The Role of Piezo1 Ion Channel in Mechanotransduction of Shear Stress: Molecular Insights into Endothelial Cell Function

Shear stress is a mechanical force exerted by blood flow, and endothelial cells that line the blood vessel walls are under constant exposure to shear stress. Endothelial cells are inherently sensitive to haemodynamic forces, which are the forces such as shear stress and cyclic stretch that arise from blood flow and changes in vascular tone, and their sensitivity to these forces play an essential role in regulating vascular function. Endothelial mechanosensitivity is regulated via a class of proteins called mechanoreceptors. These include expression of mechanosensitive ion channels which are known to play a role in sensing the shear stress and converting this information into biochemical signals to activate intracellular signalling pathways. Among the mechanosensitive ion channels that mediate endothelial mechanosensitivity, there is great interest in Piezo1, due to its plasma membrane expression and low threshold of mechanosensitivity. Piezo1 is essential for early vascular development and blood pressure regulation via vasodilation, which is dependent on intracellular calcium mechanisms (1, 2, 3). In vascular physiology, laminar high shear stress (>10 dyne/cm2) is atheroprotective in nature, whilst disturbed and low shear stress (<4 dyne/cm2) contributes to atherogenesis. To date, it is not well known how shear stress modulates Piezo1 functions under physiological and pathological shear stress levels. Therefore, the first aim of my project was to address this knowledge gap. The extracellular matrix (ECM) comprises several components which maintain cell function via communicating with membrane receptors on the endothelial cells such as integrins, or structural components such as the actin cytoskeleton. However, the basic biology of Piezo1 including the response to haemodynamic forces and interaction with extracellular matrix proteins, remains poorly understood and will be studied in this project. Thus, the second aim of this project was to examine the role of ECM in Piezo1 function and responses to physiological and pathological shear stress conditions. Vascular stiffness, haemodynamics, and endothelial cells are interrelated factors vital for cardiovascular health. Vascular stiffness as an ageing condition, is influenced by vessel wall composition, affects haemodynamics, which encompasses blood flow patterns and forces, thereby affecting endothelial physiology. Vascular stiffness is associated with endothelial damage, migration and proliferation, and arterial calcification (4) and alteration in haemodynamic forces (5), all of which contribute to endothelial dysfunction, inflammation, and the development of atherosclerosis. However, it is not known if Piezo1 contribute to vessel stiffness or how vessel stiffness affects Piezo1 function and shear stress responses. The third aim of this project was to understand these connections and elucidate the contribution of Piezo1 in endothelial responses to vascular stiffness and pathology. Overall, this project aimed to enhance our understanding of Piezo1 and its contribution to endothelial physiology, thereby contributing to potential therapeutic advancements to address vascular pathologies associated with Piezo1 impairment. In this project, I utilized microfluidic models to assess Piezo1 responses to shear stress and its agonist, Yoda1, in human aortic endothelial cells (HAECs) and a human embryonic kidney (HEK) 293 cell line stably expressing Piezo1 in presence of specific ECM proteins. To assess Piezo1 response to shear stress, I employed calcium imaging coupled with confocal microscopy, total internal reflection fluorescence microscopy (TIRFM), immunocytochemistry and small interfering ribonucleic acid (siRNA) technology to examine the contribution of Piezo1 in cellular responses to atheroprotective and pro-atherogenic shear stress levels. Furthermore, I explored the involvement of Piezo1 in endothelial responses to vascular stiffness using an innovative microfluidic model designed to mimic arterial stiffness. The results reported in this thesis reveal significant insight into the role of Piezo1 in mechanotransduction of shear stress in endothelial cells. Specifically, my research has demonstrated that shear stress sensitizes the response of Piezo1 to its agonist, Yoda1, and this shear-induced sensitization is accompanied by dynamin- and microtubule-dependent trafficking, leading to an increase in Piezo1 membrane expression. The shear-induced sensitization was rapid and dependent on the activation of the phosphoinositide 3-kinase (PI3K)/Ak strain transforming (AKT) signalling pathway. My research also found that ECM proteins regulate Piezo1 sensitivity to shear stress. Under atheroprotective flow conditions, fibronectin sensitizes the response of Piezo1 to shear stress, while under pro-atherogenic flow conditions, Piezo1 mechanosensitivity is regulated by collagens and laminin. Moreover, α5β1 and αvβ3 integrins are involved in Piezo1 sensitivity to atheroprotective flow while αvβ3 is important in regulating Piezo1 response to pro-atherogenic flow. These results suggest that the ECM/integrin interactions influence Piezo1, providing new insight into the distinct properties of Piezo1. Additionally, this project has explored the role of Piezo1 in endothelial responses to vascular stiffness. My results have demonstrated that Piezo1 mediates endothelial morphological changes and cytoskeleton remodelling in response to changes in vascular stiffness. Furthermore, the data indicates that Piezo1 regulates inflammatory responses via upregulation of intercellular adhesion molecule 1 (ICAM-1), and increased monocyte adhesion induced by stiffness and low shear stress. However, it is noteworthy that endothelial senescence induced by substrate stiffness was independent of Piezo1 expression. The findings indicate that Piezo1 contributes to atheroprotective responses in vascular pathology. This project has significantly contributed to our understanding of the multifaceted role of Piezo1 in endothelial mechanobiology. The findings from my research reveal that shear stress regulates trafficking and membrane expression of Piezo1 in endothelial cells. I have demonstrated that extracellular components modulate Piezo1 mechanotransduction of shear stress via integrin signalling and identified the role of Piezo1 in stiffness-induced endothelial inflammation. Given the crucial role of Piezo1 in vascular development and overall vascular function, gaining a comprehensive understanding of how haemodynamic forces precisely regulate the activity of Piezo1 presents significant prospects for the development of innovative therapeutic interventions.