权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

再灌注过程中与 eNOS 解偶联相关的体内和离体机制

基本信息

批准号：
7454935
负责人：
Lindon H Young
金额：
$ 22.5万
依托单位：
PHILADELPHIA COLLEGE OF OSTEOPATHIC MED
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-19 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7454935
关键词：
7,8-dihydrobiopterin Adherence Aerobic Exercise Animals Aortic Segment Biological Availability Blood Vessels Blood flow Cardiac Cell Adhesion Molecules Clinical Coronary Electrodes Enhancers Evaluation Femoral vein Functional disorder Heart Heart Injuries Hydrogen Peroxide In Vitro Infiltration Injury Ischemia Left Life Style Measurement Measures Modeling Myocardial Infarction Myocardial Ischemia Nitric Oxide Nitric Oxide Synthase Organ Organ Transplantation Oxidative Stress Patients Peptides Peroxidase Physiological reperfusion Play Public Health Quality of life Rate Rattus Reperfusion Injury Reperfusion Therapy Role Superoxides Testing Time Veins Ventricular attenuation base cofactor femoral artery human NOS3 protein in vivo indexing inhibitor/antagonist neutrophil oxidation pressure protein kinase C epsilon restoration sedentary tetrahydrobiopterin

项目摘要

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.

描述（申请人提供）：再灌注损伤是由内皮功能障碍引发的，其特征是氧化应激增加和内皮源性一氧化氮（NO）生物利用度有限，导致多形核白细胞（PMN）血管粘附和浸润增强，随后导致血管和器官功能障碍。 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.然而，当 eNOS 的必需辅助因子四氢生物蝶呤 (BH4) 在体外被氧化为二氢生物蝶呤 (BH2) 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，尚未通过实时直接测量体内再灌注过程中的氧化应激来证明控制 eNOS 解偶联的机制。最近，我们使用特定的 H2O2 或 NO 微传感器开发了一种直接测量大鼠股动脉/静脉缺血/再灌注 (I/R) 模型中股静脉体内股静脉释放的 NO 和 H2O2（SO 的间接指数）的方法。我们的初步结果表明，与同一动物中的非缺血（假）股静脉相比，I/R 股静脉中的 H2O2 显着增加。此外，BH4或eNOS抑制剂（例如蛋白激酶Cε肽抑制剂（PKCα-））减少，而BH2或eNOS增强剂（例如PKCα激活剂（PKCα+））增加再灌注期间H2O2的释放。 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.在我们的离体 PMN 诱导的心肌 I/R 损伤模型中，再灌注期间单独给予 BH4 或 PKC α-、BH2 或 PKC α+ 时，均未提供心脏保护作用。根据我们的初步结果，我们假设 BH4 氧化为 BH2 是再灌注期间 eNOS 解偶联的主要原因，并且是 PKC α+/PKC β- 对 H2O2/NO 释放和心脏保护作用相反作用的主要原因。为了检验这一假设，我们将在存在/不存在 BH4、BH2、PKC α+ 或 PKC β- 单独或不同组合的情况下执行以下具体目标：1) 和 2) 在我们的股骨 I/R 模型中体内测量股骨 I/R 静脉和假静脉的 H2O2 或 NO 释放； 3a)、3b) 和 4) 离体非缺血大鼠主动脉段的体外 NO 释放测量，再灌注后心脏收缩功能、心脏损伤和 PMN 积累的离体评估。我们预测 BH4+PKC ?+（即 eNOS 激活）而不是 BH2 +PKC ?+ 将显着降低氧化应激并保护心脏免受再灌注损伤。公共健康相关性表征再灌注损伤中 eNOS 解偶联的体内和离体机制将对确定血流再灌注期间器官移植和心脏病患者的更具选择性的治疗（例如 BH4 或 BH4 与 PKC α+ 联合）产生重大影响。与器官移植或心脏病发作后久坐的生活方式相比，临床影响将明显更好地恢复器官功能，从而带来更好的生活质量（即有氧运动）。