In vivo and ex vivo mechanisms related to eNOS uncoupling during reperfusion

描述（由申请人提供）：再灌注损伤由内皮功能障碍引发，其特征为氧化应激增加和内皮源性一氧化氮（NO）生物利用度有限，导致血管和器官功能障碍后多形核白细胞（PMN）血管粘附和浸润增强。因此，减轻内皮功能障碍可能是限制再灌注损伤的有效方法。内皮型一氧化氮合酶（eNOS）在正常内皮功能中起核心作用。然而，当eNOS的主要辅因子四氢生物蝶呤（BH 4）在体外被氧化为二氢生物蝶呤（BH 2）时，eNOS可以将其产物谱从产生NO转变为超氧化物（SO）（eNOS解偶联）。到目前为止，还没有通过实时直接测量体内再灌注期间的氧化应激来证明控制eNOS解偶联的机制。最近，我们开发了一种直接测量NO和H2 O2（SO的间接指数）释放从股静脉在体内在大鼠股动脉/静脉缺血/再灌注（I/R）模型使用特定的H2 O2或NO微传感器。我们的初步结果表明，H2 O2显着增加，在I/R股静脉相比，非缺血（假）股静脉在同一动物。此外，BH 4或eNOS抑制剂（例如，蛋白激酶C β肽抑制剂（PKC β-）降低，而BH 2或eNOS增强剂（例如，PKC？激活剂（PKC？+））增加再灌注时H2 O2的释放。此外，BH 4还显著增加了股I/R静脉的NO释放。此外，BH 4或PKC？+增加，而BH 2或PKC？减少非缺血大鼠主动脉段的NO释放。在我们的离体PMN诱导的心肌I/R损伤模型中，BH 2和PKC？+在再灌注期间单独给药时提供心脏保护。基于我们的初步结果，我们假设BH 4氧化为BH 2是再灌注过程中eNOS解偶联的主要原因，也是PKC？+/？蛋白激酶C H2 O2/NO释放和心脏保护。为了检验这一假设，我们将在存在/不存在BH 4、BH 2、PKC？+的情况下进行以下具体目标。还是PKC单独或以不同的组合：1）和2）在我们的股I/R模型中从股I/R静脉和假静脉释放的H2 O2或NO的体内测量; 3a）、3b）和4）从分离的非缺血大鼠主动脉节段的体外NO释放测量，再灌注后心脏收缩功能、心脏损伤和PMN积累的离体评价。我们预测BH 4 +PKC？+ (i.e., eNOS激活），而不是BH 2 +PKC？+可显著降低氧化应激，保护心脏免受再灌注损伤。 公共卫生相关性表征再灌注损伤中负责eNOS解偶联的体内和离体机制将对确定更具选择性的治疗方法（如BH 4或BH 4与PKC？+联合）产生重大影响，用于器官移植和心脏病患者在血流再灌注期间。临床影响将是器官功能的显著更好恢复，从而导致更好的生活质量（即，有氧运动）与器官移植或心脏病发作后久坐不动的生活方式相比。

DESCRIPTION (provided by applicant): Reperfusion injury is initiated by endothelial dysfunction, which is characterized by increased oxidative stress and limited endothelial-derived nitric oxide (NO) bioavailability, leading to enhanced polymorphonuclear leukocyte (PMN) vascular adherence and infiltration following by blood vessel and organ dysfunction. Therefore, attenuation of endothelial dysfunction may be an effective way to limit reperfusion injury. Endothelial NO synthase (eNOS) plays a central role in normal endothelial function. However, eNOS can shift its product profile from producing NO to superoxide (SO) (eNOS uncoupling) when its essential cofactor, tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2) in vitro. Until now, the mechanisms that govern eNOS uncoupling have not been demonstrated by real-time direct measuring oxidative stress during reperfusion in vivo. Recently, we developed a direct measurement of NO and H2O2 (indirect index of SO) release from femoral veins in vivo in a rat femoral artery/vein ischemia/reperfusion (I/R) model using specific H2O2 or NO microsensors. Our preliminary results show that H2O2 is significantly increased in I/R femoral veins compared to non-ischemic (sham) femoral veins in the same animal. Moreover, BH4 or an eNOS inhibitor (e.g., protein kinase C epsilon peptide inhibitor (PKC ?-)) decreased, whereas BH2 or an eNOS enhancer (e.g., PKC ? activator (PKC ?+)) increased H2O2 release during reperfusion. Additionally, BH4 also significantly increased NO release from femoral I/R veins. Furthermore, BH4 or PKC?+ increased, whereas BH2 or PKC ?- decreased NO release in non-ischemic rat aortic segments, respectively. In our ex vivo PMN-induced myocardial I/R injury model, BH4 or PKC ?-, neither BH2 nor PKC ?+ provided cardioprotection when given separately during reperfusion. Based on our preliminary results, we hypothesize that the oxidation of BH4 to BH2 is mainly responsible for eNOS uncoupling during reperfusion, and is the primary cause for opposing effects of PKC ?+/PKC ?- on H2O2/NO release and cardioprotection. To test the hypothesis, we will perform the following specific aims in the presence/absence of BH4, BH2, PKC ?+ or PKC ?- alone or in different combinations: 1) and 2) in vivo measurement of H2O2 or NO release from the femoral I/R vein and sham vein in our femoral I/R model; 3a), 3b) and 4) in vitro NO release measurement from isolated non-ischemic rat aortic segments, ex vivo evaluation of postreperfused cardiac contractile function, heart injury and PMN accumulation. We predict that BH4+PKC ?+ (i.e., eNOS activation), not BH2 +PKC ?+ will significantly decrease oxidative stress and protect heart from reperfusion injury. PUBLIC HEALTH RELEVANCE Characterizing the in vivo and ex vivo mechanism(s) responsible for eNOS uncoupling in reperfusion injury would have a significant impact in identifying more selective treatments, such as BH4 or BH4 combined with PKC ?+, for organ transplants and heart attack patients during reperfusion of blood flow. The clinical impact would be significantly better restoration of organ function resulting in a better quality of life (i.e., aerobic exercise) compared to more sedentary life-style after organ transplantation or heart attacks.