SIGNAL TRANSDUCTION BY ENDOTHELIAL XANTHINE OXIDASE

内皮黄嘌呤氧化酶的信号转导

• 批准号: 2856720
• 负责人: Gregory B. Bulkley
• 金额: $ 29.16万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2856720
• 关键词: Kupffer's cell; biological signal transduction; calcium flux; cell adhesion; cytokine receptors; enzyme activity; enzyme inhibitors; free radical oxygen; inflammation; integrins; laboratory mouse; laboratory rat; macrophage; neutrophil; nuclear factor kappa beta; orphan disease /drug; oxidative stress; phagocytosis; receptor expression; second messengers; tissue /cell culture; tumor necrosis factor alpha; vascular endothelium; xanthine dehydrogenase; xanthine oxidase

Previous studies from this project have established a critical role for reactive oxygen metabolites ("free radicals") generated from endothelial cell xanthine oxidase as triggers of neutrophil-mediated microvascular injury, and consequent organ dysfunction and failure following ischemia/reperfusion, or following shock, leading to the development of multiple organ failure. We now propose to evaluate the novel hypothesis that microvascular endothelial cell surface xanthine oxidoreductase functions physiologically as a molecular switch for the reticuloendothelial system by transducing circulating inflammatory signals (cytokines) via the limited proteolytic activation of xanthine oxidase (XO) from xanthine dehydrogenase (XD), generating hydrogen peroxide as a second messenger to trigger the integrin-mediated adhesion, trapping and activation of circulating neutrophils to effect the trapping, phagocytosis and killing of circulating microorganisms within the venules Of most organs (vestigially) and the sinusoids and central veins of the liver (functionally). In our model system of microvascular endothelial cell monolayers in vitro, gamma-interferon primes this signalling mechanism by upregulating the synthesis of xanthine dehydrogenase, while TNFalpha triggers neutrophil adhesion by mediating proteolytic XD to XO conversion. Specific Aims: 1. Confirm the necessary role for endothelial cell surface XO for the transduction of cytokine (TNFalpha) signals in endothelial cells in vitro to upregulate the expression of surface integrins by blocking XO activity with custom-made m-anti-XO, and by transfecting XO- negative endothelial cells with chicken or mutant xanthine dehydrogenase which is incapable of proteolytic activation to XO, and evaluating these cells for signalling activity. 2. Define the relationship of this novel signal transduction pathway to other, established pathways using conventional techniques. 3. Evaluate each component of the proposed pathway, including the roles of specific TNFalpha receptors, the SEKk kinase pathway, sphingomyelin/ceramide, phospholipase C, inositol triphosphate, intracellular calcium, and mu-calpain in effecting proteolytic activation of XO using established, but state-of-the art techniques. 4. Confirm the integration of the component of this system to effect phagocytic killing of microorganisms by macrophages and Kupffer cells in vitro, and by the hepatic reticuloendothelial system of the intact rat in "i"o. This project should therefore establish and characterize a novel cell signalling mechanism that is important for immune defense against circulating microorganisms, and which incidentally causes organ injury following shock or ischemia.

该项目以前的研究已经确定了以下方面的关键作用： 由内皮细胞产生的活性氧代谢物（“自由基”） 细胞黄嘌呤氧化酶作为嗜酸性粒细胞介导的微血管 损伤以及随后的器官功能障碍和衰竭 缺血/再灌注，或休克后，导致发展 多器官衰竭我们现在建议评估新的假设 微血管内皮细胞表面黄嘌呤氧化还原酶 在生理上起着分子开关的作用， 网状内皮系统通过传导循环炎症信号 （细胞因子）通过黄嘌呤氧化酶的有限蛋白水解激活 (XO)从黄嘌呤脱氢酶（XD），产生过氧化氢作为 第二信使触发整联蛋白介导的粘附、捕获和 激活循环中性粒细胞以实现捕获、吞噬作用 并杀死大多数人的微静脉内的循环微生物 器官（退化）和肝窦和中央静脉 （功能上）。在我们的微血管内皮细胞模型系统中， 在体外的单层细胞中，γ-干扰素通过 上调黄嘌呤脱氢酶的合成，而TNF α 通过介导蛋白水解XD到XO的转化触发中性粒细胞粘附。 具体目标：1。确认内皮细胞表面的必要作用 XO用于内皮细胞中细胞因子（TNF α）信号的转导 细胞在体外上调表面整合素的表达， 用定制的m-抗-XO阻断XO活性，并通过阻断XO- 鸡或突变型黄嘌呤脱氢酶阴性内皮细胞 其不能蛋白水解活化为XO，并评估这些 细胞的信号活动。2.定义这部小说的关系 信号转导途径，其他已建立的途径，使用 常规技术。3.评估拟议方案的每个组成部分 通路，包括特异性TNF α受体，SEKk 激酶途径，鞘磷脂/神经酰胺，磷脂酶C，肌醇 三磷酸盐、细胞内钙和μ-钙蛋白酶 XO的蛋白水解激活，使用已建立的，但最先进的 技术. 4.确认该系统组件的集成， 通过巨噬细胞和枯否氏菌实现对微生物的吞噬杀伤 细胞在体外，并通过肝网状内皮系统的 I“O中的完整大鼠。因此，该项目应建立和 描述了一种新的细胞信号传导机制， 对循环微生物的免疫防御，顺便说一句， 导致休克或局部缺血后的器官损伤。