Reperfusion-Induced Endothelial Cell Dysfunction

描述(申请人提供)：在心肌缺血-再灌注(I/RP)中， 产生活性氧物种(ROS)，如超氧自由基 (023)，来自血管内皮细胞(ECs)在RP过程中被认为起到了 在组织损伤中起关键作用。受伤的部分原因是 过氧亚硝酸盐(ONOO-)，是一氧化氮(NO)和亚硝酸根的反应产物 O2-，以及激活产生O2-的NAD(P)H的小GTP酶Rac1 氧化物酶。体外研究已在静态内皮细胞中使用缺氧/复氧(H/RO) 以模拟I/RP，忽略可能对细胞响应的流动影响。它 然而，已知层流剪应力的开始触发了NO和02- 产生、ONOO形成和氧化还原敏感基因的表达。因此，这一点 研究旨在探讨内源性ROS在EC功能障碍中的作用 在静态低氧内皮细胞暴露到稳定的层流(或 (振荡)剪切应力与氧气再吸收同时发生。它是 假设在RP，ECs将产生不同水平/时间分布的RO 导致与RO相比不同程度的功能障碍。生产率 NO和02-将在RP和RO期间进行测量。EC功能障碍将是 通过分析：(A)脂质过氧化和细胞凋亡/激活 促凋亡信号分子(如转录因子NF-Rb)，(B) 白细胞黏附分子及其相关的EC-中性粒细胞的表达 粘附性相互作用。在测量每个功能障碍的标记物时，抑制剂 关键的ROS来源(包括显性负性形式的腺病毒表达 和ROS清道夫(例如ONOO分解催化剂)将帮助我们 确定造成EC伤害的ROS。如果伤害被抑制的话 NO和02-抑制物，然后ONOO-被牵连及其相关变化 (细胞内和细胞外)将在使用或 没有抑制剂/清道夫的存在。胞外ONOO-被证明是 是心肌功能丧失的一个指标，所以ONOO浓度 ECS面临的风险将使用数学公式进行估计 ROS在胞外空间的运输/反应模型。总而言之，新的RP 模型将促进我们对EC在以下条件下的生存的了解 氧气张力和液体流量会发生变化。

DESCRIPTION (Provided by Applicant): In myocardial ischemia-reperfusion (I/RP), the production of reactive oxygen species (ROS), such as superoxide radicals (023, from vascular endothelial cells (ECs) during RP is thought to play a critical role in tissue injury. The injury is attributed partially to peroxynitrite (ONOO-), a product of the reaction between nitric oxide (NO) and O2-, and to the small GTPase Racl that activates the O2--producing NAD(P)H oxidase. In vitro studies have used hypoxia/reoxygenation (H/RO) in static ECs to simulate I/RP, ignoring possible flow effects on the cellular response. It is known, however, that the onset of laminar shear stress triggers NO and 02- generation, ONOO-formation and redox-sensitive gene expression. Thus, this study aims to investigate the role of endogenous ROS on EC dysfunction following the exposure of static hypoxic ECs to the onset of steady laminar (or oscillatory) shear stress concurrently with oxygen readmission. It is hypothesized that at RP, ECs will produce ROS of different levels/time profiles resulting in different extents of dysfunction compared to RO. Production rates of NO and 02- will be measured during RP vs. RO. EC dysfunction will be quantified by assaying for: (a) lipid peroxidation and apoptosis/activation of pro-apoptotic signaling molecules (such as the transcription factor NF-rB), (b) expression of leukocyte adhesion molecules and the associated EC-neutrophil adhesive interactions. While measuring each marker of dysfunction, inhibitors of key ROS sources (including adenoviral expression of a dominant negative form of Racl) and ROS scavengers (eg. a ONOO- decomposition catalyst) will help us identify the ROS responsible for the EC injury. If the injury is inhibited by both NO and 02- inhibitors, then ONOO-is implicated and its relative changes (both intracellularly and extracellularly) will be monitored during RP with or without the presence of inhibitors/scavengers. Extracellular ONOO- was shown to be an index of loss of myocardial function, so the ONOO' concentrations that the ECs are exposed to will be estimated using a mathematical transport/reaction model of ROS in the extracellular space. In sum, the new RP model will advance our knowledge on EC survival under conditions where both changes in oxygen tension and fluid flow occur.