It is now increasingly evident that hemodynamic flow has an important role in regulating vessel diameter and caliber, and this is mediated by the endothelial lining of the blood vessel wall. Two functional responses of endothelial cells that are controlled by flow are the release of endothelial-derived relaxing factor (EDRF) and the potent vasoconstrictor and mitogen, endothelin (ET-1). The mechanism of how endothelial cells sense flow is unknown. Furthermore, the responses to pulsatile flow appear to be different than to steady flow. It is known, however, that the signal transducing G-proteins mediate the responses. It is our hypothesis that fluid shear """"""""perturbs"""""""" the cell membrane and/or cytoskeleton, thereby altering the activity of G-proteins and/or associated receptors. To test this hypothesis, purified G-proteins and protein activation will be monitored under various conditions to determine if shear acts on cytoskeleton (actin, microtubule and vimentin filaments) in steady and pulsatile flow stimulation will also be assessed by perturbing each of them and measuring EDRF release. The locus of the """"""""perturbation"""""""" will also be investigated by determining the where in monitoring the location of inositol phospholipid hydrolysis. There are several responses of flow which differ from agonist stimulation in endothelial cells. An important difference is the role of calcium-calmodulin in the activation of EDRF synthase. By using molecular and biochemical techniques to characterize these differences, we may be able to shed light on the mechanism of flow-signal transduction. One of the functions of flow-induced EDRF appears to be regulation of ET-1 release. It is not clear, however, how this regulation occurs. This will be investigated by measuring the effect of EDRF and EDRF-induced cGMP on peptide intermediates of ET-1. By establishing how the fundamental mechanism by which flow stimulates endothelial cells, and its regulation of EDRF and ET-1, we will be able to understand flow-dependent control of vascular diameter and remodeling.
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