Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational ...modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.
Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided ...detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S1PR1, a receptor for the blood-borne bioactive lipid sphingosine-1-phosphate (S1P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S1PR1 leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S1PR1 signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S1PR1 signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses.
► Sphingosine-1-phosphate receptor-1 (Edg1) is a negative regulator of angiogenesis ► Loss of S1pr1 (Edg1) causes endothelial hyperplasia and derangement of the aorta ► S1PR1 (Edg1) regulates cellular adhesion, motility, and VE-cadherin localization ► S1PR1 (Edg1) regulates VEGF-induced VEGFR2 signaling and internalization
Angiogenesis, the formation of new blood vessels from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Gaengel et al. report that sphingosine-1-phosphate receptor 1 (S1PR1 or Edg1) inhibits angiogenesis and promotes acquisition of vascular stability by regulating the interplay between VE-cadherin and VEGFR2.
ECs (endothelial cells) in the developing vasculature are heterogeneous in morphology, function and gene expression. Inter-endothelial signalling via Dll4 (Delta-like 4) and Notch has recently ...emerged as a key regulator of endothelial heterogeneity, controlling arterial cell specification and tip versus stalk cell selection. During sprouting angiogenesis, tip cell formation is the default response to VEGF (vascular endothelial growth factor), whereas the stalk cell phenotype is acquired through Dll4/Notch-mediated lateral inhibition. Precisely how Notch signalling represses stalk cells from becoming tip cells remains unclear. Multiple components of the VEGFR (VEGF receptor) system are regulated by Notch, suggesting that quantitative differences in protein expression between adjacent ECs may provide key features in the formation of a functional vasculature. Computational modelling of this selection process in iterations, with experimental observation and validation greatly facilitates our understanding of the integrated processes at the systems level. We anticipate that the study of mosaic vascular beds of genetically modified ECs in dynamic interactions with wild-type ECs will provide a powerful tool for the investigation of the molecular control and cellular mechanisms of EC specification.
Platelet-derived growth factor (PDGF)-D is a PDGF receptor β (PDGFRβ)-specific ligand implicated in a number of pathological conditions, such as cardiovascular disease and cancer, but its biological ...function remains incompletely understood. In this study, we demonstrate that PDGF-D binds directly to neuropilin 1 (NRP1), in a manner that requires the PDGF-D C-terminal Arg residue. Stimulation with PDGF-D, but not PDGF-B, induced PDGFRβ-NRP1 complex formation in fibroblasts. Additionally, PDGF-D induced translocation of NRP1 to cell-cell junctions in endothelial cells, independently of PDGFRβ, altering the availability of NRP1 for VEGF-A-VEGFR2 signaling. PDGF-D showed differential effects on pericyte behavior in
sprouting assays compared to PDGF-B. Furthermore, PDGF-D-induced PDGFRβ-NRP1 interaction can occur in
between molecules located in different cells (endothelial cells and pericytes). In summary, we show that NRP1 can act as a co-receptor for PDGF-D-PDGFRβ signaling and is possibly implicated in intercellular communication in the vascular wall.
ENG (endoglin) is a coreceptor for BMP (bone morphogenetic protein) 9/10 and is strongly expressed in endothelial cells. Mutations in
lead to the inherited vascular disorder hereditary hemorrhagic ...telangiectasia characterized by local telangiectases and larger arteriovenous malformations (AVMs); but how ENG functions to regulate the adult vasculature is not understood.
The goal of the work was to determine how ENG maintains vessel caliber in adult life to prevent AVM formation and thereby protect heart function.
Genetic depletion of endothelial
in adult mice led to a significant reduction in mean aortic blood pressure. There was no evidence of hemorrhage, anemia, or AVMs in major organs to explain the reduced aortic pressure. However, large AVMs developed in the peripheral vasculature intimately associated with the pelvic cartilaginous symphysis-a noncapsulated cartilage with a naturally high endogenous expression of VEGF (vascular endothelial growth factor). The increased blood flow through these peripheral AVMs explained the drop in aortic blood pressure and led to increased cardiac preload, and high stroke volumes, ultimately resulting in high-output heart failure. Development of pelvic AVMs in this region of high VEGF expression occurred because loss of ENG in endothelial cells leads to increased sensitivity to VEGF and a hyperproliferative response. Development of AVMs and associated progression to high-output heart failure in the absence of endothelial ENG was attenuated by targeting VEGF signaling with an anti-VEGFR2 (VEGF receptor 2) antibody.
ENG promotes the normal balance of VEGF signaling in quiescent endothelial cells to maintain vessel caliber-an essential function in conditions of increased VEGF expression such as local hypoxia or inflammation. In the absence of endothelial ENG, increased sensitivity to VEGF drives abnormal endothelial proliferation in local regions of high VEGF expression, leading to AVM formation and a rapid injurious impact on heart function.
Early breach of the blood-brain barrier (BBB) and consequently extravasation of blood-borne substances into the brain parenchyma is a common hallmark of ischemic stroke. Although BBB breakdown is ...associated with an increased risk of cerebral hemorrhage and poor clinical prognosis, the cause and mechanism of this process are largely unknown. The aim of this study was to establish an imaging and analysis protocol which enables investigation of the dynamics of BBB breach in relation to hemodynamic properties along the arteriovenous axis. Using longitudinal intravital two-photon imaging following photothrombotic induction of ischemic stroke through a cranial window, we were able to study the response of the cerebral vasculature to ischemia, from the early critical hours to the days/weeks after the infarct. We demonstrate that disruption of the BBB and hemodynamic parameters, including perturbed blood flow, can be studied at single-vessel resolution in the three-dimensional space as early as 30 min after vessel occlusion. Further, we show that this protocol permits longitudinal studies on the response of individual blood vessels to ischemia over time, thus enabling detection of (maladaptive) vascular remodeling such as intussusception, angiogenic sprouting and entanglement of vessel networks. Taken together, this in vivo two-photon imaging and analysis protocol will be useful in future studies investigating the molecular and cellular mechanisms, and the spatial contribution, of BBB breach to disease progression which might ultimately aid the development of new and more precise treatment strategies for ischemic stroke.
Tissue fluid drains through blind-ended lymphatic capillaries, via smooth muscle cell (SMC)-covered collecting vessels into venous circulation. Both defective SMC recruitment to collecting vessels ...and ectopic recruitment to lymphatic capillaries are thought to contribute to vessel failure, leading to lymphedema. However, mechanisms controlling lymphatic SMC recruitment and its role in vessel maturation are unknown. Here, we demonstrate that platelet-derived growth factor B (PDGFB) regulates lymphatic SMC recruitment in multiple vascular beds. PDGFB is selectively expressed by lymphatic endothelial cells (LECs) of collecting vessels. LEC-specific deletion of
prevented SMC recruitment causing dilation and failure of pulsatile contraction of collecting vessels. However, vessel remodelling and identity were unaffected. Unexpectedly,
overexpression in LECs did not induce SMC recruitment to capillaries. This was explained by the demonstrated requirement of PDGFB extracellular matrix (ECM) retention for lymphatic SMC recruitment, and the low presence of PDGFB-binding ECM components around lymphatic capillaries. These results demonstrate the requirement of LEC-autonomous PDGFB expression and retention for SMC recruitment to lymphatic vessels, and suggest an ECM-controlled checkpoint that prevents SMC investment of capillaries, which is a common feature in lymphedematous skin.
Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood ...capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in
trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.
Spheroids of differentiating embryonic stem cells, denoted embryoid bodies, constitute a high-quality model for vascular development, particularly well suited for loss-of-function analysis of genes ...required for early embryogenesis. This review examines vasculogenesis and angiogenesis in murine embryoid bodies and discusses the promise of stem cell-based models for the study of human vascular development.
Equilibrium distribution and transport kinetics of boron between ferrosilicon and calcium silicate slag were investigated through experimental study. A 50 pct CaO-50 pct SiO
2
slag (by weight) was ...held for different durations at 1873 K (1600 °C) in a graphite tube furnace together with boron-doped ferrosilicon alloys in the range 50 to 85 wt pct Fe. The results indicate that the equilibrium partition ratio of boron between slag and metal is 2.4 ± 0.7 and largely independent of ferrosilicon alloy composition below 85 wt pct Fe. At 85 wt pct Fe, the equilibrium partition ratio is measured to be 6.2 ± 1.4. Although reported with a significant standard deviation, it is also found that the mass transfer rate of boron between ferrosilicon alloy and slag increases slightly with increasing iron content in ferrosilicon. These findings are discussed in terms of slag–metal bulk and interfacial properties.
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