Abstract

Introduction Atherosclerosis is the major underlying cause of heart attack and stroke and, as such, understanding the underlying pathological mechanisms of the disease remains a priority. Whilst neutrophils are the most abundant circulating leukocyte, they are rarely detected within developing plaques. Neutrophil microvesicles (NMVs), large (>0.1µm) extracellular vesicles derived from the plasma membrane, were shown to increase miR-155 in endothelial cells (ECs) at atheroprone sites. This led to exacerbation of plaque formation in a mouse model of atherosclerosis. We hypothesise that NMVs contain other microRNA (miRNA) that may influence atherosclerosis initiation and progression. Methods NMV were isolated from human peripheral blood neutrophils that were stimulated with 10 ng/mL native (n)LDL, 10 ng/mL oxidised (ox)LDL, or PBS only (unstimulated control). Small RNAs were isolated from NMV using miRNeasy mini kit (Qiagen, Germany) and quantity and quality of isolated RNA determined using NanoPhotometer® N60 spectrophotometer (Geneflow, UK) and a 2100 Bioanalyser (Agilent, UK). Small RNA sequencing analysis was performed using Illumina sequencing platform by Novogen, UK. Sequencing data was processed to obtain the clean reads that aligned to the human reference genome. Data was further processed and visualised using R software. Results 757 miRNAs were detected in NMVs. Of these, 527 miRNAs were expressed in all NMV groups, 55 uniquely expressed in NMVs from n/oxLDL stimulated neutrophils and 36 only in unstimulated control NMVs. The most abundant miRNA in all samples was miR-148a-3p, a miRNA previously shown to enhance plaque formation. High levels of miR-155 were also detected in all samples. Conclusion NMVs have been found to contain a high abundance of miRNAs, with some expression dependent on the stimulus used for induction of MV release. Target and pathway analysis of these data are ongoing. miRNA previously shown to play a role in plaque formation were among the most highly abundant within NMV suggesting that NMV delivery of miRNA to atherosclerotic plaques may play an important role in exacerbating plaque formation. Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this poster.