Introduction: The development of multigene modifications in pigs represents a major breakthrough in the efforts to overcome interspecies immune barriers in pig-to-human xenotransplantation. Currently, selecting the combination of genetic modification with the best functional phenotype to prevent immune rejection to xenografts remains a challenge. As the first barrier to the recipient’s immune response, endothelial cells (EC) play a crucial role in the immunological processes determining the fate of xenografts. Therefore, elucidating the impact and immunological consequences of genetic modifications on EC and their sugar-rich glycocalyx layer on the cellular level is necessary.
Method: Here, we compare the functional effect of two different genetic modification sets, GTKO/hCD46/hTBM (3GM) and GTKO/CMAHKO/hCD46/hCD55/hCD59/hHO1/hA20 (7GM), on porcine EC grown under physiological flow conditions. We perfused the EC with normal human serum and recombinant human TNF-a to simulate inflammatory and xenogeneic conditions. Thereafter, we characterized the complement activation by the deposition of C3b/c on the surface of EC. We also analyzed the clot formation after each treatment by perfusing EC with recalcified human plasma spiked with fluorescently labeled human fibrinogen. To see the influence of each genetic modification set on the carbohydrate profiles of EC glycocalyx, we performed N-glycan and glycosphingolipid profiling using a multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (CGE-LIF) detection.
Results: We showed that both transgenic ECs are protected from complement activation when perfused with human serum, but 7GM-EC showed significantly less complement deposition in comparison to 3GM-EC when simultaneously perfused with TNF-a and human serum. Interestingly, human serum-treated 7GM-EC also showed better prevention of clot formation. However, when perfused with TNF-a or human serum +TNF-a, 3GM-EC provided a more significant protection against clot formation than 7GM-EC. We also observed distinct N-glycan and glycosphingolipid profiles in the two transgenic ECs, possibly influencing the distinct complement and coagulation states of each cell type in xenotransplantation settings.
Conclusion: Taken together, we showed that genetic modification sets used to overcome rejection in xenotransplantation provide differential protection against complement activation and coagulation dysregulation. A complete set of xenoantigen deletion and expression of human complement, coagulation, and inflammation regulatory genes may be necessary to prevent rejection and prolong the survival of xenografts in xenotransplantation.