Nox Isoforms and Vascular Cell Phenotype

心血管疾病的临床表现，如心脏病发作、中风和充血性心力衰竭，是发达国家的头号死因。尽管对特定的心血管危险因素如高胆固醇血症、糖尿病和高血压有相当大的兴趣，但来自心脏研究的数据清楚地表明，心血管疾病事件的主要决定因素是衰老。 老化血管的特征在于抗氧化酶的控制降低、活性氧物质的通量增加和血管功能障碍。这种活性氧物质的通量已被证明是血管表型作为年龄的函数的主要决定因素，与衰老的“自由基理论”一致。这一理论最初由Harman提出，指出了自由基的主要来源是电子。然而，现在有令人信服的证据表明，在整个血管，特别是在内皮细胞的活性氧的多种酶源。其中的原理是与经典的中性粒细胞呼吸爆发氧化酶相关的NADPH氧化酶家族。我们和其他人已经发现，内皮细胞含有NADPH氧化酶催化亚基（Nox）的两种特异性亚型; Nox 2和Nox 4。前者与呼吸爆发氧化酶（GP 91/phox，现在是Nox 2）相同，而后者是新的。相当多的数据表明，血管疾病和衰老的特征是过量的血管活性氧部分是由于Nox酶活性。大多数关于Nox源性活性氧和血管疾病的研究都集中在Nox 2上。然而，Nox 4的作用以及它与Nox 2在血管表型方面的差异尚不清楚。考虑到Nox 4是脉管系统中的主要Nox同种型，这就更成问题了。该提议旨在弥合这一知识差距，并基于以下假设：由特定Nox亚型产生的活性氧是血管疾病和衰老特征性的适应不良血管表型的重要决定因素。因此，本申请的目的是确定特定的Nox亚型在控制内皮细胞表型中的作用，并阐明所涉及的机制。为了实现这一目标，我们将首先表征内皮细胞中NADPH氧化酶催化亚基异构体的表达、亚细胞定位和亚基需求。然后，我们将使用功能获得和功能丧失策略来确定Nox亚型在控制涉及ROS的内皮细胞表型中的作用，如NO生物活性、粘附分子表达、增殖、迁移和衰老。然后，我们将继续使用稳定的siRNA表达或细胞特异性Nox过表达来操纵小鼠中的Nox 4表达，并研究其对血管功能和动脉损伤反应的影响。这些研究的成功进行将提供有关氧化还原介导的血管细胞表型控制的机制信息，并为我们提供必要的见解，以设计新的治疗策略，重点是Nox衍生的活性氧。

Clinical manifestations of cardiovascular disease, such as heart attack, stroke, and congestive heart failure, are the number one cause of death in the developed world. Despite considerable interest in specific cardiovascular risk factors such as hypercholesterolemia, diabetes, and hypertension, data from the Framingham Heart Study indicate clearly the main determinant of incident cardiovascular disease is aging. Aged blood vessels are characterized by reduced control of antioxidant enzymes, an increased flux of reactive oxygen species, and vascular dysfunction. This flux of reactive oxygen species has proven to be a major determinant of vascular phenotype as a function of age, consistent with the "Free Radical Theory" of aging. This theory, originally proposed by Harman/1, pointed to the mitochondrion as the principal source of free radicals. However, there is now compelling evidence for multiple enzymatic sources of reactive oxygen species throughout blood vessels and particularly in the endothelium. Principle among these are the NADPH oxidase family of enzymes that are related to the classical neutrophil respiratory burst oxidase. We and others have found that endothelial cells contain two specific isoforms of the NADPH oxidase catalytic subunit (Nox); Nox2 and Nox4. The former is identical to the respiratory burst oxidase (GP91/phox, now Nox2), whereas the latter is novel. Considerable data indicate that excess vascular reactive oxygen species that are characteristic of vascular disease and aging are due, in part, from Nox enzymatic activity. Most investigation on Nox-derived reactive oxygen species and vascular disease has focused on Nox2. However, the role of Nox4 and how it differs with Nox2 with regard to vascular phenotype is not known. This is all the more problematic considering that Nox4 is the major Nox isoform in the vasculature. This proposal is designed to bridge this gap in knowledge and is based upon the hypothesis that reactive oxygen species produced by specific Nox isoforms is an important determinant of the maladaptive vascular phenotype that is characteristic of vascular disease and aging. The objective of this application, therefore, is to determine the role of specific Nox isoforms in controlling endothelial cell phenotype and elucidate the mechanisms involved. In order to achieve this objective, we will first characterize the NADPH oxidase catalytic subunit isoforms in endothelial cells with expect to their expression, subcellular localization, and subunit requirements. We will then use gain-of-function and loss-of-function strategies to determine the role of Nox isoforms in controlling endothelial cell phenotypes involving ROS such as NO bioactivity, adhesion molecule expression, proliferation, migration, and senescence. We will then go on to manipulate Nox4 expression in mice using stable siRNA expression or cell-specific Nox overexpression and examine the implications for vascular function and the response to arterial injury. Successful prosecution of these studies will provide mechanistic information on redox-mediated control of vascular cell phenotype and afford us the necessary insight to design new therapeutic strategies that focus on Nox-derived reactive oxygen species.