Abstract 4355626: Endothelial CD39 Deficiency Alters Purinergic Signaling and Increases Susceptibility to Ischemia-Reperfusion Injury

Introduction: Endothelial cells (ECs) regulate vascular tone and thrombosis through purinergic signaling, with CD39 (ENTPD1) hydrolyzing extracellular nucleotides. While elevated CD39 activity has been linked to both pro- and anti-thrombotic effects, our prior work showed that endothelial-specific CD39 deletion enhances vasoreactivity and confers thromboresistance. We hypothesized that this altered purinergic signaling may predispose to increased ischemia-reperfusion (I/R) injury. Methods: We analyzed heart tissue from wild-type and EC-specific CD39 knockout (EC-cKO) mice. CD31-enriched cardiac cells underwent single-cell RNA sequencing (scRNA-seq; 10X Genomics). Data were analyzed using MAST for differential gene expression, and Ingenuity Pathway Analysis (IPA) was used for pathway enrichment. Surface marker expression was validated by flow cytometry. I/R injury was induced by 45-minute coronary ligation followed by 24-hour reperfusion. Infarct size was measured with Coomassie blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining and quantified by blinded observers using Fiji (ImageJ). Results: scRNA-seq revealed significant transcriptional changes in EC-cKO endothelial cells, including upregulation of P2ry2 , Nos3 (eNOS), Klf2 , and Klf4 . Vascular smooth muscle cells showed no significant changes. IPA predicted the activation of hypoxia and vasodilation pathways and inhibition of endothelial adhesion. Flow cytometry confirmed increased endothelial P2y2 surface expression. Following I/R injury, EC-cKO mice exhibited significantly larger infarcts than controls, indicating heightened vulnerability to ischemic damage. Conclusions: Endothelial CD39 deletion disrupts purinergic signaling, upregulating P2y2 and activating vasodilatory and hypoxia-related pathways while reducing endothelial adhesion. Despite baseline thromboresistance, these changes may impair endothelial integrity and increase susceptibility to I/R injury. Our findings underscore a dual role for endothelial CD39 in maintaining vascular homeostasis and protecting against ischemic stress.