Abstract 512: Regulation of Neutrophil Extracellular Traps (NETs) by Mesenchymal Stromal Cell Derived Adenosine

Abstract

Introduction: Mesenchymal stromal cells (MSCs) reduce the infiltration of leukocytes into the heart after ischemia reperfusion injury (MI/R). Inhibition of the surface ectonucleotidase CD73 prevents this anti-inflammatory effect. CD73 is a key step in a pathway that converts adenine nucleotides (ATP, ADP, AMP) into adenosine, which in turn exerts vasodilatory and immunosuppressive effects. Neutrophil extracellular trap (NET) formation is a thrombotic and inflammatory process involved in coronary occlusion and myocardial damage. We hypothesized that adenosine may be a key regulator of NET formation and that CD73-dependant adenosine production by MSCs reduces NETs after MI/R. Methods: Neutrophils isolated from venous blood of healthy donors by percoll gradient density centrifugation were cultured with MSCs for in vitro studies. MSCs were encapsulated in aliginate by electrostatic encapsulation and secured to the epicardium with PEG-maleimide hydrogel at the time of reperfusion. This biomaterial strategy supports and maintains viable cells in the vicinity of injury for 1 week. 24 hours after injury, hearts were perfused, excised, and digested and NETs quantified by flow cytometry. Results: Treatment of neutrophils with adenosine or the adenosine receptor agonist NECA reduces NET formation in vitro . Co-culture of MSCs with neutrophils also prevents NET formation. Inhibition of CD73 activity on MSCs with the antagonist APCP prevents the anti-NET activities of MSCs. In vivo , encapsulation and placement of MSCs on the epicardial surface at the time of MI/R reduces myocardial NETs 24 hours after MI/R. Treatment of MSCs with APCP prior to implantation reduces the anti-NET effects of MSCs, as well as functional benefits. Conclusions: There is growing evidence of the importance of NETs in cardiac ischemia-reperfusion injury but little is known of the regulation of NETs in vivo . Our data suggests that adenosine exerts a strong anti-NET effect and that CD73-generated adenosine from MSCs may be capable of in vivo anti-inflammatory and anti-NET regulation. Future studies are focused on genetic techniques to knock down CD73 activity on MSCs, direct implication of NETs on cardiovascular pathophysiology, and non-cellular methods to deliver CD73 in MI/R.