Over-Expression of Ecto-5'-Nucleotidase in Pig Endothelial Cells

Ecto-5'-nucleotidase (E5'N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides into immunosuppressive adenosine. Species differences and especially lO-fold higher activity of E5'N in human endothelial cells (EC) than in pig EC could be important barrier for xenotransplantation.The major aim ofmy thesis is evaluation whether expression of human E5'N on pig EC is able to attenuate cell death mediated by components ofhuman blood responsible for delayed xenograft rejection (DXR). A pig cell line was transfected with human E5'N and efficiency assessed with flow cytometry and nucleotide breakdown assays using cell monolayers and lysates. Transfected cells were >95% positive for human E5'N/There was a massive increase in E5'N activity in transfected pig EC lysates and intact cells using extracellu~ar AMP as the substrate. Adenosine production from the breakdown of ATP was also significantly higher in transfected cells, proving E5'N to be the rate-limiting enzyme in adenosine production by pig EC. Incubation of transfected cells with AMP showed a time-dependent induction of the antiapoptotic protein Bcl-2 mediated via AI receptors, and subsequent protection of these cells from hydrogen peroxide-mediated apoptosis through AI receptors. Human natural killer (NK) cells were significantly less cytotoxic towards transfected than non-transfected pig EC. This effect was abrogated by an inhibitor of E5'N and mimicked by prior incubation of NK cells with adenosine. Supernatants from transfected cells also significantly inhibited platelet aggregation and expression of E5'N attenuated platelet adhesion to EC compared to nontransfected cells. However, transfection with E5'N did not protect cells from antibody and complement-mediated cytotoxicity. Functional expression of human E5'N in pig EC provided significant protection from apoptosis, NK cell-mediated lysis and platelet adhesion and aggregation, the main mechanisms of xenograft r~jection once hyperacute rejection has been overcome. E5'N is thus a potential candidate for. engineering of transgenic animals for xenotransplantation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo