LEUKOREGULATION BY CD39 IN LUNG ISCHEMIA AND INFLAMMATI*

描述(由申请人提供)： 在环境或缺血应激条件下，肺表现出较高的 免疫反应程度和形成促凝血剂表型，导致 白细胞募集和微血管血栓形成。我们假设 跨膜蛋白CD39在内皮细胞中的表达 (胞外apyrase)，它在激活的细胞释放过程中代谢ATP和ADP。 细胞，提供了ECs抑制血管内的关键机制 血栓形成(通过抑制ADP介导的血小板/血小板募集)和 调节白细胞运输(通过减少黏附受体的表达)。老鼠 CD39基因为空，通过删除包含 Apyrase保守结构域，表现出潜在的血栓前状态和白质黏附 单侧肺缺血/再灌流背景下的表型 表明气体交换和生存能力受损。重组可溶性CD39， 缺乏跨膜区，但有效地保留了apyrase活性 抑制小鼠血小板聚集和白细胞与EC单层的黏附 体外培养。试点数据显示，可溶性CD39重组了CD39缺失的小鼠 使其表型正常化，并给予功能挽救。缺氧性CD39-/-肺 ECS还表现出对单核细胞和中性粒细胞的粘附性增加， 可被可溶性CD39逆转。这项计划的目标是：(1) 建立CD39在肺缺血后的血栓调节作用， 低氧，或炎性应激，侧重于识别特定的 可由CD39调节的血栓或纤溶范例；(2) 确定CD39在肺缺血后的白血球调节作用， 低氧或炎性应激，以及CD39+/+和CD39-/-肺 微血管内皮细胞暴露在低氧或炎症介质中。互动 还将研究白细胞黏附和血栓形成机制之间的关系；以及(3) 探讨ECCD39介导的细胞增殖抑制机制(S) 炎症性、缺氧性或缺氧性肺白细胞计数 应激，关注EC黏附受体或其同源配体 白细胞。总体而言，实验将确定内源性CD39如何调节 血栓形成和特定白细胞群的募集并鉴定 体内模型中可溶性CD39给药的功能后果 肺缺血、缺氧性或炎性应激。建议进行的研究 应确定以CD39为基础的肺血管新机制 血栓调节和白细胞调节可能导致一种新的治疗方法 治疗肺缺血、脓毒症、急性呼吸窘迫的方法 综合症，或其他炎症性/血栓疾病。

DESCRIPTION (provided by applicant): Under conditions of environmental or ischemic stress, the lungs exhibit a high degree of immunoreactivity and develop a procoagulant phenotype, leading to leukocyte recruitment and microvascular thrombosis. We hypothesized that endothelial cell (EC) expression of a transmembrane protein, CD39 (ectoapyrase), which metabolizes ATP and ADP in the releasate of activated cells, provides a critical mechanism by which ECs inhibit intravascular thrombosis (by inhibiting ADP-mediated platelet/platelet recruitment) and modulate leukocyte traffic(by reducing adhesion receptor expression). Mice null for the CD39 gene, created by deleting exons 4-6 containing the apyrase-conserved domains, exhibit a latent prothrombotic and leukoadhesive phenotype in the setting of unilateral lung ischemia/reperfusion, demonstrating impaired gas exchange and survival. Recombinant soluble CD39, lacking the transmembrane region but retaining apyrase activity, potently suppresses platelet aggregation and leukocyte adhesion to EC monolayers in vitro. Pilot data show that soluble CD39 "reconstituted" the CD39 null mice to normalize their phenotype and confer functional rescue. Hypoxic CD39 -/-pulmonary ECs also showed increased adhesivity for monocytes and neutrophils, which was reversed by soluble CD39. The Aims of this project are: (1) To establish the thromboregulatory role of CD39 in the lungs following ischemic, hypoxic, or inflammatory stress, focusing on identification of specific thrombotic or fibrinolytic paradigms which may be modulated by CD39; (2) To determine the leukoregulatory role of CD39 in the lungs following ischemic, hypoxic, or inflammatory stress, and in CD39 +/+ and CD39 -/- pulmonary microvascular ECs exposed to hypoxia or inflammatory mediators. Interactions between leukoadhesive and thrombotic mechanisms will also be studied; and (3) To identify the mechanisms(s) underlying EC CD39-mediated suppression of pulmonary leukosequestration following inflammatory, hypoxic, or ischemic stress, focusing on EC adhesion receptors or their cognate ligands on leukocytes. Overall, experiments will determine how endogenous CD39 modulates thrombosis and recruitment of specific leukocyte cell populations and identify the functional consequences of soluble CD39 administration in in vivo models of pulmonary ischemic, hypoxic, or inflammatory stress. The proposed studies should identify a new CD39-based mechanism of pulmonary vascular thromboregulation and leukoregulation which may lead to a novel therapeutic approach to treat pulmonary ischemia, sepsis, acute respiratory distress syndrome, or other inflammatory/thrombotic diatheses.