Fbxw7 Controls Angiogenesis by Regulating Endothelial Notch Activity

Abstract

Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7 iECKO mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.

FBW7 regulates endothelial functions by targeting KLF2 for ubiquitination and degradation

“…Previous studies using global knockout mice suggested that degradation of Notch by FBW7 is responsible for the development of normal blood vessels [28,53]. Results from a recent study using an endothelial cell-specific Fbw7 knockout mouse model support the notion that FBW7 is a potent positive regulator of angiogenesis by targeting Notch for degradation in endothelial cells [31]. Consistent with our study, they also showed that knockdown of FBW7 results in the inhibition of HUVEC proliferation [31].…”

“…This result is consistent with a previous report that both VEGR2 and eNOS are downstream targets of KLF2 [7,33]. Moreover, FBW7 has also been demonstrated as an efficient E3 ubiquitin ligase for Notch, which may regulate the expression of VEGFR2 in endothelial cells [31,35]. To exclude the effect of Notch, HUVECs transfected with control or FBW7 siRNA were treated with γ-secretase inhibitor DAPT for 24 h to block the Notch signaling.…”

“…Fbw7-deficient mice show defects in vascular development and embryonic lethality at day E10.5 (E10.5) [28]. Recent studies indicate that FBW7 may regulate angiogenesis by targeting Notch, NF-1 or HIF1 [29][30][31]. However, the role of FBW7 in vascular biology remains to be investigated.…”

Müller glia cells regulate Notch signaling and retinal angiogenesis via the generation of 19,20-dihydroxydocosapentaenoic acid

“…The results of the present study show that the genetic deletion and inhibition of the sEH activates Notch signaling and attenuates physiological sprouting angiogenesis in the murine Welcker et and Clurman, 2008), and endothelial cell-specific and inducible Fbxw7 mutant (Fbxw7 iECKO ) mice demonstrate retinal blood vessel growth impairment that is attributable to Notch activation (Izumi et al, 2012). To confirm the in vitro data suggesting that 19,20-DHDP inhibits -secretase activity, the lipid was given intravitrealy to Fbxw7 iECKO mice.…”

“…The finding that 19,20-DHDP also affected NICD generation in this model indicated that its molecular target was the -secretase. To confirm this in vivo, we assessed the consequences of the intravitreal application of 19,20-EPD or 19,20-DHDP to 5-d-old mice with hyper-activated endothelial cell Notch signaling, i.e., animals lacking the E3 ubiquitin ligase involved in the ubiquitination and degradation of the NICD (Izumi et al, 2012). In the latter Fbxw7 iECKO animals, which demonstrate markedly impaired retinal blood vessel growth impairment that is attributable to Notch activation (Izumi et al, 2012), 19,20-DHDP increased vessel branching as well as tip cell and filopodia numbers.…”