A Bioengineered Approach to Study The Mechanobiology of Endothelial Cells in Response to Cyclic Stretch and Substrate Stiffness

Vascular ageing results in mechanical and structural changes to the vascular wall causing loss of vessel elasticity and decreased vessel compliance. Arterial stiffening is a major risk factor associated with the progression of age-related health conditions such as cardiovascular diseases and dementia. Increasing vessel stiffness significantly impacts the intensity of flow-induced cyclic stretch applied on the vascular endothelium. However, to date, less is known on the cooperative effects of cyclic stretch (CS) and vessel stiffness on endothelial cell biology. Advancements in the field of endothelial biology has been considerably hampered by a lack of technology that can mimic vessel wall elasticity in the presence of cyclic stretch. Chapter 1 presents a summary on vascular ageing and ageing-associated vascular dysfunction. This literature review explains the physiological and pathological effects of arterial stiffening on vascular morphology. It includes a brief outline of the biomechanical effects of vascular ageing on vascular endothelial cells and its surrounding extracellular matrix. It also incorporates a review of in vitro bioengineered vascular models used to study vascular ageing and age-associated cardiovascular disorders. I summarized the recent progress in in vitro platforms including traditional two-dimensional (2D) and newly emerging advanced three-dimensional (3D) vascular platforms. This review identified a clear gap in the current available platforms and motivated me to develop a novel 3D in vitro vascular model for exploring age- associated vascular dysfunctions. Chapter 2 presents my first research contribution. Here, I designed and characterised a 3D-printed vascular simulation model that can mimic vascular endothelial cells (VECs) in young and aged blood vessels with adjustable stiffness, thus combining the physiological relevance of animal models with the simplicity of in vitro models. I demonstrated a cam-driven system for cyclic stretch of aortic endothelial cells and used this versatile system for studying the cytoskeletal structure and morphology of aortic endothelial cells in response to cyclic stretch. Using this unique model and a combination of fluorescent microscopy and image processing approaches, I further demonstrated that cyclic stretch leads to the changes in cytoskeletal alignment of the endothelial actin stress fibres, increases the cell area and aspect ratio in a dose- and time- dependent manner. This new model enabled me to successfully show the effects of ageing-associated diseased blood vessels on endothelial cell morphology and how cyclic stretch contributes to endothelial dysfunction. Chapter 3 presents my second research contribution. To outline the molecular and mechanical pathways involved in complex stretch patterns during the development of vascular pathologies, a greater understanding of the effects of stiffness and CS on VEC signalling, gene expression, structure, and function is crucial. Here, I developed a versatile model to investigate the synergistic effect of CS and substrate stiffness on endothelial cells. I cultured endothelial cells on elastomeric wells covered with fibronectin-coated polyacrylamide gel and by varying the concentrations of acrylamide and bisacrylamide; this enabled me to develop soft and stiff substrates to mimic the stiffness and stretch levels that endothelial cells experience in young and aged arteries. I investigated the effect of cyclic stretch and substrate stiffness on endothelial cytoskeleton remodelling, cell and nuclear morphology, and NF-kB nuclear translocation. Experiments indicated that exposure of endothelial cells to cyclic stretch affects the cytoskeleton remodelling and the cell morphology depending on the substrate stiffness. Chapter 4 presents my third research contribution. Here, I investigated the complex signalling pathways involving mechanosensitive ion channels which govern endothelial responses to change in mechanical forces associated with vessel stiffening. Here, I used a novel method of modifying the surfaces of the elastomeric wells with different ratios of PDMS thereby generating different stiffness levels to simulate young and ageing artery stiffness level. I demonstrated the suitability of this novel technique for studying changes in the orientation of actin stress fibres, nuclear area and circularity as well as NF-kB and senescence activity within endothelial cells in response to both cyclic stretch and substrate stiffness. Using this method, I focused on elucidating the role of mechanosensitive cation permeable ion channels TRPV4, which modulated the calcium ion (Ca2+) influx into VECs. I further studied the contribution of TRPV4 in endothelial cell cytoskeletal remodelling, nuclear morphology, senescence and NF-kB nuclear translocation. Experiments deduced that TRPV4 plays a protective role in stiff substrates to decrease premature senescence and inflammation of endothelial cells. TRPV4 mechanosignalling also appeared to mediate endothelial cell reorientation, senescence and inflammation. Chapter 5 presents a general discussion of the significant findings of this study. This novel and unique model provides opportunities for a better understanding of the vascular endothelium during age-associated vessel stiffening. This provides avenues for the development of new therapies for targeting age-related conditions such as CVD and dementia. Here, I also discussed potential future work for progression in this field to improve the current model.