NEU1 Sialidase Disrupts CD31-Driven Angiogenesis in Human Lung Endothelia

描述（由申请人提供）：

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.