A role for Wnt via the Frizzled (Fzd) cell surface receptors has recently emerged in vascular cell development. Among the Fzd members, Frizzled4 (Fzd4) was recently implicated in vessel formation in adult retinal vascular network. Consistent with this finding, we aimed to evaluate the function of Fzd4 in adult vascular setting.
MicroCT and immunohistochemistry approaches revealed that Fzd4 deletion altered dramatically coronary and arterial network formations in the heart and in the kidney with a significant decrease of the arterial branching. Micro-array analysis revealed that in hearts of Fzd4 KO neonate mice, the primary effect of Fzd 4 deletion was the down-regulation of the cell cycle-regulating transcription factor E2F1, with a decreased expression of a large number of E2F target genes. This observation indicated that loss of Fzd4 lead to cell cycle arrest.
We conducted in vitro studies to confirm the role of Fzd4 on cell cyle response on murine embryonic fibroblasts (MEF) and endothelial cells (EC) isolated from KO Fzd4 vs Wt mice. Fzd4 KO reduced MEF and EC proliferation. Fzd4 siRNA-mediated knockdown (KD) in EC delayed G1 to S cell cycle entry compared to that in control EC (transition G1 to S at 12H, 3,5 % vs 14,5 % vs control EC). In vitro matrigel assay showed that Fzd4 KD reduced the differentiation of HUVEC to form capillary structures under VEGF A-stimulation (43±4 in Fzd4 KD EC vs 28± 7, in control EC, P<0.005) but did not modify EC migration compared to control EC in a transwell migration assay.
These findings are the first to show that a frizzled member Fzd4 can repress E2F1 cell cycle progression in EC independently of the Wnt/β catenin canonical pathway and affects deeply vascular formation and branching.Le texte complet de cet article est disponible en PDF.