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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL066301-03
Application #
6979735
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Project Start
2003-03-17
Project End
2008-02-29
Budget Start
2004-08-01
Budget End
2005-02-28
Support Year
3
Fiscal Year
2004
Total Cost
$218,147
Indirect Cost
Name
Temple University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Rizzo, Victor (2009) Enhanced interstitial flow as a contributing factor in neointima formation: (shear) stressing vascular wall cell types other than the endothelium. Am J Physiol Heart Circ Physiol 297:H1196-7
Radel, Chris; Carlile-Klusacek, Maryellen; Rizzo, Victor (2007) Participation of caveolae in beta1 integrin-mediated mechanotransduction. Biochem Biophys Res Commun 358:626-31
Rizzo, Victor (2007) Lights, camera, actin! The cytoskeleton takes center stage in mechanotransduction. Focus on ""Mapping the dynamics of shear stress-induced structural changes in endothelial cells."". Am J Physiol Cell Physiol 293:C1771-2
Yang, Baohua; Rizzo, Victor (2007) TNF-alpha potentiates protein-tyrosine nitration through activation of NADPH oxidase and eNOS localized in membrane rafts and caveolae of bovine aortic endothelial cells. Am J Physiol Heart Circ Physiol 292:H954-62
Carlile-Klusacek, Maryellen; Rizzo, Victor (2007) Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae. Am J Physiol Heart Circ Physiol 293:H366-75
Yang, Baohua; Oo, Tin N; Rizzo, Victor (2006) Lipid rafts mediate H2O2 prosurvival effects in cultured endothelial cells. FASEB J 20:1501-3
Radel, C; Rizzo, V (2005) Integrin mechanotransduction stimulates caveolin-1 phosphorylation and recruitment of Csk to mediate actin reorganization. Am J Physiol Heart Circ Physiol 288:H936-45
Gertzberg, Nancy; Neumann, Paul; Rizzo, Victor et al. (2004) NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha. Am J Physiol Lung Cell Mol Physiol 286:L37-48
Cowden, Karen D; Simon, M Celeste (2002) The bHLH/PAS factor MOP3 does not participate in hypoxia responses. Biochem Biophys Res Commun 290:1228-36