VEGFRs and Notch: a dynamic collaboration in vascular patterning

2009 ◽  
Vol 37 (6) ◽  
pp. 1233-1236 ◽  
Author(s):  
Lars Jakobsson ◽  
Katie Bentley ◽  
Holger Gerhardt
Keyword(s):  
Selection Process ◽  
Cell Formation ◽  
Computational Modelling ◽  
Stalk Cell ◽  
Cell Phenotype ◽  
Vegf Receptor ◽  
Cellular Mechanisms ◽  
Dynamic Interactions ◽  
Receptor System ◽  
Molecular Control

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.

scholarly journals The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis

Angiogenesis ◽  
2021 ◽  
Author(s):  
Lanette Kempers ◽  
Yuki Wakayama ◽  
Ivo van der Bijl ◽  
Charita Furumaya ◽  
Iris M. De Cuyper ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Target Genes ◽  
Cell Formation ◽  
Stalk Cell ◽  
Vegf Receptor ◽  
Tip Cell ◽  
Sprouting Angiogenesis ◽  
Vegf Signaling

AbstractSprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic ‘tip’ and ‘stalk’ cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

scholarly journals The Role of Heparan Sulfate and Neuropilin 2 in VEGFA Signaling in Human Endothelial Tip Cells and Non-Tip Cells during Angiogenesis In Vitro

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 926
Author(s):  
Marchien G. Dallinga ◽  
Yasmin I. Habani ◽  
Alinda W. M. Schimmel ◽  
Geesje M. Dallinga-Thie ◽  
Cornelis J. F. van Noorden ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Heparan Sulfate ◽  
Splice Variant ◽  
Cell Formation ◽  
Stalk Cell ◽  
Vegf Receptor ◽  
Tip Cell ◽  
Sprout Formation ◽  
Tip Cells

During angiogenesis, vascular endothelial growth factor A (VEGFA) regulates endothelial cell (EC) survival, tip cell formation, and stalk cell proliferation via VEGF receptor 2 (VEGFR2). VEGFR2 can interact with VEGFR2 co-receptors such as heparan sulfate proteoglycans (HSPGs) and neuropilin 2 (NRP2), but the exact roles of these co-receptors, or of sulfatase 2 (SULF2), an enzyme that removes sulfate groups from HSPGs and inhibits HSPG-mediated uptake of very low density lipoprotein (VLDL), in angiogenesis and tip cell biology are unknown. In the present study, we investigated whether the modulation of binding of VEGFA to VEGFR2 by knockdown of SULF2 or NRP2 affects sprouting angiogenesis, tip cell formation, proliferation of non-tip cells, and EC survival, or uptake of VLDL. To this end, we employed VEGFA splice variant 121, which lacks an HSPG binding domain, and VEGFA splice variant 165, which does have this domain, in in vitro models of angiogenic tip cells and vascular sprouting. We conclude that VEGFA165 and VEGFA121 have similar inducing effects on tip cells and sprouting in vitro, and that the binding of VEGFA165 to HSPGs in the extracellular matrix does not seem to play a role, as knockdown of SULF2 did not alter these effects. Co-binding of NRP2 appears to regulate VEGFA–VEGFR2-induced sprout initiation, but not tip cell formation. Finally, as the addition of VLDL increased sprout formation but not tip cell formation, and as VLDL uptake was limited to non-tip cells, our findings suggest that VLDL plays a role in sprout formation by providing biomass for stalk cell proliferation.

Abstract 57: The Epigenetic Enzyme Kdm6b Controls the Pro-Fibrotic Transcriptome Signature of Foam Cells

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Annette E Neele ◽  
Koen H Prange ◽  
Marten A Hoeksema ◽  
Saskia van der Velden ◽  
Tina Lucas ◽  
...  
Keyword(s):  
Foam Cell ◽  
Smooth Muscle Actin ◽  
Cell Formation ◽  
Atherosclerotic Lesion ◽  
Foam Cells ◽  
Foam Cell Formation ◽  
Cell Phenotype ◽  
Sequencing Analysis ◽  
Dependent Manner ◽  
Alpha Smooth Muscle Actin

Aim: Foam cells are a key hallmark of atherosclerotic lesion formation. Within the atherosclerotic lesion macrophages scavenge modified lipoproteins and thereby acquire their foam cell characteristics. Besides their foam cell phenotype, macrophages can have specific inflammation regulatory functions in atherosclerotic lesions. Epigenetic pathways are crucial for monocyte to macrophage differentiation and activation. The H3K27 demethylase Kdm6b (also known as Jmjd3) is regulated in response to various triggers and regulates several modes of macrophage activation. Given the crucial role of macrophage foam cells in atherosclerosis, we here studied Kdm6b in peritoneal foam cells in order to identify regulated pathways. Material and Methods: A myeloid deficient Kdm6b mice (LysMCre-Kdm6b fl/fl ) was generated and bone marrow of Kdm6b wt or Kdm6b del mice was transplanted to irradiated Ldlr -/- mice which were fed a high fat diet for 9 weeks to induce foam cell formation. Peritoneal foam cells from Kdm6b del or Kdm6b wt mice were isolated and used for RNA-sequencing analysis. Results: Among the list of downregulated genes many genes involving fibrosis were affected in Kdm6b deficient foam cells including Collagen genes ( Col1a1 , Col1a2 ), Alpha smooth muscle actin ( Acta2 ) and Fibronectin-1 ( Fn1 ). Pathway analysis on downregulated genes ( P -value < 0.05) indicated that pathways involved in epithelial to mesenchymaltransition (EMT) ( q- value=10 -13 ) and extracellular matrix organization ( q- value=10 -4 ) were significantly downregulated. Pro-fibrotic pathways were thus strongly suppressed in Kdm6b deleted foam cells. Analysis of published datasets of foam cells showed that foam cell formation induces these pro-fibrotic characteristics. Overlay of both data sets indicated that fibrotic genes which are induced upon foam cell formation, are reduced in the absence of Kdm6b. These data suggest that foam cell formation induces a pro-fibrotic gene signature in a Kdm6b-dependent manner. Conclusion: We identified Kdm6b as a novel regulator of the pro-fibrotic signature of peritoneal foam cells.

High-content Analysis in Monastrol Suppressor Screens

2011 ◽  
Vol 50 (03) ◽  
pp. 265-272 ◽  
Author(s):  
Y. Ge ◽  
D. Zhang ◽  
X. Zhou ◽  
Z. Zhang
Keyword(s):  
Feature Extraction ◽  
Feature Selection ◽  
Image Segmentation ◽  
Selection Process ◽  
Fluorescent Microscopy ◽  
Back Propagation ◽  
Principal Component ◽  
Current Approach ◽  
Bipolar Cells ◽  
Cell Phenotype

SummaryObjectives: High-content screening (HCS) via automated fluorescent microscopy is a powerful technology for the effective expression of cellular processes. However, HCS will generally produce tremendous image datasets, which leads to difficulties of handling and analyzing. We proposed an automatic classification approach for simultaneous feature extraction and cell phenotype recognition of monoaster and bipolar cells in HCS system.Methods: The proposed approach was composed of image segmentation, feature extraction, and classification. The image segmentation was based on the Laplacian of Gaussian (LoG) edge detection method. For the reduction of noise effect on cellular images, we employed an adaptive threshold in microtubule channel. The principal component analysis was used in the feature selection process. The classification was performed with a back-propagation neural network (BPNN). Using the current approach, the cell phases were distinguished from three-channel acquisitions of cellular images and the numbers of bipolar and monoaster cells were automatically counted.Results: The validity of this approach was examined by the application of screening the response of drug compounds in suppressing Monastrol. Our results indicate that the proposed algorithm could improve the recognition rates of monoaster and bipolar cells to 97.98% and 93.12%, respectively, compared with 97.02% and 86.96% obtained from the same samples by multi-phenotypic mitotic analysis (MMA).Conclusions: We have shown that BPNN is a valuable tool to classify cell phenotype. To further improve the classification performance, more test data, more optimized feature selection approaches, and advanced classifier may be required and will be investigated in future works.

scholarly journals Cryptococcal Titan Cell Formation Is Regulated by G-Protein Signaling in Response to Multiple Stimuli

2011 ◽  
Vol 10 (10) ◽  
pp. 1306-1316 ◽  
Author(s):  
Laura H. Okagaki ◽  
Yina Wang ◽  
Elizabeth R. Ballou ◽  
Teresa R. O'Meara ◽  
Yong-Sun Bahn ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
G Protein ◽  
Pathogenic Fungus ◽  
Cell Formation ◽  
Cell Phenotype ◽  
Protein Signaling ◽  
Human Pathogens ◽  
Morphological Form ◽  
Content Type ◽  
Oxidative Stresses

ABSTRACT The titan cell is a recently described morphological form of the pathogenic fungus Cryptococcus neoformans . Occurring during the earliest stages of lung infection, titan cells are 5 to 10 times larger than the normal yeast-like cells, thereby resisting engulfment by lung phagocytes and favoring the persistence of infection. These enlarged cells exhibit an altered capsule structure, a thickened cell wall, increased ploidy, and resistance to nitrosative and oxidative stresses. We demonstrate that two G-protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3 a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation through elements of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. This conserved signaling pathway, in turn, mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G 1 cyclin Pcl103, the Rho104 GTPase, and two GTPase-activating proteins, Gap1 and Cnc1560. These observations support developing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens.

scholarly journals Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

2020 ◽  
Vol 21 (13) ◽  
pp. 4804
Author(s):  
Vincent van Duinen ◽  
Wendy Stam ◽  
Eva Mulder ◽  
Farbod Famili ◽  
Arie Reijerkerk ◽  
...  
Keyword(s):  
Drug Screening ◽  
Cell Formation ◽  
Cell Types ◽  
Culture Conditions ◽  
In Vitro Assays ◽  
Vegf Receptor ◽  
Screening Assay ◽  
Angiogenesis Assay

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

scholarly journals Computational modelling of T-cell formation kinetics: output regulated by initial proliferation-linked deferral of developmental competence

2013 ◽  
Vol 10 (78) ◽  
pp. 20120774 ◽  
Author(s):  
Erica Manesso ◽  
Vijay Chickarmane ◽  
Hao Yuan Kueh ◽  
Ellen V. Rothenberg ◽  
Carsten Peterson
Keyword(s):  
T Cell ◽  
Computational Models ◽  
Cell Formation ◽  
Computational Modelling ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Yield Data ◽  
Substantial Progress ◽  
A Cell

Bone-marrow-derived progenitors must continually enter the thymus of an adult mouse to sustain T-cell homeostasis, yet only a few input cells per day are sufficient to support a yield of 5 × 10 7 immature T-cells per day and an eventual output of 1–2 × 10 6 mature cells per day. While substantial progress has been made to delineate the developmental pathway of T-cell lineage commitment, still little is known about the relationship between differentiation competence and the remarkable expansion of the earliest (DN1 stage) T-cell progenitors. To address this question, we developed computational models where the probability to progress to the next stage (DN2) is related to division number. To satisfy differentiation kinetics and overall cell yield data, our models require that adult DN1 cells divide multiple times before becoming competent to progress into DN2 stage. Our findings were subsequently tested by in vitro experiments, where putative early and later-stage DN1 progenitors from the thymus were purified and their progression into DN2 was measured. These experiments showed that the two DN1 sub-populations divided with similar rates, but progressed to the DN2 stage with different rates, thus providing experimental evidence that DN1 cells increase their commitment probability in a cell-intrinsic manner as they undergo cell division. Proliferation-linked shifts in eligibility of DN1 cells to undergo specification thus control kinetics of T-cell generation.

scholarly journals Computational modelling suggests dynamic interactions between Ca2+, IP3 and G protein-coupled modules are key to robust Dictyostelium aggregation

2009 ◽  
Vol 5 (6) ◽  
pp. 612 ◽  
Author(s):  
Najl V. Valeyev ◽  
Jung-Su Kim ◽  
J. S. (Pat) Heslop-Harrison ◽  
Ian Postlethwaite ◽  
Nicolay V. Kotov ◽  
...  
Keyword(s):  
G Protein ◽  
Computational Modelling ◽  
Dynamic Interactions ◽  
G Protein Coupled

scholarly journals Density enhanced phosphatase-1 down-regulates urokinase receptor surface expression in confluent endothelial cells

Blood ◽  
2011 ◽  
Vol 117 (15) ◽  
pp. 4154-4161 ◽  
Author(s):  
Patrick M. Brunner ◽  
Patricia C. Heier ◽  
Judit Mihaly-Bison ◽  
Ute Priglinger ◽  
Bernd R. Binder ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Endothelial Cell Migration ◽  
Cell Surface Receptor ◽  
Migration And Invasion ◽  
Cell Behavior ◽  
Vegf Receptor ◽  
Receptor System ◽  
Upar Expression

Abstract VEGF165, the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis. We found that uPAR expression on the surface of confluent endothelial cells was down-regulated compared with subconfluent proliferating endothelial cells. Regulation of uPAR expression was most probably affected by extracellular signal-regulated kinase 1/2 (ERK1/2) activation, a downstream signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR. That DEP-1 plays a biologic role in angiogenic endothelial cell behavior was demonstrated in endothelial cell migration, proliferation, and capillary-like tube formation assays in vitro.

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