D008 First evidences of frizzled-7 involvement in vessel formation

Recent evidence links the Wnt/frizzled pathway to proper vascular growth but the molecular players are still unknown. Recently, we evidenced that frizzled7 (Fzd7) interacted with a Wnt pathway modulator sFRP1 and was involved on endothelial cell morphogenesis. Here, we investigated whether Fzd7 may be involved in vessel formation. Knocking down (KD) of Fzd7 by injecting morpholino oligonucleotides impaired intersomitic endothelial cells migration in zebrafish embryo. Phenotypes were rescued by the injection of Fzd7 mRNA. To elucidate a role of Fzd7 in vascular formation, we set up a model of vessel formation using a murine embryonic stem (ES) cell model. Embryoid bodies (EBs) differentiating under VEGF induction in vitro were stained for CD31 to visualize the formation of vascular structures. We showed that Fzd7 expression was modulated during the kinetic of EB differentiation and that Fzd7 was expressed in EC assembled in vascular structures. Fzd7 was highly localized in cell-cell junctions, colocalized with VE-cadherin and beta catenin-positive junctions. In order to analyze the role of Fzd7 in endothelial cell function, effect of Fzd7 knock down by small interfering RNAs was investigated in human umbilical vein endothelial cells (HUVEC). KD of Fzd7 had no effect on cell proliferation whereas a strong inhibition of HUVEC migration and tube formation was observed. Indeed, Fzd7 siRNA decreased cell migration to 59±17 % (n=3, p<0.0001) compared to control siRNA in transwell assay. We also found that HUVEC transfected with control siRNA form well-connected tubular structures in Matrigel assay after 18h, whereas Fzd7 siRNA treated HUVEC showed a strong delay in tubular structures formation (43±5 tubes/field in control vs 15±7 in Fzd7 siRNA, p<0.0001). This finding suggests that Fzd7 may play a crucial role during vascular formation. Fzd7 is essential for zebrafish vascular development in vivo and regulates endothelial cells functions involved in angiogenesis in vitro.