title: Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.
abstract: BACKGROUND
  The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay.
  
  
  METHODS
  Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC.
  
  
  RESULTS
  Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and antithrombin was determined. Microthrombi could not be detected histologically.
  
  
  CONCLUSIONS
  These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation.
authors.affiliation: Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Petersen
authors.forename: Wolf
authors.initials: W
authors.lastname: Ramackers
authors.forename: Andrea
authors.initials: A
authors.lastname: Lucas-Hahn
authors.forename: Erika
authors.initials: E
authors.lastname: Lemme
authors.forename: Petra
authors.initials: P
authors.lastname: Hassel
authors.forename: Anna-Lisa
authors.initials: AL
authors.lastname: Queisser
authors.forename: Doris
authors.initials: D
authors.lastname: Herrmann
authors.forename: Brigitte
authors.initials: B
authors.lastname: Barg-Kues
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Carnwath
authors.forename: Johannes
authors.initials: J
authors.lastname: Klose
authors.forename: Andreas
authors.initials: A
authors.lastname: Tiede
authors.forename: Lars
authors.initials: L
authors.lastname: Friedrich
authors.forename: Wiebke
authors.initials: W
authors.lastname: Baars
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schwinzer
authors.forename: Michael
authors.initials: M
authors.lastname: Winkler
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D006084:Graft Rejection / Q000276:immunology / Q000517:prevention & control*; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000098:blood supply / Q000276:immunology* / Q000502:physiology; D010477:Perfusion; D013552:Swine; D019076:Transgenes; D014183:Transplantation, Heterologous / Q000276:immunology*
chemical_list: D051547:Heme Oxygenase-1