Human Endothelial Protein C Receptor Overexpression Protects Intraportal Islet Grafts in Mice.

Islet transplantation can potentially cure type 1 diabetes mellitus, but it is limited by a shortage of human donors as well as by islet graft destruction by inflammatory and thrombotic mechanisms. A possible solution to these problems is to use genetically modified pig islets. Endothelial protein C receptor (EPCR) enhances protein C activation and regulates coagulation, inflammation, and apoptosis. We hypothesized that human EPCR (hEPCR) expression on donor islets would improve graft survival and function. Islets from an hEPCR transgenic mouse line strongly expressed the transgene, and hEPCR expression was maintained after islet isolation. Islets were transplanted from hEPCR mice and wild-type (WT) littermates into diabetic mice in a marginal-dose syngeneic intraportal islet transplantation model. The blood glucose level normalized within 5 days in 5 of 7 recipients of hEPCR islets, compared with only 2 of 7 recipients of WT islets (P < .05). Transplanted hEPCR islets had better preserved morphology and more intense insulin staining than WT grafts, and they retained transgene expression. The improved engraftment compared with WT islets suggests that inflammation and coagulation associated with the transplant process can be reduced by hEPCR expression on donor tissue.

Altered glycosylation in donor mice causes rejection of strain-matched skin and heart grafts.

Differential protein glycosylation in the donor and recipient can have profound consequences for transplanted organs, as evident in ABO-incompatible transplantation and xenotransplantation. In this study, we investigated the impact of altered fucosylation on graft acceptance by using donor mice overexpressing human α1,2-fucosyltransferase (HTF). Skin and heart grafts from HTF transgenic mice were rapidly rejected by otherwise completely matched recipients (median survival times 16 and 14 days, respectively). HTF skin transplanted onto mice lacking T and B cells induced an natural killer cell-mediated innate rejection crisis that affected 50-95% of the graft at 10-20 days. However, in the absence of adaptive immunity, the residual graft recovered and survived long-term (>100 days). Experiments using "parked" grafts or MHC class II-deficient recipients suggested that indirect rather than direct antigen presentation plays a role in HTF skin graft rejection, although the putative antigen(s) was not identified. We conclude that altered glycosylation patterns on donor tissue can trigger a powerful rejection response comprising both innate and adaptive components. This has potential implications for allotransplantation, in light of increasing recognition of the variability of the human glycome, and for xenotransplantation, where carbohydrate remodeling has been a lynchpin of donor genetic modification.

Protective effects of transgenic human endothelial protein C receptor expression in murine models of transplantation.

Thrombosis and inflammation are major obstacles to successful pig-to-human solid organ xenotransplantation. A potential solution is genetic modification of the donor pig to overexpress molecules such as the endothelial protein C receptor (EPCR), which has anticoagulant, anti-inflammatory and cytoprotective signaling properties. Transgenic mice expressing human EPCR (hEPCR) were generated and characterized to test this approach. hEPCR was expressed widely and its compatibility with the mouse protein C pathway was evident from the anticoagulant phenotype of the transgenic mice, which exhibited a prolonged tail bleeding time and resistance to collagen-induced thrombosis. hEPCR mice were protected in a model of warm renal ischemia reperfusion injury compared to wild type (WT) littermates (mean serum creatinine 39.0 ± 2.3 µmol/L vs. 78.5 ± 10.0 µmol/L, p < 0.05; mean injury score 31 ± 7% vs. 56 ± 5%, p < 0.05). Heterotopic cardiac xenografts from hEPCR mice showed a small but significant prolongation of survival in C6-deficient PVG rat recipients compared to WT grafts (median graft survival 6 vs. 5 days, p < 0.05), with less hemorrhage and edema in rejected transgenic grafts. These data indicate that it is possible to overexpress EPCR at a sufficient level to provide protection against transplant-related thrombotic and inflammatory injury, without detrimental effects in the donor animal.

Protective effects of recombinant human antithrombin III in pig-to-primate renal xenotransplantation.

Delayed rejection of pig kidney xenografts by primates is associated with vascular injury that may be accompanied by a form of consumptive coagulopathy in recipients. Using a life-supporting pig-to-baboon renal xenotransplantation model, we have tested the hypothesis that treatment with recombinant human antithrombin III would prevent or at least delay the onset of rejection and coagulopathy. Non-immunosuppressed baboons were transplanted with transgenic pig kidneys expressing the human complement regulators CD55 and CD59. Recipients were treated with an intravenous infusion of antithrombin III eight hourly (250 units per kg body weight), with or without low molecular weight heparin. Antithrombin-treated recipients had preservation of normal renal function for 4-5 days, which was twice as long as untreated animals, and developed neither thrombocytopenia nor significant coagulopathy during this period. Thus, recombinant antithrombin III may be a useful therapeutic agent to ameliorate both early graft damage and the development of systemic coagulation disorders in pig-to-human xenotransplantation.

Hyperacute rejection of vascularized heart transplants in BALB/c Gal knockout mice.

Pig-to-primate vascularized xenografts undergo hyperacute rejection (HAR). This results from pre-formed xenoreactive antibodies directed against galactose-alpha1,3-galactose (alphaGal) in the donor organ and activation of the complement cascade. We describe an in vivo murine model of HAR using a BALB/c mice system devoid of histocompatibility or complement differences between donor and recipient to investigate in isolation, the effects of alphaGal epitope and anti-alphaGal antibody interactions in causing rejection of vascularized heart transplants. Gal KO mice were immunized with rabbit red blood cell membranes to induce high anti-alphaGal antibody titers that were predominantly IgM by ELISA (enzyme-linked immunosorbent assay). When alphaGal-expressing mice hearts were transplanted heterotopically into these recipients (n= 12), 67% of grafts rejected within 24 h, the majority within 16 h with histological features of HAR. In contrast, none of the grafts in the non-immunized Gal KO recipient control group (n=11) underwent HAR. Interestingly, approximately 50% of the remaining grafts in both the immunized and non-immunized Gal KO recipient group were rejected between 7 and 27 days by a rejection process characterized by a dense infiltrate of macrophage/monocytes, perivascular cuffing and tissue destruction similar to recent descriptions of delayed xenograft rejection (DXR). In addition, some grafts (21.5%) continued to survive in the immunized Gal KO recipients despite the presence of anti-alphaGal antibody and normal complement activity and these showed well-preserved myocardium when harvested whilst still functioning well at days 30 or 90. No rejection was seen when Gal KO donors were used in this system (n=4), nor when alphaGal-expressing BALB/c hearts were transplanted into alphaGal-expressing BALB/c recipients (n=5). This in vivo small animal model offers the opportunity to test a variety of strategies to overcome HAR prior to more resource intensive pig-to-primate studies, and may provide insights into the processes similar to DXR and accommodation.

Naturally acquired anti-alpha Gal antibodies in a murine allograft model similar to delayed xenograft rejection.

Antibodies directed against galactose-alpha1,3-galactose (alphaGal) are believed to play an important role in the pathogenesis of delayed xenograft rejection (DXR). This study was designed to determine whether alpha1,3-galactosyltransferase-deficient (Gal KO) mice can naturally acquire a sufficient anti-alphaGal titre to cause the delayed type rejection of alphaGal-expressing hearts. Gal KO mice of various ages were assessed for anti-alphaGal antibody levels. alphaGal-expressing hearts were transplanted heterotopically into these mice and monitored daily. Rejecting and surviving hearts were evaluated histologically. In Gal KO mice greater than 6-month-old, 64% had an anti-alphaGal antibody titre above the background level. When wild-type alphaGal-expressing hearts were transplanted into this group, 45% of grafts rejected within 5 to 13 days. Histological examination of the rejected hearts displayed marked tissue damage and an inflammatory infiltrate of predominantly macrophage/monocytes. Surviving grafts showed preserved morphology. Like humans, Gal KO mice naturally develop anti-alphaGal antibodies with age. The titre in these mice was sufficient to cause a "delayed-type" rejection of a significant proportion of alphaGal-expressing cardiac grafts. This model thus provides an opportunity to investigate the role of naturally acquired anti-alphaGal antibodies in the pathogenesis of DXR.