In recent years, reactive oxygen species (ROS) have been shown to have critical roles in normal vascular function and the pathogenesis of vascular disease. These molecules have profound effects on vascular smooth muscle cell (VSMC) growth, migration and, as we now show, differentiation. In the previous grant period, we focused on NAD(P)H oxidases (Nox), enzymes that are major sources of 02 ?"""""""" in vascular cells. We found that Nox1 is localized to caveolae and mediates growth of VSMCs, while Nox4 is found in focal adhesions and the nucleus. Our preliminary data indicate that Nox4 may be important in post-natal differentiation, stress fiber formation and focal adhesion assembly. These data indicate that the subcellular localization of Nox enzymes may be critical to their function. We now propose to examine in more detail the molecular mechanisms linking Nox4 to the regulation of the actin cytoskeleton and differentiation marker gene expression in adult cells. Our proposal centers on Nox4 regulation of the small molecular weight G protein Rho. In the first specific aim, we will define the molecular mechanisms by which Nox4 regulates Rho expression and activity. We hypothesize that Nox4 induces RhoA transcription, and leads to its activation by promoting its release via a guanine dissociation inhibitor, RhoGDI.
In Aim 2, we plan to determine the specific molecular pathways by which Nox4 mediates the expression of differentiation marker genes, F-actin polymerization, stress fiber formation and the conversion of focal complexes to mature focal adhesions. We hypothesize that Nox4 activation of Rho stimulates its effectors, Rho kinase and mDia, leading to actin polymerization, focal adhesion assembly and translocation of smooth muscle-specific transcription factors to the nucleus. The sequential molecular pathways responsible for the link between Nox4 and these physiological endpoints will be investigated in detail. In the final aim, we will test some of these molecular relationships in vivo, using unique mouse models of vascular injury and smooth muscle redifferentiation in which Nox4 is upregulated by genetic manipulation of its binding partner. Delineating the functional consequences of Nox4 activation will lead to a better understanding of the mechanisms controlling vascular smooth muscle function in health and disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Method to Extend Research in Time (MERIT) Award (R37)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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Thrasher, Terry N
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Emory University
Internal Medicine/Medicine
Schools of Medicine
United States
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Vukelic, Sasa; Xu, Qian; Seidel-Rogol, Bonnie et al. (2018) NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase ?-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion. Arterioscler Thromb Vasc Biol 38:2423-2434
Hernandes, Marina S; Lassègue, Bernard; Hilenski, Lula L et al. (2018) Polymerase delta-interacting protein 2 deficiency protects against blood-brain barrier permeability in the ischemic brain. J Neuroinflammation 15:45
Xu, Qian; Huff, Lauren P; Fujii, Masakazu et al. (2017) Redox regulation of the actin cytoskeleton and its role in the vascular system. Free Radic Biol Med 109:84-107
Hernandes, Marina S; Lassègue, Bernard; Griendling, Kathy K (2017) Polymerase ?-interacting Protein 2: A Multifunctional Protein. J Cardiovasc Pharmacol 69:335-342
Amanso, Angelica; Lyle, Alicia N; Griendling, Kathy K (2017) NADPH Oxidases and Measurement of Reactive Oxygen Species. Methods Mol Biol 1527:219-232
Fujii, Masakazu; Amanso, Angélica; Abrahão, Thalita B et al. (2016) Polymerase delta-interacting protein 2 regulates collagen accumulation via activation of the Akt/mTOR pathway in vascular smooth muscle cells. J Mol Cell Cardiol 92:21-9
Di Marco, Elyse; Gray, Stephen P; Kennedy, Kit et al. (2016) NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis. Free Radic Biol Med 97:556-567
Griendling, Kathy K; Touyz, Rhian M; Zweier, Jay L et al. (2016) Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association. Circ Res 119:e39-75
Lee, Minyoung; San Martín, Alejandra; Valdivia, Alejandra et al. (2016) Redox-Sensitive Regulation of Myocardin-Related Transcription Factor (MRTF-A) Phosphorylation via Palladin in Vascular Smooth Muscle Cell Differentiation Marker Gene Expression. PLoS One 11:e0153199
Fernandez, Isabel; Martin-Garrido, Abel; Zhou, Dennis W et al. (2015) Hic-5 Mediates TGF?-Induced Adhesion in Vascular Smooth Muscle Cells by a Nox4-Dependent Mechanism. Arterioscler Thromb Vasc Biol 35:1198-206

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