Background: Previous work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular\nendothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of\nexplant airway growth and branching that was rescued by the addition of Vegf165. The aims of this study were to\ndetermine protective pathways by which VEGF isoforms attenuate hyperoxic lung growth retardation and to\nidentify the target cell for VEGF action.\nMethods: Timed pregnant CD-1 or fetal liver kinase (FLK1)-eGFP lung explants cultured in 3% or 50% oxygen were\ntreated �± Vegf121, VEGF164/Vegf165 or VEGF188 in the presence or absence of anti-rat neuropilin-1 (NRP1) antibody\nor GO6983 (protein kinase C (PKC) pan-inhibitor) and lung growth and branching quantified. Immunofluorescence\nstudies were performed to determine apoptosis index and location of FLK1 phosphorylation and western blot studies\nof lung explants were performed to define the signaling pathways that mediate the protective effects of VEGF.\nResults: Heparin-binding VEGF isoforms (VEGF164/Vegf165 and VEGF188) but not Vegf121 selectively reduced\nepithelial apoptosis and partially rescued lung bud branching and growth. These protective effects required\nNRP1-dependent FLK1 activation in endothelial cells. Analysis of downstream signaling pathways demonstrated\nthat the VEGF-mediated anti-apoptotic effects were dependent on PKC activation.\nConclusions: Vegf165 activates FLK1-NRP1 signaling in endothelial cells, leading to a PKC-dependent paracrine\nsignal that in turn inhibits epithelial cell apoptosis
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