Abstract
Vascular smooth muscle cell (VSMC) ageing involves osteo-chondrogenic differentiation, vascular calcification, and increased extracellular matrix (ECM) stiffness. SRY-Box Transcription Factor 9 (Sox9), expressed in the vasculature, regulates ECM related gene expression and is mechanosensitive in chondrocytes. The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprising Nesprin 1/2, SUN1/2, and Lamin A/C, plays a vital role in mechanosignaling and its dysregulation has been associated with accelerated vascular ageing. In this study, the relationship between VSMC aging, Sox9-driven ECM regulation, and the impact of LINC complex integrity on ECM composition was investigated both in vitro and in vivo.Examination of aortic patient tissue samples revealed a strong correlation between Sox9 in the aortic media and the senescence marker p16. Though not a direct regulator of senescence, Sox9 exhibited mechanosensitivity in senescent VSMCs on rigid matrices. Modifying Sox9 levels in VSMCs influenced ECM stiffness and composition, prompting senescent cell reversion to a proliferative state with reduced senescent markers p16, p21, and inflammatory marker IL6. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a novel Sox9 target that increases ECM stiffness during VSMC senescence through increased extracellular vesicle (EV) release.
The LINC complex showed disruption in vascular senescence, characterized by an upregulation of the Kash domain of Nesprin 1 (Kash1), downregulation of SUN2, and Kash1 mechanosensitivity in senescent VSMCs. Experimental LINC disruption through Kash1 overexpression (Kash1 OE) resulted in Sox9 dimerization but did not lead to significant changes in ECM composition.
However, it did induce alterations in heterochromatin organization, reduced cellular contractility, and increased cellular proliferation. Assay for Transposase- accessible chromatin with sequencing (ATAC)- and RNA-seq analysis of young Kash1 OE VSMCs suggested that increased cell proliferation might be driven by the upregulation of Forkhead Protein M1 (Foxm1), a cell cycle related transcription factor. Foxm1 was found to be upregulated in both young and senescent Kash1 OE VSMCs, as well as in the neointima areas of older patients scoring higher American Heart Association (AHA) plaque scores. Additionally, Kash1 OE increased mineral deposition and osteogenic marker expression in young VSMCs.
These findings underscore Sox9's role in increasing ECM stiffness during VSMC aging through LH3 upregulation and enhanced EV release, emphasizing ECM's significance in governing VSMC phenotype. LINC complex disruption during VSMC aging, along with increased cell proliferation and pro-osteochondrogenic changes driven by Kash1 OE, provides insights into previously unexplored mechanisms behind vascular remodelling.
| Date of Award | 1 Mar 2024 |
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| Original language | English |
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| Supervisor | Cathy Shanahan (Supervisor) & Susan Cox (Supervisor) |