Abstract
BackgroundPrevious work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165. The aims of this study were to determine protective pathways by which VEGF isoforms attenuate hyperoxic lung growth retardation and to identify the target cell for VEGF action.MethodsTimed pregnant CD-1 or fetal liver kinase (FLK1)-eGFP lung explants cultured in 3% or 50% oxygen were treated ± Vegf121, VEGF164/Vegf165 or VEGF188 in the presence or absence of anti-rat neuropilin-1 (NRP1) antibody or GO6983 (protein kinase C (PKC) pan-inhibitor) and lung growth and branching quantified. Immunofluorescence studies were performed to determine apoptosis index and location of FLK1 phosphorylation and western blot studies of lung explants were performed to define the signaling pathways that mediate the protective effects of VEGF.ResultsHeparin-binding VEGF isoforms (VEGF164/Vegf165 and VEGF188) but not Vegf121 selectively reduced epithelial apoptosis and partially rescued lung bud branching and growth. These protective effects required NRP1-dependent FLK1 activation in endothelial cells. Analysis of downstream signaling pathways demonstrated that the VEGF-mediated anti-apoptotic effects were dependent on PKC activation.ConclusionsVegf165 activates FLK1-NRP1 signaling in endothelial cells, leading to a PKC-dependent paracrine signal that in turn inhibits epithelial cell apoptosis.
Highlights
Previous work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165
Using a Fetal liver kinase (FLK1)-eGFP transgenic mouse we show that the heparin binding VEGF isoforms activate FLK1 on developing lung endothelial cells in a NRP1 dependent manner, leading to protein kinase C (PKC) activation that mediates a paracrine signal to suppress hyperoxia-induced apoptosis in nearby airway epithelial cells
Heparin-binding VEGF isoforms partially rescue lung growth in hyperoxic explant culture We have previously shown that all VEGF isoforms are downregulated and lung development severely impaired following explant exposure to 50% oxygen
Summary
Previous work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165. VEGF-A is a well-characterized endothelial growth factor that is secreted by several cell types including airway epithelium and is essential for vasculogenesis and angiogenesis [1]. Both FLT1 and FLK1 have been localized to endothelial cells and are expressed throughout. VEGF120/Vegf121 is diffusible, whereas VEGF164/ Vegf165 and VEGF188/Vegf189 are variably sequestered by binding to heparan sulfate moieties on the cell surface and in the extracellular matrix. These isoforms are expressed in distinct temporo-spatial patterns, suggesting that each may serve a specific developmental function. VEGF188 becomes the predominant isoform after E16, remaining high throughout adulthood [5]
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