Vascular smooth muscle NAD(P)H oxidases

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Reactive oxygen species play essential roles in vascular physiology and pathophysiology. Some of the most important sources of reactive oxygen species in the vessel wall are the nonphagocytic NAD(P)H oxidases. This proposal is centered on the noxl-based oxidase, a homologue of the neutrophil respiratory burst oxidase that is expressed specifically in vascular smooth muscle cells. Noxl was cloned in this laboratory and is the first of what is now a family of gp91phox-like proteins. It has been shown to be activated by the vasoactive agonists angiotensin II and platelet-derived growth factor, and to be functionally important in growth. In vascular smooth muscle cells, two other neutrophil subunits, p22phox and p47phox have been shown to be essential to agonist-induced oxidase activity; however, whether they interact with noxl to form a functional oxidase is not known. In fact, virtually nothing is known about the regulation and assembly of the presumably multisubunit noxl-based oxidase. The major goal of this project is thus to define the molecular mechanisms by which the noxl-based NAD(P)H oxidase complex is regulated in vascular smooth muscle and to determine its role in the abnormal smooth muscle growth that is a hallmark of hypertension and restenosis. The first specific aim is to determine the functional role of p47phox in activation of the noxl-p22phox oxidase in vascular smooth muscle cells. A series of biochemical and molecular techniques will be used to assess the agonist-sensitive association of p47phox with noxl and p22phox and its potential phosphorylation and translocation by angiotensin II. In the second aim, we will examine the phenotypic consequences of noxl, p22phox and noxl/p22phox overexpression on hypertrophic and hyperplastic growth of smooth muscle cells in vivo. At Emory, we have already generated transgenic mice that overexpress noxl or p22phox specifically in smooth muscle cells, and we will use these mice, together with double overexpressors obtained by crossing these mice, to investigate the effects of oxidase overexpression on smooth muscle growth in a model of hypertension and restenosis. Finally, in the third specific aim, we will determine whether deletion of p47phox ameliorates the effect of noxl overexpression in vivo. Noxl or p22phox overexpressors will be crossed with mice bearing a genetic deletion of p47phox and used to study hyperplastic and hypertrophic smooth muscle growth in vivo. Understanding the factors controlling the activation of the noxl-based NAD(P)H oxidase provides a rationale for therapeutic intervention that is likely to affect many of the factors contributing to vascular disease.