Abstract 251: The Role of Adenosine Derived From Mesenchymal Stromal Cell on Neutrophil Extracellular Trap Formation in Myocardial Ischemia Reperfusion

Abstract

Introduction: In response to tissue damage or infection, neutrophils expel their chromatin into the extracellular space in a thrombotic and inflammatory process called neutrophil extracellular traps(NETs). There is increasing evidence that NETs play a role in myocardial ischemia/reperfusion (MI/R) injury. NETs are found in thrombi aspirated from coronary arteries, atherosclerotic plaques, and deep vein thrombosis. However, nothing is known about the regulation of NETs in vivo. We hypothesized that adenosine, well know for anti-inflammatory effects, reduces NET formation. After MI/R, adenosine is made from conversion of ATP released from damaged cells. Methods: Neutrophils were isolated from human venous blood and stimulated to produce NETs by PMA, a PKC agonist and is well known to induce robust NET formation and serves as a positive control. NETs were quantified by Sytox green fluorescent to quantify extracellular DNA and immunohistochemical staining for citrullinated histone H3, a specific marker of NETs. Results: Neutrophils treated with PMA increased NET production (1.81fold vs unstimulate groups, p<0.001, n=27-28). Neutrophils treated with adenosine (10uM) had a 30.2% decrease NET formation (p<0.001, n=15). There are four adenosine receptors called A1, A2a, A2b, and A3. We used agonists and antagonists’ strategy to check probable receptors involved in suppression NETs signal. We find both A2a receptor agonist-CGS 21680 and A2b agonist-BAY 60-6583 can suppress NETs formation (29.6% and 29.3% respectively p<0.001, n=5-6). But only A2a antagonist-ZM241385 can reverse adenosine’s inhibition (13.8%v.s.24.7%, p<0.05, n=6). Conclusion: Adenosine can downregulate NET formation in vitro likely through A2a receptor activation. Future studies will investigate A2a receptor using genetic knock out, as well as in vivo significance of NET inhibition in MI/R.