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. 1440The Nonstandard abbreviations used: endothelial cell (EC); galactose α1,3-galactose (αGal); nucleoside triphosphate diphosphohydrolase (NTPDase).

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 wildtype mice were then transplanted into streptozotocininduced 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. Diabetes 54:2109 -2116, 2005

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.

Injury to nerve tissue in the peripheral nervous system (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss of sensation from the tissue innervated by the harvested nerve. The challenges that persist with nerve repair have resulted in development of nerve guides or conduits from non-neural biological tissues and various polymers to improve the prognosis for the repair of damaged nerves in the PNS. This study describes the design and fabrication of a multimodal controlled pore size nerve regeneration conduit using polylactic acid (PLA) and (PLA):poly(lactic-coglycolic) acid (PLGA) fibers within a neurotrophin-enriched alginate hydrogel. The nerve repair conduit design consists of two types of PLGA fibers selected specifically for promotion of axonal outgrowth and Schwann cell growth (75:25 for axons; 85:15 for Schwann cells). These aligned fibers are contained within the lumen of a knitted PLA sheath coated with electrospun PLA nanofibers to control pore size. The PLGA guidance fibers within the nerve repair conduit lumen are supported within an alginate hydrogel impregnated with neurotrophic factors (NT-3 or BDNF with LIF, SMDF and MGF-1) to provide neuroprotection, stimulation of axonal growth and Schwann cell migration. The conduit was used to promote repair of transected sciatic nerve in rats over a period of 4 weeks. Over this period, it was observed that over-grooming and self-mutilation (autotomy) of the limb implanted with the conduit was significantly reduced in rats implanted with the full-configuration conduit compared to rats implanted with conduits containing only an alginate hydrogel. This indicates return of some feeling to the limb via the fully-configured conduit. Immunohistochemical analysis of the implanted conduits removed from the rats after the four-week implantation period confirmed the presence of myelinated axons within the conduit and distal to the site of implantation, further supporting that the conduit promoted nerve repair over this period of time. This study describes the design considerations and fabrication of a novel multicomponent, multimodal bio-engineered synthetic conduit for peripheral nerve repair. AbstractInjury to nerve tissue in the Peripheral Nervous System (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss ...

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.

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