Tyrosine phosphorylation of vascular endothelial cadherin and the regulation of microvascular permeability.
BACKGROUND
Adherens junctions (AJ), by their association with the endothelial cytoskeleton, maintain microvascular barrier integrity. Phosphorylation states of AJ proteins, such as vascular endothelial (VE) cadherin, can potentially alter the interactions between component AJ proteins. Furthermore, AJ protein phosphorylation is susceptible to regulation by inflammatory mediators. We previously demonstrated the importance of VE cadherin in tumor necrosis factor (TNF)-mediated endothelial permeability. We now postulate that TNF-induced endothelial permeability is associated with tyrosine phosphorylation of VE cadherin.


METHODS
Confluent monolayers of human umbilical vein endothelial cells were exposed to saline solution, TNF-alpha (100 U/mL) or TNF and the Src-tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Permeability was assessed by fluorescein isothiocyanate-dextran flux. VE-cadherin phosphorylation was determined by immunoprecipitation and immunoblotting with antiphosphotyrosine antibody. Data are expressed as mean +/- SEM and analyzed by analysis of variance.


RESULTS
TNF-alpha-induced tyrosine phosphorylation of VE cadherin and increased intercellular gap formation. These changes were associated with increased endothelial-cell monolayer permeability, all of which were prevented by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Exposure to an inactive tyrphostin, AG9 (negative control), did not prevent TNF-induced endothelial permeability.


CONCLUSIONS
We conclude that tyrosine phosphorylation of VE cadherin is an important regulatory pathway associated with TNF-induced endothelial barrier dysfunction. Modulating AJ protein phosphorylation may provide targets for therapy during inflammation.
Nwariaku
Fiemu E
FE
Department of Surgery, University of Texas Southwestern Medical Center, Dallas, USA.
Liu
Zijuan
Z
Zhu
Xudong
X
Turnage
Richard H
RH
Sarosi
George A
GA
Terada
Lance S
LS
D015703:Antigens, CD; D000970:Antineoplastic Agents / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003911:Dextrans / Q000493:pharmacokinetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D006801:Humans; D010766:Phosphorylation; D011743:Pyrimidines / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors; D000099152:Cadherin 5
D015703:Antigens, CD; D000970:Antineoplastic Agents; D015820:Cadherins; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate; D011743:Pyrimidines; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; D019061:src-Family Kinases; D000099152:Cadherin 5; C412373:AG 1879; C015219:fluorescein isothiocyanate dextran