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Endothelial cells, which line the luminal surface of all blood vessels, exert a controlling influence on many vessel properties and play key roles in the development of vascular disease. The endothelial cells are themselves influenced by the shear stress (frictional force per unit area) imposed on them by the flow of blood. Indeed, a central tenet of vascular biology is that arterial endothelial cells are centrally involved in the negative feedback regulation of this shear stress: they control vessel diameter and vessel wall remodelling to keep the shear stress they experience at around 10-15 dynes/cm2.Recent theoretical work by Weinberg and Ethier demonstrates that this central tenet is incorrect. Scaling arguments show that mean aortic blood flow velocity is approximately constant across mammalian species, a deduction supported by much experimental evidence, but the aortic diameter is smaller, and hence velocity gradients (which determine haemodynamic wall shear stress) are steeper, in smaller animals. More specifically, the theory predicts an inverse 3/8ths power dependence of wall shear stress on body mass. The predicted differences are substantial - 20 fold between mice and people - and have been quantitatively confirmed by experiment.Since endothelial cell behaviour is highly dependent on shear, it might be expected to vary substantially between animals of different size. This is not the case, however; remarkably, although shear-dependent properties such as cell shape vary spatially within each animal at aortic branch points, where the time-averaged shear stress is known also to vary, the shape of aortic endothelial cells away from branch points does not vary with body size. These observations demonstrate the existence of a mechanism that allows arterial endothelial cells to adjust to the global level of wall shear stress occurring at a given body size, but also show that the mechanism cannot depend on the magnitude of the time-averaged shear experienced by each individual cell since this would give preclude any spatial variation in cell properties at branch points. The proposed project would test the hypothesis that endothelial cells adjust by sensing and responding to the fundamental frequency of fluctuations in shear stress. This frequency is the heart rate, which scales inversely with size raised to the 1/4 power. In order to test this hypothesis, aortic endothelial cells would be isolated from pig or mouse aortas and exposed ex vivo to the mean shear stress and frequency appropriate either to the species from which they were derived, or appropriate to the other species. It would be necessary to construct an apparatus (from commercially-available components, although the entire apparatus is not commercially available) capable of exposing the cells to the appropriate shear stress patterns under sterile conditions. Shear-dependent endothelial behaviours that would be examined are (i) rate of cell turnover, (ii) cell shape, (iii) cytoskeletal structure, (iv) strength of cellular adherence to the substrate, (v) NO production, (vi) expression and location of the transcription factor NF-?B, and (vii) production of reactive oxygen species. If the results do not support the hypothesis, preliminary experiments will be conducted to investigate the alternative hypotheses that humoral factors or cyclic stretch are involved.Ethier and Weinberg collectively have broad experience of culturing endothelial cells, measuring shear-dependent cell behaviour, and developing perfusion apparatus for culture systems, which ensure the feasibility of the proposed work. The 1-year project would initiate a continuing long-term collaborative programme. It would be timely, resting as it does on work published in the last few months, has the potential to revolutionise thinking about biomechanical influences on endothelial cells, and would have important implications for understanding of human vascular physiology and disease.
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