Glycocalyx dynamics of genetically modified arterial and venous porcine endothelial cells after xenogeneic activation
Anastasia Milusev1,2, Alain Despont1, Mitra Gultom1, Jane Shaw1, Nikolai Klymiuk3,4, Elisabeth Kemter4, Eckhard Wolf4, Nicoletta Sorvillo1, Robert Rieben1.
1Department for BioMedical Research, Medical Faculty, University of Bern, Bern, Switzerland; 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; 3Technical University Munich, Munich, Germany; 4Ludwig Maximilian University Munich, Munich, Germany
Introduction: Organs from genetically modified pigs have been successfully used in pre-clinical xenotransplantation research, but rejection is still a major hurdle to xenograft survival. Endothelial cells (ECs) and their sulfated, sugar-rich surface known as the glycocalyx are the first barrier encountered by the recipient’s immune system, making them a target for rejection. While glycocalyx shedding is known to occur in different inflammatory conditions and during transplant rejection, it is currently not well known how the glycocalyx of genetically modified ECs changes in a xenotransplantation environment. We therefore aimed to characterize the glycocalyx composition and particularly heparan sulfate dynamics of arterial and venous genetically modified porcine ECs.
Methods: We investigated the glycocalyx dynamics, particularly heparan sulfate (HS), and complement activation on genetically modified porcine arterial and venous ECs (Galα1,3Gal KO, transgenic for human CD46 and thrombomodulin, GTKO/hCD46/hTM) in vitro in a 2D microfluidic model. Cells were activated with human serum, human TNFα or both stimuli to evaluate HS shedding and complement C3b/c deposition during xenogeneic endothelial activation.
Results: We have previously shown that wild type arterial but not venous porcine ECs shed HS after xenogeneic activation with human serum or human TNFα. We now show that both arterial and venous GTKO/hCD46/hTM ECs are protected against HS shedding and complement activation, as shown by unscathed HS and 75-85% reduction in C3b/c deposition compared to wild type ECs. Interestingly, this protection was abolished on arterial GTKO/hCD46/hTM ECs after simultaneous perfusion with both human serum and human TNFα. HS shedding and increased C3b/c deposition were observed under these conditions. Although venous GTKO/hCD46/hTM ECs also showed increased C3b/c deposition there was still no HS shedding.
Conclusions: In an inflammatory xenotransplantation setting, GTKO/hCD46/hTM ECs revert to a proinflammatory phenotype which is similar to that of wild type ECs. This suggests that during transplant rejection GTKO/hCD46/hTM ECs are more susceptible to inflammation and strategies to prevent inflammation could help to reduce endothelial activation and help to prevent xenograft rejection.
