Frizzled 4 Regulates Arterial Network Organization Through Noncanonical Wnt/Planar Cell Polarity Signaling

Abstract

Rationale: A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, but the components that mediate this regulation remain elusive.Objective: We investigated the involvement of one of the receptors, Frizzled4 (Fzd4), in this process because its role has been implicated in retinal vascular development. Methods and Results:We found that loss of fzd4 function in mice results in a striking reduction and impairment of the distal small artery network in the heart and kidney. We report that loss of fzd4 decreases vascular cell proliferation and migration and decreases the ability of the endothelial cells to form tubes. We show that fzd4 deletion induces defects in the expression level of stable acetylated tubulin and in Golgi organization during migration. Deletion of fzd4 favors Wnt noncanonical AP1-dependent signaling, indicating that Fzd4 plays a pivotal role favoring PCP signaling. Our data further demonstrate that Fzd4 is predominantly localized on the top of the plasma membrane, where it preferentially induces Dvl3 relocalization to promote its activation and ␣-tubulin recruitment during migration. In a pathological mouse angiogenic model, deletion of fzd4 impairs the angiogenic response and leads to the formation of a disorganized arterial network. Key Words: blood vessels Ⅲ imaging Ⅲ ischemia Ⅲ transgenic mice Ⅲ vascular biology D uring development, blood vessel formation ensures tissue growth and organ function in the entire organism. The essential role of Wnt/Frizzled signaling in the development of the vascular network was established when it was demonstrated that deletion of distinct Wnt genes caused embryonic lethality with severe phenotypes. A growing body of evidence supports the hypothesis that the Wnt/planar cell polarity (PCP) pathway regulates endothelial cell proliferation and angiogenesis, 1-3 but the components that mediate this regulation remain elusive. Embryo-specific deletion of Wnt7b/7a, which bypassed early lethality because of Wnt7b effect on placenta formation, demonstrated a role of Wnt7a/7b ligands in blood-brain barrier formation through Wnt canonical signaling. 4 These models also indicated that Fzd4 is a prominent receptor involved in vascular formation. Fzd4 has been linked to genetic diseases altering retinal vascular development in Norrie disease, familial exudative vitreoretinopathy, 5,6 and osteoporosis-pseudoglioma. 7 In mice, Fzd4 controls retinal vascular growth and organization, 8 and blood-brain barrier formation in the cerebellum. 9 Moreover, Fzd4 is linked to sterility. 10 We have previously demonstrated that the action of sFRP1, a secreted regulator of the Wnt pathway, is mediated in part by Fzd4 in endothelial cells. 2 The sFRP1 stimulates angiogenesis in vivo and in vitro 11 via a noncanonical Wnt-dependent mechanism and activates downstream signaling factors such as GSK3␤ and Rac1. There is growing evidence of a link between noncanonical Wnt/PCP signaling and angi...

Endothelial miR-17∼92 cluster negatively regulates arteriogenesis via miRNA-19 repression of WNT signaling

“…The enhanced baseline and stimulated arteriogenesis in mice lacking the miR-17∼92 cluster in ECs is reminiscent of a recent report of reduced basal and ischemia-stimulated arterial density in mice lacking the WNT receptor FZD4 (25). In addition, ECs derived from these FZD4 KO mice failed to form cords in vitro (21,25). Given the inverse correlations between the phenotypes that we observed and the vascular phenotypes in the FZD4 KO mice, we examined whether endothelial-derived miR-17∼92 can potentially regulate FZD4 and the signaling pathway downstream of its activation.…”

“…The enhanced baseline and stimulated arteriogenesis in mice lacking the miR-17∼92 cluster in ECs is reminiscent of a recent report of reduced basal and ischemia-stimulated arterial density in mice lacking the WNT receptor FZD4 (25). In addition, ECs derived from these FZD4 KO mice failed to form cords in vitro (21,25).…”

“…Aberrant activation of components of this pathway in ECs may produce severe vascular phenotypes and result in embryonic lethality in some cases (18)(19)(20)(21)(22)(23). Of relevance, FZD4 and LRP5, which has high homology to LRP6 and shares many biochemical properties (24), have been linked to vascular disorders, including defective intraretinal and cerebellar vascular patterning and arterial network formation (21,23,25).…”

The Wnt pathway and the roles for its antagonists, DKKS, in angiogenesis

“…Recently, the formin Dvl-associated activator of morphogenesis-1, an essential player in cytoskeletal organization, was found to be a downstream intermediate of the Wnt/PCP pathway in regulating endothelial cell proliferation and angiogenesis (45). It has been recently demonstrated that Fz-4 acts in arterial formation and organization through a Wnt/ PCP pathway, altering Golgi organization and tubulin acetylation (46). Fz-4 activates Dvl3 on the apical cell membrane promoting a-tubulin recruitment and reorganization.…”

Wnt5a uses CD146 as a receptor to regulate cell motility and convergent extension

“…CD146 activated JNK efficiently in the absence of all those receptors except Fz4, which is involved in regulating Wnt/PCP signalling ( Supplementary Fig. S7a) 24 . Knockdown of CD146 does not affect Fz5, Fz7 and Ror2-activated JNK ( Supplementary Fig.…”