Abstract 356: C/EBP α Mediates Shear Stress-Dependent Downregulation of the Angiotensin Type 1 Receptor
The renin-angiotensin system, through the actions of angiotensin II, has significant proinflammatory actions on the vessel wall that can contribute to the progression of atherosclerosis. Hemodynamic forces are major determinants of atherosclerotic plaque localization. Plaques tend to form at arterial bifurcations, branch points, and curvatures where blood flow is low and oscillatory, whereas blood vessels exposed to high laminar shear stress remain comparatively devoid of plaques. Immunostaining of C57BL/6 mouse aortic arches showed a distinctive endothelial angiotensin type 1 receptor (AT1R) staining at the athero-prone inner curvature of the aortic arch but not in the adjoining outer curvature. In HUVECs exposed to laminar shear stress, we found that the AT1R was downregulated within 3 hours. The objective of the present study was to determine the mechanisms underlying shear stress-dependent downregulation of AT1R. We designed luciferase plasmids driven by different constructs of the AT1R promoter, spanning -1934 to +101 from the transcription start site, and transfected them into mouse primary endothelial cells. Using luciferase reporter assay, we found that shear stress reduced expression of the reporter construct by 42% (static = 3.00±0.51%, shear = 1.73±0.30%, P<0.05) after 6 hours. Using truncated constructs of the AT1R promoter, we identified the region -492 to -657 as necessary for the shear stress-dependent downregulation of AT1R (static = 1.20±0.23%, shear = 1.11±0.22%). Based on sequence conservation among human, mouse, rat and chimp, transcription factor binding site analysis pointed to a well-conserved site for C/EBP alpha. We used chromatin immunoprecipitation followed by qPCR analysis to demonstrate that C/EBP alpha was more strongly bound to the AT1R promoter under shear stress (3.21±0.83%) compared with static conditions (0.99±0.29%, P<0.05). Immunofluorescence also showed that C/EBP alpha was more abundant in the nucleus of endothelial cells under shear stress (12.7±3.0%) than static (1.9±1.6%) conditions. Our results suggest that the shear-stress dependent downregulation of AT1R is, at least in part, mediated through C/EBP alpha. This is, to our knowledge, the first indication of a role for C/EBP alpha in shear stress-dependent gene regulation.
- © 2012 by American Heart Association, Inc.