- To determine properties, structure, modulation and the functional role of mechanically-gated chloride channels in vascular endothelium
- To determine properties and modulation of mechano-sensitive Ca2+ entry in endothelial cells.
- To determine changes in the expression and presentation of P and E selectins and changes in leukocytes adhesion in endothelium after mechanical stimulation
Vascular endothelial cells (EC) are constantly exposed to mechanical shear and strain forces. These forces induce a variety of EC responses, such as changes in the intracellular Ca2+ concentration, pH, phosphoinositol turnover, tyrosine phosphorylation, NO release and secretion of a variety of vasoactive compounds finally leading to changes in gene expression and cytoskeletal remodelling. The cellular and molecular mechanisms responsible for the signal transduction between mechanical stimuli and EC responses are not known. This research proposal therefore is focused on mechano-sensitive signalling, i.e. the elucidation of mechanisms by which mechanical forces can be transduced into cell responses. Three major mechanisms will be investigated: 1. Properties of mechano-sensitive Cl channels (Icl, mech) including their molecular biological characterisation of Icl mech their modulation via the cytoskeleton, annexins, via tyrosine phosphorylation, their functional role as transporters of metabolites for NO synthesis, as modulators for EC proliferation, and as modulator of Cl-dependent protein kinases.
2. Properties of the mechanically activated Ca2+ release and Ca2+ entry which will comprise an electrophysiological characterisation of the mechanically induced Ca2+ entry including modulation by depletion of intracellular Ca2+ store and a characterisation of the Ca2+ influx in EC through stretch-activated non-selective cation channels.
3. Cellular responses of EC activated by mechanical stimulation such as peroxide formation, expression and presentation of P and E selectins and functional consequences for leukocyte adhesion. The final aim is to identify mechano-sensor molecules in EC and their main mechanism of modulation. Elucidation of these mechanism may lead to the identification of novel modulator sites of EC function and provide novel targets in the treatment of hypertension and atherosclerosis.