that NE activates protease-activated receptor-2 (PAR2), stimulates actin filament (F-actin) formation, decreases intercellular junction molecule VE-cadherin expression, and increases the permeability of human arterial endothelial cells (hECs). NE also prompts degradation of VE-cadherin and its binding proteins p120- and β-catenins via MG132-sensitive proteasomes. NE stimulates phosphorylation of myosin light-chain (MLC) and its regulator myosin phosphatase target subunit-1 (MYPT1), a target of Rho kinase (ROCK). Inhibitors of PAR2 and ROCK prohibit NE-induced F-actin formation, MLC phosphorylation, and VE-cadherin reduction in hECs, and impede monocyte transmigration through hEC monolayer pretreated with either neutrophils or NE. Further, administration of an NE inhibitor GW311616A significantly attenuates vascular leakage, leukocyte infiltration, and the expression of proinflammatory cytokines in the white adipose tissue from high-fat diet (HFD)-induced obese mice. Likewise, NE-deficient mice are resistant to HFD-induced vascular leakage in the heart. Together, NE regulates actomyosin cytoskeleton activity and VE-cadherin expression by activating PAR2 signaling in the endothelial cells, leading to increased vascular permeability and leukocyte extravasation. Hence, inhibition of NE is a potential approach to mitigate vascular injury and leukocyte infiltration in obesity-related systemic inflammation.

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23

Nottebaum, Astrid F., Giuseppe Cagna, Mark Winderlich, Alexander C. Gamp, Ruth Linnepe, Christian Polaschegg, Kristina Filippova et al. "VE-PTP maintains the endothelial barrier via plakoglobin and becomes dissociated from VE-cadherin by leukocytes and by VEGF". Journal of Experimental Medicine 205, n.º 12 (17 de noviembre de 2008): 2929–45. http://dx.doi.org/10.1084/jem.20080406.

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We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)–cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885–4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin–mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor α–activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, β-catenin, and plakoglobin. Surprisingly, only plakoglobin but not β-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not β-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not β-catenin.

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24

Csortos, Csilla, Istvan Czikora, Natalia V. Bogatcheva, Djanybek M. Adyshev, Christophe Poirier, Gabor Olah y Alexander D. Verin. "TIMAP is a positive regulator of pulmonary endothelial barrier function". American Journal of Physiology-Lung Cellular and Molecular Physiology 295, n.º 3 (septiembre de 2008): L440—L450. http://dx.doi.org/10.1152/ajplung.00325.2007.

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TGF-β-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the β-isoform of the catalytic subunit of PP1 (PP1cβ) from pulmonary artery EC. As PP1cβ, but not PP1cα, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

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Liu, Tiegang, Oscar E. Guevara, Rod R. Warburton, Nicholas S. Hill, Matthias Gaestel y Usamah S. Kayyali. "Regulation of vimentin intermediate filaments in endothelial cells by hypoxia". American Journal of Physiology-Cell Physiology 299, n.º 2 (agosto de 2010): C363—C373. http://dx.doi.org/10.1152/ajpcell.00057.2010.

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Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood. We have previously shown that hypoxia-activated signaling in endothelial cells alters their contractility and adhesiveness. We have also linked p38-MAP kinase signaling pathway leading to HSP27 phosphorylation and increased actin stress fiber formation to endothelial barrier augmentation. We now show that vimentin, a major intermediate filament protein in endothelial cells, is regulated by hypoxia. Our results indicate that exposure of endothelial cells to hypoxia causes vimentin filament networks to initially redistribute perinuclearly. However, by 1 hour hypoxia these networks reform and appear more continuous across cells than under normoxia. Hypoxia also causes transient changes in vimentin phosphorylation, and activation of PAK1, a kinase that regulates vimentin filament assembly. In addition, exposure to 1 hour hypoxia increases the ratio of insoluble/soluble vimentin. Overexpression of phosphomimicking mutant HSP27 (pmHSP27) causes changes in vimentin distribution that are similar to those observed in hypoxic cells. Knocking-down HSP27 destroys the vimentin filamentous network, and disrupting vimentin filaments with acrylamide increases endothelial permeability. Both hypoxia- and pmHSP27 overexpression-induced changes are reversed by inhibition of phosphatase activity. In conclusion hypoxia causes redistribution of vimentin to a more insoluble and extensive filamentous network that could play a role in endothelial barrier stabilization. Vimentin redistribution appears to be mediated through altering the phosphorylation of the protein and its interaction with HSP27.

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Su, Kai, Jianguo Wang, Yang Lv, Ming Tian, You-Yang Zhao, Richard D. Minshall y Guochang Hu. "YAP expression in endothelial cells prevents ventilator-induced lung injury". American Journal of Physiology-Lung Cellular and Molecular Physiology 320, n.º 4 (1 de abril de 2021): L568—L582. http://dx.doi.org/10.1152/ajplung.00472.2020.

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Ventilator-induced lung injury is associated with an increase in mortality in patients with respiratory dysfunction, although mechanical ventilation is an essential intervention implemented in the intensive care unit. Intrinsic molecular mechanisms for minimizing lung inflammatory injury during mechanical ventilation remain poorly defined. We hypothesize that Yes-associated protein (YAP) expression in endothelial cells protects the lung against ventilator-induced injury. Wild-type and endothelial-specific YAP-deficient mice were subjected to a low (7 mL/kg) or high (21 mL/kg) tidal volume ( VT) ventilation for 4 h. Infiltration of inflammatory cells into the lung, vascular permeability, lung histopathology, and the levels of inflammatory cytokines were measured. Here, we showed that mechanical ventilation with high VT upregulated YAP protein expression in pulmonary endothelial cells. Endothelial-specific YAP knockout mice following high VT ventilation exhibited increased neutrophil counts and protein content in bronchoalveolar lavage fluid, Evans blue leakage, and histological lung injury compared with wild-type littermate controls. Deletion of YAP in endothelial cells exaggerated vascular endothelial (VE)-cadherin phosphorylation, downregulation of vascular endothelial protein tyrosine phosphatase (VE-PTP), and dissociation of VE-cadherin and catenins following mechanical ventilation. Importantly, exogenous expression of wild-type VE-PTP in the pulmonary vasculature rescued YAP ablation-induced increases in neutrophil counts and protein content in bronchoalveolar lavage fluid, vascular leakage, and histological lung injury as well as VE-cadherin phosphorylation and dissociation from catenins following ventilation. These data demonstrate that YAP expression in endothelial cells suppresses lung inflammatory response and edema formation by modulating VE-PTP-mediated VE-cadherin phosphorylation and thus plays a protective role in ventilator-induced lung injury.

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27

Stephan, Delphine, Anais Roger, Jehanne Aghzadi, Sylvie Carmona, Christophe Picard, Jean-Philippe Dales y Sophie Desplat-Jégo. "TWEAK and TNFα, Both TNF Ligand Family Members and Multiple Sclerosis-Related Cytokines, Induce Distinct Gene Response in Human Brain Microvascular Endothelial Cells". Genes 13, n.º 10 (24 de septiembre de 2022): 1714. http://dx.doi.org/10.3390/genes13101714.

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Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family involved in various diseases including brain inflammatory pathologies such as multiple sclerosis. It has been demonstrated that TWEAK can induce cerebrovascular permeability in an in vitro model of the blood–brain barrier. The molecular mechanisms playing a role in TWEAK versus TNFα signaling on cerebral microvascular endothelial cells are not well defined. Therefore, we aimed to identify gene expression changes in cultures of human brain microvascular endothelial cells (hCMEC/D3) to address changes initiated by TWEAK exposure. Taken together, our studies highlighted that gene involved in leukocyte extravasation, notably claudin-5, were differentially modulated by TWEAK and TNFα. We identified differential gene expression of hCMEC/D3 cells at three timepoints following TWEAK versus TNFα stimulation and also found distinct modulations of several canonical pathways including the actin cytoskeleton, vascular endothelial growth factor (VEGF), Rho family GTPases, and phosphatase and tensin homolog (PTEN) pathways. To our knowledge, this is the first study to interrogate and compare the effects of TWEAK versus TNFα on gene expression in brain microvascular endothelial cells.

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Liu, Hui, Kavi Devraj, Kerstin Möller, Stefan Liebner, Markus Hecker y Thomas Korff. "EphrinB-mediated reverse signalling controls junctional integrity and pro-inflammatory differentiation of endothelial cells". Thrombosis and Haemostasis 112, n.º 07 (2014): 151–63. http://dx.doi.org/10.1160/th13-12-1034.

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SummaryThe EphB/ephrinB receptor-ligand system is pivotal for the development of the embryonic vasculature and for angiogenesis in the adult organism. We observed that (i) the expression of ephrinB2 and ephrinB1 is up-regulated in capillaries during inflammation, that (ii) these ligands are localised on the luminal endothelial surface, and that (iii) they interact with the ephrinB-receptor EphB2 on monocyte/macrophages. This study delineates the impact of ephrinB-mediated reverse signalling on the integrity and proinflammatory differentiation of the endothelium. To this end, in vitro analyses with human cultured endothelial cells reveal that knockdown of ephrinB2 or ephrinB1 impairs monocyte transmigration through the endothelium. While ephrinB2 but not ephrinB1 interacts with PECAM-1 (CD31) in this context, reverse signalling by ephrinB1 but not ephrinB2 elicits a c-Jun N-terminal kinase (JNK)-dependent up-regulation of E-selectin expression. Furthermore, treatment of endothelial cells with soluble EphB2 receptor bodies or EphB2-overexpressing mouse myeloma cells links ephrinB2 to PECAM-1 and induces its Src-dependent phosphorylation while diminishing Src homology phosphotyrosyl phosphatase-2 (SHP-2) activity and increasing endothelial cell permeability. We conclude that extravasation of EphB2 positive leukocyte populations is facilitated by lowering the integrity of endothelial cell junctions and enhancing the pro-inflammatory phenotype of the endothelium through activation of ephrinB ligands.

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Eklund, Lauri, Jaakko Kangas y Pipsa Saharinen. "Angiopoietin–Tie signalling in the cardiovascular and lymphatic systems". Clinical Science 131, n.º 1 (9 de diciembre de 2016): 87–103. http://dx.doi.org/10.1042/cs20160129.

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Endothelial cells that form the inner layer of blood and lymphatic vessels are important regulators of vascular functions and centrally involved in the pathogenesis of vascular diseases. In addition to the vascular endothelial growth factor (VEGF) receptor pathway, the angiopoietin (Ang)–Tie system is a second endothelial cell specific ligand–receptor signalling system necessary for embryonic cardiovascular and lymphatic development. The Ang–Tie system also regulates postnatal angiogenesis, vessel remodelling, vascular permeability and inflammation to maintain vascular homoeostasis in adult physiology. This system is implicated in numerous diseases where the vasculature has an important contribution, such as cancer, sepsis, diabetes, atherosclerosis and ocular diseases. Furthermore, mutations in the TIE2 signalling pathway cause defects in vascular morphogenesis, resulting in venous malformations and primary congenital glaucoma. Here, we review recent advances in the understanding of the Ang–Tie signalling system, including cross-talk with the vascular endothelial protein tyrosine phosphatase (VE-PTP) and the integrin cell adhesion receptors, focusing on the Ang–Tie system in vascular development and pathogenesis of vascular diseases.

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Napione, Lucia, Simona Pavan, Andrea Veglio, Andrea Picco, Guido Boffetta, Antonio Celani, Giorgio Seano, Luca Primo, Andrea Gamba y Federico Bussolino. "Unraveling the influence of endothelial cell density on VEGF-A signaling". Blood 119, n.º 23 (7 de junio de 2012): 5599–607. http://dx.doi.org/10.1182/blood-2011-11-390666.

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Abstract Vascular endothelial growth factor-A (VEGF) is the master determinant for the activation of the angiogenic program leading to the formation of new blood vessels to sustain solid tumor growth and metastasis. VEGF specific binding to VEGF receptor-2 (VEGFR-2) triggers different signaling pathways, including phospholipase C-γ (PLC-γ) and Akt cascades, crucial for endothelial proliferation, permeability, and survival. By combining biologic experiments, theoretical insights, and mathematical modeling, we found that: (1) cell density influences VEGFR-2 protein level, as receptor number is 2-fold higher in long-confluent than in sparse cells; (2) cell density affects VEGFR-2 activation by reducing its affinity for VEGF in long-confluent cells; (3) despite reduced ligand-receptor affinity, high VEGF concentrations provide long-confluent cells with a larger amount of active receptors; (4) PLC-γ and Akt are not directly sensitive to cell density but simply transduce downstream the upstream difference in VEGFR-2 protein level and activation; and (5) the mathematical model correctly predicts the existence of at least one protein tyrosine phosphatase directly targeting PLC-γ and counteracting the receptor-mediated signal. Our data-based mathematical model quantitatively describes VEGF signaling in quiescent and angiogenic endothelium and is suitable to identify new molecular determinants and therapeutic targets.

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Yin, Jun, Lu Lv, Peng Zhai, Tao Long, Qiang Zhou, Huiwen Pan, Godwin Botwe et al. "Connexin 40 regulates lung endothelial permeability in acute lung injury via the ROCK1-MYPT1- MLC20 pathway". American Journal of Physiology-Lung Cellular and Molecular Physiology 316, n.º 1 (1 de enero de 2019): L35—L44. http://dx.doi.org/10.1152/ajplung.00012.2018.

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Increased pulmonary vascular permeability is a hallmark of acute lung injury (ALI). Connexin 40 (Cx40) is a gap junctional protein abundantly present in the lung microvascular endothelium. Yet, the role of Cx40 in the regulation of lung vascular permeability and its underlying mechanisms are unclear. Here, we tested the hypothesis that Cx40 participates in regulation of lung endothelial permeability via a mechanism involving a Rho-associated protein kinase (ROCK) dependent regulation of myosin light chain (MLC). In murine models of intratracheal acid- or LPS-induced lung injury, genetic deficiency of Cx40 attenuated key features of ALI including vascular barrier failure. In human pulmonary microvascular endothelial cells (PMVECs), thrombin-induced loss of transendothelial electrical resistance was attenuated by a Cx40-inhibiting mimetic peptide (40GAP27), Cx40-specific shRNA, or ROCK inhibitor Y27632. In isolated perfused mouse lungs, platelet-activating factor-induced lung weight gain was abrogated by gap junction blocker carbenoxolone, 40GAP27, Y27632, or genetic deficiency of Cx40. Phosphorylation of MLC20 increased drastically in both LPS-treated PMVECs and HCl-treated mouse lungs. Expression of ROCK1 was increased in both LPS-treated PMVECs and HCl-treated mouse lungs, and paralleled by phosphorylation of MLC20. Coimmunoprecipitation experiments revealed protein-protein interaction between ROCK1 and Cx40. LPS-induced upregulation of ROCK1 and phosphorylation of MLC20 were blocked by knockdown of Cx40. LPS caused phosphorylation of myosin phosphatase targeting subunit 1, which could be abrogated by Y27632 or Cx40-shRNA. Our findings reveal a role of Cx40 in regulation of ROCK1 and MLC20 that contributes critically to lung vascular barrier failure in ALI.

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Cho, Taewoo, Rocco Romagnuolo, Corey Scipione, Michael B. Boffa y Marlys L. Koschinsky. "Apolipoprotein(a) stimulates nuclear translocation of β-catenin: a novel pathogenic mechanism for lipoprotein(a)". Molecular Biology of the Cell 24, n.º 3 (febrero de 2013): 210–21. http://dx.doi.org/10.1091/mbc.e12-08-0637.

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Lipoprotein(a) (Lp(a)) is associated with cardiovascular disease risk. This may be attributable to the ability of Lp(a) to elicit endothelial dysfunction. We previously reported that apolipoprotein(a) (apo(a); the distinguishing kringle-containing component of Lp(a)) elicits cytoskeletal rearrangements in vascular endothelial cells, resulting in increased cellular permeability. These effects require a strong lysine-binding site (LBS) in apo(a). We now report that apo(a) induces both nuclear β-catenin–mediated cyclooxygenase-2 (COX-2) expression and prostaglandin E2 secretion, indicating a proinflammatory role for Lp(a). Apo(a) caused the disruption of VE-cadherin/β-catenin complexes in a Src-dependent manner, decreased β-catenin phosphorylation, and increased phosphorylation of Akt and glycogen synthase kinase-3β, ultimately resulting in increased nuclear translocation of β-catenin; all of these effects are downstream of apo(a) attenuation of phosphatase and tensin homologue deleted on chromosome 10 activity. The β-catenin–mediated effects of apo(a) on COX-2 expression were absent using a mutant apo(a) lacking the strong LBS. Of interest, the normal and LBS mutant forms of apo(a) bound to human umbilical vein endothelial cells in a similar manner, and the binding of neither was affected by lysine analogues. Taken together, our findings suggest a novel mechanism by which apo(a) can induce proinflammatory and proatherosclerotic effects through modulation of vascular endothelial cell function.

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Ubogu, Eroboghene E., Nejla Yosef y Robin H. Xia. "Development of a human in vitro blood-nerve barrier model (94.20)". Journal of Immunology 182, n.º 1_Supplement (1 de abril de 2009): 94.20. http://dx.doi.org/10.4049/jimmunol.182.supp.94.20.

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Abstract Phenotypic and functional differences exist between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells from the same tissue. Little is known about cellular interactions at the blood-nerve barrier (BNB). An in vitro BNB (IVBNB) model was developed using primary human endoneurial endothelial cells (pHEndECs) freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion and density centrifugation. pHEndECs are spindle shaped, contact inhibited, differentiate to form capillary-like tubes, are Ulex Europaeus Agglutinin 1 and von Willebrand Factor positive and endocytose acetylated low density lipoprotein. They also express alkaline phosphatase, γ-glutamyl transpeptidase, glucose transporter-1, p-glycoprotein and low levels of cellular adhesion molecules. Culturing pHEndECs on collagen coated transwell inserts was used to develop the IVBNB. High transendothelial electrical resistances (~160Ω.cm2; maximal 12 days after seeding) and low solute permeability to fluoresceinated high molecular weight (70 kDa) dextran (~0.7%) were seen. Electron microscopy demonstrated intercellular tight junctions and 50-100 nm-diameter pinocytic vesicles. These features are consistent with the BNB. This model provides an avenue to study mechanisms of leukocyte entry into peripheral nerves.

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Zhou, Gang, George Kamenos, Suresh Pendem, John X. Wilson y Feng Wu. "Ascorbate protects against vascular leakage in cecal ligation and puncture-induced septic peritonitis". American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 302, n.º 4 (15 de febrero de 2012): R409—R416. http://dx.doi.org/10.1152/ajpregu.00153.2011.

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Vascular leakage in multiple organs is a characteristic pathological change in sepsis. Our recent study revealed that ascorbate protects endothelial barrier function in microvascular endothelial cell monolayers through inhibiting serine/threonine protein phosphatase 2A (PP2A) activation (Han M, Pendem S, Teh SL, Sukumaran DK, Wu F, Wilson JX. Free Radic Biol Med 48: 128–135, 2010). The present study addressed the mechanism of protection by ascorbate against vascular leakage in cecal ligation and puncture (CLP)-induced septic peritonitis in mice. CLP caused NADPH oxidase activation and endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide, increased NO production by inducible NOS (iNOS) and neuronal NOS (nNOS) activity, and elevated 3-nitrotyrosine (a product of peroxynitrite) formation and PP2A activity in the hindlimb skeletal muscles at 12 h after CLP. The increase in PP2A activity was associated with decreased levels of phosphorylated serine and threonine in occludin, which was immunoprecipitated from freshly harvested endothelial cells of the septic skeletal muscles. Moreover, CLP increased the vascular permeability to fluorescent dextran and Evans blue dye in skeletal muscles. An intravenous bolus injection of ascorbate (200 mg/kg body wt), given 30 min prior to CLP, prevented eNOS uncoupling, attenuated the increases in iNOS and nNOS activity, decreased 3-nitrotyrosine formation and PP2A activity, preserved the phosphorylation state of occludin, and completely inhibited the vascular leakage of dextran and Evans blue. A delayed ascorbate injection, given 3 h after CLP, also prevented the vascular permeability increase. We conclude that ascorbate injection protects against vascular leakage in sepsis by sequentially inhibiting excessive production of NO and superoxide, formation of peroxynitrite, PP2A activation, and occludin dephosphorylation. Our study provides a scientific basis for injection of ascorbate as an adjunct treatment for vascular leakage in sepsis.

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Verin, Alexander D., Robert Batori, Anita Kovacs-Kasa, Mary Cherian-Shaw, Sanjiv Kumar, Istvan Czikora, Vijaya Karoor, Derek Strassheim, Kurt R. Stenmark y Evgenia V. Gerasimovskaya. "Extracellular adenosine enhances pulmonary artery vasa vasorum endothelial cell barrier function via Gi/ELMO1/Rac1/PKA-dependent signaling mechanisms". American Journal of Physiology-Cell Physiology 319, n.º 1 (1 de julio de 2020): C183—C193. http://dx.doi.org/10.1152/ajpcell.00505.2019.

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The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small-interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.

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Schweitzer, Kelly S., Steven X. Chen, Sarah Law, Mary Van Demark, Christophe Poirier, Matthew J. Justice, Walter C. Hubbard et al. "Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures". American Journal of Physiology-Lung Cellular and Molecular Physiology 309, n.º 2 (15 de julio de 2015): L175—L187. http://dx.doi.org/10.1152/ajplung.00411.2014.

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The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1–20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10–20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

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DiPaolo, Brian C. y Susan S. Margulies. "Rho kinase signaling pathways during stretch in primary alveolar epithelia". American Journal of Physiology-Lung Cellular and Molecular Physiology 302, n.º 10 (15 de mayo de 2012): L992—L1002. http://dx.doi.org/10.1152/ajplung.00175.2011.

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Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.

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Abu Helal, Raghd, Harrison T. Muturi, Abraham D. Lee, Wei Li, Hilda E. Ghadieh y Sonia M. Najjar. "Aortic Fibrosis in Insulin-Sensitive Mice with Endothelial Cell-Specific Deletion of Ceacam1 Gene". International Journal of Molecular Sciences 23, n.º 8 (14 de abril de 2022): 4335. http://dx.doi.org/10.3390/ijms23084335.

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(1) Background: Mice with global Ceacam1 deletion developed plaque-like aortic lesions even on C57BL/6J background in the presence of increased endothelial cell permeability and insulin resistance. Loss of endothelial Ceacam1 gene caused endothelial dysfunction and reduced vascular integrity without affecting systemic insulin sensitivity. Because endothelial cell injury precedes atherosclerosis, we herein investigated whether the loss of endothelial Ceacam1 initiates atheroma formation in the absence of insulin resistance. (2) Methods: Endothelial cell-specific Ceacam1 null mice on C57BL/6J.Ldlr−/− background (Ldlr−/−VECadCre+Cc1fl/fl) were fed an atherogenic diet for 3–5 months before metabolic, histopathological, and en-face analysis of aortae were compared to their control littermates. Sirius Red stain was also performed on liver sections to analyze hepatic fibrosis. (3) Results: These mice displayed insulin sensitivity without significant fat deposition on aortic walls despite hypercholesterolemia. They also displayed increased inflammation and fibrosis. Deleting Ceacam1 in endothelial cells caused hyperactivation of VEGFR2/Shc/NF-κB pathway with resultant transcriptional induction of NF-κB targets. These include IL-6 that activates STAT3 inflammatory pathways, in addition to endothelin-1 and PDGF-B profibrogenic effectors. It also induced the association between SHP2 phosphatase and VEGFR2, downregulating the Akt/eNOS pathway and reducing nitric oxide production, a characteristic feature of endothelial dysfunction. Similarly, hepatic inflammation and fibrosis developed in Ldlr−/−VECadCre+Cc1fl/fl mice without an increase in hepatic steatosis. (4) Conclusions: Deleting endothelial cell Ceacam1 caused hepatic and aortic inflammation and fibrosis with increased endothelial dysfunction and oxidative stress in the presence of hypercholesterolemia. However, this did not progress into frank atheroma formation. Because these mice remained insulin sensitive, the study provides an in vivo demonstration that insulin resistance plays a critical role in the pathogenesis of frank atherosclerosis.

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39

Clarke, H., A. P. Soler y J. M. Mullin. "Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets". Journal of Cell Science 113, n.º 18 (15 de septiembre de 2000): 3187–96. http://dx.doi.org/10.1242/jcs.113.18.3187.

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Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10(−7) M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10(−7) M TPA for up to 2 hours. Immunofluorescence microscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPA-treated cells. Immunoblot analysis indicated that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remained unchanged for at least 2 hours in cells treated with 10(−7) M TPA compared to their corresponding control cells. As the phosphorylation state of occludin is thought to be important in both tight junction assembly and regulation, the effect of phorbol ester treatment on the phosphorylation of occludin was investigated. Surprisingly, activation of protein kinase C with 10(−7) M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithelial electrical resistance. This dephosphorylation of occludin, occurring after activation of a serine/threonine kinase by TPA, suggested that protein kinase C was not acting directly on this tight junction target protein. If occludin dephosphorylation is involved in increasing tight junction permeability, then protein kinase C is apparently further upstream in the signaling pathway regulating epithelial barrier function, with a downstream serine/threonine phosphatase acting upon occludin.

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40

Bae, Jong-Sup y Alireza R. Rezaie. "Glutamic Acid 192 of Activated Protein C Restricts the Specificity of PAR-1 Cleavage in Endothelial Cells." Blood 110, n.º 11 (16 de noviembre de 2007): 1752. http://dx.doi.org/10.1182/blood.v110.11.1752.1752.

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Abstract It is known that residue 192 (chymotrypsin numbering) plays a key role in determining the P3 binding specificity of coagulation proteases. This residue is a Glu in both thrombin and activated protein C. Previous studies have indicated that the substrates containing an acidic residue at the P3 position are not recognized efficiently by either thrombin or APC in the absence of a cofactor. Protease activated receptor 1 (PAR-1), a common substrate for both thrombin and APC contains an acidic Asp at the P3 position. In this study we examined the ability of Glu-192 to Gln (E192Q) substitution mutants of both thrombin and APC to recognize and cleave PAR-1 in human umbilical vein endothelial cells (HUVEC) transfected with a PAR-1 cleavage reporter plasmid in which the exodomain of the receptor has been fused to a cDNA fragment encoding the soluble alkaline phosphatase. Thrombin E192Q cleaved PAR-1 with a catalytic efficiency that was approximately 5-fold higher than that of wild-type thrombin. On the other hand, the activity of APC E192Q toward PAR-1 was improved approximately 100-fold. Thus, unlike an approximately 1000-fold lower activity for APC in cleaving PAR-1 relative to thrombin, the activity of APC E192Q toward PAR-1 was only 10-fold lower than that of thrombin. These results suggest that the inhibitory interaction of Glu-192 of APC with P3-Asp of PAR-1 is responsible for its poor activity toward PAR-1. It is known that the cleavage of PAR-1 by thrombin in endothelial cells elicits a proinflammatory response. However, the cleavage of the same receptor by APC in complex with endothelial protein C receptor (EPCR) invokes a protective antiinflammatory response. The mechanism of the paradoxical effect of PAR-1 signaling by the two proteases is not known. We used these mutants to investigate the possibility that the level of PAR-1 activation by either thrombin or APC dictates the type of the response in endothelial cells. Furthermore, we used these mutants in TNF-a-stimulated endothelial cell permeability and apoptosis assays to understand the mechanism by which EPCR enables APC to activate PAR-1 in endothelial cells. We discovered that the dose of receptor activation is not responsible for the paradoxical effect of PAR-1 signaling by APC and thrombin in endothelial cells. Furthermore, the interaction of APC with EPCR is not associated with an improvement in the catalytic efficiency of the protease toward PAR-1. The possible EPCR and PAR-1 dependent recognition and signaling mechanism of APC and thrombin is discussed.

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Veronesi, Bellina, Kent Carlsón y Marion Ehrich. "An In Vitro Model of the Blood-Brain Barrier: The Response of Madin-Darby Canine Kidney Cells to Triethyl Tin". Alternatives to Laboratory Animals 24, n.º 3 (junio de 1996): 349–57. http://dx.doi.org/10.1177/026119299602400308.

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The development of a cell culture model which simulates the properties of the blood–brain barrier (BBB) is necessary for the detection of neurotoxic chemicals that can disrupt the barrier, and to provide a more “risk relevant” in vitro screening battery. The present study evaluates the Madin-Darby canine kidney (MDCK) epithelial cell line for this purpose. Changes in electrical resistance and enzyme activities were correlated in confluent MDCK cells exposed to the neurotoxic metal, triethyl tin (TET). Concentrations of TET (0.001–10μM) were established that produced depression in electrical resistance of the MDCK cells after exposure for 8 hours or caused fluorescein leakage after exposure for 72 hours. Confluent cultures of MDCK cells were then exposed to these concentrations of TET and assayed after exposure for 24 hours and 72 hours for changes in those enzymes common to both epithelial and cerebral endothelial cells. The results indicated that increased alkaline phosphatase (APP), γ-glutamyl transpeptidase (GGTP) and superoxide dismutase (SOD) characterised the loss of electrical resistance and permeability disruption in TET-exposed MDCK confluent cultures. Relative increases in APP and decreases in GGTP activities preceded cytotoxicity, which was associated with a high SOD activity. Such enzyme changes may be predictive endpoints of barrier cell disruption by neurotoxic metals in this cell line and support the additional evaluation of the MDCK cell line as an in vitro “screen” for chemicals that disrupt the BBB.

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42

Hansen, Kristoffer B., Christian Staehr, Palle D. Rohde, Casper Homilius, Sukhan Kim, Mette Nyegaard, Vladimir V. Matchkov y Ebbe Boedtkjer. "PTPRG is an ischemia risk locus essential for HCO3–-dependent regulation of endothelial function and tissue perfusion". eLife 9 (21 de septiembre de 2020). http://dx.doi.org/10.7554/elife.57553.

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Acid-base conditions modify artery tone and tissue perfusion but the involved vascular-sensing mechanisms and disease consequences remain unclear. We experimentally investigated transgenic mice and performed genetic studies in a UK-based human cohort. We show that endothelial cells express the putative HCO3–-sensor receptor-type tyrosine-protein phosphatase RPTPγ, which enhances endothelial intracellular Ca2+-responses in resistance arteries and facilitates endothelium-dependent vasorelaxation only when CO2/HCO3– is present. Consistent with waning RPTPγ-dependent vasorelaxation at low [HCO3–], RPTPγ limits increases in cerebral perfusion during neuronal activity and augments decreases in cerebral perfusion during hyperventilation. RPTPγ does not influence resting blood pressure but amplifies hyperventilation-induced blood pressure elevations. Loss-of-function variants in PTPRG, encoding RPTPγ, are associated with increased risk of cerebral infarction, heart attack, and reduced cardiac ejection fraction. We conclude that PTPRG is an ischemia susceptibility locus; and RPTPγ-dependent sensing of HCO3– adjusts endothelium-mediated vasorelaxation, microvascular perfusion, and blood pressure during acid-base disturbances and altered tissue metabolism.

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43

Zhu, Xiaolong, Cong Qiu, Yiran Wang, Yuanqing Jiang, Yefeng Chen, Linge Fan, Ruizhe Ren et al. "FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis". Proceedings of the National Academy of Sciences 119, n.º 26 (21 de junio de 2022). http://dx.doi.org/10.1073/pnas.2202631119.

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Angiogenesis contributes fundamentally to embryonic development, tissue homeostasis, and wound healing. Basic fibroblast growth factor (FGF2) is recognized as the first proangiogenic molecule discovered, and it facilitates angiogenesis by activating FGF receptor 1 (FGFR1) signaling in endothelial cells. However, the precise roles of FGFR and the FGF/FGFR signaling axis in angiogenesis remain unclear, especially because of the contradictory phenotypes of in vivo FGF and FGFR gene deficiency models. Our previous study results suggested a potential role of posttranslational small ubiquitin-like modifier modification (SUMOylation), with highly dynamic regulatory features, in vascular development and disorder. Here, we identified SENP1-regulated endothelial FGFR1 SUMOylation at conserved lysines responding to proangiogenic stimuli, while SENP1 functioned as the deSUMOylase. Hypoxia-enhanced FGFR1 SUMOylation restricted the tyrosine kinase activation of FGFR1 by modulating the dimerization of FGFR1 and FGFR1 binding with its phosphatase PTPRG. Consequently, it facilitated the recruitment of FRS2α to VEGFR2 but limited additional recruitment of FRS2α to FGFR1, supporting the activation of VEGFA/VEGFR2 signaling in endothelial cells. Furthermore, SUMOylation-defective mutation of FGFR1 resulted in exaggerated FGF2/FGFR1 signaling but suppressed VEGFA/VEGFR2 signaling and the angiogenic capabilities of endothelial cells, which were rescued by FRS2α overexpression. Reduced angiogenesis and endothelial sprouting in mice bearing an endothelial-specific, FGFR1 SUMOylation-defective mutant confirmed the functional significance of endothelial FGFR1 SUMOylation in vivo. Our findings identify the reversible SUMOylation of FGFR1 as an intrinsic fine-tuned mechanism in coordinating endothelial angiogenic signaling during neovascularization; SENP1-regulated FGFR1 SUMOylation and deSUMOylation controls the competitive recruitment of FRS2α by FGFR1 and VEGFR2 to switch receptor-complex formation responding to hypoxia and normoxia angiogenic environments.

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Panchatcharam, Manikandan, Sumitra Miriyala, Pooja Patil, Adrienne Nguyen, Manjula Sunkara, Andrew Morris, Diana Escalante-Alcalde y Susan S. Smyth. "Abstract 382: Mice with Endothelial-Targeted Inactivation of PPAP2B (Lipid Phosphate Phosphatase 3) Display Enhanced Vascular Inflammation and Permeability". Arteriosclerosis, Thrombosis, and Vascular Biology 32, suppl_1 (mayo de 2012). http://dx.doi.org/10.1161/atvb.32.suppl_1.a382.

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Meta-analysis of data from a series of genome-wide association studies (GWAS) revealed a striking association between the PPAP2B loci and coronary artery disease. The PPAP2B gene encodes for the lipid phosphate phosphatase 3 (LPP3) integral membrane enzyme, that dephosphorylates lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and related bioactive lipids. While emerging evidence suggests a role for both LPA and S1P in experimental models of atherosclerosis, essentially nothing is understood about the pathophysiologic role of LPP3, largely due to the fact that genetic inactivation results embryonic lethality. We report that constitutive inactivation of LPP3 in vascular endothelial cells accomplished by breeding mice with floxed Ppap2b to mice expressing Cre recombinase under control of the Tie1 promoter results in embryonic lethality, indicating that dysregulation of LPP3-dependent vascular endothelial cell function likely underlies the developmental phenotype observed in Ppap2b -null embryos. Using an estrogen inducible Cre transgene under control of the Tie1 promoter, we have inactivated Ppap2b expression in endothelial cells in adult mice and confirmed the lack of vascular endothelial LPP3 by immunologic and biochemical approaches. The absence of vascular endothelial LPP3 results in a 2.2±0.5 fold increase in basal vascular permeability, as assessed by Evans blue dye extravasation in lung tissue. The permeability difference is exaggerated following an inflammatory challenge (LPS; 2 mg/kg), and LPS-induced expression of plasma inflammatory markers IL6 and KC were 3.3±0.5 and 1.9±0.6 fold higher in mice lacking endothelial LPP3. LPS-enhanced permeability in the absence of LPP3 was attenuated by administration of an LPA receptor antagonist. Mice lacking LPA receptors 1 and 2 (LPA1, LPA2) or LPA receptor 4 (LPA4) were protected from LPS-mediated permeability. These results demonstrate a fundamental role for LPP3, possibility via modulation of local LPA levels and signaling through LPA1, LPA2, and LPA4 receptors, in maintaining the integrity of the vascular endothelium.

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Sigaud, Romain, Nadège Dussault, Caroline Berenguer-Daizé, Christine Vellutini, Zohra Benyahia, Mylène Cayol, Fabrice Parat et al. "Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors". Frontiers in Oncology 11 (8 de octubre de 2021). http://dx.doi.org/10.3389/fonc.2021.753244.

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VE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. We have previously shown that adrenomedullin (AM) blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherinY731) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. In this article, we demonstrate that the AM-mediated dephosphorylation of pVE-cadherinY731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2Y542) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherinY731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM-receptors-blocking antibodies showed a decrease in pSHP-2Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts.Our findings show that AM acts on VE-cadherin dynamics through pSHP-2Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature.

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Park, Hyeri, Jin Seok, Jun Hyeong You, Jae Yeon Kim, Ja-Yun Lim y Gi Jin Kim. "Increased phosphatase regenerating liver-1 trigger vascular remodeling in injured ovary via platelet-derived growth factor signaling pathway". Stem Cell Research & Therapy 13, n.º 1 (7 de marzo de 2022). http://dx.doi.org/10.1186/s13287-022-02772-9.

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Abstract Background Vascular abnormalities in the ovary cause infertility accompanied by ovarian insufficiency due to a microenvironment of barren ovarian tissues. Placenta-derived mesenchymal stem cells (PD-MSCs, Naïve) treatment in ovarian dysfunction shows angiogenic effect, however, the therapeutic mechanism between ovarian function and vascular remodeling still unclear. Therefore, we examined whether by phosphatase regenerating liver-1 (PRL-1), which is correlated with angiogenesis in reproductive systems, overexpressed PD-MSCs could maximize the angiogenic effects in an ovarian tissues injured of rat model with partial ovariectomy and their therapeutic mechanism by enhanced vascular function via PDGF signaling. Methods PD-MSCsPRL-1 (PRL-1) were generated by nonviral AMAXA gene delivery system and analyzed the vascular remodeling and follicular development in ovary. One week after Sprague–Dawley (SD) rats ovariectomy, Naïve and PRL-1 was transplanted. The animals were sacrificed at 1, 3 and 5 weeks after transplantation and vascular remodeling and follicular development were analyzed. Also, human umbilical vein endothelial cells (HUVECs) and ovarian explantation culture were performed to prove the specific effects and mechanism of PRL-1. Results Vascular structures in ovarian tissues (e.g., number of vessels, thickness and lumen area) showed changes in the Naïve and PRL-1-overexpressed PD-MSC (PRL-1) transplantation (Tx) groups compared to the nontransplantation (NTx) group. Especially, PRL-1 induce to increase the expression of platelet-derived growth factor (PDGF), which plays a role in vascular remodeling as well as follicular development, compared to the NTx. Also, the expression of genes related to pericyte and vascular permeability in arteries was significantly enhanced in the PRL-1 compared to the NTx (p < 0.05). PRL-1 enhanced the vascular formation and permeability of human umbilical vein endothelial cells (HUVECs) via activated the PDGF signaling pathway. Conclusions Our results show that PRL-1 restored ovarian function by enhanced vascular function via PDGF signaling pathway. These findings offer new insight into the effects of functionally enhanced stem cell therapy for reproductive systems and should provide new avenues to develop more efficient therapies in degenerative medicine.

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47

Kam, Julia Y., Tina Cheng, Danielle C. Garland, Warwick J. Britton, David M. Tobin y Stefan H. Oehlers. "Inhibition of infection-induced vascular permeability modulates host leukocyte recruitment to Mycobacterium marinum granulomas in zebrafish". Pathogens and Disease, 19 de abril de 2022. http://dx.doi.org/10.1093/femspd/ftac009.

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Abstract Mycobacterial granuloma formation involves significant stromal remodeling including the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as anti-angiogenic or vascular normalizing therapies are beneficial host-directed therapies in pre-clinical models of tuberculosis across host-mycobacterial pairings. Using the zebrafish-Mycobacterium marinum infection model, we demonstrate that vascular normalization by inhibition of vascular endothelial protein tyrosine phosphatase (VE-PTP) decreases granuloma hypoxia, the opposite effect of hypoxia-inducing anti-angiogenic therapy. Inhibition of VE-PTP decreased neutrophil recruitment to granulomas in adult and larval zebrafish and decreased the proportion of neutrophils that extravasated distal to granulomas. Furthermore, VE-PTP inhibition increased the accumulation of T cells at M. marinum granulomas. Our study provides evidence that, similar to the effect in solid tumors, vascular normalization during mycobacterial infection increases the T cell:neutrophil ratio in lesions which may be correlates of protective immunity.

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48

Kunimura, Kayo, Satomi Miki, Miyuki Takashima y Jun-ichiro Suzuki. "S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway". Cell Communication and Signaling 19, n.º 1 (15 de febrero de 2021). http://dx.doi.org/10.1186/s12964-020-00692-w.

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Abstract Background Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway. Methods Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively. Results We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell–cell contact regions and the increase of actin stress fibers induced by TNF-α. Conclusions The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.

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Lalatsa, Aikaterini, Yujiao Sun, Jose Ignacio Gamboa y Shira Knafo. "Preformulation Studies of a Stable PTEN-PDZ Lipopeptide Able to Cross an In Vitro Blood-Brain-Barrier Model as a Potential Therapy for Alzheimer’s Disease". Pharmaceutical Research 37, n.º 10 (4 de septiembre de 2020). http://dx.doi.org/10.1007/s11095-020-02915-8.

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Abstract Purpose Amyloid β (Aβ) drives the accumulation of excess Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) at synapses, inducing synaptic depression and perturbing memory. This recruitment of PTEN to synapses in response to Aβ drives its interaction with PSD95/Disc large/Zonula occludens-1 (PDZ) proteins and, indeed, we previously showed that an oligo lipopeptide (PTEN-PDZ) capable of blocking such PTEN:PDZ interactions rescues the synaptic and cognitive deficits in a mouse model of Alzheimer’s disease. Hence, the PTEN:PDZ interaction appears to be crucial for Aβ-induced synaptic and cognitive impairment. Here we have evaluated the feasibility of using PTEN-PDZ lipopeptides based on the human/mouse PTEN C-terminal sequence, testing their stability in biological fluids, their cytotoxicity, their ability to self-assemble and their in vitro blood-brain barrier (BBB) permeability. Myristoyl or Lauryl tails were added to the peptides to enhance their cell permeability. Methods Lipopeptides self assembly was assessed using electron microscopy and the thioflavin T assay. Stability studies in mouse plasma (50%), intestinal washing, brain and liver homogenates as well as permeability studies across an all human 2D blood-brain barrier model prepared with human cerebral endothelial cells (hCMEC/D3) and human astrocytes (SC-1800) were undertaken. Results The mouse lauryl peptide displayed enhanced overall stability in plasma, ensuring a longer half-life in circulation that meant there were larger amounts available for transport across the BBB (Papp0-4h: 6.28 ± 1.85 × 10−6 cm s−1). Conclusion This increased availability, coupled to adequate BBB permeability, makes this peptide a good candidate for therapeutic parenteral (intravenous, intramuscular) administration and nose-to-brain delivery. Graphical Abstract

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Zhao, Hongyan, Peng Wang, Xiaohong Wang, Yutian Li, Qianqian Li, Jing Chen, Chengen Ma, Chunting Wang y Guo-Chang Fan. "Abstract 386: Lipocalin 10 Protects Against Sepsis-triggered Vascular Leakage Through Activation Of The Ssh1-cofilin Signaling Pathway". Arteriosclerosis, Thrombosis, and Vascular Biology 42, Suppl_1 (mayo de 2022). http://dx.doi.org/10.1161/atvb.42.suppl_1.386.

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Background: The increased vascular leakage seen in sepsis patients is well-recognized to be responsible for sepsis-triggered organ failure and patient mortality. Prior work suggests that lipocalin 10 (Lcn10), a member of the lipocalin superfamily, is significantly downregulated in the blood of non-survival septic patients when comparing to the survival group. Nonetheless, whether Lcn10 affects endothelial barrier integrity in sepsis remains unknown. Methods and Results: First, we observed that the expression of Lcn10 was remarkably down-regulated in the hearts of both endotoxin LPS- and cecal ligation/puncture (CLP)-induced septic mice, compared to their controls. Interestingly, further analysis of Lcn10 expression in different cell types isolated from LPS- and CLP-hearts showed that reduction of Lcn10 occurred only in cardiac endothelial cells (ECs) but not in cardiomyocytes or fibroblasts. Using a global Lcn10-knockout (KO) mouse model, we found that loss of Lcn10 greatly increased vascular permeability, which correlated with more severe cardiac depression and higher mortality following LPS challenge or CLP surgery, compared to LPS- or CLP-treated wild-type (WT) mice. By contrast, in vitro overexpression of Lcn10 in ECs provided greater resistance to LPS-caused monolayer leak, compared to control cells. A mechanistic analysis by RNA-sequencing and RT-qPCR revealed that both endogenous and exogenous elevation of Lcn10 in ECs could greatly upregulate slingshot homolog 1 (Ssh1) expression. Ssh1 is a phosphatase known to activate Cofilin, a key actin-binding protein that plays an essential role in controlling actin filament dynamics. Accordingly, phosphorylated Cofilin levels were significantly reduced and thereby, reorganized F-actin to cortical actin for stabilizing tight junction molecules in Lcn10-treated ECs, compared to control cells. Finally, knockdown of Ssh1 in ECs by siRNA greatly offsets Lcn10-induced reduction of monolayer leakage upon LPS insult. Conclusions: Endothelial Lcn10 is critical for protecting against sepsis-induced cardiovascular leakage, and its underlying mechanism involves the activation of Ssh1-Cofilin pathway. Our study suggests that Lcn10 could be a novel regulator of vascular barrier integrity.

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