scholarly journals Reciprocal Association between the Apical Junctional Complex and AMPK: A Promising Therapeutic Target for Epithelial/Endothelial Barrier Function?

2019 ◽  
Vol 20 (23) ◽  
pp. 6012 ◽  
Author(s):  
Kazuto Tsukita ◽  
Tomoki Yano ◽  
Atsushi Tamura ◽  
Sachiko Tsukita
Keyword(s):  
Barrier Function ◽  
Intracellular Signaling ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Cell Polarization ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Junctional Complex ◽  
Endothelial Barrier Function ◽  
Apical Junctional Complex

Epithelial/endothelial cells adhere to each other via cell–cell junctions including tight junctions (TJs) and adherens junctions (AJs). TJs and AJs are spatiotemporally and functionally integrated, and are thus often collectively defined as apical junctional complexes (AJCs), regulating a number of spatiotemporal events including paracellular barrier, selective permeability, apicobasal cell polarity, mechano-sensing, intracellular signaling cascades, and epithelial morphogenesis. Over the past 15 years, it has been acknowledged that adenosine monophosphate (AMP)-activated protein kinase (AMPK), a well-known central regulator of energy metabolism, has a reciprocal association with AJCs. Here, we review the current knowledge of this association and show the following evidences: (1) as an upstream regulator, AJs activate the liver kinase B1 (LKB1)–AMPK axis particularly in response to applied junctional tension, and (2) TJ function and apicobasal cell polarization are downstream targets of AMPK and are promoted by AMPK activation. Although molecular mechanisms underlying these phenomena have not yet been completely elucidated, identifications of novel AMPK effectors in AJCs and AMPK-driven epithelial transcription factors have enhanced our knowledge. More intensive studies along this line would eventually lead to the development of AMPK-based therapies, enabling us to manipulate epithelial/endothelial barrier function.

The protective effects of wine pomace products on the vascular endothelial barrier function

2020 ◽  
Vol 11 (9) ◽  
pp. 7878-7891 ◽  
Author(s):  
Gisela Gerardi ◽  
Mónica Cavia-Saiz ◽  
María D. Rivero-Pérez ◽  
María L. González-SanJosé ◽  
Pilar Muñiz
Keyword(s):  
Barrier Function ◽  
Endothelial Permeability ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Vascular Endothelial ◽  
Protective Effects ◽  
Endothelial Barrier Function ◽  
E Cadherin

The Wine Pomace Products (WPP) prevent the increase of endothelial permeability induced by INF-γ and increase E-cadherin expression in the cell junctions.

scholarly journals The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation

2012 ◽  
Vol 23 (21) ◽  
pp. 4212-4225 ◽  
Author(s):  
Ilse Timmerman ◽  
Mark Hoogenboezem ◽  
Anton M. Bennett ◽  
Dirk Geerts ◽  
Peter L. Hordijk ◽  
...  
Keyword(s):  
Barrier Function ◽  
Adherens Junctions ◽  
Tyrosine Phosphatase ◽  
Endothelial Permeability ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Src Homology 2 Domain ◽  
Cell Cell

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.

CCM2 and PAK4 act downstream of atrial natriuretic peptide signaling to promote cell spreading

2017 ◽  
Vol 474 (11) ◽  
pp. 1897-1918 ◽  
Author(s):  
Koichi Miura ◽  
Takashi Nojiri ◽  
Yoshiharu Akitake ◽  
Koji Ando ◽  
Shigetomo Fukuhara ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Barrier Function ◽  
Molecular Mechanisms ◽  
Cell Spreading ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Cavernous Malformations ◽  
Atrial Natriuretic

Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. Via its receptor, guanylyl cyclase-A (GC-A), ANP maintains cardiovascular homeostasis by exerting diuretic, natriuretic, and hypotensive effects mediated, in part, by endothelial cells. Both in vivo and in vitro, ANP enhances endothelial barrier function by reducing RhoA activity and reorganizing the actin cytoskeleton. We established mouse endothelial cells that stably express GC-A and used them to analyze the molecular mechanisms responsible for actin reorganization. Stimulation by ANP resulted in phosphorylation of myosin light chain (MLC) and promotion of cell spreading. p21-activated kinase 4 (PAK4) and cerebral cavernous malformations 2 (CCM2), a scaffold protein involved in a cerebrovascular disease, were required for the phosphorylation of MLC and promotion of cell spreading by ANP. Finally, in addition to the GC domain, the kinase homology domain of GC-A was also required for ANP/GC-A signaling. Our results indicate that CCM2 and PAK4 are important downstream mediators of ANP/GC-A signaling involved in cell spreading, an important initial step in the enhancement of endothelial barrier function.

Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment

2021 ◽  
pp. 247255522110309
Author(s):  
Oleksii Dubrovskyi ◽  
Erica Hasten ◽  
Steven M. Dudek ◽  
Michael T. Flavin ◽  
Leo Li-Ying Chan
Keyword(s):  
Drug Discovery ◽  
Barrier Function ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Cell Monolayer ◽  
Endothelial Permeability ◽  
Image Cytometry ◽  
Endothelial Barrier ◽  
Published Data ◽  
Endothelial Barrier Function

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.

TNF-induced endothelial barrier disruption: beyond actin and Rho

2014 ◽  
Vol 112 (12) ◽  
pp. 1088-1102 ◽  
Author(s):  
Beatriz Marcos-Ramiro ◽  
Diego García-Weber ◽  
Jaime Millán
Keyword(s):  
Endothelial Cell ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Transendothelial Migration ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Adhesion Receptors ◽  
Barrier Disruption ◽  
Leukocyte Transendothelial Migration

SummaryThe decrease of endothelial barrier function is central to the long-term inflammatory response. A pathological alteration of the ability of endothelial cells to modulate the passage of cells and solutes across the vessel underlies the development of inflammatory diseases such as atherosclerosis and multiple sclerosis. The inflammatory cytokine tumour necrosis factor (TNF) mediates changes in the barrier properties of the endothelium. TNF activates different Rho GTPases, increases filamentous actin and remodels endothelial cell morphology. However, inhibition of actin-mediated remodelling is insufficient to prevent endothelial barrier disruption in response to TNF, suggesting that additional molecular mechanisms are involved. Here we discuss, first, the pivotal role of Rac-mediated generation of reactive oxygen species (ROS) to regulate the integrity of endothelial cell-cell junctions and, second, the ability of endothelial adhesion receptors such as ICAM-1, VCAM-1 and PECAM-1, involved in leukocyte transendothelial migration, to control endothelial permeability to small molecules, often through ROS generation. These adhesion receptors regulate endothelial barrier function in ways both dependent on and independent of their engagement by immune cells, and orchestrate the crosstalk between leukocyte transendothelial migration and endothelial permeability during inflammation.

RhoA inactivation enhances endothelial barrier function

1999 ◽  
Vol 277 (5) ◽  
pp. C955-C964 ◽  
Author(s):  
José M. Carbajal ◽  
Richard C. Schaeffer
Keyword(s):  
Barrier Function ◽  
Focal Adhesions ◽  
Protein S ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Rho Proteins ◽  
Endothelial Barrier Function ◽  
Selective Permeability ◽  
Permeability Assay ◽  
Pulmonary Artery Endothelial Cell

The modulation of endothelial barrier function is thought to be a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA with Clostridium botulinum exoenzyme C3 transferase (C3), an inhibitor of the small GTP-binding protein RhoA. Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using a size-selective permeability assay and quantitative digital imaging measurements of SF/FA. C3 treatment disassembled SF/FA, stimulated diffuse myosin II immunostaining, and reduced the phosphotyrosine (PY) content of paxillin and 130- to 140-kDa proteins that included p125FAK. C3-treated monolayers displayed a 60–85% decline in F-actin content and a 170–300% increase in EC surface area with enhanced endothelial barrier function. This activity correlated with reorganization of F-actin and PY protein(s) to β-catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation of myosin ribbons, SF/FA, and the increased PY content of proteins, these characteristics were Rho dependent. Our data show that C3 inhibition of Rho proteins leads to cAMP-like characteristics of reduced SF/FA and enhanced endothelial barrier function.

Regulation of vascular endothelial barrier function

1994 ◽  
Vol 267 (3) ◽  
pp. L223-L241 ◽  
Author(s):  
H. Lum ◽  
A. B. Malik
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Mediators ◽  
Barrier Function ◽  
Cell Junctions ◽  
Paracellular Transport ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Cell Rounding ◽  
Cell Cell

The increase in endothelial permeability in response to inflammatory mediators such as alpha-thrombin and histamine is accompanied by cell rounding and interendothelial gap formation, implicating that the predominant transport pathway is a diffusive one [i.e., via cellular junctions (paracellular transport)]. However, the possible contribution by vesicle-mediated transport (i.e., via albumin binding protein gp60) to the overall permeability increase needs investigation. Regulation of paracellular transport in endothelial cells is associated with modulation of actin-based systems which anchor the cell to its neighbor or extracellular matrix, thus maintaining endothelial integrity. At the cell-cell junctions, actin is linked indirectly to the plasma membrane by linking proteins (e.g., vinculin, catenins, alpha-actinin) to cadherins, which function in homophilic intercellular adhesion. Cadherins may also play a role in regulating the formation of tight junctions, which also may be associated with actin. At endothelial focal contacts, the transmembrane receptors (integrins) for matrix proteins are linked to actin via linking proteins (i.e., vinculin, talin, alpha-actinin). In response to inflammatory mediators, second messengers signal two regulatory pathways which modulate the actin-based systems, which may lead to impairment of the endothelial barrier integrity. One pathway is based on protein kinase C (PKC) isozyme-specific phosphorylation of linking proteins at the cell-cell and cell-matrix junctions. The increased phosphorylation is associated with actin reorganization, cell rounding, and increased paracellular transport. The other is the activation of myosin light-chain kinase, (MLCK), which causes an actin-myosin-based contraction that may lead to a centripetal retraction of endothelial cells. Current research is in the identification of protein substrates of PKC isozymes, the specific role of their phosphorylation in barrier function, and determining the precise role of MLCK in modulation of endothelial barrier function.

scholarly journals Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium

2010 ◽  
Vol 298 (3) ◽  
pp. L361-L370 ◽  
Author(s):  
K. L. Grinnell ◽  
B. Casserly ◽  
E. O. Harrington
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Barrier Function ◽  
Adherens Junction ◽  
Fiber Formation ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Protein Tyrosine

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.

Tyrosine phosphorylation and the small GTPase rac cross-talk in regulation of endothelial barrier function

2005 ◽  
Vol 94 (09) ◽  
pp. 620-629 ◽  
Author(s):  
Jochen Seebach ◽  
Hans-Jürgen Mädler ◽  
Beata Wojciak-Stothard ◽  
Hans-Joachim Schnittler
Keyword(s):  
Tyrosine Phosphorylation ◽  
Barrier Function ◽  
Rho Gtpase ◽  
Small Gtpase ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Protein Tyrosine Phosphorylation ◽  
Endothelial Barrier Function ◽  
Protein Tyrosine ◽  
Junctional Proteins

SummaryEndothelial barrier function depends on the integrity of intercellular adherens junctions controlled by the association of VEcadherin/ catenin complex with cortical actin filaments. Both tyrosine phosphorylation/dephosphorylation of junctional proteins and actin reorganization mediated by rho-GTPases regulate barrier function but the relationship between these regulatory mechanisms is unclear. Here we studied the effects of factors increasing protein tyrosine phosphorylation, pervanadate (PV) and VEGF, on distribution of VE-cadherin, F-actin polymerization and transendothelial electrical resistance (TER) in human umbilical vein endothelial cells (HUVECs). Changes in protein tyrosine phosphorylation of cytoplasmic and junctional proteins, as well as the activity of rho-GTPase rac1, were also measured. We report for the first time that PV and VEGF induced a rapid transient increase in endothelial barrier function accompanied by rac1 activation, a differentiated tyrosine phosphorylation of theVE-cadherin/catenin complex, recruitment of actin filament to cell junctions and ruffle formation. A sustained decrease in endothelial barrier function was observed at later times of PV and VEGF treatment. Expression of dominant negative rac1, N17rac1 abolished the barrier-enhancing effects of PV andVEGF, while the sustained decrease in barrier function was unaffected. These observations bring into focus early shortterm effects of protein tyrosine phosphorylation in cells, often overshadowed by more pronounced and long-lasting later effects and may play an important role in the regulation of endothelial barrier function.

scholarly journals The PI3K p110α isoform regulates endothelial adherens junctions via Pyk2 and Rac1

2010 ◽  
Vol 188 (6) ◽  
pp. 863-876 ◽  
Author(s):  
Robert J. Cain ◽  
Bart Vanhaesebroeck ◽  
Anne J. Ridley
Keyword(s):  
Barrier Function ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Endothelial Permeability ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Regulatory Subunit ◽  
Nucleotide Exchange ◽  
Endothelial Barrier Function ◽  
Junction Protein

Endothelial cell–cell junctions control efflux of small molecules and leukocyte transendothelial migration (TEM) between blood and tissues. Inhibitors of phosphoinositide 3-kinases (PI3Ks) increase endothelial barrier function, but the roles of different PI3K isoforms have not been addressed. In this study, we determine the contribution of each of the four class I PI3K isoforms (p110α, -β, -γ, and -δ) to endothelial permeability and leukocyte TEM. We find that depletion of p110α but not other p110 isoforms decreases TNF-induced endothelial permeability, Tyr phosphorylation of the adherens junction protein vascular endothelial cadherin (VE-cadherin), and leukocyte TEM. p110α selectively mediates activation of the Tyr kinase Pyk2 and GTPase Rac1 to regulate barrier function. Additionally, p110α mediates the association of VE-cadherin with Pyk2, the Rac guanine nucleotide exchange factor Tiam-1 and the p85 regulatory subunit of PI3K. We propose that p110α regulates endothelial barrier function by inducing the formation of a VE-cadherin–associated protein complex that coordinates changes to adherens junctions with the actin cytoskeleton.

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