Extensive evidence from animal models and human studies supports the hypothesis that NADPH oxidase derived oxidative stress is a major contributor to the pathophysiology of cardiovascular diseases. In particular, dysregulation of Nox1 NADPH oxidase has been implicated in many vascular diseases including atherosclerosis, aortic dissection, hypertension and stroke. Although molecular mechanisms activating the Nox2 (gp91phox) NADPH oxidase have been elucidated, the events that lead to dysregulation of Nox1 NADPH oxidase activity are not well understood. Our prior studies and preliminary data support the notion that NoxA1, the homologue of p67phox, is expressed in mouse and human vascular smooth muscle cells (VSMC) and is a key regulator of Nox1 NADPH oxidase activity. NoxA1-dependent reactive oxygen species (ROS) play an essential role in agonist-induced VSMC proliferation and growth-promoting, redox-sensitive protein kinase activation. NoxA1 is upregulated in aortas and atherosclerotic lesions of ApoE-/- mice and in intimal and medial VSMC of human early carotid atherosclerotic lesions. In this proposal we will investigate how NoxA1 and p47phox interaction regulates Nox1 NADPH oxidase activity in VSMC, the mechanisms that regulate NoxA1 expression and post translational modifications and their role in Nox1 NADPH oxidase activation. We will also determine the role of NoxA1-dependent ROS production in restenosis and atherosclerosis. We will screen a small molecule library to identify potential inhibitors of Nox1 NADPH oxidase activity to complement the genetic approaches. To accomplish these goals, we have assembled a unique set of reagents and methodologies including NoxA1-/-, ApoE-/-/NoxA1-/- and [SM22α-CreKI/+/NoxA1f/+] mice, Nox1y/-, p47phox-/- and NoxA1-/- mouse aortic VSMC cultures, adenoviral NoxA1 overexpression and shRNA constructs and a functional AlphaScreen and a 100,000 member library of small molecules for identifying potential Nox1 NADPH oxidase inhibitors. Our research strategy includes the following three specific aims.
Aim 1 : Define the molecular mechanisms of NoxA1-dependent NADPH oxidase activity in agonist-induced ROS generation in mouse and human aortic VSMC.
Aim 2 : Determine the effect of NoxA1 deficiency in restenosis and atherosclerosis.
Aim 3 : Identify novel and specific NoxA1 and p47phox interaction inhibitors as means to regulating Nox1 activity. Together, these studies will establis the role of VSMC Nox1 NADPH oxidase in vascular pathophysiology and potentially identify strategies/compounds for the regulation of this enzyme.
Oxidative stress generated from dysregulated Nox1 NADPH oxidase activity has been implicated in a growing number of vascular diseases including atherosclerosis, aortic dissection, hypertension and stroke. Elucidation of molecular composition and activation mechanisms of this VSMC NADPH oxidase will yield valuable insight to develop novel approaches for the treatment of various oxidative stress-dependent vascular diseases.
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