Search

The BMP pathway regulates developmental processes including angiogenesis, yet its signaling outputs are complex and context-dependent. Recently, we showed that SMAD6, an intracellular BMP inhibitor expressed in endothelial cells, decreases vessel sprouting and branching both in vitro and in zebrafish. Genetic deletion of SMAD6 in mice results in poorly characterized cardiovascular defects and lethality. Here, we analyzed the effects of SMAD6 loss on vascular function during murine development. SMAD6 was expressed in a subset of blood vessels throughout development, primarily in arteries, while expression outside of the vasculature was largely confined to developing cardiac valves with no obvious embryonic phenotype. Mice deficient in SMAD6 died during late gestation and early stages of postnatal development, and this lethality was associated with vessel hemorrhage. Mice that survived past birth had increased branching and sprouting of developing postnatal retinal vessels and disorganized tight and adherens junctions. In vitro, knockdown of SMAD6 led to abnormal endothelial cell adherens junctions and increased VE-cadherin endocytosis, indicative of activated endothelium. Thus, SMAD6 is essential for proper blood vessel function during murine development, where it appears to stabilize endothelial junctions to prevent hemorrhage and aberrant angiogenesis.

Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation., The mechanism underlying endothelial cell responses to BMP signals is unknown. Here, the authors show that the endothelial response to pro-angiogenic BMP ligands is regulated by Notch via its effect on SMAD6, a known inhibitor of BMP intracellular signaling cascade.

Objective— Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-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 and flt-1 −/− mouse embryonic stem 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 −/− embryonic stem cell–derived vessel branching dysmorphogenesis and endothelial hyperproliferation, and rescue of flt-1 −/− vessels was accompanied by a reduction in elevated Notch targets. Surprisingly, wild-type vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in wild-type endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels but not caudal vein plexuses. In contrast, exogenous VEGF caused wild-type embryonic stem cell–derived vessel and zebrafish intersegmental vessel 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.

Approximately 30% of tumor endothelial cells have over-duplicated (>2) centrosomes, which may contribute to abnormal vessel function and drug resistance. Elevated levels of vascular endothelial growth factor A induce excess centrosomes in endothelial cells, but how other features of the tumor environment affect centrosome over-duplication is not known. To test this, we treated endothelial cells with tumor-derived factors, hypoxia, or reduced p53, and assessed centrosome numbers. We found that hypoxia and elevated levels of bone morphogenetic protein 2, 6 and 7 induced excess centrosomes in endothelial cells through BMPR1A and likely via SMAD signaling. In contrast, inflammatory mediators IL-8 and lipopolysaccharide did not induce excess centrosomes. Finally, down-regulation in endothelial cells of p53, a critical regulator of DNA damage and proliferation, caused centrosome over-duplication. Our findings suggest that some tumor-derived factors and genetic changes in endothelial cells contribute to excess centrosomes in tumor endothelial cells.