title: Alpha-galactosyl-mediated activation of porcine endothelial cells: studies on CD31 and VE-cadherin in adhesion and signaling.
abstract: BACKGROUND
  Ligation of alpha-galactosyl epitopes on endothelial cells by naturally occurring human antibodies causes hyperacute rejection in porcine-to-human xenotransplantation. The alpha-galactosyl-specific lectin Bandeiraea simplicifolia isolectin B4 (IB4) has been reported to trigger endothelial "gap" formation and tyrosine phosphorylation of an unidentified 130-kDa protein. We have studied two 130-kDa junctional adhesion molecules, CD31 and VE-cadherin, in porcine aortic endothelial cells (PAECs) during IB4-mediated activation. The cellular distribution of these molecules, their susceptibility to tyrosine phosphorylation, and their capacity to bind IB4 or natural human antibodies have been determined.
  
  
  METHODS
  Porcine CD31 and VE-cadherin were cloned. Recombinant proteins and monoclonal antibodies were prepared. The distribution and phosphorylation of CD31 and VE-cadherin in confluent PAECs activated with IB4 or human serum were studied by confocal microscopy and Western blotting, respectively.
  
  
  RESULTS
  IB4 caused rapid redistribution of CD31 and VE-cadherin away from cell junctions and tyrosine-phosphorylation of CD31 but not VE-cadherin. A monoclonal antibody to CD31 also triggered tyrosine phosphorylation of this molecule, but brief exposure of PAECs to normal human serum did not. Tyrosine-phosphorylated CD31 complexed with SHP2 and other unidentified phosphoproteins. Both IB4 and natural human antibodies bound to porcine CD31 but not to VE-cadherin. Cell adhesion tests showed that porcine and human CD31 are functionally incompatible.
  
  
  CONCLUSIONS
  Endothelial cell retraction during IB4-mediated activation of PAECs is associated with rapid loss of CD31 and VE-cadherin from cell junctions. CD31 becomes strongly tyrosine-phosphorylated and forms a cell signaling complex, which may have a significant role in the response of the xenograft vascular endothelium.
authors.affiliation: The Babraham Institute, Cambridge, United Kingdom.
authors.forename: K
authors.initials: K
authors.lastname: Nasu
authors.forename: A
authors.initials: A
authors.lastname: Whyte
authors.forename: S J
authors.initials: SJ
authors.lastname: Green
authors.forename: P C
authors.initials: PC
authors.lastname: Evans
authors.forename: P J
authors.initials: PJ
authors.lastname: Kilshaw
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D015703:Antigens, CD; D015820:Cadherins / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D037102:Lectins / Q000276:immunology / Q000494:pharmacology; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine; D014183:Transplantation, Heterologous / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D007074:Immunoglobulin G; D037102:Lectins; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D014409:Tumor Necrosis Factor-alpha; D000099152:Cadherin 5; C045475:Griffonia simplicifolia lectins