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Identification of a vascular smooth muscle cell transition associated with fibrous cap formation and induced by thrombin receptor activation https://doi.org/10.17863/CAM.119870 Taylor, James Jorgensen, Helle Introduction: Vascular smooth muscle cells (VSMCs) accumulate in atherosclerotic plaques and display phenotypic plasticity that influences both plaque growth and stability. The fibrous cap, a stabilising feature of plaques, contains an abundance of VSMC-derived cells. However, the cellular transitions and regulatory mechanisms underlying fibrous cap formation remain incompletely understood. The overarching aim of my thesis was to delineate the phenotypic transitions of VSMCs in atherosclerosis, with focus on transitions associated with fibrous cap formation. Methods and results: Single-cell RNA sequencing (scRNA-seq) of lineage-traced VSMCs revealed a disease-specific VSMC state characterised by the co-expression of contractile genes, fibrous cap-associated extracellular matrix (ECM) components (including fibrillar collagens and elastin) and NOTCH3, a factor that has recently been linked to fibrous cap formation through VSMC regulation. Computational trajectory analysis inferred that this fibrous cap-associated VSMC (fcVSMC) state is derived from a plastic intermediate VSMC population marked by Ly6a/SCA1, Vcam1 and Lgals3 expression – a population that I term “intermediate modulated VSMCs” (imVSMCs). Multi-colour VSMC lineage tracing and immunostaining analyses indicated that NOTCH3+ fcVSMCs and VCAM1+ imVSMCs were present in the same clonal VSMC populations in both atherosclerotic and injured arteries, suggesting that transition between these states occurs in vivo and is a conserved mechanism across disease models. Further, kinetic analysis in vascular injury suggested that VCAM1+ imVSMCs give rise to NOTCH3+ fcVSMCs. In atherosclerosis, fcVSMCs were predominantly localised to fibrous caps rather than lesion cores, supporting a role in cap formation and plaque stability. To identify regulatory pathways of the imVSMC to fcVSMC transition, I combined scRNA-seq trajectory analysis with spatial transcriptomics of human atherosclerotic plaques. From this, protease-activated receptor-1 (PAR1) emerged as a candidate regulator of fcVSMC generation. PAR1 was expressed in VSMCs within the fibrous caps of human plaques and its activation by thrombin in cultured human VSMCs induced expression of contractile, ECM and other genes characteristic of the fcVSMC state. Conclusions: My findings uncover a VSMC transition associated with fibrous cap formation in atherosclerosis that is also present during vascular injury-induced remodelling. This study identifies PAR1 as a regulator of this transition and thus as a promising therapeutic target for enhancing plaque stability by promoting the transition to a matrix-producing, fibrous cap- associated VSMC state. This work advances our understanding of VSMC plasticity and its potential in stabilising atherosclerotic plaques. University of Cambridge 2024-11-21 Thesis Doctoral Doctor of Philosophy (PhD) British Heart Foundation https://www.repository.cam.ac.uk/handle/1810/386848 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/dd378539-9632-4b30-ac6e-6160f814b587/download e43d907c6f4ba200c8daa7d8b969289e https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2d99c258-9907-43ac-bd3f-98f2b08a36bf/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Cardiovascular