Transference of recombinant VE-cadherin cytoplasmic domain alters endothelial junctional integrity and porcine microvascular permeability.
VE-cadherin constitutes endothelial adherens junctions through a homophilic binding of its extracellular domain and by the anchoring of its intracellular domain to actin cytoskeleton via catenins. The aim of this study was to determine the functional importance of VE-cadherin-cytoskeleton association in the maintenance of endothelial junctional integrity. A recombinant VE-cadherin cytoplasmic domain (rVE-cad CPD) was expressed in E. coli and purified through Ni-NTA spin columns. Immunoprecipitation assays showed that rVE-cad CPD was able to bind beta-catenin in vitro and to compete with endogenous VE-cadherin for binding of beta-catenin in human umbilical vein endothelial cells. A significant increase in the transendothelial flux of albumin was observed in the endothelial cell monolayers transfected with rVE-cad CPD. Importantly, transfection of rVE-cad CPD into intact isolated coronary venules markedly elevated the albumin permeability of the venular endothelium. In addition, immunofluorescence microscopic analysis revealed a conformational change of VE-cadherin from a uniform, continuous distribution along the cell membrane under control conditions to a diffuse, stitch-like pattern after rVE-cad CPD transfection. The effects were likely due to an attenuated anchorage of endogenous VE-cadherin to the cytoskeleton, as evidenced by a decreased partitioning of VE-cadherin in the detergent-insoluble cytoskeletal pool. The results suggest that the intracellular association of VE-cadherin with beta-catenin-linked cytoskeleton is essential to the maintenance of endothelial junctional integrity and microvascular permeability.
Guo
Mingzhang
M
Cardiovascular Research Institute, Department of Surgery, Texas A&M University System Health Science Center, 702 Southwest H. K. Dodgen Loop, Temple, TX 76504, USA.
Wu
Mack H
MH
Granger
Harris J
HJ
Yuan
Sarah Y
SY
D000818:Animals; D015703:Antigens, CD; D015820:Cadherins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002199:Capillary Permeability / Q000502:physiology*; D002478:Cells, Cultured; D003593:Cytoplasm; D003599:Cytoskeleton / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008833:Microcirculation / Q000502:physiology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013552:Swine; D014471:Umbilical Veins / Q000166:cytology; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D011994:Recombinant Proteins; D000099152:Cadherin 5