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作用。血管壁中活性氧的一些最重要来源是非吞噬细胞NAD（P）H氧化酶。该提议集中在基于noxl的氧化酶上，其是在血管平滑肌细胞中特异性表达的嗜中性粒细胞呼吸爆发氧化酶的同源物。Noxl是在这个实验室中克隆的，它是现在gp 91 phox样蛋白家族中的第一个。它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，并在生长中发挥重要作用。在血管平滑肌细胞中，另外两种中性粒细胞亚基p22 phox和p47 phox已被证明是激动剂诱导的氧化酶活性所必需的;然而，它们是否与noxl相互作用形成功能性氧化酶尚不清楚。事实上，几乎没有什么是已知的调控和装配的推测多亚基noxl为基础的氧化酶。因此，本项目的主要目标是确定血管平滑肌中基于noxl的NAD（P）H氧化酶复合物调节的分子机制，并确定其在血管平滑肌中的作用。 异常平滑肌生长是高血压和再狭窄的标志。第一个具体的目的是确定p47 phox在血管平滑肌细胞中noxl-p22 phox氧化酶激活中的功能作用。一系列的生物化学和分子技术将被用来评估激动剂敏感的关联p47 phox与noxl和p22 phox和其潜在的磷酸化和易位的血管紧张素II。在第二个目标中，我们将研究noxl，p22 phox和noxl/p22 phox过表达对体内平滑肌细胞肥大和增生生长的表型后果。在埃默里大学，我们已经产生了在平滑肌细胞中特异性过表达noxl或p22 phox的转基因小鼠，我们将使用这些小鼠，以及通过杂交这些小鼠获得的双过表达者，研究氧化酶过表达对高血压和再狭窄模型中平滑肌生长的影响。最后，在第三个具体目标中，我们将确定p47 phox的缺失是否改善体内noxl过表达的效果。 将Noxl或p22 phox过表达者与携带p47 phox遗传缺失的小鼠杂交， 用于研究体内增生和肥大平滑肌生长。了解控制基于noxl的NAD（P）H氧化酶的激活的因素为可能影响导致血管疾病的许多因素的治疗干预提供了理论基础。

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.