Exploring the role of substrate stiffness in endothelial senescence

Romagnoli, Camilla (2025) Exploring the role of substrate stiffness in endothelial senescence. PhD thesis, University of Glasgow.

Abstract

The last two centuries have witnessed a remarkable rise in average human life expectancy, which has doubled in the most advanced countries. Cardiovascular diseases are among the leading causes of death in older adults. As the proportion of elderly people grows, the number of those affected by some form of cardiovascular disease is expected to continue increasing; hence, narrowing the gap between total and healthy lifespan becomes essential. One of the key hallmarks of ageing is cellular senescence, a stable state of cell cycle arrest. Endothelial cells are among the first to undergo senescence, and tissues with a high density of endothelial cells exhibit the highest levels of senescence. It is now widely recognised that the mechanical properties of the extracellular matrix strongly influence cellular behaviour and that these properties change with ageing. Arterial stiffness progressively increases with age, contributing to a higher risk of cardiovascular diseases. While various studies have explored the role of physical stimuli in senescence, much remains to be uncovered regarding the effect of stiffness on endothelial senescence.

We aim to address this gap by studying the role of stiffness on endothelial senescence and exploring the differences in response depending on the senescence inducer. To do so, we worked with human umbilical vein endothelial cells (HUVECs), growing them to monolayer state and inducing senescence either through treatment with the chemotherapeutic drug Doxorubicin or by passaging the cells to mimic therapy-induced senescence and replicative senescence, respectively. To assess senescence induction, we examined various hallmarks, including DNA damage, β-gal staining, cytokine secretion, cell cycle arrest, and proliferation. To evaluate the effect of substrate stiffness on the phenotype, we seeded the cells onto collagen-coated polyacrylamide hydrogels of two different stiffnesses (3 kPa and 30 kPa) as well as glass. Additionally, we analysed cellular morphology and adhesion, nuclear morphology and lamina properties, YAP nuclear translocation, and cell-cell adhesion proteins. Finally, we investigated the mechanical properties of these cells to determine their mechanical fingerprint.

In this study, we first established two effective methods for inducing senescence in HUVECs in vitro. For therapy-induced senescence using Doxorubicin, we optimised our protocol by initially testing a wide range of doses. The doses were first narrowed down based on toxicity and then further refined by assessing senescence hallmarks. For replicative senescence (RS), we first evaluated population doubling before performing the senescence assays.

Next, we produced and characterised polyacrylamide hydrogels to examine senescence markers as a function of substrate stiffness. Overall, we observed a substantial increase in senescence markers at increasing stiffness. In senescent populations on glass, we detected an increase in cell cycle arrest markers, β-gal staining, DNA damage, and cytokine secretion. RNA sequencing further revealed an upregulation of genes associated with cell-adhesion and leukocyte migration. In Doxorubicin-treated cells, the senescence-associated secretory phenotype (SASP) was more pronounced compared to RS cells, whereas the opposite was observed for p53 and DNA damage. Interestingly, on softer substrates, SASP was significantly reduced in Doxorubicin-treated cells compared to those on stiffer substrates.

To further characterise our model, we investigated how mechanosensing and mechanotransductive elements were altered by senescence in endothelial cells. In Doxorubicin-treated cells, we observed an increased aspect ratio and a larger cellular area compared to the control. The nuclear area appeared particularly enlarged, alongside increased YAP nuclear translocation. Regarding the nuclear lamina, lamin A/C invaginations were more pronounced, while lamin B intensity was reduced. Replicative senescent cells exhibited increased cellular area and stiffness. They also displayed a greater number of focal adhesions and reduced VE-cadherin and CD31 intensity. Additionally, they showed increased YAP nuclear translocation, decreased lamin B intensity, and a higher prevalence of lamin B and lamin A/C invaginations.

In summary, we propose a system for modelling endothelial senescence using two distinct methods and investigating the impact of substrates’ stiffness on senescence. We successfully modelled senescence and confirmed the detrimental effect of matrix stiffness on the senescent phenotype. Furthermore, we demonstrated that senescence not only affects cellular proliferation but also influences cellular morphology and adhesion, nuclear morphology and lamina properties, cell-cell interactions, and the mechanical fingerprint of cells. We also showed that the level of expression in the senescent markers, as well as the mechanobiology related changes, depend on the senescence inducer.

We believe this work provides an insightful overview of the impact of substrate stiffness on endothelial senescence. To the best of our knowledge, it is the first time this type of study compares two different inducers of senescence. We hope the results obtained will enable further research on the complex relationship between arterial stiffening and endothelial senescence, which may eventually help prevent cardiovascular disorders.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QR Microbiology; T Technology > T Technology (General)
• Colleges/Schools: College of Science and Engineering > School of Engineering > Biomedical Engineering
• Supervisor's Name: Vassalli, Professor Massimo, Gourdon, Professor Delphine and Salmeron-Sanchez, Professor Manuel
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85236
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 20 Jun 2025 13:39
• Last Modified: 20 Jun 2025 13:44
• Thesis DOI: 10.5525/gla.thesis.85236
• URI: https://theses.gla.ac.uk/id/eprint/85236