Opposing roles of Nox 1 and Nox 4 in vascular physiology and pathophysiology

活性氧簇(ROS)与正常的血管功能和 血管疾病的发病机制。多年来，我们实验室一直在研究NADPH氧化酶(NOx)家族，这些酶是血管系统中ROS的重要来源。我们已经证明，管壁中的细胞表达多种NOx蛋白，这些蛋白受到不同的调控，产生不同比例的超氧化物和过氧化氢，具有不同的细胞内位置，重要的是，似乎具有不同的细胞功能。NOX1被生长因子激活，并参与血管平滑肌的增殖，而NOX4似乎调节结构性ROS的产生和维持细胞分化。我们认为，虽然NOXL在病理生理情况下被激活，但NOX4控制着细胞的“氧化还原设定点”，并受到维持分化表型的激动剂的调节。我们有耐人寻味的新初步数据表明 形态发生蛋白-4(BMP4)可能通过激活NOX4来阻止血小板衍生生长因子(PDGF)诱导的增殖。因此，该项目的总体目标是更好地确定NOX1和NOX4的不同调控机制，并确定它们在细胞增殖控制中的不同角色。在目标1中，我们将定义PDGF调控NOXL和NOX4表达的机制。我们推测，PDGF激活的转录机制包括MEF2诱导NOXL和ERK1/2介导的FoxMI激活抑制NOX4。目的2研究NOX4在骨形态发生蛋白(BMP)介导的血管平滑肌细胞增殖抑制中的作用及其抑制作用。我们的初步数据显示，BMP4抑制PDGF诱导的增殖，部分是通过 抑制NoXL。BMP4还增加了过氧化氢的产生，导致我们认为它激活了NOX4，导致H202诱导细胞周期抑制因子p21^“的上调。在目标3中，我们计划确定NOX1和NOX4在侧支形成中的作用。我们的模型预测，NOX4的表达和活性影响生长因子的反应性，而PDGF诱导的增殖需要NoXL。为了达到这个目的，我们将使用与生理相关的刺激--缺血诱导的侧支形成--来检验这一假说。最后，在目标4中，我们将确定NOX4在体内新生内膜形成中的作用。我们假设，敲除NOX4将减少生理上必要的过氧化氢的产生，并允许NoXL信号不受抑制地进行，导致血管损伤后损伤的加剧。这 结合体外和体内研究，我们将能够深入了解NOXL和NOX4在正常血管功能和血管疾病中的不同和潜在相反的作用。这些研究将提供重要的基本信息，以此为基础开发新的治疗方法，只针对那些有助于病理的氧化信号方面。

Reactive oxygen species (ROS) have been implicated in both normal vascular function and in the pathogenesis of vascular disease. For a number of years, our laboratory has been studying the NADPH oxidase (Nox) family of enzymes, which are important sources of ROS in the vasculature. We have shown that cells in the vessel wall express multiple Nox proteins, and that these proteins are differentially regulated, produce different ratios of superoxide and hydrogen peroxide, have distinct intracellular locations, and importantly, appear to serve distinct cellular functions. Nox1 is activated by growth factors and is implicated in vascular smooth muscle proliferation, while Nox4 appears to regulate constitutive ROS production and to maintain cell differentiation. We suggest that while Noxl is activated in pathophysiological situations, Nox4 controls the "redox set point" of the cell, and is regulated by agonists that maintain the differentiated phenotype. We have intriguing new preliminary data suggesting that bone morphogenic protein-4 (BMP4) prevents platelet-derived growth factor (PDGF)-induced proliferation, potentially by activating Nox4. The overall goal of this project is thus to better define the mechanisms that differentially regulate Nox1 and Nox4 and to determine their contrasting roles in the control of cell proliferation. In Aim 1, we will define the mechanisms by which Noxl and Nox4 expression are regulated by PDGF. We hypothesize that the transcriptional mechanisms activated by PDGF include MEF2 to induce Noxl and ERK1/2-mediated activation of FoxMI to repress Nox4. Aim 2 is focused on determining the functional role of Nox4 and inhibition of Noxl in BMP-mediated attenuation of vascular smooth muscle cell proliferation. Our preliminary data show that BMP4 inhibits PDGF-induced proliferation, in part by inhibiting Noxl. BMP4 also increases H2O2 production, leading us to propose that it activates Nox4, resulting in H202-induced upregulation of the cell cycle inhibitor p21^"'. In Aim 3, we plan to determine the role of Noxl and Nox4 in collateral formation. Our model predicts that the expression and activity of Nox4 influence growth factor responsiveness, while PDGF-induced proliferation requires Noxl. In this Aim, we will test this hypothesis using a physiologically relevant stimulus; that of ischemia-induced collateral formation. Finally, in Aim 4, we will determine the role of Nox4 in neointimal formation in vivo. We hypothesize that knockout of Nox4 will reduce physiologically necessary H2O2 production and allow Noxl signaling to proceed unchecked, leading to exacerbated lesion formation after vessel injury. This combination of in vitro and in vivo studies will allow Us to gain insight into the distinct and potentially opposing roles of Noxl and Nox4 in normal vascular function and in vascular disease. These studies will provide import;ant basic information on which to base the development of new therapies that target only those aspects of oxidative signaling that contribute to pathology.