The vascular endothelium has evolved a set of adaptive responses to the changing hemodynamic forces that continually challenge them. Inadequate or inappropriate adjustments to alterations in flow often result in pathophysiology such as hypertension and atherosclerosis. Although past research has deciphered many of the mechanisms involved in transducing hemodynamic forces into chemical signals within the cell, a key missing element is the mechanoreceptor purported to exist in the endothelial cell. By using a unique methodology that allows for the purification of luminal endothelial cell plasma membranes, we recently demonstrated that significant mechano-signaling can be initiated by enhancing fluid flow in situ in specialized invaginated, microdomains on the cell surface called caveolae. We hypothesize that caveolae represent unique structures through which endothelial cells can discriminate amongst changing hemodynamic forces. In order to test this hypothesis, we propose to manipulate cell surface density of caveolae through over-expression and anti-sense depletion of caveolin-1. These cells will be subjected to precisely defined patterns and magnitudes of physiological flow in a parallel plate apparatus and measured for established biochemical and morphological endpoints that characterize the temporal nature of the mechanotransduction response. Satisfying this aim will serve to spatially define mechanotransduction however, the mechanism of shear-induced signal propagation within caveolae remains unclear. Emerging evidence indicates that nitric oxide (NO) and/or reactive oxygen species (ROS) serve as import early responding second messengers that may participate in the mechanotransduction process. We propose that NO and/or ROS are generated within caveolae in response to sheer-stress and serve a key mechanotransducing second messengers. The following aims are proposed to test this hypothesis: i) characterize the role of nitric oxide (NO) as a mechano-signaling mediator in caveolae, ii) determine the spatial location of shear-stress induced superoxide formation specifically within the endothelial plasma membrane and iii) examine the function of ROS generation in the caveolae mediated acute mechanotransduction process. Molecular and pharmacological approaches will be used to investigate the specific contribution of shear stress induced NO and ROS to the mechanotransduction process. Results from these studies will extend our insight into the basic mechanisms by which endothelial cells respond to changes in fluid mechanical forces generated by flowing blood. Such information would greatly add to our understanding of both normal cardiovascular function and the pathophysiology seen in atherosclerosis.
