Abstract
Purpose: :
Apelin together with its cognate G-protein coupled receptor, APJ has been implicated in retinal angiogenesis and also in retinal vascular remodelling. There have been recent reports of apelin signalling being important in the OIR model of angiogenesis, whereas in the CNV and VLDLR-/- mouse models of pathological angiogenesis, apelin has no discernable role. The aim of this study was to investigate how apelin-APJ signalling regulates vascular remodelling and angiogenesis in the context of other pro-angiogenic factors.
Methods: :
In order to assess the angiogenic response to apelin under different conditions we have employed various in-vitromodels including the metatarsal ex-vivo angiogenesis assay and the organotypic co-cultureangiogenesis assay. The effect of treating these assays with apelin and the effect of apelin depletion in the presence and absence of other pro-angiogenic factors such as VEGF was determined. Endothelial tip cell formation, tube formation, vessel bifurcation and calibre were determined and quantified using Imaris and angiosys software.
Results: :
Targeted deletion of Apl resulted in a significant reduction in angiogenesis in the metatarsal angiogenesis assay. In an organotypic co-culture assay where HUVEC were grown on human dermal fibroblasts, siRNA knockdown of apelin and APJ led to a significant decrease in endothelial tubule formation. Exogenous addition of apelin led to an increase in vessel branching in the co-culture assay. We then used these angiogenesis models to dissect the effects of apelin on downstream signalling from other pro-angiogenic molecules such as VEGF and Wnt. Addition of VEGF significantly increased vessel branchformation (4 fold) and total tubule length in the co-cultures in comparison to the modest increase (1.4 fold) of these parameters upon apelin treatment. When VEGF and apelin were applied together, we observed up to a 6 fold increase in vessel branch formation. These results suggest that apelin and VEGF have a synergistic effect on vessel branch formation.
Conclusions: :
Angiogenic factors that regulate vessel morphogenesis are tightly regulated in their expression and co-operation in signalling. However, in diseases such as diabetic retinopathy, age-related macular degeneration or macular telangiectasia, such regulation is disrupted resulting in abnormal vessel growth. The study here suggests that apelin, which we have previously shown to be involved in vessel remodelling, can also modify the angiogenic outcome of VEGF signalling and could play a role in disease pathology.
Keywords: neovascularization • cell-cell communication • vascular endothelial growth factor