The long and winding road: detecting and quantifying Notch activation in endothelial cells

We have been investigating molecular mechanisms of vascular development and diversification using embryonic stem (ES) cells. Previously, we established an ES cell differentiation system that reproduces early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors (Nature, 2000). Endothelial cells (ECs) were induced from VEGFR2 + progenitor cells with VEGF. VEGF alone mainly induced venous ECs, which are negative for arterial EC markers, ephrinB2 and CXCR4. Interestingly, addition of 8bromo-cAMP with VEGF activated Notch signaling in ECs and substantially induced arterial ECs. Nevertheless, activation of Notch using notch intracellular domain (NICD)-estrogen receptor fusion protein failed to induce arterial ECs (Arterioscler Thromb Vasc Biol, 2006). These results indicate that Notch activation was not sufficient to induce arterial ECs from vascular progenitors. Then we further investigated the downstream of cAMP, which would suffice signals for constructive induction of arterial ECs. We found that phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, completely inhibited cAMP-induced arterial EC differentiation as well as Notch activation. GSK3β inhibitor, Bio, partially reversed the inhibitory effects of LY294002. Whereas activation of β-catenin signaling alone using Doxycycline-induced constitutive active form of β-catenin expression showed only a weak effect on arterial EC induction, simultaneous activation of β-catenin and Notch signaling completely restored arterial EC induction even in the absence of cAMP. Thus, cAMP-PI3K-GSK3β-β-catenin pathway plays a critical role in arterial EC differentiation. Furthermore, chromatin immunoprecipitation assay on the two RBP-J binding sites of mouse EphrinB2 revealed that RBP-J, NICD, and β-catenin formed a protein complex on these two sites only in arterial ECs, but not in venous ECs. These findings suggest that Notch and β-catenin signaling converged to form a novel arterial EC-specific protein complex to induce arterial ECs. This study would provide novel understandings of cellular and molecular mechanisms for arterial-venous specification and novel molecular targets for drug discovery.

Download Full-text

EHBP1 and EHD2 regulate Dll4 caveolin-mediated endocytosis during blood vessel development

10.1101/2020.05.19.104547 ◽

2020 ◽

Author(s):

Amelia M Webb ◽

Caitlin R Francis ◽

Jayson M Webb ◽

Hayle Kincross ◽

Keanna M Lundy ◽

...

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Blood Vessel ◽

Notch Signaling ◽

Blood Vessel Development ◽

Absolute Requirement ◽

Membrane Bound ◽

Vessel Development ◽

Notch Activation

ABSTRACTDespite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear. Here, we identify EHD2 and EHBP1 as novel regulators of Notch activation in endothelial cells through controlling endocytosis of Delta-like ligand 4 (Dll4). Knockout of EHBP1 and EHD2 in zebrafish produced a significant increase in ectopic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. In vitro, EHBP1 and EHD2 localized to plasma membrane-bound Dll4 and actin independently of clathrin. Disruption of caveolin endocytosis resulted in EHBP1 and EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization in endothelial cells. Overall, we demonstrate that EHBP1 and EHD2 regulate Dll4 endocytosis by anchoring caveolar endocytic pits to the actin cytoskeleton.

Download Full-text

Faculty Opinions recommendation of Adrenomedullin/cyclic AMP pathway induces Notch activation and differentiation of arterial endothelial cells from vascular progenitors.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1033026.374638 ◽

2006 ◽

Author(s):

David Bates

Keyword(s):

Endothelial Cells ◽

Cyclic Amp ◽

Arterial Endothelial Cells ◽

Notch Activation

Download Full-text

Notch Activation Induces Endothelial Cell Cycle Arrest and Participates in Contact Inhibition: Role of p21Cip1 Repression

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.8813-8822.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 8813-8822 ◽

Cited By ~ 145

Author(s):

Michela Noseda ◽

Linda Chang ◽

Graeme McLean ◽

Jonathan E. Grim ◽

Bruce E. Clurman ◽

...

Keyword(s):

Cell Cycle ◽

Endothelial Cells ◽

Vascular Function ◽

Nuclear Translocation ◽

Notch Pathway ◽

Contact Inhibition ◽

Cyclin D ◽

A Cell ◽

Rb Phosphorylation ◽

Notch Activation

ABSTRACT Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.

Download Full-text

High Content Analysis uncovers functionally relevant metastable phenotypic states in human Endothelial Cells monolayers

10.1101/2020.11.17.362277 ◽

2020 ◽

Author(s):

Francois Chesnais ◽

Juliette Le Caillec ◽

Errin Roy ◽

Davide Danovi ◽

Lorenzo Veschini

Keyword(s):

Endothelial Cells ◽

Phenotypic Variance ◽

Human Endothelial Cells ◽

Relational Information ◽

Distinct Cell ◽

Endothelial Junctions ◽

High Content Analysis ◽

Functional Relevance ◽

Emergent Behaviours ◽

Notch Activation

SummaryEndothelial cells (EC) present distinct cell properties in different tissues. Heterogeneity within the same vascular bed has been proposed to underpin emergent behaviours in EC monolayers. Quantification and functional relevance of EC phenotypic variance are challenging to assess. Here we developed an EC profiling tool (ECPT) to uniquely enable single EC profiling within a monolayer providing spatial and relational information regarding cell proliferation, inter-endothelial Junctions and NOTCH activation. We used ECPT to characterise differential phenotypes in arterial, venous and microvascular EC populations. Our analysis highlighted extensive heterogeneity within individual monolayers and revealed VEGF-modulable metastability of NOTCH signalling which in turn regulates inter-endothelial junction’s stability. We suggest that accounting for adaptive emerging endothelial behaviours is necessary to develop revascularisation strategies for regenerative medicine and to design more effective EC-targeting drugs for cardiovascular diseases and cancer.

Download Full-text

Shear stress induces expression, intracellular reorganization and enhanced Notch activation potential of Jagged1

Integrative Biology ◽

10.1039/c8ib00036k ◽

2018 ◽

Vol 10 (11) ◽

pp. 719-726 ◽

Cited By ~ 8

Author(s):

R. C. H. Driessen ◽

O. M. J. A. Stassen ◽

M. Sjöqvist ◽

F. Suarez Rodriguez ◽

J. Grolleman ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Activation Potential ◽

Notch Activation

Shear stress induces a ligand specific Jagged1 response and enhanced signal sending potential in endothelial cells.

Download Full-text

Endothelial Jagged1 Antagonizes Dll4/Notch Signaling in Decidual Angiogenesis during Early Mouse Pregnancy

International Journal of Molecular Sciences ◽

10.3390/ijms21186477 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6477

Author(s):

Nicole M. Marchetto ◽

Salma Begum ◽

Tracy Wu ◽

Valerie O’Besso ◽

Christina C. Yarborough ◽

...

Keyword(s):

Endothelial Cells ◽

Notch Signaling ◽

Pregnancy Loss ◽

Endothelial Cell Proliferation ◽

Spiral Artery ◽

Capillary Endothelial Cells ◽

Early Mouse ◽

Post Implantation ◽

Notch Activation

Maternal spiral arteries and newly formed decidual capillaries support embryonic development prior to placentation. Previous studies demonstrated that Notch signaling is active in endothelial cells of both decidual capillaries and spiral arteries, however the role of Notch signaling in physiologic decidual angiogenesis and maintenance of the decidual vasculature in early mouse pregnancy has not yet been fully elucidated. We used the Cdh5-CreERT2;Jagged1(Jag1)flox/flox (Jag1∆EC) mouse model to delete Notch ligand, Jag1, in maternal endothelial cells during post-implantation, pre-placentation mouse pregnancy. Loss of endothelial Jag1 leads to increased expression of Notch effectors, Hey2 and Nrarp, and increased endothelial Notch signaling activity in areas of the decidua with remodeling angiogenesis. This correlated with an increase in Dll4 expression in capillary endothelial cells, but not spiral artery endothelial cells. Consistent with increased Dll4/Notch signaling, we observed decreased VEGFR2 expression and endothelial cell proliferation in angiogenic decidual capillaries. Despite aberrant Dll4 expression and Notch activation in Jag1∆EC mutants, pregnancies were maintained and the decidual vasculature was not altered up to embryonic day 7.5. Thus, Jag1 functions in the newly formed decidual capillaries as an antagonist of endothelial Dll4/Notch signaling during angiogenesis, but Jag1 signaling is not necessary for early uterine angiogenesis.

Download Full-text

Endotoxin Alteration of the Mucopolysaccharide Blood Vessel Coating in Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050755 ◽

1974 ◽

Vol 32 ◽

pp. 148-149

Author(s):

D. E. Philpott ◽

A. Takahashi

Keyword(s):

Skeletal Muscle ◽

Endothelial Cells ◽

Salivary Gland ◽

Carotid Artery ◽

Basement Membrane ◽

Coronary Vessels ◽

Ruthenium Red ◽

E Coli ◽

Old Rats ◽

The Brain

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

Download Full-text