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

Abstract

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 HO-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 HO-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 HO-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.

CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells

“…See also Figures S3 A and S3C. (C) Adenosine production in OC primary cells, previously FACS-sorted into CD73 high and CD73 low cells, was measured as described by Chisci et al. (2017) .…”

The Role of Hydrogen Peroxide in Redox-Dependent Signaling: Homeostatic and Pathological Responses in Mammalian Cells

“…It restricts the infiltration of neutrophils into tissues, and thus protects it from inflammatory damage. Therefore, the coordinated action of CD39/CD73 axis results in the rapid conversion of extracellular ATP to adenosine, leading to the activation of a protective change of metabolism of cells, as we reported in a model of endothelial cells exposed to H 2 O 2 stimulus [ 95 ]. This protective metabolic change could have impact on immunological response: adenosine mainly mediates anti-inflammatory effects, while ATP in the extracellular space acts as a strong pro-inflammatory stimulus on T cells, dendritic cells, and neutrophils [ 85 ].…”

How to influence the mesenchymal stem cells fate? Emerging role of ectoenzymes metabolizing nucleotides

“…The P2Y2 and P2X2 receptor activities have been shown to be important for the differentiation of pluripotent stem cells into neurons expressing cholinergic and glutamatergic receptors (Jiang et al, 2017), and the function of the P2Y1 receptors has been attributed to stem cells mobilization and migration. The P2X7 receptor inhibition is suggested to limit the cell death of transplanted neural progenitor cells and to favor neurogenesis (Oliveira, Illes, & Ulrich, 2016 On the contrary, Chisci et al (2017) However, the molecular mechanisms underlying MSCs immunomodulation are numerous and incompletely understood. Some evidence exists that the immunomodulatory properties of MSCs may, at least partially, be mediated via the components of purinergic signaling (Caplan & Sorrell, 2015;Lee et al, 2014).…”