*Purpose: The thromboregulatory and anti-inflammatory potential of human CD39 (hCD39) makes it a gene of interest in xenotransplantation. In porcine endothelium, CD39 activity is generally lower than in human cells and membrane bioactivity can be further lost with inflammatory stress. We hypothesized that transgenic hCD39 overexpression in endothelial cells (EC) could modulate cell activation and death.

*Methods: ECs from wild type (WT; n=1), TKO.hCD39 (EGEN-1331; n=3), TKO (EGEN-1536; n=3) pigs and one human deceased donor were cultured in vitro. EC were activated for 3 hours using LPS (1ug/mL), Ionomycin, and PMA, and/or ATP, and 1 hour ATP (200µM) at 37°C. FACS measured hCD39 expression and viability, malachite green assay measured ATP hydrolysis, and porcine-specific ELISA measured IL-6, IL-27, and IL-1β.

*Results: TKO.hCD39 ECs expressed higher levels of hCD39 when compared to hECs at baseline. After 3 hours activation, hCD39 expression by TKO.hCD39 ECs substantively decreased from baseline, while human ECs exhibited no loss (Fig 1). High levels of IL-27 were detected in supernatants after EC activation, but hCD39 transgene did not limit IL-6 and IL-1β release (Fig 2). Interestingly, IL-27 production by porcine EC did not further impact hCD39 transgene expression in activated TKO.hCD39 cultures.

*Conclusions: hCD39 expression by TKO.hCD39 ECs rapidly decreases after exposure to LPI/ATP and cannot improve cell death rates, nor stimulate production of IL-27, IL-6 and IL-1β under these conditions. Further work will focus on the effects of similar transgenes in genetically engineered pigs to determine immunomodulatory pathways in-vivo.

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