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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL053601-04
Application #
2609351
Study Section
Pathology A Study Section (PTHA)
Project Start
1994-12-01
Project End
1999-11-30
Budget Start
1997-12-01
Budget End
1999-11-30
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Pathology
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Park, H; Go, Y M; Darji, R et al. (2000) Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase. Am J Physiol Heart Circ Physiol 278:H1285-93
Go, Y M; Patel, R P; Maland, M C et al. (1999) Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH(2)-terminal kinase. Am J Physiol 277:H1647-53
Moellering, D; Mc Andrew, J; Patel, R P et al. (1999) The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase. FEBS Lett 448:292-6
Go, Y M; Park, H; Maland, M C et al. (1999) In vitro system to study role of blood flow on nitric oxide production and cell signaling in endothelial cells. Methods Enzymol 301:513-22
Park, H; Go, Y M; St John, P L et al. (1998) Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase. J Biol Chem 273:32304-11
Moellering, D; McAndrew, J; Patel, R P et al. (1998) Nitric oxide-dependent induction of glutathione synthesis through increased expression of gamma-glutamylcysteine synthetase. Arch Biochem Biophys 358:74-82
Go, Y M; Park, H; Maland, M C et al. (1998) Phosphatidylinositol 3-kinase gamma mediates shear stress-dependent activation of JNK in endothelial cells. Am J Physiol 275:H1898-904
Engelman, J A; Chu, C; Lin, A et al. (1998) Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Lett 428:205-11
Jo, H; Sipos, K; Go, Y M et al. (1997) Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2- and Gbeta/gamma-dependent signaling pathways. J Biol Chem 272:1395-401