Endothelial cells (EC) exposed to oscillatory shear stress (OS) experience oxidative stress as a signature of atherosclerosis. Conversely, unidirectional laminar shear stress (LS) reduces reactive oxygen species (ROS) levels and inflammatory responses. Peroxiredoxins (PRX) are antioxidant enzymes that reduce hydrogen peroxide, but have yet to be investigated in response to shear stress. We hypothesized that LS, compared to OS, promotes increased expression of PRX, which in turn influences the balance of ROS in EC. In this study, we identified all six PRX family members in bovine aortic endothelial cells (BAEC). Furthermore, we revealed that PRX are regulated by shear stress in EC. When compared to OS and static culture (ST), exposure to chronic LS upregulated PRX1 levels intracellularly. LS also upregulated PRX5 relative to ST, but not OS. In addition, PRX exhibited broad subcellular localization in BAEC, but these patterns did not change in response to shear stress. To establish the functional importance of PRX1 in shear stress-dependent redox balance, we next examined the role of PRX1 in LS-mediated hydrogen peroxide regulation. Here, Amplex Red assay was used to measure ROS levels in BAEC. Depletion of PRX1 using siRNA resulted in significantly higher ROS levels following LS, OS, and ST, while PRX5 depletion did not. These findings indicated that chronic exposure to LS upregulates PRX1 expression to keep ROS levels low in EC. To identify the pathway by which atheroprotective LS stimulates PRX1 protein production, we also undertook gene expression studies. We discovered that LS upregulates Prdx1 gene in a time-dependent manner compared to OS or ST. However, this increase in expression was not due to stabilization of Prdx1 mRNA. In addition, Prdx1 promoter analysis revealed a Nrf2 transcription factor binding site 160bp upstream of the gene. Nrf2 overexpression promoted basal PRX1 protein production, while Nrf2 depletion reduced Prdx1 mRNA following exposure to LS. Collectively, our work illustrated that LS affects PRX1 by inducing the Prdx1 gene, in part via the transcription factor Nrf2. Moreover, this discovery of PRX1 as a mechanosensitive antioxidant may contribute important insights into endothelial cell biology and provide a novel therapeutic target for vascular diseases.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/24779 |
| Date | 19 May 2008 |
| Creators | Mowbray, Amy Leigh |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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