Angiotensin II uncoupling of eNOS in hypertension

描述（由申请人提供）：越来越多的证据表明，氧化应激有助于高血压和动脉粥样硬化的发病机制。血管NAD（P）H氧化酶在高血压中被激活，主要参与血管活性氧（ROS）的产生。事实上，用抗氧化酶或血管NAD（P）H氧化酶抑制剂清除ROS可显著恢复内皮功能以降低血压。然而，这些治疗未能使内皮功能或血压完全正常化，这意味着涉及其他机制，这些机制不容易通过清除ROS而逆转。近年来的研究表明，在动脉粥样硬化或高血压血管中，内皮型一氧化氮合酶（eNOS）从抗氧化酶转变为产生ROS而不是一氧化氮的促氧化酶。eNOS解偶联的原因之一似乎是eNOS辅因子四氢生物蝶呤（H4 B）缺乏。有趣的是，推测一些抗氧化剂治疗的不满意结果部分是由于它们在重新偶联eNOS中无效。H4 B在疾病条件下如何持续缺乏的确切机制仍不清楚。我们和其他人最近已经表明血管紧张素II在体外和体内解偶联eNOS（图1-3，Mollnau等Cir Res 90：E58-65）。在这个项目中，我们将充分表征这种现象，并确定它是否是由血管NAD（P）H氧化酶衍生的过氧化氢和内皮H4 B缺乏介导的（目的1）。在目标2中，我们将确定血管紧张素II对内皮H4 B补救酶的调节是否有助于内皮H4 B缺乏。将在血管紧张素II输注小鼠中研究H4 B前体/类似物对eNOS再偶联、内皮功能和血压的影响、血管NAD（P）H氧化酶的抑制、H4 B补救酶的过表达或2种或更多种的组合。在目标3中，我们将研究eNOS单体化，苏氨酸磷酸化和热休克蛋白90（HSP 90）的解离在eNOS的血管紧张素II解偶联及其与H4 B缺乏的相互依赖性中的潜在作用。将研究促进eNOS二聚化的试剂对eNOS再偶联的影响及其与HSP 90的关联。这些拟议的实验可能最终导致新的治疗方法恢复从解偶联eNOS产生一氧化氮，预计这将降低血压和阻止动脉粥样硬化。我们还将确定eNOS解偶联是否增加高血压（目的4，将研究H4 B缺陷型hph-1小鼠）。这将解决问题：eNOS解偶联仅仅是高血压的后果/标志物还是原因之一？

DESCRIPTION (provided by applicant): Accumulating evidence suggests that oxidant stress contributes to the pathogenesis of hypertension and atherosclerosis. Vascular NAD(P)H oxidases are activated in hypertension, predominantly contribute to vascular production of reactive oxygen species (ROS). Indeed, scavenging of ROS with either antioxidant enzymes or inhibitors of vascular NAD(P)H oxidases significantly restores endothelial function to reduce blood pressure. Nevertheless, these treatments failed to completely normalize endothelial function or blood pressure, implicating involvement of other mechanisms that are not readily reversible by scavenging ROS. Recent studies demonstrated that in atherosclerotic or hypertensive blood vessels, endothelial nitric oxide synthase (eNOS) transformed from an antioxidant enzyme into a pro-oxidant enzyme producing ROS rather than nitric oxide. 1 of the causes for this uncoupling of eNOS seems to be a deficiency in eNOS cofactor tetrahydrobiopterin (H4B). It is interesting to speculate that unsatisfactory outcomes of some antioxidant therapies are partially due to their ineffectiveness in recoupling eNOS. The precise mechanisms as to how H4B becomes persistently deficient under disease conditions remain unclear. We and others have recently shown that angiotensin II uncouples eNOS in vitro and in vivo (Figures 1-3, Mollnau et al Cir Res 90: E58-65). In this project we will fully characterize this phenomenon and determine whether it is mediated by vascular NAD(P)H oxidase-derived hydrogen peroxide and an endothelial H4B deficiency (aim 1). In aim 2 we will determine whether angiotensin II modulation of endothelial H4B salvage enzymes contributes to endothelial H4B deficiency. Effects on eNOS recoupling, endothelial function and blood pressure of H4B precursors/analogues, inhibition of vascular NAD(P)H oxidases, overexpression of H4B salvage enzymes or combination of 2 or more will be studied in angiotensin II infused mice. In aim 3 we will examine potential roles of eNOS monomerization, threonine phosphorylation and dissociation from heat shock protein 90 (HSP90) in angiotensin II uncoupling of eNOS and their interdependence with H4B deficiency. Effects on eNOS recoupling of agents promoting eNOS dimerization and its association with HSP90 will be studied. These proposed experiments could ultimately lead to novel therapeutics restoring nitric oxide production from uncoupled eNOS, which are anticipated to reduce blood pressure and impede atherosclerosis. We will also determine whether eNOS uncoupling augments hypertension (aim 4, H4B-deficient hph-1 mice will be studied). This will address the question: is eNOS uncoupling a mere consequence/marker of hypertension or 1 of the causes?