title: Investigating the effects of platelet derived growth factor on the microRNA cargo content and function of vascular smooth muscle cell-derived extracellular vesicles
Creators: name: Pashova, Antoniya
Publisher: name: University of Glasgow
Year: 2023

description: Invasive revascularisation procedures such as coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) are used to treat advanced atherosclerosis. The CABG procedure is the most common type of cardiac surgery performed in the UK and the great saphenous vein (SV) remains the most widely used conduit largely due to availability and length. However, recent reports suggest that approximately 50% of all SV grafts fail at 10 years following CABG and the incidence of arterial restenosis, following drug-eluting stent (DES) implantation remains around 10%. Vascular smooth muscle cells (VSMCs) proliferation- and migration- driven neointimal formation post CABG- and/or PCI- induced vascular injury has been considered a major pathological driver of both vein graft disease (VGD) and in-stent restenosis (ISR) ultimately leading to treatment failure. Accumulating evidence suggests that extracellular vesicles (EVs) play a significant role in intercellular communication in both physiological and pathophysiological conditions, and due to their promising therapeutic potential, EVs are currently being extensively studied as disease mediators and therapeutic delivery vehicles. Understanding the different mechanisms that may be involved in the regulation of VSMC-driven neointimal formation remains an important step towards successfully developing therapeutic strategies that could improve clinical outcomes associated with both CABG and PCI. Therefore, the primary aim of this thesis was to study the effect of VSMC-derived EVs on recipient cell responses with a particular focus on EV-mediated autocrine regulation of VSMC. Since abnormal signalling mediated by platelet-derived growth factor (PDGF), has been implicated in the development of neointimal formation post vascular injury, prolonged PDGF stimulation of human SV smooth muscle cells (HSVSMCs) was used to model the pathological conditions under which neointimal lesions develop in an in vitro setting. Following the optimisation of an EV purification method, EVs were obtained from the conditioned culture media (CCM) of HSVSMCs +/- PDGF stimulation and successfully characterised in terms of size, concentration, protein content and morphological appearance. It was found that while EV size and morphology remained unaltered, PDGF stimulation of HSVSMCs resulted in increased EV secretion. Further studies determined that PDGF was not packaged into EVs after prolonged PDGF treatment of HSVSMCs. Next, following small RNA sequencing (RNAseq) analysis, it was found that PDGF stimulation of HSVSMCs induced significant changes in their EV cargo. Six known differentially expressed miRNAs: miR-24-3p, miR-409-3p, miR-21-5p, let-7A-5p, miR-1-3p and miR-224-5p, were found to be significantly upregulated in PDGF EVs compared to control EVs. Four out of six differentially expressed miRNAs (miR-24-3p, miR-224-5p, miR-409-3p and, let-7A-5p) were also successfully validated by qRT-PCR analysis. Gene set enrichment analysis (GSEA) revealed that miR-24-3p and miR-224-5p miRNAs may be involved in the regulation of biological processes such as cell proliferation, migration, and apoptosis - all previously implicated in the development of neointimal formation. Next, the effects of miR-24-3p or miR-224-5p EVs on HSVSMC proliferation, migration and cell viability were assessed. It was found that, while neither miR24-3p EVs nor miR-224-5p EVs had any significant effect on PDGF-induced HSVSMC proliferation and cell viability compared to naïve EVs, miR-224-5p EVs significantly inhibited EV-depleted foetal bovine serum (FBS)-induced HSVSMC migration compared to both naïve EVs and miR-24-3p EVs through an unknown mechanism. Finally, it was found that serum EVs from mice with carotid artery ligation induced vascular injury do not significantly differ compared to EVs from control mice in terms of size and concentration. However, qRT-PCR analysis of miR-24- 3p and miR-224-5p suggested that the expression of both miRNAs was significantly upregulated in serum EVs isolated from injured mice at day 14 and day 5 after surgery respectively compared to control mice. Overall, these studies provide evidence that prolonged PDGF signalling in HSVSMCs significantly alters the EV population secreted by those cells in terms of concentration of particles released and miRNA expression profile. Additionally, the demonstrated ability of miR-224-5p EVs to supress HSVSMC migration compared to naïve EVs and miR-24-3p EVs provides valuable insights into an alternative mechanism of EV-mediated regulation of VSMCs with promising potential for future therapeutic studies.