NEU1 Sialidase Disrupts CD31-Driven Angiogenesis in Human Lung Endothelia

描述(由申请人提供)： 背景/原理：我们已证实NEU1唾液酸酶在人肺微血管内皮细胞S中表达，它与CD31结合，并与CD31结合，也称为血小板内皮细胞黏附分子-1。此外，NEU1干扰CD31驱动的HPMEC毛细血管样管形成或体外血管生成。NEU1需要与它自己的伴侣//运输蛋白，保护蛋白/组织蛋白A(PPCA)结合，才能充分发挥催化活性。我们假设，在屏障形成和血管生成过程中，当HPMECs相互作用和CD31ED域(ED)S同源相互作用时，细胞质适配蛋白TNFa受体相关因子(TRAF6)与一个或多个src家族激酶S物理结合并激活，进而增加p120连环蛋白的酪氨酸磷酸化。这种磷酸蛋白促进NEU1的募集，很可能伴随着PPCA，使CD31-ED上的唾液酸残基被清除，并破坏CD31驱动的血管生成。NEU1表达增加可能参与了包括特发性肺纤维化(IPF)在内的肺部疾病的发病机制。目的(S)：1.确定CD31域(S)在人肺微血管内皮细胞中募集NEU1和/或PPCA所需的结构域。2.明确接头蛋白TRAF6与一个或多个SFK(S)共同调节NEU1向CD31募集的机制(S)。3.明确NEU1唾液酸酶在体外调控内皮细胞毛细血管样管形成和体内血管生成的机制(S)。4.建立NEU1在正常人和特发性肺纤维化(IPF)患者肺组织中的表达模式及催化活性。方法：建立原代HPMEC系统，其中NEU1、PPCA、ST6GAL-I、CD31、SFKs、TRAF6和p120ctn将通过siRNA技术或编码野生型和催化死亡的NEU1、PPCA、ST6GAL-I和野生型和显性阴性TRAF6的腺病毒载体感染而被操纵。从野生型、NEU1-/-和CD31-/-小鼠中获得的肺微血管内皮细胞也将被研究。蛋白质之间的相互作用将通过免疫共沉淀、GST融合蛋白下拉试验和远蛋白质印迹/凝胶覆盖分析来研究。最后，将在Matrigel的体外和植入NEU1、CD31、SFK(S)、TRAF6和/或p120ctn表达已被操纵的HPMEC的小鼠身上测量血管生成。将定量检测IPF患者肺组织中NEU1mRNA和蛋白的表达。结果：我们发现在融合后的HPMECs中，NEU1唾液酸酶被募集到CD31，并且需要CD31。NEU1-CD31的关联依赖于TRAF6和SFK，并涉及p120ctn。NEU1过表达减少了EC与细胞外基质的黏附，抑制了EC向伤口的迁移，并以CD31依赖的方式损害了体外血管生成。ST6GAL-I可抵消NEU1效应。IPF患者肺血管内皮细胞NEU1表达增加。现状：这个项目将探索不是任何以前资助的项目的延伸的新概念(S)。影响：如果我们建议的研究成功完成，他们将产生一个范式转变，即NEU1可以选择性地招募到特定的受体(S)，以调节其唾液酸化状态(S)和反应性。NEU1表达增加可能在IPF的病理生理机制中起重要作用。

DESCRIPTION (provided by applicant): Background/Rationale: We now have established that NEU1 sialidase is expressed in human lung microvascular endothelial cell (HPMEC)s, where it associates with and desialylates CD31, also known as platelet endothelial cell adhesion molecule (PECAM)-1. Further, NEU1 disrupts CD31-driven HPMEC capillary- like tube formation or in vitro angiogenesis. NEU1 requires association with its own chaperone//transport protein, protective protein/cathepsin A (PPCA), for full catalytic activity. We hypothesize that during barrier formation and angiogenesis, as HPMECs engage each other and the CD31-ectodomain (ED)s homophilically interact, the cytoplasmic adapter protein, TNFa Receptor-Associated Factor (TRAF)6, physically associates with and activates one or more src family kinase (SFK)s, which in turn, increases tyrosine phosphorylation of p120 catenin. This phosphoprotein facilitates recruitment of NEU1, most likely accompanied by PPCA, to CD31, permitting removal of sialic acid residues from the CD31-ED and disruption of CD31-driven angiogenesis. Increased NEU1 expression may contribute to the pathogenesis of lung diseases, including Idiopathic pulmonary Fibrosis (IPF). Objective(s): 1. To define the CD31 domain(s) required for its recruitment of NEU1 and/or PPCA in human lung microvascular endothelia. 2. To define the mechanism(s) through which the adapter protein, TRAF6, in concert with one or more SFK(s), regulate NEU1 recruitment to CD31. 3. To define the mechanism(s) through which NEU1 sialidase regulates in vitro EC capillary-like tube formation and in vivo angiogenesis. 4. To establish patterns of NEU1 expression and catalytic activity in the lung tissues of normal subjects and patients with Idiopathic Pulmonary Fibrosis (IPF). Methods: A primary HPMEC system in which NEU1, PPCA, ST6GAL-I, CD31, SFKs, TRAF6, and p120ctn will be manipulated through siRNA technology or infection with adenoviral vectors encoding for wild-type and catalytically-dead NEU1, PPCA, ST6GAL-I, and wild-type and dominant-negative TRAF6. Lung microvascular ECs harvested from wild-type, NEU1-/-, and CD31-/- mice with and without gene rescue will also be studied. Protein-protein interactions will be studied with co-immunoprecipitation, GST-fusion protein pull-down, and Far Western blotting/gel-overlay assays. Finally, angiogenesis will be measured on Matrigel in vitro, and in mice implanted with HPMECs in which expression of NEU1, CD31, SFK(s), TRAF6, and/or p120ctn, have been manipulated. NEU1 mRNA and protein expression will be quantified in lung tissues from IPF patients. Findings/Results: We have established that in postconfluent HPMECs, NEU1 sialidase is recruited to and desialylates CD31.The NEU1-CD31 association is both TRAF6- and SFK-dependent and p120ctn is involved. NEU1 overexpression reduces EC adhesion to extracellular matrix, restrains EC migration into a wound, and impairs in vitro angiogenesis in a CD31-dependent manner. ST6GAL-I counteracts the NEU1 effect. NEU1 expression is increased in the pulmonary vascular endothelium of IPF patients. Status: This project will explore new concepts that are not an extension of any previously funded project(s). Impact: If our proposed studies are successfully completed, they will generate a paradigm shift in which NEU1 can be selectively recruited to specific receptor(s), to regulate their sialylation state(s) and responsiveness. Increased NEU1 expression may mechanistically contribute to IPF pathophysiology.