Reperfusion-Induced Endothelial Cell Dysfunction

描述（由申请人提供）：在心肌缺血-再灌注（I/RP）中， 产生活性氧物质（ROS），如超氧化物自由基 (023在RP过程中，血管内皮细胞（EC）的作用被认为是 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-），一氧化氮（NO）和 O2-和小的GT3-Racl，其激活产生O2-的NAD（P）H 氧化酶。体外研究使用静态EC缺氧/复氧（H/RO） 模拟I/RP，忽略可能的流动对细胞反应的影响。它 然而，已知层流剪切应力的开始触发NO和O2-。 代、ONOO形成和氧化还原敏感基因表达。因此， 本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EC暴露于稳定层流（或 振荡）剪切应力与氧气再吸收同时进行。是 假设在RP时，EC将产生不同水平/时间曲线的ROS 导致与RO相比不同程度的功能障碍。生产率 将在RP与RO期间测量NO和O2的浓度。EC功能障碍将是 通过测定以下来定量：（a）脂质过氧化和细胞凋亡/活化 促凋亡信号分子（如转录因子NF-r B），（B） 白细胞粘附分子和相关EC-中性粒细胞的表达 粘附相互作用在测量功能障碍的每一个标志物时， 关键的ROS来源（包括显性阴性形式的腺病毒表达） 的Racl）和ROS清除剂（例如，ONOO-分解催化剂）将帮助我们 确定负责EC损伤的ROS。如果损伤被抑制， NO和O2-抑制剂，那么ONOO-是牵连和它的相对变化 (both细胞内和细胞外）将在RP期间进行监测， 而不存在抑制剂/清除剂。细胞外ONOO-被证明是 是心肌功能丧失的指标，因此， 将使用数学公式估计EC暴露于 ROS在细胞外空间的运输/反应模型。总之，新RP 模型将推进我们的知识EC生存的条件下， 发生氧张力和流体流动的变化。

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.