SIGNAL TRANSDUCTION BY ENDOTHELIAL XANTHINE OXIDASE

这个项目之前的研究已经确立了一个关键作用 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中性粒细胞介导的微血管的触发物 损伤，以及随后的器官功能障碍和衰竭 缺血/再灌注，或继发休克，导致 多器官衰竭。我们现在建议评估这一新的假设 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 转导循环炎症信号的网状内皮系统 (细胞因子)通过黄嘌呤氧化酶有限的蛋白水解性激活 (XO)从黄嘌呤脱氢酶(XD)产生过氧化氢，作为一种 第二信使触发整合素介导的黏附、捕获和 激活循环中的中性粒细胞以影响捕获、吞噬 以及杀死大多数人小静脉内的循环微生物 器官(残留的)以及肝窦和中央静脉 (功能上)。在我们的微血管内皮细胞模型系统中 在体外单层，γ-干扰素通过以下方式启动这一信号机制 上调黄嘌呤脱氢酶的合成，而肿瘤坏死因子α 通过介导蛋白水解性XD到XO的转化来触发中性粒细胞的黏附。 具体目标：1.确定内皮细胞表面的必要作用 XO在内皮细胞细胞因子(TNFpha)信号转导中的作用 体外培养细胞上调表面整合素的表达 用定制的m-anti-XO阻断XO活性，并通过转导XO- 鸡肉或突变黄嘌呤脱氢酶阴性内皮细胞 不能对XO进行蛋白水解性激活，并对这些 用于信号活动的细胞。2.界定这部小说的关系 到其他已建立的通路的信号转导通路 传统的技术。3.评估建议中的每个组成部分 途径，包括特定的肿瘤坏死因子α受体，SEKk的作用 激酶途径，神经鞘蛋白/神经酰胺，磷脂酶C，肌醇 三磷酸、细胞内钙和肌钙蛋白在细胞内的作用 使用已建立但最先进的XO蛋白水解物激活 技巧。4.确认该系统的组件集成到 巨噬细胞和枯否细胞对微生物的吞噬杀灭作用 细胞在体外，并通过肝脏网状内皮系统 完整的老鼠在“I”o。因此，这个项目应该建立和 描述一种新的细胞信号机制，该机制对 对循环微生物的免疫防御，顺便说一句 在休克或缺血后导致器官损伤。

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.