scholarly journals The Cx43 Carboxyl-Terminal Mimetic Peptide αCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1192
Author(s):  
Randy E. Strauss ◽  
Louisa Mezache ◽  
Rengasayee Veeraraghavan ◽  
Robert G. Gourdie
Keyword(s):  
Barrier Function ◽  
Therapeutic Potential ◽  
Endothelial Barrier ◽  
Cell Periphery ◽  
Endothelial Barrier Function ◽  
Mimetic Peptide ◽  
Zonula Occludens ◽  
Junction Protein ◽  
Zonula Occludens 1 ◽  
Cell Cell

The Cx43 carboxyl-terminus (CT) mimetic peptide, αCT1, originally designed to bind to Zonula Occludens 1 (ZO1) and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing (ECIS), a TRITC-dextran Transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell–cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular chiral orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell–cell border localization of endothelial VE-cadherin, the tight junction protein Zonula Occludens 1 (ZO1), and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1 binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema.

scholarly journals The Cx43 Carboxyl-Terminal Mimetic Peptide alphaCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner

2021 ◽  
Author(s):  
Randy E Strauss ◽  
Louisa Mezache ◽  
Rengasayee Veeraraghavan ◽  
Robert G. Gourdie
Keyword(s):  
Barrier Function ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Endothelial Barrier ◽  
Cell Periphery ◽  
Endothelial Barrier Function ◽  
Mimetic Peptide ◽  
Junction Protein ◽  
Cell Cell

The Cx43 CT mimetic peptide, αCT1, originally designed to bind to ZO1 and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing(ECIS), a FITC-dextran transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell-cell border localization of endothelial VE-cadherin, the Tight Junction protein Zonula Occludens 1 (ZO1) , and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1-binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema.

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.

Regulation of vascular endothelial barrier function by Epac, a cAMP-activated exchange factor for Rap GTPase

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 1950-1955 ◽  
Author(s):  
Xavier Cullere ◽  
Sunil K. Shaw ◽  
Lorna Andersson ◽  
Junichi Hirahashi ◽  
Francis W. Luscinskas ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Barrier Function ◽  
Rho Gtpase ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Cortical Actin ◽  
Cell Contacts ◽  
Exchange Factor ◽  
Gtpase Activation ◽  
Cell Cell

Abstract Endothelial cell-cell junctional proteins and cortical actin are of central importance for regulating vascular permeability. Rap1, a member of the Ras family of GTPases, is enriched at endothelial cell-cell contacts and activated by cyclic AMP (cAMP) through a PKA-independent pathway. Activation of a cAMP-inducible guanine-exchange factor for Rap, Epac, results in markedly enhanced basal endothelial barrier function by increasing cortical actin and subsequent redistribution of adherens and tight junctional molecules to cell-cell contacts. Activation of Epac also counteracts thrombin-induced hyperpermeability through down-regulation of Rho GTPase activation, suggesting cross-talk between Rap and Rho GT-Pases. Thus, Epac/Rap activation represents a new pathway for regulating endothelial cell 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.

Histamine alters endothelial barrier function at cell-cell and cell-matrix sites

2000 ◽  
Vol 278 (5) ◽  
pp. L888-L898 ◽  
Author(s):  
Alan B. Moy ◽  
Michael Winter ◽  
Anant Kamath ◽  
Ken Blackwell ◽  
Gina Reyes ◽  
...  
Keyword(s):  
Barrier Function ◽  
Umbilical Vein ◽  
Inducible Promoter ◽  
Endothelial Barrier ◽  
Cell Contact ◽  
Cell Resistance ◽  
Endothelial Barrier Function ◽  
Cell Matrix ◽  
Contact Exposure ◽  
Cell Cell

To determine how histamine regulates endothelial barrier function through an integrative cytoskeletal network, we mathematically modeled the resistance across an endothelial cell-covered electrode as a function of cell-cell, cell-matrix, and transcellular resistances. Based on this approach, histamine initiated a rapid decrease in transendothelial resistance predominantly through decreases in cell-cell resistance in confluent cultured human umbilical vein endothelial cells (HUVECs). Restoration of resistance was characterized by initially increasing cell-matrix resistance, with later increases in cell-cell resistance. Thus histamine disrupts barrier function by specifically disrupting cell-cell adhesion and restores barrier function in part through direct effects on cell-matrix adhesion. To validate the precision of our technique, histamine increased the resistance in subconfluent HUVECs in which there was no cell-cell contact. Exposure of confluent monolayers to an antibody against cadherin-5 caused a predominant decrease in cell-cell resistance, whereas the resistance was unaffected by the antibody to cadherin-5 in subconfluent cells. Furthermore, we observed an increase predominantly in cell-cell resistance in ECV304 cells that were transfected with a plasmid containing a glucocorticoid-inducible promoter controlling expression of E-cadherin. Transmission electron microscopy confirmed tens of nanometer displacements between adjacent cells at a time point in which histamine maximally decreased cell-cell resistance.

scholarly journals Podosome formation impairs endothelial barrier function by sequestering zonula occludens proteins

2019 ◽  
Vol 235 (5) ◽  
pp. 4655-4666 ◽  
Author(s):  
Yan‐Ning Rui ◽  
Yawen Chen ◽  
Yichen Guo ◽  
Caroline E. Bock ◽  
John P. Hagan ◽  
...  
Keyword(s):  
Barrier Function ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Zonula Occludens

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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