title: Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-β1-induced permeability of centrally derived vascular endothelium.
abstract: Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.
authors.affiliation: Save Sight Institute, the University of Sydney, Australia.
authors.forename: Weiyong
authors.initials: W
authors.lastname: Shen
authors.forename: Shiying
authors.initials: S
authors.lastname: Li
authors.forename: Sook Hyun
authors.initials: SH
authors.lastname: Chung
authors.forename: Ling
authors.initials: L
authors.lastname: Zhu
authors.forename: Jason
authors.initials: J
authors.lastname: Stayt
authors.forename: Tao
authors.initials: T
authors.lastname: Su
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Couraud
authors.forename: Ignacio A
authors.initials: IA
authors.lastname: Romero
authors.forename: Babette
authors.initials: B
authors.lastname: Weksler
authors.forename: Mark C
authors.initials: MC
authors.lastname: Gillies
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002199:Capillary Permeability / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D062507:Claudin-5; D004730:Endothelium, Vascular / Q000254:growth & development / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D062507:Claudin-5; D008565:Membrane Proteins; D017027:Protein Tyrosine Phosphatases; D053773:Transforming Growth Factor beta1; D014443:Tyrosine; D014638:Vanadates; D000099152:Cadherin 5; C107466:CLDN5 protein, human