Thrombo-Inflammatory Role of CD39 In Vascular Stasis

 描述（由申请人提供）：骨科手术后或长时间旅行后肢体固定可能引发深静脉血栓形成（DVT），从而导致栓塞和死亡。该提案模拟体外血管流动，并利用两种DVT手术模型来检查血管停滞、缺氧和在血管界面起作用以维持或恢复稳态的关键外核苷酸酶（CD 39）之间的关系。CD 39是一种跨膜内皮细胞（和白细胞）胞外酶，催化细胞外ATP和ADP的磷酸化水解。通过代谢删除这些由受伤和发炎的血管释放的促血栓形成和促炎“危险信号”，CD 39可以防止血管中血小板聚集和白细胞募集的爆炸性级联反应，这些血管易受流动冲击和/或静脉瓣膜袋的低氧张力环境特征的影响。使用我们通过floxed CD 39/Ella Cre小鼠杂交产生的整体缺陷型CD 39小鼠，我们的初步数据首次显示天然CD 39在DVT小鼠模型中起到阻碍血栓和炎症发展的作用。此外，可以在小鼠血浆和脱落的外泌体中检测到CD 39抗原，其中它可以作为下游血栓形成危险信号参与。待检验的总体假设是，CD 39是静脉血栓形成/炎症的关键内源性抑制因子，并且携带CD 39的细胞外囊泡可能脱落以标记DVT中的“危险”并保护下游血管节段。本课题的具体目的是：（1）阐明静态或低流量流体剪切下内皮细胞CD 39表达的分子动力学。这些实验将使用最先进的分子技术来确定内皮CD 39基因-启动子相互作用，其驱动对层流剪切或停滞的反应;（2）确定血管停滞伴或不伴全身缺氧对（i）局部静脉壁CD 39表达和活性的影响;和（ii）CD 39释放到白细胞或携带CD 39的细胞外囊泡的循环中。研究将利用我们已经开发的新测定来量化脱落到循环中的携带CD 39的外来体的CD 39抗原和核苷酸酶活性;（3）确定在静脉停滞或低流量条件下，在有或没有全身性缺氧的情况下，CD 39在原位血栓增生和血管壁炎症中的作用。这些实验将使用我们独特的全局和细胞谱系（巨噬细胞，内皮细胞）特异性CD 39基因缺失和过表达小鼠品系，并在DVT的稳健静脉淤滞和低流量模型中对其进行测试。CD 39将通过过继转移的白细胞或施用重组可溶性CD 39或免疫纯化的携带CD 39的外来体来增加（或在敲除小鼠中重建）。这些研究应该为DVT的发病机制提供新的见解，并为理解我们的发现转化为新的治疗方法以减轻对血管淤滞和低氧血症的血栓炎症反应提供一个窗口。

 DESCRIPTION (provided by applicant): Limb immobilization after orthopedic surgery or prolonged travel can trigger deep vein thrombosis (DVT), which can result in embolization and death. This proposal models vascular flow in vitro and utilizes two surgical models of DVT to examine the relationship between vascular stasis, hypoxia, and a critical ectonucleotidase (CD39) which acts at the blood-vessel interface to maintain or restore homeostasis. CD39 is a membrane-spanning endothelial (and leukocyte) ectoenzyme, which catalyzes the phosphohydrolysis of extracellular ATP and ADP. By metabolically deleting these prothrombotic and proinflammatory "danger signals" released by injured and inflamed vessels, CD39 may prevent an explosive cascade of platelet aggregation and leukocyte recruitment in vessels made susceptible by flow impingment and/or the low oxygen tension environment characteristic of venous valve pockets. Using globally-deficient CD39 mice we have created through floxed CD39/EIIa Cre mouse intercrosses, our preliminary data shows for the first time that native CD39 acts to impede development of thrombus and inflammation in a murine model of DVT. Furthermore, CD39 antigen can be detected in mouse plasma and shed exosomes, where it may participate as a downstream thrombosis danger signal. The overall hypothesis to be tested is that CD39 is a critical endogenous suppressor of venous thrombosis/inflammation and that CD39-bearing extracellular vesicles may be shed to mark "danger" in DVT and protect downstream vascular segments. The Specific Aims of this project are: (1) To elucidate the molecular dynamics of endothelial CD39 expression under static or low-flow fluid shear. These experiments will use state-of-the art molecular techniques for ascertaining endothelial CD39 gene-promoter interactions driving response to laminar fluid shear or stasis; (2) To determine the effects of vascular stasis with or without systemic hypoxia on (i) local vein wall CD39 expression and activity; and (ii) release of CD39 into the circulation on leukocytes or CD39-bearing extracellular vesicles. Studies will leverage novel assays we have developed to quantify both CD39 antigen and nucleotidase activity of CD39-bearing exosomes shed into the circulation; (3) To ascertain the role for CD39 in in situ thrombus accretion and vascular wall inflammation under conditions of venous stasis or low flow, with or without systemic hypoxia. These experiments will use our unique strains of global- and cell lineage (macrophage, endothelial)-specific CD39 gene-deleted and - overexpressing mice, and test them in robust venous stasis and low-flow models of DVT. CD39 will be augmented (or reconstituted in knockout mice) via adoptively transferred leukocytes, or administration of recombinant soluble CD39 or immunopurified CD39-bearing exosomes. Studies should provide new insights into DVT pathogenesis and provide a window for understanding potential translation of our discoveries into new therapeutic approaches to mitigate the thromboinflammatory response to vascular stasis and hypoxemia.