Overexpression of human endothelial protein C receptor to regulate thrombosis and inflammation in solid organ and cellular xenotransplantation

© 2012 Dr. Kwong Fei Eddy LeeClinical xenotransplantation using pig solid organ and cellular grafts is a potential solution to the critical shortage of human donors for transplantation. Preclinical studies have shown that genetic modification of the donor pig will be essential for clinical success. The current genetically modified (GM) pig platform lacks αGal gene expression to delete the predominant porcine xenoantigen and has increased expression of complement regulatory proteins to successfully overcome hyperacute rejection (HAR). However, organ xenografts still succumb to acute vascular rejection (AVR). AVR is associated with coagulation dysfunction causing thrombotic microangiopathy and a frequently intractable consumptive coagulopathy. Contributing factors include failure to inhibit the strong humoral immune response against the xenograft, upregulation of tissue factor expression on circulating recipient cells, and molecular incompatibilities affecting key regulatory mechanisms. In the case of pig pancreatic islets, the prothrombotic surface of the xenograft triggers an ‘instant blood mediated inflammatory reaction’ (IBMIR), destroying up to 70% of the transplanted islets. It is postulated that overexpression of anti-coagulant and anti-inflammatory molecules on the endothelium of organ xenografts and on the surface of islet xenografts could protect them against thrombosis and inflammation. The technology is available to build such modifications onto the GM pig platform, and the current challenge is to identify the most effective molecules that could either work synergistically and/or have multiple regulatory functions. The candidate molecule examined in this thesis is the endothelial protein C receptor (EPCR), which has documented anti-coagulant, anti-inflammatory and cytoprotective properties. The aim of the project was to investigate whether overexpressing human EPCR on donor tissue has protective effects in clinically relevant small animal models of solid organ and islet transplantation. Whether co-expressing EPCR with thrombomodulin confers additional graft protection was also investigated. hEPCR transgenic mice were generated and characterised, revealing a measurable anti-coagulant and anti-inflammatory phenotype. The mice were then investigated in models of renal ischaemia-reperfusion injury (IRI), cardiac transplantation, and intraportal islet transplantation, covering a variety of mechanisms relevant to inflammation and thrombosis. The results demonstrated that hEPCR overexpression is effective in reducing renal IRI, provides some protection to cardiac xenografts, and protects intraportal islet grafts. Overall, therefore, this thesis supports hEPCR overexpression as a beneficial addition to the GM pig platform for clinical xenotransplantation