Introduction: Multigene modifications in donor pigs have significantly advanced xenotransplantation by overcoming interspecies innate immune barriers. A prominent model includes α1,3-galactosyltransferase-knockout pigs expressing human CD46 and thrombomodulin (3GM), which have demonstrated promising life-supporting graft function in preclinical settings. As the initial interface between graft and host, endothelial cells (ECs) are critical regulators of complement activation, coagulation, and inflammation. Here, we investigate how these genetic modifications modulate complement responses in pig ECs during xenogeneic and inflammatory conditions.
Methods: Primary ECs from 3GM pigs were cultured under physiological shear stress and perfused with normal human serum (NHS) or recalcified citrated human plasma to assess complement and coagulation activation, respectively. Inflammatory conditions were simulated using recombinant human TNF-α (rhTNF-α) perfusion. Antibody (IgG, IgM) and complement deposition (C1q, C3b/c, and C5b-9) and coagulation markers (clot formation time) were assessed, and bulk RNA-seq was performed to investigate transcriptomic changes.
Results: 3GM ECs showed significantly reduced antibody and complement deposition following NHS perfusion, as well as better anticoagulant properties upon citrated human plasma exposure, indicating effective regulation of xenogeneic complement and coagulation pathways. However, this protection was reduced under rhTNF-α-induced inflammation conditions. Interestingly, the increased complement deposition under inflammatory conditions was not attributed to increased deposition of IgG, IgM, or C1q, although differences in endothelial glycocalyx dynamics were observed. Transcriptomic analysis revealed that 3GM ECs exhibit reduced expression of key proinflammatory, coagulation, and complement-related genes (e.g., VCAM1, CCL2, C1QBP, SERPINE1) compared to wild-type (WT) ECs. In addition, upon xenogeneic activation, 3GM ECs displayed minimal transcriptomic changes and distinct responses to that of WT ECs. In contrast, rhTNF-α-induced inflammation led to more aligned gene expression profiles between 3GM and WT ECs, where upregulation of immune and defense response pathways was observed, potentially contributing to increased complement and coagulation dysregulation.
Conclusion: Our findings highlight that 3GM pig ECs were able to effectively prevent complement activation in xenogeneic settings. However, this protection is compromised under inflammatory conditions. These results suggest that incorporating additional anti-inflammatory transgenes may be necessary to maintain immune quiescence and improve graft survival in clinical xenotransplantation.