Identifying post-translational modifications in vascular smooth muscle cell ageing and matrix stiffness response

Steward, Oliver (2025) Identifying post-translational modifications in vascular smooth muscle cell ageing and matrix stiffness response. Masters thesis, University of East Anglia.

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Abstract

We find ourselves in an increasingly ageing population and this has brought about an increase in the prevalence of age-related diseases. Cardiovascular disease (CVD) is the deadliest age-related disease in the world. The aorta is the largest artery in the body and carries oxygenated blood from the heart to the rest of the body. It changes shape in response to blood pressure to maintain a hydrostatic pressure reservoir and ensure there is enough pressure to move the blood around the entire body. This ability to change in shape is called compliance. Compliance is facilitated by the composition of the extracellular matrix (ECM), elastin provides the ability to stretch and recoil and collagen provides strength to prevent the aorta from over-stretching. However, as we age, the aorta undergoes ECM remodelling which increases aortic wall stiffness and reduces its compliance. Vascular smooth muscle cells (VSMCs) are the predominant cell type in the aortic wall, they sense increased aortic wall stiffness and further accelerate aortic dysfunction. Proteins are a critical component of all cell types, they are responsible for almost all cellular functions including regulating cell structure and support, metabolism, transport within cells and cell signalling. Proteins can be post-translationally modified (PTM), this is the process of chemical changes in the proteins that occur alter protein modification. This can result in changes to the protein structure and function.

This study aims to identify key post-translational modifications involved in both matrix stiffness response and ageing. This was achieved by growing VSMCs on 2, 12 and 72 kPa hydrogels to model matrix stiffness and using passage 8 versus passage 16 to model ageing. Additionally, to mimic ageing in cells an SiRNA knockdown of FACE1, which converts pre-lamin A into lamin A was carried out to mimic the pre-lamin A accumulation that occurs in ageing. Key genes upregulated in matrix stiffness response were chosen and validated in VSMCs, these genes were chosen as they were upregulated by matrix stiffness in a published bulk mRNA dataset of human aortic VSMCs. These were NAT10, CHEK2, ANXA3 and PDLIM1. Western blot analysis of these genes in cultured human aortic VSMCs showed that there was a significant increase in NAT10 and PDLIM protein levels in response to increased stiffness. ANXa3 and Chk2 also trended towards upregulation in response to stiffness increases. NAT10 and Chk2 were also upregulated in aged VSMCs.

Microtubules can withstand the mechanical pressures exerted on the aorta and provide mechanical support to VSMCs. They are post-translationally modified to increase stability enhancing their ability to withstand pressure changes. Microtubules are made of an alpha and beta heterodimer, and these can be acetylated to increase stability. Total and acetylated alpha-tubulin were investigated in both 12 vs 72 kPa hydrogels to observe stiffness response changes and ageing through passage 8 vs passage 16 cells. There was no change in acetylation levels in stiffness response or ageing. Additionally, the microtubule stabilising protein Tau was investigated in mimicked ageing and there were no observed changes in Tau 100276251 phosphorylation levels. Finally, modifications of H3K9 were investigated. These were investigated due to their involvement in regulating inflammation-specific response genes which respond to MMPs that are involved in the remodelling of the ECM.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
Depositing User:	Nicola Veasy
Date Deposited:	24 Jun 2025 09:32
Last Modified:	24 Jun 2025 09:32
URI:	https://ueaeprints.uea.ac.uk/id/eprint/99692
DOI:

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