2379Endothelial cell derived extracellular vesicles mediate immune cell deployment from the spleen and transcriptional programming following acute myocardial infarction

Abstract

Abstract Background Acute myocardial infarction (AMI) induces transcriptional activation of monocyte en route to the injured myocardium, possibly through interactions involving plasma liberated extracellular vesicles (EVs), which are enriched for proteins and microRNAs (miRNAs) post-AMI. Neutrophils are the first immune cells to arrive at sites of injury and mediate further damage to the ischaemic myocardium. Here, we describe neutrophil-deployment from the spleen in AMI and by endothelial cell (EC) derived-EVs. Methods Patients presenting AMI provided informed consent as part of the Oxford Acute Myocardial Infarction Study (OxAMI). Plasma EV were isolated by differential ultra-centrifugation (120,000g, 2 hours) followed by washing and characterised for: morphology using transmission electron microscopy (TEM), size and concentration profiling by Nanoparticle Tracking Analysis, EV markers (TSG101, ALIX, CD9, HSP70) by western blot, and miRNAs by RT-qPCR. Mouse and human EC were used in vitro to derive EC-EV under control conditions or after inflammatory stimulation with tumour necrosis factor-alpha (TNF-α) (10ng/mL) and from CRISPR-edited miRNA-126 knock-out ECs. EC-EVs were tail vein injected into wild-type mice or exposed to primary human peripheral blood neutrophils in vitro. Results Patients presenting with AMI (N=15) have significantly more plasma EV at time of injury vs a 6-month follow-up measurement (2.2-fold more, P=0.008). Plasma EVs at the time of AMI presentation correlate significantly with the extent of ischaemic injury (R=0.046, P=0.006) and plasma neutrophils (R=0.37, P=0.017). Experimental AMI in wild-type mice induced a significant increase in peripheral blood neutrophils and a simultaneous reduction in splenic-neutrophils, suggesting splenic-neutrophil deployment (P=0.004). Human plasma EV are enriched for vascular cell adhesion molecule-1 (VCAM-1) and EC-associated miR-126 post-AMI (Akbar et al 2017). miRNA-126-mRNA targets are significantly over represented when compared to neutrophil Gene Ontology terms for: degranulation (P<0.001), activation (P<0.001), chemotaxis (P=0.008) and migration (P=0.008). Human and mouse EC release more EV after inflammatory stimulation and show enrichment for miRNA-126. CRISPR-edited miRNA-126 deficient human EC express more VCAM-1 (P<0.001) and release more EC-EVs (P<0.001). EC-EV exposure to primary human neutrophils alters inflammatory gene expression (IL-6 (P<0.05), CCL7 (P<0.001) and CCL18 (P<0.001)). EC-EV tail vein injected into wild-type mice mobilise splenic-neutrophils to peripheral blood (P<0.001). Conclusions Neutrophil deployment from the spleen is a novel finding in acute injury and interactions with EC-EV may mediate their splenic liberation and transcriptional programming following AMI, en route to the injured myocardium. The splenic neutrophil reserve may be a novel therapeutic target in AMI to modulate the inflammatory response before recruitment of cells to sites of injury. Acknowledgement/Funding British Heart Foundation Project Grant and Centre for Research Excellence Awards (RE/13/1/30181), Nuffield Benefaction for Medicine and ISSF