Let-7d-5p miRNA/statin Dual therapy: A Novel Approach to Targeting Smooth Muscle Cell Dysfunction in Atherosclerosis

Atherosclerotic Cardiovascular Disease (AsCVD) remains a significant global health concern and a major contributor to morbidity and mortality, even with advancements in drug therapies and lifestyle interventions. AsCVD is characterised by progressive dyslipidaemia and inflammation, leading to the deposition of fatty streaks within the blood vessel wall. A combination of environmental exposures, genetic risk factors and different cellular processes are believed to contribute to AsCVD progression. Current therapies for the management of AsCVD include the use of statins which promote an overall reduction in cholesterol burden, additional LDL/HDL modifying therapies, anti- platelet and anti-hypertensive agents. However, a significant proportion of the population remain at a substantial risk of AsCVD as they cannot achieve the desirable LDL-C levels despite these therapies. This thesis investigates whether a combined statin and miRNA-based therapy can synergise to reduce AsCVD risk, with a particular focus on the smooth muscle cell (SMC) layer. SMC-derived cells are among the most abundant cell populations within atherosclerotic plaque lesions, with SMC plasticity believed to be a key player in driving atherogenesis and plaque stability. Therefore, therapies targeting SMC plasticity represent a novel approach to manage this disease. miRNA therapy has gained popularity and been explored to study the prevention of different diseases, and our group has previously explored the role of let-7 miRNA in prevention and progression of AsCVD. We previously demonstrated that let-7 miRNA can regulate inflammatory pathway activation in vascular cells and tissues. Building on these earlier studies, the current aims of this thesis were to explore let-7d-5p miRNA therapy (overexpression and inhibition) in combination with statin treatment in human aortic SMCs (HAoSMCs). Detailed global transcriptomics analysis was performed to study the downstream networks regulated by let-7d-5p miRNA and statin treatments in HAoSMCs. Further investigations were performed in SMCs cultured from patient carotid plaque explants as well as iPSC-derived blood vessel organoids (BVOs). As described in the thesis, let-7d-5p miRNA overexpression led to a significant attenuation of key inflammatory genes in HAoSMCs including IL-6, IL-1β, ICAM1, VCAM1 that were upregulated in response to TNF-α. Statin treatments (atorvastatin and lovastatin) also exhibited a significant attenuation of key8 inflammatory genes as well as led to an upregulation of let-7d-5p miRNA levels in HAoSMCs. RNA-seq studies showed that a dual let-7d-5p miRNA and statin treatment had the greatest inhibitory effect and led to the attenuation of inflammatory pathways including TNF-α, IL-6 and IL-1β. Studies using iPSC-derived BVO cultures and treatments showed that BVOs respond to the pro-inflammatory stimulus TNF-α as well as statin treatments, suggestive of a potential tool to study and understand AsCVD. In summary, the data presented here shows that miRNA-based therapies can synergise alongside conventional statin treatment to ameliorate inflammation within vascular SMCs. We also provide evidence that iPSC-derived BVOs can be exploited as a tool to understand therapeutic responses to drugs in a complex multicellular model system, paving the way for BVOs to be utilised as a tool to study AsCVD disease progression.