Vascular smooth muscle NAD(P)H oxidases

活性氧在血管生理和病理生理中起着重要的作用。血管壁上活性氧最重要的来源是非吞噬性NAD(P)H氧化酶。这一建议集中在基于诺克的氧化酶上，这是一种在血管平滑肌细胞中特异性表达的中性粒细胞呼吸爆发氧化酶的同源物。Noxl是在该实验室克隆的，是目前gp91phox样蛋白家族中的第一个。它已被血管活性激动剂血管紧张素II和血小板衍生生长因子激活，并在生长中具有重要的功能。在血管平滑肌细胞中，另外两种中性粒细胞亚基p22phox和p47phox已被证明对激动剂诱导的氧化酶活性至关重要；然而，它们是否与诺克l相互作用形成功能性氧化酶尚不清楚。事实上，人们对多亚基诺克氧化酶的调节和组装几乎一无所知。因此，本项目的主要目标是确定血管平滑肌中基于nox的NAD(P)H氧化酶复合物的调节分子机制，并确定其在血管平滑肌中的作用

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.