Shear stress increases vascular production of nitric oxide by acute activation of the endothelial nitric oxide synthase (eNOS) enzyme and also by upregulation of eNOS expression. Our laboratory has demonstrated that laminar shear stress activates eNOS mRNA transcription and mRNA stabilization through divergent signal transduction pathways. Increased eNOS mRNA transcription in response to shear depends on activation of c-Src, RAS, MEK1/2 and ERK1/2. Furthermore, we have identified the region within the eNOS promoter that is instrumental for transcription in response to shear stress. The transcription factor NFkappaB binds to this region in a shear stress-dependent manner, and seems to be critically important in the induction of eNOS promoter activity in response to shear. NFkappaB activates a variety of target genes in response to diverse stimuli in the vessel wall that are mostly linked to the onset of atherosclerosis. In contrast, NO has an atheroprotective effect in the vessel wall, and has been shown to inhibit NFkappaB activation. We propose that this represents a classical negative feedback loop, whereby shear activates NFkappaB which stimulates NO production and eNOS expression and that NO inhibits NFkappaB. In conditions where NO is not produced, we hypothesize that unbridled activation of NFkappaB may occur. In this project, several aims are directed toward understanding this concept.
In aim 1, we will examine molecular signals responsible for activation of NFkappaB in response to shear.
In aim 2, we will examine the hypothesis that NO production in response to shear provides a negative feedback to inhibit NFkappaB-binding to the eNOS promoter, inhibiting eNOS promoter activity and reducing eNOS transcription. To manipulate levels NO, cells will be treated with the NOS inhibitor L-NAME and we will study cells from eNOS -/- mice.
In aim 3, we will determine if NFkappaB is activated in vivo during exercise training, if its activation is modulated by NO and if NFkappaB activation mediates the increase in eNOS expression during exercise. In a final aim, we will determine if an absence of NO promotes vascular inflammation during exercise. These studies will be performed in eNOS -/- mice that do not produce NO. In addition, we have preliminary data showing that exercise training has a heterogeneous effect in young otherwise healthy humans, and in about 40% of subjects, flow-mediated vasodilatation is worsened by exercise training. We plan studies to determine if the NFkappaB driven gene products, VCAM-1 and ICAM-1, are increased by exercise training in these subjects. Overall, these studies will provide novel information regarding the regulation of eNOS gene expression and the role of NO to prevent NFkappaB-mediated vascular inflammation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL075209-02
Application #
7062767
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
2005-01-01
Project End
2008-12-31
Budget Start
2005-01-01
Budget End
2005-12-31
Support Year
2
Fiscal Year
2005
Total Cost
$259,653
Indirect Cost
Name
Emory University
Department
Type
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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