Cardiovascular disease is characterized by increased generation of reactive oxygen species (ROS) in the vessel wall, which results in activation of signaling pathways that ultimately promote cell growth and neointimal formation. ROS derived from smooth muscle cells (SMCs) is a major contributing factor in the development of vascular disease, though antioxidant therapies have achieved only limited therapeutic benefit. Therefore, it is necessary to identify targeted approaches to prevent ROS generation. NADPH oxidases are the predominant source of ROS in the vasculature, with Nox1 being the primary catalytic NADPH oxidase expressed in SMCs. Nox1-derived ROS have been linked to atherosclerosis as well as neointimal formation and SMC migration following injury, but the precise mechanisms by which Nox1 activates redox- dependent signaling pathways remain incompletely defined. In order to develop targeted therapeutics against Nox1, it is first necessary to understand the parameters that must be met for Nox1 activation. Previous studies demonstrate that cytokine activation of the pro- inflammatory factor nuclear factor-?B (NF-?B) requires internalization of Nox1 via endocytosis. The objective of this proposal is to identify novel regions within Nox1 that regulate its activatio and redox signaling to mediate migration and neointimal formation. The hypothesis is that membrane trafficking and phosphorylation of Nox1 are necessary for cytokine-induced Nox1 activation in SMCs. The following aims are proposed to test the central hypothesis: 1) Examine the functional consequences of cytokine-induced Nox1 trafficking in SMCs. 2) Define how phosphorylation of Nox1 regulates its trafficking and activation. 3) Determine whether the inhibition of Nox1 phosphorylation or trafficking provides therapeutic benefit in the prevention of neointimal hyperplasia. Proposed studies for the first aim will utilize site-directed mutagenesis o canonical internalization motifs within Nox1 to define how cytokine stimulation affects Nox1 trafficking as well Nox1-dependent ROS generation, NF-?B activation, and SMC migration. For the second aim, a systems biology approach will be applied to quantitate the dynamic changes in Nox1 phosphorylation at specific amino acid residues. Next, these phosphorylation sites will be mutated to evaluate the importance of phosphorylation in the mechanisms of Nox1 activation.
The third aim will use an in vivo model of injury to examine the role of Nox1 internalization and/or phosphorylation in neointimal formation. These studies have the potential to identify additional signaling events and motifis within Nox1 that are necessary for activation i SMCs. An immediate clinical impact of these studies is the potential to uncover alternative approaches to generate Nox1-targeted therapeutics for vascular pathologies.
Since cardiovascular disease is the leading cause of mortality in the US, it is critical to identif new therapeutic strategies to reduce the health care burden. Reactive oxygen species (ROS) are well-known to promote vascular disease by stimulating neointimal hyperplasia following injury, and Nox1 NADPH oxidase is the primary source of ROS in vascular smooth muscle cells. Proposed studies aim to identify regions within Nox1 that regulate its trafficking and activation i response to injury. Results from these studies will provide important new insights into Nox1 signaling. Importantly, these findings will serve as a platform for the development of new therapeutic targets to modify the maladaptive pathways involved in vascular disease with the potential to reduce cardiovascular morbidity and mortality.
| Thiel, William H; Esposito, Carla L; Dickey, David D et al. (2016) Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation. Mol Ther 24:779-87 |
| Xu, Shaoping; Chamseddine, Ali H; Carrell, Samuel et al. (2014) Nox4 NADPH oxidase contributes to smooth muscle cell phenotypes associated with unstable atherosclerotic plaques. Redox Biol 2:642-50 |
| Steyers 3rd, Curtis M; Miller Jr, Francis J (2014) Endothelial dysfunction in chronic inflammatory diseases. Int J Mol Sci 15:11324-49 |
| Csányi, Gábor; Miller Jr, Francis J (2014) Oxidative stress in cardiovascular disease. Int J Mol Sci 15:6002-8 |
| Streeter, Jennifer; Schickling, Brandon M; Jiang, Shuxia et al. (2014) Phosphorylation of Nox1 regulates association with NoxA1 activation domain. Circ Res 115:911-8 |
| Streeter, Jennifer; Thiel, William; Brieger, Kate et al. (2013) Opportunity nox: the future of NADPH oxidases as therapeutic targets in cardiovascular disease. Cardiovasc Ther 31:125-37 |
| Makki, Nader; Thiel, Kristina W; Miller Jr, Francis J (2013) The epidermal growth factor receptor and its ligands in cardiovascular disease. Int J Mol Sci 14:20597-613 |
| Dayal, Sanjana; Wilson, Katina M; Motto, David G et al. (2013) Hydrogen peroxide promotes aging-related platelet hyperactivation and thrombosis. Circulation 127:1308-16 |
| Kinkade, Kara; Streeter, Jennifer; Miller, Francis J (2013) Inhibition of NADPH oxidase by apocynin attenuates progression of atherosclerosis. Int J Mol Sci 14:17017-28 |
