Angiotensin II uncoupling of eNOS in hypertension

描述(申请人提供)：越来越多的证据表明，氧化应激导致高血压和动脉粥样硬化的发病。血管NAD(P)H氧化酶在高血压中被激活，主要参与血管中活性氧物种(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可以显著恢复内皮功能，从而降低血压。然而，这些治疗未能使内皮功能或血压完全正常化，这意味着参与了其他机制，这些机制不容易通过清除ROS而逆转。最近的研究表明，在动脉粥样硬化或高血压的血管中，内皮型一氧化氮合酶(ENOS)从抗氧化酶转变为产生ROS的促氧化酶，而不是一氧化氮。内皮型一氧化氮合酶解偶联的原因之一似乎是内皮型一氧化氮合酶辅因子四氢生物蝶呤(H4B)的缺乏。有趣的是，推测一些抗氧化剂治疗效果不佳的部分原因是它们在重新偶联eNOS方面无效。关于H4B在疾病条件下如何变得持续缺陷的确切机制仍不清楚。我们和其他人最近已经证明血管紧张素II在体外和体内解偶联eNOS(图1-3，Mollnau等人循环90：E58-65)。在这个项目中，我们将充分描述这一现象，并确定它是否由血管NAD(P)H氧化酶衍生的过氧化氢和内皮H4B缺陷(目标1)所介导。在目标2中，我们将确定血管紧张素II对内皮H4B挽救酶的调节是否与内皮H4B缺陷有关。在血管紧张素II注射的小鼠中，将研究对eNOS重新偶联、H4B前体/类似物的内皮功能和血压、血管NAD(P)H氧化酶的抑制、H4B挽救酶的过度表达或两种或两种以上的组合的影响。在目标3中，我们将研究eNOS单体、苏氨酸磷酸化和热休克蛋白90(HSP90)解离在血管紧张素II解偶联eNOS中的潜在作用，以及它们与H4B缺乏症的相互依赖。将研究促进eNOS二聚化的试剂对eNOS重新偶联的影响及其与HSP90的关系。这些拟议的实验最终可能导致新的治疗方法，恢复非偶联eNOS产生一氧化氮，有望降低血压和阻止动脉粥样硬化。我们还将确定eNOS去偶联是否会增加高血压(目标4，H4B缺陷HPH-1小鼠将被研究)。这将解决这个问题：eNOS去偶联仅仅是高血压的后果/标志，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?