Heart disease continues to be the leading cause of death in the US. Atherosclerosis is the major underlying cause of heart disease. Dysfunction of endothelial cells has been clearly linked to atherosclerosis. Endothelial cells are constantly subjected to hemodynamic shear stress and respond to shear stress by producing local paracine and autocrine mediators to maintain vascular tone and coagulation equilibrium via poorly understood signal transduction pathways. Dysfunction of endothelial cells has been clearly linked to atherosclerosis. Arterial branching and curvatures induce complex blood flow patterns resulting in altered fluid shear stresses on the endothelium. The focal nature of early atherosclerotic lesions at sites of arterial branching highlights the important role of shear stress both in normal endothelial cell function and in the atherogenic process. Therefore, it is important to understand the signal transduction mechanism regulated by shear stress in endothelial cells. However, it is not known: 1) how endothelial cells sense shear stress; 2) how they trigger early signal transduction events; and 3) how these events are coupled to endothelial cell responses under normal and pathologic conditions. Elucidating these important questions are my long term goals. It has been hypothesized by many that shear stress exerts its effects on endothelial cells through as yet unidentified flow sensing systems (FSS). G-proteins have been implicated in may shear-activated cellular functions in endothelial cells. I propose that G-proteins are the key signaling node mediating shear-induced responses in endothelial cells. I hypothesize that shear-stimulated FSS activates 1) a G-proteins(s) [which I shall call endothelial cells. I hypothesize that shear-stimulated FSS activates 1) a G-protein(s) [which I shall call """"""""G Flow-protein(s)""""""""] and, 2) the G Flow-protein(s) then regulates intracellular signal transduction pathways to activate effector systems that are responsible for cellular responses. The studies proposed in this application are designed to test this hypothesis. First, I shall identify G Flow-protein(s) in endothelial cells. Secondly, I shall identify and characterize the role of G Flow- protein(s) in shear-activated signaling pathways.
Specific Aim 1 : IDENTIFY AND CHARACTERIZE SHEAR-SENSITIVE G FLOW- PROTEIN(S) IN ENDOTHELIAL CELLS. The effect of shear stress on G-proteins will be determined by : 1) Western blots [32P] GTP crosslinking; 2)GTP/GDP binding; and 3)phosphorylation.
Specific Aim 2 : CHARACTERIZE THE ROLE OF G FLOW-PROTEIN(S) IN SHEAR- DEPENDENT SIGNALING PATHWAYS IN ENDOTHELIAL CELLS. 1. Effect of G- proteins on shear-dependent signaling pathways. I shall determine the effect of G-protein inhibitors (GDP-beta-S, pertussis toxin and C3 exoenzyme) on shear stress-induced signaling pathways: 1)inositol triphosphate (IP3); 2) adenylate cyclase; 3) MAP kinases and tyrosine kinases. 2. The effect of inhibitors of shear-sensitive signaling pathways on G FLOW-protein(s). K+ channel blockers, Ca++ chelators and cytoskeleton inhibitors will be used to characterize the flow of G Flow- protein(s) in relation to these specific pathways. The effect of these inhibitors on G Flow-protein(s) will be determined by using selected analytical techniques described in Specific Aim 1.
