The transgenic expression of human CD39 on murine islets inhibits clotting of human blood.

Platelet activation is believed to play an important role in the triggering of thrombosis of human blood by pig islets. We used a transgenic mouse model to investigate whether overexpression of CD39 (ecto nucleoside triphosphate diphosphohydrolase 1 [ENTPD1], EC 3.6.1.5), an ectonucleotidase that degrades the platelet agonists ATP, could interfere with this process. Islets isolated from CD39 transgenic mice showed 2.4-fold higher NTPDase activity than wild-type controls. When incubated with human blood, these islets significantly delayed clotting time compared to wild type islets (7.9 +/- 0.89 min versus 4.3 +/- 0.77 min, P = 0.007). Importantly, expression of human CD39 in the islets of transgenic mice had no deleterious effect on glucose metabolism. These results suggest that transgenic expression of human CD39 does not interfere with islet function and may be a useful strategy to inhibit thrombosis induced by intraportal administration of islet xenografts.

Overexpression of glutathione peroxidase with two isoforms of superoxide dismutase protects mouse islets from oxidative injury and improves islet graft function.

Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD + Gpx-1 + Cu/Zn SOD > extracellular SOD + Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/reoxygenation. Islets from transgenic or control wild-type mice were then transplanted into streptozotocin-induced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single- and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts.

Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.

Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.

Enhanced expression of glutathione peroxidase protects islet beta cells from hypoxia-reoxygenation.

The survival of pancreatic islet beta-cell xenografts and allografts may be affected by damaging reactive oxygen and nitrogen species generated during hypoxia-reoxygenation. Peroxynitrite, which is formed from superoxide and nitric oxide, appears to be an important mediator of beta-cell destruction. The intracellular antioxidant enzymes glutathione peroxidase-1 (Gpx-1) and copper-zinc superoxide dismutase (CuZn SOD) detoxify peroxynitrite and superoxide, respectively. The aim of this study was to examine whether enhanced expression of Gpx-1 and/or CuZn SOD protected NIT-1 mouse insulinoma cells from hypoxia-reoxygenation injury. Stable transfectants expressing human Gpx-1 or CuZn SOD were isolated and tested for their resistance to hydrogen peroxide (H(2)O(2)) and menadione, which generates superoxide intracellularly. Clones expressing one or both enzymes were subjected to hypoxia in glucose-free medium for 18 h, followed by reoxygenation in complete medium for 1.5 h. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction assay. Increases of up to two fold in Gpx or total SOD activity protected NIT-1 cells from H(2)O(2) and menadione. Expression of Gpx-1 significantly increased NIT-1 survival following hypoxia-reoxygenation (viability 65 +/- 9% vs. control 15 +/- 3%, P < 0.001) but CuZn SOD expression had no effect (15 +/- 1%). Expression of both enzymes was no more protective (60 +/- 6%) than expression of Gpx-1 alone. Genetic manipulation of islet beta cells to increase expression of Gpx-1 may protect them from oxidative injury associated with the transplantation procedure.