Flt-1 (Vascular Endothelial Growth Factor Receptor-1) Is Essential for the Vascular Endothelial Growth Factor–Notch Feedback Loop During Angiogenesis

Abstract

Objective Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-1 (VEGFR-1) negatively modulates VEGF signaling. Therefore, we tested the hypothesis that disrupted Flt-1 regulation of VEGF signaling causes Notch pathway defects that contribute to dysmorphogenesis of Flt-1 mutant vessels. Approach and Results Wild-type (WT) and flt-1−/− mouse embryonic stem (ES) cell-derived vessels were exposed to pharmacological and protein-based Notch inhibitors with and without added VEGF. Vessel morphology, endothelial cell proliferation, and Notch target gene expression levels were assessed. Similar pathway manipulations were performed in developing vessels of zebrafish embryos. Notch inhibition reduced flt-1−/− ES cell-derived vessel branching dysmorphogenesis and endothelial hyper-proliferation, and rescue of flt-1−/− vessels was accompanied by a reduction of elevated Notch targets. Surprisingly, WT vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in WT endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels (ISVs) but not caudal vein plexuses (CVPs). In contrast, exogenous VEGF caused WT ES cell-derived vessel and zebrafish ISV dysmorphogenesis that was rescued by Notch blockade. Conclusions Elevated Notch signaling downstream of perturbed VEGF signaling contributes to aberrant flt-1−/− blood vessel formation. Notch signaling may be dispensable for blood vessel formation when VEGF signaling is below a critical threshold.

Blood vessel anastomosis is spatially regulated by Flt1 during angiogenesis

“…Thus, cells with reduced Flt1, which are likely to have higher levels of VEGF signaling, are favored as sites of stable connection, and this bias depends on the expression of the membrane-bound decoy VEGF receptor, mFlt1. Heterogeneous Flt1 expression and Flk1 activity influence sprout initiation and extension (Chappell et al, 2009;Arima et al, 2011;Jakobsson et al, 2010;Chappell et al, 2013), and our data extend the regulatory impact of heterogeneous VEGF signaling in the patterning of blood vessels to anastomosis. Membrane-bound Flt1 appears essential for the anastomosis selectivity, which is consistent with its cellautonomous role in endothelial cells, relative to sFlt1 that is secreted and has potential to affect aspects of sprouting non-cellautonomously (Chappell et al, 2009;.…”

Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6

“…1h–j). Moreover, the BMP-induced branching phenotype was remarkably well- organized, in contrast to the effects of elevated vascular endothelial growth factor (VEGF)-A signalling, which often lead to gross dysmorphogenesis2223. Thus, BMP6 and BMP2 are pro-angiogenic and increase vascular density by inducing new branch formation.…”

Von Hippel-Lindau mutations disrupt vascular patterning and maturation via Notch

“…In the present study, we show that conditional genetic loss of Vhl (UBC ) have differential effects on vascular development via downstream perturbations in Notch pathway signaling and in conjunction with other pathway misregulation. Endothelial Notch signaling plays a critical role in sprouting angiogenesis (10)(11)(12), in vessel maturation through arterial-venous specification (16), and in the induction of contractile vascular SMC differentiation (17, 18, 20, 21). Our observations suggest Vhl lesions disrupt Notch signaling such that processes underlying vessel maturation are accelerated; most prominent among these ).…”

“…The Notch pathway intersects with VEGF-A signaling to coordinate endothelial cell behaviors such that endothelial "tip" cells emigrate outward from parent blood vessels, while "stalk" cells largely divide to promote vessel elongation (10,11). Genetic loss of delta-like 4 (Dll4) or pharmacological blockade of Notch1 receptor signaling, such as through N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment, leads to a hypersprouting phenotype and the formation of excessive vessel branches (10,12), though this nascent vasculature is not always functional (13,14).…”