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βIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling

ABSTRACT: Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report βIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial specific βIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, βIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, βIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Endothelial Cell

SUBMITTER: Paul Langlais

LAB HEAD: Nam Lee

PROVIDER: PXD026618 | Pride | 2022-02-04

REPOSITORIES: Pride

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BMP/Taz-Hippo signaling modulates VEGF-mediated angiogenesis

Project description:The BMP/TGFβ-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.

2020-10-07 | GSE82106 | GEO

CD34 marks angiogenic tip cells in human vascular endothelial cell cultures.

Project description:The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34- negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis. Four HUVEC donors were FACS-sorted by CD34 and divided in CD34-positive and CD34-negative fractions.

2012-01-05 | E-GEOD-34850 | biostudies-arrayexpress

Identification of the RNA-bound proteome in endothelial cells

Project description:Most new blood vessels emerge through sprouting angiogenesis, an intricate biological process that entails the coordinated migration of groups of endothelial cells branching from parental vessels. Critical guidance cues such as the Vascular Endothelial Growth Factor secreted by the surrounding tissues regulate the molecular and cellular responses triggered during angiogenesis. Similarly to other collective cell migration systems, endothelial cells within sprouting vessels are hierarchically organised into leader tip cells and follower stalk cells. Endothelial tip cells are highly polarised, exhibiting a leading edge that extends filopodia-containing protrusions and that interacts with the extracellular matrix, whilst their rear engages in cell-to-cell contacts with stalk cells. Polarisation of cytoplasmic components contributes to dynamic membrane remodelling phenomena that are constrained to the cell-leading front during migration. Amongst other factors, asymmetric distribution of mRNA is a well-established and crucial determinant of cell polarity. The active transport of transcripts is mediated by mRNA-binding proteins (mRBPs). Subcellular distribution of transcript messages results in restricted synthesis of the proteins they encode to particular compartments within the cell. This mode of post-transcriptional control of gene expression contributes to rapid and localised cellular responses to dynamic environmental stimuli with implications in development and disease. Although the involvement of RNA interacting proteins in angiogenesis has been previously reported, whether mRBPs play a role in asymmetric transcript localisation crucial for endothelial cell migration is yet to be fully explored.

2024-03-28 | PXD046994 | Pride

CD34 marks angiogenic tip cells in human vascular endothelial cell cultures.

2012-01-05 | GSE34850 | GEO

Slit2 modifies VEGF-induced angiogenic responses in rabbit skeletal muscle by inducing capillary sprouting and decreasing vascular permeability via reduced eNOS activity

Project description:Rationale: Slit2 is a possible modulator of vascular endothelial growth factor (VEGF) - induced angiogenesis, but its effects have not been tested in large animal models. Objective: We studied the effect of Slit2 on therapeutic angiogenesis induced by VEGF receptor 2 (VEGFR2) ligands Vammin and VEGF-DΔNΔC in vivo in rabbit skeletal muscles. The Slit2 target genes were also studied by RNA sequencing (RNA-Seq) in endothelial cells. Methods and Results: Adenoviral intramuscular gene transfers were performed into rabbit hindlimbs. Confocal and multiphoton microscopy were used for blood vessel imaging. Signaling experiments and gene expression analyses were performed to study mechanisms of Slit2 action. Slit2 decreased VEGFR2-mediated vascular permeability. It also reduced VEGFR2-mediated increase in blood perfusion and capillary enlargement, whereas sprouting of the capillaries was increased. Slit2 gene transfer alone did not have any effects on vascular functions or morphology. VEGFR2 activation was not affected by Slit2, but eNOS phosphorylation was diminished. The transcriptome profiling showed Slit2 downregulating angiogenesis-related genes such as nuclear receptor subfamily 4 group A member 1 (NR4A1) and Stanniocalcin-1 (STC-1) as well as genes related to endothelial cell migration and vascular permeability. Conclusions: Combining Slit2 with VEGFs adjusts VEGFR2-mediated angiogenic effects into a more physiological direction. This possibly allows the use of higher VEGF vector doses to achieve a more widespread vector and VEGF distribution in the target tissues leading to a better therapeutic outcome while reducing excess vascular permeability. HUVEC mRNA profiles after adenoviral vector gene transfers in duplicate.

2015-05-26 | E-GEOD-63283 | biostudies-arrayexpress

Pro-angiogenic transcriptome of zebrafish endothelial cells

Project description:To identify previously unknown determinants of endothelial cell sprouting, we defined and exploited a pharmacological strategy for the manipulation of angiogenic cell behavior in vivo. Whereas high vascular endothelial growth factor receptor (Vegfr) signaling is known to promote tip cell (TC) specification, activation of the Notch receptor via its ligand Delta-like 4 (Dll4) represses the TC phenotype to promote stalk cell (SC) fate. Conversely, suppression of Notch activity upon antagonistic interaction with its ligand Jagged1 pro- motes TC formation. Hence, specification of TCs involves tight spatiotemporal control of Vegfr/Notch signaling. Consequently, we hypothesized that the pharmacological manipulation of Vegfr/Notch signaling selectively during zebrafish intersegmental vessel (ISV) angiogenesis would enable the precise control of angiogenic EC behavior and sprouting- associated gene expression in vivo. For more information, see Herbert et al., 2012, PMID 22921365.

2019-05-09 | GSE130889 | GEO

Microarray Analysis of Retinal Endothelial Tip Cells Identifies CXCR4 as a Mediator of Tip Cell Morphology and Branching

Project description:The development of the vertebrate vascular system is mediated by both genetic patterning of vessels and by angiogenic sprouting in response to hypoxia. Both of these processes depend on the detection of environmental guidance cues by endothelial cells. A specialized subtype of endothelial cell known as the tip cell is believed to be involved in the detection and response to these cues, but the molecular signaling pathways utilized by tip cells to mediate tissue vascularization remain largely uncharacterized. To identify genes critical to tip cell function, we have developed a method to isolate them using laser capture microdissection, permitting comparison of RNA extracted from endothelial tip cells to that of endothelial stalk cells using microarray analysis. Genes enriched in tip cells include ESM-1, Angiopoietin-2, and SLP-76. CXCR4, a receptor for the chemokine SDF-1, was also identified as a tip cell-enriched gene, and we provide evidence for a novel role for this receptor in mediating tip cell morphology and vascular patterning in the neonatal retina.

2010-03-17 | GSE19284 | GEO

Slit2 modifies VEGF-induced angiogenic responses in rabbit skeletal muscle by inducing capillary sprouting and decreasing vascular permeability via reduced eNOS activity

2015-05-26 | GSE63283 | GEO

Endothelin2 signaling in the neural retina promotes the endothelial tip cell state and inhibits angiogenesis

Project description:Endothelin signaling is required for neural crest migration and homeostatic regulation of blood pressure. Here we report that constitutive over-expression of Endothelin-2 (Edn2) in the mouse retina perturbs vascular development by inhibiting endothelial cell (EC) migration across the retinal surface and subsequent EC invasion into the retina. Developing endothelial cells exist in one of two states: tip cells at the growing front, and stalk cells in the vascular plexus behind the front. This division of endothelial cell states is one of the central organizing principle of angiogenesis. In the developing retina, Edn2 over-expression leads to over-production of endothelial tip cells by both morphologic and molecular criteria. Spatially localized over-expression of Edn2 produces a correspondingly localized endothelial response. Edn2 over-expression in the early embryo inhibits vascular development at mid-gestation, but Edn2 over-expression in developing skin and brain has no discernable effect on vascular structure. Inhibition of retinal angiogenesis by Edn2 requires expression of Endothelin receptor A (Ednra) but not Ednrb in the neural retina. Taken together, these observations imply that the neural retina responds to Edn2 by synthesizing one or more factors that promote the endothelial tip cell state and inhibit angiogenesis. The response to Edn2 is sufficiently potent that it over-rides the activities of other homeostatic regulators of angiogenesis, such as vascular endothelial growth factor. Z/Edn2 females were crossed to Six3-Cre; Six3-Cre males. Postnatal P8 pups were genotyped for the Z/Edn2 allele by detection of Laz-Z activity in tail clips. Retinas from 2 - 3 pups were pooled for each data point.

2014-01-01 | E-GEOD-50059 | biostudies-arrayexpress

Endothelin2 signaling in the neural retina promotes the endothelial tip cell state and inhibits angiogenesis

2014-01-01 | GSE50059 | GEO

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