Simultaneous overexpression of human E5NT and ENTPD1 protects porcine endothelial cells against H2O2-induced oxidative stress and cytotoxicity in vitro

Ischemia-reperfusion injury (IRI) and oxidative stress still limit the survival of cells and organs in xenotransplantation models. Ectonucleotidases play an important role in inflammation and IRI in transplantation settings. We tested the potential protective effects derived by the co-expression of the two main vascular ectonucleotidases, ecto-5’-nucleotidase (E5NT) and ecto nucleoside triphosphate diphosphohydrolase 1 (ENTPD1), in an in vitro model of H2O2-induced oxidative stress and cytotoxicity. We produced a dicistronic plasmid (named pCX-DI-2A) for the co-expression of human E5NT and ENTPD1 by using the F2A technology. pCX-DI-2A-transfected porcine endothelial cells simultaneously overexpressed hE5NT and hENTPD1, which were correctly processed and localized on the plasma membrane. Furthermore, such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 as shown by the efficient catabolism of pro-inflammatory and pro-thrombotic extracellular adenine nucleotides along with the enhanced production of the anti-inflammatory molecule adenosine. Interestingly, we found that the hE5NT/hENTPD1 co-expression system conferred protection to cells against H2O2-induced oxidative stress and cytotoxicity. pCX-DI-2A-transfected cells showed reduced activation of caspase 3/7 and cytotoxicity than mock-, hE5NT- and hENTPD1-transfected cells. Furthermore, pCX-DI-2A-transfected cells showed decreased H2O2-induced production of ROS as compared to the other control cell lines. The cytoprotective phenotype observed in pCX-DI-2A-transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Our data add new insights to the protective effects of the combination of hE5NT and hENTPD1 against oxidative stress and constitute a proof of concept for testing this new genetic combination in pig-to-non-human primates xenotransplantation models