Acute myocardial infarction rapidly increases blood neutrophils (<2 hours). Release of neutrophils from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 minutes) release of extracellular vesicles (EVs), which are enriched in vascular cell adhesion molecule-1 (VCAM-1) and miRNA-126, and are thus a potential mechanism for communicating with remote tissues. Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to their arrival at injured tissue. Ischemic myocardium leads to the generation and release of EC-derived-EVs bearing VCAM-1. EC-EV delivery to the spleen alters inflammatory gene and chemokine protein expression, and mobilises neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EV and showed that its deletion removed the ability of EC-EV to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Our findings show a novel mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve, independent of classical chemokine signalling, and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Background Myocardial infarction (MI) induces activation of immune cells and alters their gene expression en route to the injured myocardium but the underlying mechanisms coordinating immune cell programming following MI remain unknown. Plasma extracellular vesicle (EV) numbers are elevated in MI, correlate with the extent of myocardial injury and mobilises immune cells from the splenic reserve to peripheral blood. Here, we describe the role of plasma EV-microRNAs (miRs) in the modulation of peripheral blood mononuclear cell (PBMC) transcriptomes post-MI. Methods PBMCs were exposed to plasma EVs followed by whole transcriptome RNA-sequencing. Plasma EVs were isolated by size-exclusion chromatography and ultra-centrifugation (2 hours at 120,000 x g) from patients presenting with ST-segment elevation MI (STEMI) (N=9) and non-STEMI (NSTEMI) (N=11) control patients. Plasma EVs were characterised by western blot and Nanoview for EV markers CD9 and CD63, transmission electron microscopy (TEM) for morphology and Nanoparticle Tracking Analysis for size and concentration. High sensitive C-reactive protein (hs-CRP) and PCSK9 were determined in plasma by ELISA and compared to plasma EV number using Pearson's correlation. Plasma EV-miRs were measured by Agilent microarray and miR-mRNA putative targets assessed by TargetScanHuman. Results Plasma EVs were positive for EV markers CD9 and CD63, displayed typical EV morphology by TEM and had a heterogeneous size and concentration distribution profile as determined by Nanoparticle Tracking Analysis. Plasma EV number correlated significantly with hs-CRP at presentation (R2= 0.20 and P&lt;0.05). miRNA array analysis revealed STEMI plasma-EVs contained significantly more miR-4487 (P&lt;0.001), miR-6511b-5p (P&lt;0.001), miR-4508 (P&lt;0.001) vs NSTEMI control plasma-EVs at the time of injury. STEMI-plasma-EVs induced differential gene expression in PBMCs vs. NSTEMI-control-plasma-EVs. Gene set enrichment analysis (GSEA) showed STEMI-plasma-EVs upregulated pro-inflammatory pathways including: interferon-α (IFN-α) (P&lt;0.01), IFN-γ (P&lt;0.01), tumour necrosis factor-α (TNF-α) (P&lt;0.01) and interleukin-6 (IL-6)-STAT3 signalling of the acute phase response (P&lt;0.05). miR-4487 (P&lt;0.001) and miR-6511-5p (P&lt;0.05) predicted mRNA targets were significantly enriched in PBMC transcriptomes following treatment with STEMI plasma-EVs. Conclusions Plasma EVs mediating immune cell transcriptional programming following MI by promoting inflammatory pathways in PBMCs is a novel finding. Targeting PBMCs with EVs may allow modulation of the immune response following myocardial injury, to perturb inflammatory immune mediated damage following ischaemic injury. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation Centre of Research Excellence Awards, British Heart Foundation Project Grant, Novo Nordisk Fonden the Tripartite Immunometabolism Consortium and Wellcome Institutional Strategic Support Fund (ISSF)

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Background The myocardium of neonatal mice is able to regenerate after myocardial infarction (MI), whereas in adults the formation of scar predominantly occurs following heart injury. Macrophages are involved in the fibrotic response in adult mouse hearts, but also required for successful regeneration in neonates. Recent work has demonstrated that macrophages directly contribute collagen to scar formation following MI. Furthermore, neonatal and adult cardiac macrophages have divergent transcriptional responses to injury. Here, we describe differential transcriptomes and signalling pathways of these functionally distinct neonatal resident cardiac macrophages. Methods Hearts from neonatal P1, P7 and adult CD1 mice (n=3 per group) were digested with collagenase to produce a single cell suspension. Macrophages were isolated by FACS and identified as Ly6G, F4/80+, LyChi/lo cells. Macrophage whole transcriptomes were measured by Illumina RNA-sequencing. Transcript abundance was quantified from raw reads by Salmon and analysis of differentially expressed (DE) genes was carried out with DESeq2. Gene Ontology (GO) enrichment analysis of DE genes was performed with PANTHER. Genes were ranked according to p-value for differential expression, then these ranked genes were used for Gene Set Enrichment Analysis (GSEA) to detect enriched gene sets from the Molecular Signatures Database. Results RNA-sequencing of transcriptomes from neonatal P1, P7 and adult mouse macrophages from hearts highlighted distinct gene expression profiles. The greatest differences were between P1 vs. adult (4,494 differentially expressed (DE) genes at p&lt;0.05) and P7 vs. adult (3,347 DE genes), whereas P1 and P7 macrophages were relatively similar (478 DE genes). A set of 171 genes was found to be DE in P1 vs. P7 and adult macrophages. This P1-specific gene set was highly enriched for GO terms including matrix disassembly (29-fold enrichment, p&lt;0.05) and regulation of chemokine production (12-fold enrichment, p&lt;0.05). GSEA analysis highlighted key functional pathways that were differentially regulated in P1 macrophages, including oxidative phosphorylation and glycolysis, the E2F transcription factors and cell cycle regulators Myc and p53. Conclusions We highlight key genes and pathways distinct to resident neonatal cardiac macrophages to determine the basis for the regenerative capacity of these cells. Interestingly, while genes associated with extracellular matrix were previously shown to be altered after MI in neonatal macrophages, similar differences were also observed here in the basal state of resident neonatal macrophages. We also identified transcriptional and cell cycle regulators linked to the programming and regenerative capacity of these macrophages. The functional differences found in neonatal macrophages might represent potential targets for novel therapeutics. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Novo Nordisk Fonden the Tripartite Immunometabolism Consortium (NNF15CC0018486), British Heart Foundation Centre of Research Excellence Awards (RE/13/1/30181)

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