LEUKOREGULATION BY CD39 IN LUNG ISCHEMIA AND INFLAMMATI*

描述（由申请人提供）： 在环境或缺血应激的条件下，肺表现出高的 免疫反应性的程度，并形成促凝血表型，导致 白细胞募集和微血管血栓形成。我们假设 内皮细胞（EC）表达跨膜蛋白CD 39 （外腺苷三磷酸双磷酸酶），其在活化的腺苷三磷酸的释放物中代谢ATP和ADP。 细胞，提供了一个关键的机制，通过它EC抑制血管内 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（通过减少粘附受体表达）。小鼠 对于CD 39基因为空，通过删除包含CD 39基因的外显子4-6而产生。 腺苷三磷酸双磷酸酶保守结构域，表现出潜在的血栓前和白细胞粘附 在单侧肺缺血/再灌注的情况下的表型， 表明气体交换和存活受损。重组可溶性CD 39， 缺乏跨膜区但保留腺苷三磷酸双磷酸酶活性， 抑制血小板聚集和白细胞粘附于EC单层， 体外初步数据显示可溶性CD 39“重建”了CD 39缺失小鼠， 以使其表型正常化并赋予功能性拯救。低密度CD 39-/-肺 内皮细胞对单核细胞和中性粒细胞的粘附性也增加， 可溶性CD 39可逆转该效应。本项目的目标是：（1） 建立CD 39在缺血后肺中的血栓调节作用， 缺氧或炎症应激，重点是识别特定的 血栓形成或纤维蛋白溶解范例，其可由CD 39调节;（2） 确定CD 39在缺血后肺中的白细胞调节作用， 缺氧或炎症应激，以及CD 39 +/+和CD 39-/-肺 微血管内皮细胞暴露于缺氧或炎症介质。相互作用 还将研究白细胞粘附和血栓形成机制之间的关系;以及（3） 为了确定EC CD 39介导的抑制细胞增殖的潜在机制， 炎症、缺氧或缺血后肺白细胞隔离 应力，重点是EC粘附受体或其同源配体上 白细胞总之，实验将确定内源性CD 39如何调节 血栓形成和特定白细胞群募集，并鉴定 在体内模型中给予可溶性CD 39的功能结果 肺缺血缺氧或炎症性应激。拟议的研究 应该确定一个新的基于CD 39的肺血管 血栓调节和白细胞调节，这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 综合征或其他炎性/血栓性素质。

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.