A complete understanding of endothelial calcium (Ca²⁺) signalling has been hampered by the inability to directly image the endothelium in arteries exposed to physiological conditions. To date, most studies have been performed in cultured cell models, or in preparations where arteries have been sliced open and flattened out to expose the endothelium to microscopic investigation. Such techniques have provided much useful information, but cannot fully replicate in vivo responses because of the unphysiological arrangements. The focus of the work presented in this thesis is the investigation of endothelial Ca²⁺ signalling in a physiologically relevant model. This thesis describes a novel microscopic imaging system that enables direct visualisation of the endothelium from within the lumen of intact and pressurised arteries. The system incorporates a custom-built, side-viewing imaging probe (consisting of a gradient-index relay lens and an aluminium-coated micro-optical prism), and permits direct visualisation of an area of the endothelium encompassing ~200 cells with subcellular resolution. The system allows arteries to be pressurised within a normal physiological range, and cytosolic Ca²⁺ concentration ([Ca²⁺]i) to be measured in endothelial cells labelled with fluorescent chemical Ca²⁺ indicators. Using this system, the endothelial Ca²⁺ response to haemodynamic stimuli (chemical and mechanical) and its alterations in ageing were investigated. The data indicate that coordinated Ca²⁺ signalling contributes to the physiological endothelial response, and that sustained mechanical stimulation dramatically alters endothelial Ca²⁺ signals. Moreover, endothelial Ca²⁺ signalling is significantly altered in ageing. Histological analysis and computational modelling provide evidence that changes in cell geometry may regulate endothelial Ca²⁺ signalling, and highlight the importance of studying the endothelium in a physiologically relevant model. Furthermore, gradientindex imaging provides parallel access to hundreds of endothelial cells in intact arteries, permitting examination of macroscopic endothelial regulatory functions that are inaccessible by traditional microscopic approaches.
|Date of Award
|22 Apr 2015
- University Of Strathclyde
|EPSRC (Engineering and Physical Sciences Research Council)