Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys

Petersen, Björn GND; Ramackers, W.; Lucas-Hahn, Andrea GND; Lemme, Erika GND; Hassel, Petra GND; Queißer, Anna-Lisa GND; Herrmann, Doris GND; Barg-Kues, Brigitte GND; Carnwath, Joseph Wallace GND; Klose, J.; Tiede, A.; Friedrich, L.; Baars, W.; Schwinzer, R.; Winkler, M.; Niemann, Heiner GND

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-a-mediated apoptosis assay. Methods: Transgenic and non-transgenic PAEC were tested in a TNF-a-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- 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-a-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-? 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.

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Petersen, Björn / Ramackers, W. / Lucas-Hahn, Andrea / et al: Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. 2011.

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