Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation

In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes

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

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

Mutation in utp15 Disrupts Vascular Patterning in a p53-Dependent Manner in Zebrafish Embryos

Angiogenesis is the process by which the highly branched and functional vasculature arises from the major vessels, providing developing tissues with nutrients, oxygen, and removing metabolic waste. During embryogenesis, vascular patterning is dependent on a tightly regulated balance between pro- and anti-angiogenic signals, and failure of angiogenesis leads to embryonic lethality. Using the zebrafish as a model organism, we sought to identify genes that influence normal vascular patterning.In a forward genetic screen, we identified mutant LA1908, which manifests massive apoptosis during early embryogenesis, abnormal expression of several markers of arterial-venous specification, delayed angiogenic sprouting of the intersegmental vessels (ISV), and malformation of the caudal vein plexus (CVP), indicating a critical role for LA1908 in cell survival and angiogenesis. Genetic mapping and sequencing identified a G to A transition in the splice site preceding exon 11 of utp15 in LA1908 mutant embryos. Overexpression of wild type utp15 mRNA suppresses all observed mutant phenotypes, demonstrating a causative relationship between utp15 and LA1908. Furthermore, we found that injecting morpholino oligonucleotides inhibiting p53 translation prevents cell death and rescues the vascular abnormalities, indicating that p53 is downstream of Utp15 deficiency in mediating the LA1908 phenotypes.Taken together, our data demonstrate an early embryonic effect of Utp15 deficiency on cell survival and the normal patterning of the vasculature and highlight an anti-angiogenic role of p53 in developing embryos

Mutation in utp15 disrupts vascular patterning in a p53-dependent manner in zebrafish embryos.

BackgroundAngiogenesis is the process by which the highly branched and functional vasculature arises from the major vessels, providing developing tissues with nutrients, oxygen, and removing metabolic waste. During embryogenesis, vascular patterning is dependent on a tightly regulated balance between pro- and anti-angiogenic signals, and failure of angiogenesis leads to embryonic lethality. Using the zebrafish as a model organism, we sought to identify genes that influence normal vascular patterning.Methodology and principal findingsIn a forward genetic screen, we identified mutant LA1908, which manifests massive apoptosis during early embryogenesis, abnormal expression of several markers of arterial-venous specification, delayed angiogenic sprouting of the intersegmental vessels (ISV), and malformation of the caudal vein plexus (CVP), indicating a critical role for LA1908 in cell survival and angiogenesis. Genetic mapping and sequencing identified a G to A transition in the splice site preceding exon 11 of utp15 in LA1908 mutant embryos. Overexpression of wild type utp15 mRNA suppresses all observed mutant phenotypes, demonstrating a causative relationship between utp15 and LA1908. Furthermore, we found that injecting morpholino oligonucleotides inhibiting p53 translation prevents cell death and rescues the vascular abnormalities, indicating that p53 is downstream of Utp15 deficiency in mediating the LA1908 phenotypes.Conclusions and significanceTaken together, our data demonstrate an early embryonic effect of Utp15 deficiency on cell survival and the normal patterning of the vasculature and highlight an anti-angiogenic role of p53 in developing embryos

Hypoxia induces excess centrosomes in EC independent of cell-autonomous VEGF-A signaling.

<p>(A) Frequency of excess centrosomes in HUVEC after treatment with 100 μM hypoxic-mimetic agent desferrioxamine (DFO) for 4 days. (B) Frequency of excess centrosomes in HUVEC after 4 days of incubation in 2% oxygen. (C) Frequency of excess centrosomes in HUVEC after incubation in 20% or 2% oxygen for 4 days and indicated treatments. Error bars, standard deviation from mean. Statistics: two-tailed unpaired Student’s t-test. *, p≤0.05; ns, not significant.</p

Tumor-Derived Factors and Reduced p53 Promote Endothelial Cell Centrosome Over-Duplication

<div><p>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.</p></div

BMP-induced centrosome over-duplication is dependent on BMPR1A.

<p>(A, B) Frequency of excess centrosomes in indicated siRNA-treated HUVEC cultured with vehicle or 200 ng/ml of BMP6 (A) or BMP2 (B) for 4 days. C, non-targeting control siRNA; R1A, BMPR1A siRNA; R1B, BMPR1B siRNA; R2, BMPR2 siRNA. (C) Representative images of HUVEC treated with indicated siRNA and vehicle or BMP6 and stained for phospho-SMAD1/5 (pSMAD1/5, green) and nucleus (DRAQ7, blue). Cells were starved in Opti-MEM for 4 hr, followed by 30 min treatment with vehicle or BMP6. Only the nuclear pSMAD1/5 is shown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168334#sec002" target="_blank">Methods</a> for details of mask). (D) Quantification of nuclear pSMAD1/5 in HUVEC treated as indicated. (E) Western blot of phospho-SMAD1/5 (pSMAD) and total SMAD1 in HUVEC treated as indicated. Cells were starved in Opti-MEM for 4 hr, then treated with vehicle or BMP6 for 30 min. Error bars, standard deviation from mean. Statistics: two-tailed paired (A, B) or unpaired (D) Student’s t-test. ns, not significant; *, p≤0.05; **, p≤0.01; ***, p≤0.001. Scale bars: 10 μm.</p

Down-regulation of p53 induces excess centrosomes in EC.

<p>(A) Frequency of excess centrosomes in HUVEC infected with human p53 shRNA. (B) Frequency of excess centrosomes in normal mouse endothelial cells (NEC) infected with mouse p53 shRNA. Error bars, standard deviation from mean. Statistics: two-tailed unpaired Student’s t-test. *, p≤0.05.</p

BMP2 and BMP7 induce excess centrosomes in EC.

<p>(A) Representative images of HUVEC with normal (#1 and #2) and over-duplicated centrosomes (#3). HUVEC were stained with γ-tubulin for centrosomes (green) and DRAQ7 for nuclei (blue). (B, C) Frequency of excess centrosomes in HUVEC after treatment with 200 ng/ml BMP2 (B) or BMP7 (C) for 4 days. Error bars, standard deviation from mean. Statistics: two-tailed unpaired Student’s t-test. *, p≤0.05. Scale bars: 1 μm unless indicated otherwise.</p

Inflammatory mediators do not induce excess centrosomes in EC.

<p>(A) Frequency of excess centrosomes in HUVEC after treatment with indicated factors for 4 days. (B) HUVEC incubated with 10 ng/ml LPS for 4 days prior to determination of excess centrosome frequency. Results are shown in fold of increase, and each frequency was normalized to its respective control. Error bars, standard deviation from mean. Statistics: Two-tailed unpaired Student’s t-test (A), Χ² test (B). *, p≤0.05; ns, not significant.</p