OXIDANTS AND ENDOTOXIN INDUCED ENDOTHELIAL INJURY

描述（改编自申请人摘要）：申请人将 进一步观察到细胞内产生反应性的 氧物种（ROS）有助于内毒素刺激的前列腺素类 合成和从肺内皮释放。 两个密切相关的 根据初步数据提出假设， 内毒素导致细胞内产生ROS，而抗氧化剂， DMSO，以及细胞外产生的一氧化氮（NO）和 过氧亚硝基阴离子（ONOO）下调内毒素刺激的前列腺素释放 是细胞内产生ROS的结果，包括产生和 细胞内NO和ONOO的可用性和（ii）抗氧化剂， 超正常水平的细胞内NO和ONOO抑制内毒素刺激的 通过影响ecNOS和考克斯-2 mRNA来调节前列腺素的合成。 均不 和ONOO通常被认为介导氧化剂组织损伤，但 最近有报道称，它们的过度繁殖会使 有益和有害的影响。 拟议的实验代表了 三个目标综合利用细胞技术 生物学、生物化学和分子生物学。 实验被提议为 测定：1）在以下时间过程中诱导ecNOS和考克斯-2 暴露于内毒素以及这是否是细胞内 氧化应激，2）外源性产生的NO和ONOO的影响， 内毒素反应以及观察到的有益作用是否是 其细胞内水平改变的结果，以及，3）是否 经基因工程改造以高表达NO的内皮细胞受到保护 内毒素诱导的前列腺素合成和释放。 以来 初步数据还表明，在牛的iNOS活性的差异， 肺动脉内皮细胞与牛肺微血管 细胞（BMVEC），申请方将在BPAEC和BMVEC中检查1）和2）; 也将检查从这两个相同部位培养的人类细胞， 必要 提高了对内毒素诱导的 内皮细胞的变化将导致改善和新的策略， 治疗成人呼吸窘迫综合征。

DESCRIPTION (Adapted from the applicant's abstract): The applicant will further pursue the observation that intracellular generation of reactive oxygen species (ROS) contributes to endotoxin-stimulated prostanoid synthesis and release from pulmonary endothelium. Two closely associated hypotheses are proposed based on preliminary data demonstrating that endotoxin causes intracellular generation of ROS, and that the antioxidant, DMSO, as well as extracellularly generated nitric oxide (NO) and peroxynitrite (ONOO) downregulate endotoxin-stimulated prostaglandin release is the result of intracellular generation of ROS, including generation and availability of intracellular NO and ONOO and (ii) antioxidants and supranormal levels of intracellular NO and ONOO inhibit endotoxin-stimulated prostaglandin synthesis through effects on ecNOS and COX-2 mRNA. Both NO and ONOO have been generally thought to mediate oxidant tissue injury, but their excessive generation has recently been reported to confer both beneficial and deleterious effects. The proposed experiments represent a comprehensive approach to three specific aims and utilize techniques of cell biology, biochemistry and molecular biology. Experiments are proposed to determine: 1) the time course of induction of ecNOS and COX-2 following exposure to endotoxin and whether this is a consequence of intracellular oxidant stress, 2) the effects of exogenously generated NO and ONOO on the endotoxin response and whether the observed beneficial effects are the result of alterations in their intracellular level and, 3) whether endothelial cells genetically engineered to hyperexpress NO are protected from endotoxin-induced prostaglandin synthesis and release. Since the preliminary data also indicate differences in iNOS activity in bovine pulmonary artery endothelial cells (BPAEC) and bovine lung microvascular cells (BMVEC), the applicant will examine 1) and 2) in both BPAEC and BMVEC; human cells cultured from those same two sites will also be examined, as necessary. Improved understanding of the mechanism(s) of endotoxin-induced endothelial changes will result in improved and novel strategies for treatment of patients with the adult respiratory distress syndrome.