Objective
Endothelial cells in the blood vessels wall respond to mechanical forces by a variety of biological events (NO synthesis, PGI2 secretion, tPA expression, endothelin mRNA modulation, mitosis, etc.). The signalling mechanism between physical stimulus and biological event is still completely unknown.
Mechano-sensitive pathways in Ca2+ signalling in human endothelial cells will be studied by a combination of cyto- and molecular biological techniques with simultaneous patch-clamp and intracellular Ca2+ measurements. Mechanical stimulation activates a Cl- conductance in endothelial cells.
A study will be made of whether this conductance is related to the mdr-1 gene product P-glycoprotein. Also a possible G-protein mediated activation of this channel will be investigated. Another mechanism to provide mechano-sensitivity to endothelial cells is a mechanically induced release of intracellular calcium which depletes intracellular stores for calcium. A study will be made of whether the mechanical activation of PLC or PLA2 is involved in this mechano-sensitive signalling pathway.
A possible role of the cytoskeleton will be approached by modulating the mechano-response with compounds that interfere with the actin-cytoskeleton. Mechanical stimulation of endothelial cells induces also Ca2+ entry. A study will be made of whether this entry is related to the filling state of intracellular Ca2+ stores.
Contribution of a directly gated Ca2+ entry will be studied especially in relation to the possibly channel forming, actin binding protein, annexin 2. For this reason, endothelial cells will be transfected with a annexin-p11 binding construct. This construction forms large p11-annexin II aggregates which may immobilise the functional active annexins, and modulate the mechano-responses on Ca2+ entry.
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Programme(s)
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Topic(s)
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Funding Scheme
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Coordinator
3000 Leuven
Belgium
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