NOXs, eNOS Uncoupling and Diabetic Atherogenesis

摘要 本申请的中心焦点是解决三个假设：1)NAD(P)H氧化酶1(NOX1) 2)叶酸(FA)依赖的eNOS辅因子修复 四氢生物蝶呤(H4B)抢救酶二氢叶酸还原酶(DHFR)在糖尿病中重新偶联eNOS；3) 内皮型一氧化氮合酶的重新偶联阻碍糖尿病动脉粥样硬化形成。 内皮型一氧化氮合酶(ENOS)通过产生一氧化氮合酶(NO)是血管稳态的主要保护者 具有强大的抗炎和抗动脉粥样硬化作用的氧化物。过去十年的研究表明 然而，已经确定eNOS可以解偶联而产生超氧化物(O2？-)，而不是NO，当其 辅因子H4B缺乏，即由于过氧亚硝酸根介导的氧化。这种转变可能会潜在地 持续氧化应激，这与糖尿病的病因和心血管并发症的加速有关。 事实上，其他人和我们已经报道了糖尿病小鼠主动脉产生eNOS衍生的、L名称敏感的O2 或者老鼠。我们进一步证明糖尿病eNOS的解偶联是由血管紧张素II(Ang II)介导的，AS Ang II信号衰减剂坎地沙坦或卡托普利有效地重新偶联eNOS以恢复主动脉H4B含量和 NO产生，同时eNOS产生的O2-减少。糖尿病内皮型一氧化氮合酶的解偶联也与 H4B抢救酶二氢叶酸还原酶(DHFR)缺失，介导eNOS Ang II解偶联 培养内皮细胞。 我们先前已经证明，Ang II通过NOX依赖的H_2O_2产生和H_2O_2-来解偶联eNOS。 培养的主动脉内皮细胞依赖的dhfr缺陷。有待澄清的是哪种特定的NOX 亚型位于糖尿病未偶联eNOS的上游(目标1)，DHFR缺乏在糖尿病中是否起重要作用 糖尿病内皮型一氧化氮合酶的解偶联及叶酸能否恢复dhfr的表达和活性 ENOS(目标2)。在初步实验中，我们发现了有趣的证据，表明FA在培养的主动脉中重新偶联eNOS 血管内皮细胞和血管紧张素Ⅱ输注小鼠。在具体目标3中，我们将考察eNOS的再挂钩是否有效 在阻止糖尿病小鼠的动脉粥样硬化形成方面。 总体假设是内皮细胞NOX1在体内被高血糖/糖尿病激活，导致 ROS的初始产生(Ang II依赖)，随后的DHFR缺乏，以及eNOS的解偶联，这在 反过来，夸大氧化应激以加速糖尿病动脉粥样硬化的形成。

ABSTRACT The central focus of this application is to address three hypotheses: 1) NAD(P)H oxidase 1 (NOX1) mediates eNOS uncoupling in diabetes; 2) Folic acid (FA)-dependent restoration of eNOS cofactor tetrahydrobiopterin (H4B) salvage enzyme dihydrofolate reductase (DHFR) recouples eNOS in diabetes; 3) Recoupling of eNOS impedes diabetic atherogenesis. Endothelial nitric oxide synthase (eNOS) is a major protector of vascular homeostasis by producing nitric oxide (NO¿) that has potent anti-inflammatory and anti-atherosclerotic effects. Studies in the past decade have however established that eNOS can become uncoupled to produce superoxide (O2¿-) rather than NO¿, when its cofactor H4B was deficient, i.e. consequent to peroxynitrite mediated oxidation. This transformation may potentially sustain oxidant stress that has been implicated in diabetic etiology and acceleration of cardiovascular complications. Indeed, others and we have reported eNOS-derived, L-NAME-sensitive O2¿- production from aortas of diabetic mice or rats. We have further demonstrated that diabetic uncoupling of eNOS is mediated by angiotensin II (Ang II), as Ang II signaling attenuators Candesartan or Captopril effectively recoupled eNOS to restore aortic H4B content and NO¿ production, while diminishing eNOS-derived O2¿- production. Diabetic uncoupling of eNOS is also associated with a loss in H4B salvage enzyme dihydrofolate reductase (DHFR), which mediates Ang II uncoupling of eNOS in cultured endothelial cells. We have previously shown that Ang II uncouples eNOS via NOX-dependent H2O2 production and H2O2- dependent DHFR deficiency in cultured aortic endothelial cells. What remain to be elucidated is which specific NOX isoform lies upstream of uncoupled eNOS in diabetes (Aim 1), whether DHFR deficiency plays an important role in diabetic uncoupling of eNOS and whether folic acid (FA) can restore DHFR expression and activity to recouple eNOS (Aim 2). In preliminary experiments we found intriguing evidence that FA recoupled eNOS in cultured aortic endothelial cells and Ang II infused mice. In specific aim 3 we will examine whether recoupling of eNOS is effective in impeding atherogenesis in diabetic mice. The overall hypothesis is that endothelial NOX1 is activated by hyperglycemia/diabetes in vivo, resulting in an initial production of ROS (Ang II-dependent), consequent DHFR deficiency, and uncoupling of eNOS, which in turn, exaggerates oxidant stress to accelerate diabetic atherogenesis.