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Mechanisms of Ca2+ entry and mechanical sensation in the vasculature

Hou, Bing (2014) Mechanisms of Ca2+ entry and mechanical sensation in the vasculature. PhD thesis, University of Leeds.

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Both Ca2+ entry and shear stress sensation of vascular cells play important roles not only in vascular physiology, but also pathology. Therefore, understanding the mechanism of these processes could greatly help the therapeutic strategies to treat vascular diseases like atherosclerosis. The overall aim of this research was to develop a better understanding of molecular mechanisms of Ca2+ entry and shear stress sensation in the vasculature. Transient Receptor Potential Canonical (TRPC) proteins assemble to form channels for calcium and sodium ion entry. TRPC5 readily forms functional homomers, whereas TRPC1 forms functional heteromers with TRPC5 but has weak or no channel function on its own. In this study, impact of cholesterol on these proteins was investigated, because it is an important membrane constituent and driver of cardiovascular and other diseases. I found that TRPC5-mediated calcium entry is suppressed by cholesterol due to internalization of TRPC5 via the caveolin-1-dependent retraction mechanism but that TRPC1 prevents the internalization by heteromerising with TRPC5 and causing segregation to a membrane raft rich in GM1 ganglioside and dissociated from caveolin-1. Endogenous TRPC5 containing channels of vascular smooth muscle cells are stimulated by exogenous GM1 gangliosides and resistant to inhibition by cholesterol. The data suggest that a previously unrecognized purpose of incorporating a subunit in a heteromer is to segregate the protein complex to a membrane raft that is protected against cholesterolevoked internalization. Force sensors used by endothelial cells to detect fluid shear stress are pivotal in vascular physiology and disease but there is lack of clarity about their identity. Here I show that a recently-discovered calcium-permeable channel formed by Piezo1 is important in sensing physiological shear stress and in driving a key downstream functional event. Calcium influx evoked by physiological shear stress depended on endogenous Piezo1. Exogenous Piezo1 confers sensitivity to shear stress on otherwise resistant cells. Real-time subcellular tracking studies showed accumulation of Piezo1 at the endothelial cell leading edge. Depletion of endogenous Piezo1 prevented endothelial cell alignment to shear stress. Calpain activation and focal adhesion is the mechanism of Piezo1-dependent alignment of endothelial cells to shear stress. Piezo1 also crosstalks with CD31, which is a component of a known shear stress sensory complex. The data suggest that Piezo1 has the dual function of sensing physiological shear stress and driving downstream endothelial cell alignment through calcium ion entry and a calpain-focal adhesion mechanism. Alignment of endothelial cells has major roles in physiology and various disease processes which include protection against atherosclerosis. In summary, this research has generated new knowledge and hypotheses about molecular mechanism of Ca2+ entry and mechanical sensation of vascular cells under physiological and pathological conditions, which may help generate new strategies to treat cardiovascular diseases such as atherosclerosis.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
Identification Number/EthosID: uk.bl.ethos.638899
Depositing User: Mr Bing Hou
Date Deposited: 26 Feb 2015 13:08
Last Modified: 06 Oct 2016 14:42
URI: http://etheses.whiterose.ac.uk/id/eprint/7803

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