Ridger, VictoriaKiss-Toth, EndreAlmalki, Salman2025-12-302025https://hdl.handle.net/20.500.14154/77753Thesis Abstract Cardiovascular disease (CVD) remains the leading cause of mortality worldwide, with atherosclerosis representing the primary underlying pathological process. While monocytes are known to play a crucial role in atherosclerosis initiation and progression through their recruitment to the vessel wall and differentiation into macrophages, the contribution of neutrophil to this process remains poorly understood. Our group has recently shown that neutrophils can exacerbate atherosclerosis through the release of neutrophil-derived microvesicles (NMVs). This thesis investigated the hypothesis that NMVs deliver specific miRNAs to monocytes, altering their function in atherosclerosis. Through small RNA sequencing, we identified 321 distinct miRNAs within NMVs, with 153 showing high expression levels > 100 transcripts per million (TPM). Comparison between NMVs produced from native low-density lipoprotein (nLDL) and oxidised LDL (oxLDL) stimulation revealed minimal differential expression, with only one miRNA (hsa-miR-23a-3p) showing significant difference (FDR < 0.05). Using confocal microscopy, we confirmed rapid internalisation of NMVs by monocytes within one hour of exposure. Integration of NMV miRNA profiles with published monocyte transcriptome data identified 18 key regulatory miRNAs highly abundant in NMVs but showing low or absent expression in monocytes. Target prediction analysis using TargetScan and miRTarBase revealed these miRNAs could potentially regulate thousands of genes, including those associated with atherosclerosis pathways. To investigate functional consequences of NMV uptake, we examined monocyte responses following NMV treatment. Inflammatory cytokines (IL-6, IL-8, IL-10) measured by ELISA and RT-qPCR showed no significant changes. Furthermore, transcriptome analysis revealed no differentially expressed genes in monocytes following NMV treatment (FDR < 0.05), indicating transcriptional homeostasis is maintained despite NMV internalisation. These findings demonstrate that NMV miRNA cargo remains remarkably stable regardless of neutrophil activation state, and that resting monocytes do not mount immediate transcriptional responses to NMV exposure. This suggests NMV mediated effects may operate through post- transcriptional mechanisms or require additional inflammatory signals, providing new insights into extracellular vesicle-mediated communication in atherosclerosis and identifying potential regulatory networks for future investigation in cardiovascular disease181enAtherosclerosisCardiovascular diseaseNeutrophil microvesiclesMonocyteRNA SequencingmiRNA SequencingThesis