title: Gene transfer of the adaptor Lnk (SH2B3) prevents porcine endothelial cell activation and apoptosis: implication for xenograft's cytoprotection.
abstract: BACKGROUND
  Targeting protective gene expression to porcine endothelium by genetic modification of the donor could improve xenograft survival by controlling cell activation and death. We previously found that, in endothelial cells (EC), the molecular adaptor Lnk (SH2B3) is a negative regulator of cytokine signaling. We also have shown that Lnk is upregulated in pig EC (PAEC) in response to tumor necrosis factor-α (TNF) and xenoreactive natural antibodies (XNA) binding. The present study investigated whether ectopic expression of human Lnk using gene transfer may be efficient to control signaling pathways associated with inflammation and apoptosis in porcine aortic endothelial cells (PAEC).
  
  
  METHODS
  Endothelial cells cultures were established from WT and Gal(-/-) pigs and transduced with a recombinant adenovirus encoding human Lnk. Phenotype and functions of transduced PAEC expressing Lnk were analyzed by flow cytometry, western blot and XNA and complement-dependent assays. The regulatory functions of Lnk toward inflammation were assessed in TNF-activated EC, and the protective functions were tested toward TNF-induced apoptosis and anoïkis. Apoptosis assays included DNA content analysis and caspase-3/7 activity.
  
  
  RESULTS
  First, we found that as a result of adenoviral transduction, human Lnk was efficiently and similarly expressed in EC from WT or Gal(-/-) pigs. Lnk expression or EC transduction caused no significant change in the binding of XNA (IgG and IgM) to PAEC and has no effect on complement activation and C5b-9 formation. We demonstrated that expression of human Lnk efficiently inhibits TNF signaling in PAEC and decreases VCAM-1 induction by 46.3 ± 1.2% compared to controls (n = 6, **P < 0.01). Furthermore, expression of Lnk was associated with a significant decrease in the percentage of caspase-3/7-dependent apoptosis caused by TNF in the presence of actinomycin D and also reduces cell death by anoïkis by 25.0 ± 1.9% compared to controls (n = 5, **P < 0.01).
  
  
  CONCLUSIONS
  Together, these findings indicate that the signaling adaptor Lnk is effective to reduce PAEC activation and apoptosis. Thus, Lnk is a potential candidate for the modulation of signaling pathways to protect vascular EC from inflammation in xenotransplantation.
authors.affiliation: INSERM, UMR634, Nantes, France.
authors.forename: Mathias
authors.initials: M
authors.lastname: Chatelais
authors.forename: Julie
authors.initials: J
authors.lastname: Devallière
authors.forename: Cesare
authors.initials: C
authors.lastname: Galli
authors.forename: Béatrice
authors.initials: B
authors.lastname: Charreau
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D023102:Anoikis; D017209:Apoptosis; D002478:Cells, Cultured; D019610:Cytoprotection; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology*; D005700:Galactosyltransferases / Q000235:genetics; D055786:Gene Knockout Techniques; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013552:Swine; D014161:Transduction, Genetic / Q000379:methods*; D014183:Transplantation, Heterologous / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C490778:SH2B3 protein, human; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine