Abstract
BackgroundPorcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.Methodology/Principal FindingsConfluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm2 and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001).Conclusions/SignificanceThis novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.
Highlights
Human antibodies against the porcine galactose 1,3α-galactose (Gal) antigen and subsequent complement activation trigger endothelial injury and thrombosis, resulting in hyperacute rejection of wild type (WT) porcine organs. [1,4,5,6,7,8,9,10] Organs from pigs that do not express Gal (GalTKO) have significantly improved survival,[1,4,11,12,13] which is further enhanced by transgenic expression of human proteins, such as the complement regulatory protein CD46 (GalTKO.hCD46)
Initial experiments were performed by perfusing WT 14259 cell line endothelia with heparinized human blood to recapitulate hyperacute xenograft injury mediated by anti-Gal antibodies, and develop a reference condition for maximal thrombosis to serve as a positive control (Fig. 1A & S1 Video)
Primary WT porcine aortic endothelial cell (PAEC) resulted in even more rapid thrombosis and obstruction of the proximal channel; loss of flow due to rapid channel occlusion presumably accounts for significantly lower overall adhesion and total thrombus volume in assays using WT primary PAECs (S3 Table)
Summary
Pig to human xenotransplantation is a potential means of addressing the critical shortage of organs available for transplantation.[1,2,3,4] human antibodies against the porcine galactose 1,3α-galactose (Gal) antigen and subsequent complement activation trigger endothelial injury and thrombosis, resulting in hyperacute rejection of wild type (WT) porcine organs. [1,4,5,6,7,8,9,10] Organs from pigs that do not express Gal (GalTKO) have significantly improved survival,[1,4,11,12,13] which is further enhanced by transgenic expression of human proteins, such as the complement regulatory protein CD46 (GalTKO.hCD46). [14] Despite these advances, thrombosis remains a critical process associated with xenograft injury.[2,4,12,13,15,16,17,18,19,20] Current strategies to control acute thrombosis include adding human thromboregulatory transgenes, such as for endothelial protein C receptor, to the existing genetic background (GalTKO. hCD46.hEPCR).[1,4,21] As such, the ability to mechanistically characterize thrombus formation is critical to studying the effects of genetic and pharmacologic interventions on xenograft injury.Previously described models used for xenotransplantation and general thrombosis research have limited ability to investigate the mechanisms contributing to thrombotic xenograft injury under physiologic conditions. While ex- and in-vivo whole organ studies are valuable and clinically translatable models of pig-to-human transplantation, they involve multiple incompletely understood pathways, and lack the cellular resolution to dynamically study events occurring at the blood—endothelial interface.[11,12,13,15,22] static in-vitro assays lack physiologic shear-flow,[10,23,24,25,26] which is a critical condition for a range of interactions occurring at the endothelial interface,[27,28,29,30] including many involved in thrombus formation, stabilization and resolution.[27,29,31,32,33,34,35].
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