Abstract 11662: Extracellular Vesicle (EV)-Derived PiRNA, ImEX-Pi, is Protective in a Rat Model of Ischemia/Reperfusion Injury

Abstract

Introduction: Cardiosphere-derived cells (CDCs ) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. CDCs exert their effect through the secretion of EVs, lipid-bilayer nanoparticles laden with bioactive molecules. Among the bioactive cargo identified in these immortalized CDC (imCDC)-derived EVs (imCDC-EV) are piwi RNAs (PiRNAs). PiRNAs bind Piwi proteins (members of the argonaute family) and regulate both the epigenome and transcriptome. The role of piRNAs (in particular EV-derived piRNAs) in modulating gene expression remains undescribed. Here we describe the role of a PiRNA highly enriched in imCDC-EVs (imEX-Pi). Methods: EVs were prepared by 24 hours hypoxic conditioning followed by normoxic recovery for three cycles. EVs were isolated from conditioned media using ultrafiltration. Fractions were analyzed for particle size, concentration and piRNA content. In vitro, bone marrow derived-macrophages (BMDM) were exposed to imCDC-EV, imEX-Pi and vehicle for transcriptomic analysis. In vivo, myocardial ischemia-reperfusion (I/R) injury was induced in 8-10-week-old Wistar-Kyoto female rats by LAD coronary ligation for 45 minutes followed by reperfusion. Twenty minutes later, imCDC-EV, imEX-Pi, scramble or vehicle were injected into the left ventricle with an aortic cross-clamp. Cardiac Troponin I levels, infarct size and circulating monocytes were assessed at 24 and 48 hrs post I/R. Results: RNA sequencing showed that piRNAs are enriched in both CDC-EV and imCDC-EV. ImEX-Pi was one of the most highly-expressed non-coding RNAs. In vitro , imEX-Pi altered the transcriptome of BMDMs compared to vehicle. This included upregulation of inflammatory and cell death pathways. In a rat model of I/R injury, imEX-Pi reduced infarct size and cardiac troponin levels compared to vehicle- and scrambled- treated animals. ImEX-Pi also significantly decreased circulating monocytes at 24 hours followed by a significant increase at 48hrs. Conclusions: We show that imEX-Pi is a key mediator of imCDC-EV therapeutic efficacy and that the principle target cells may be macrophages. This is further rationalized by the critical role that macrophages play in the injury and resolution response.