Spatial regulation of VEGF receptor endocytosis in angiogenesis

Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, and University of Muenster, Faculty of Medicine, D-48149 Muenster, Germany.
Nature Cell Biology (Impact Factor: 19.68). 01/2013; 15(3). DOI: 10.1038/ncb2679
Source: PubMed


Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.

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    • "In contrast, it has been demonstrated that VEGF induces VEGFR2 desensitization or downregulation rapidly and actively by two distinct mechanisms: trafficking to the endosome and interaction with VEcadherin [Bruns et al., 2010]. The activated and phosphorylated VEGFR2 is internalized within the initial 30–45 min and ubiquitinated for lysosomal degradation [Bruns et al., 2010; Nakayama et al., 2013]. This receptor Fig. 4. The effects of stimulation with PRP and VEGF on focal adhesion and actin fiber formation. "
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    ABSTRACT: Platelet-rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previous in vitro and in vivo studies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP-induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time-lapse quantitative imaging demonstrated that in the initial phase (∼2 h) of treatment, PRP substantially stimulated cell migration in a wound-healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45 ± 0.16 mm and 0.82 ± 0.23 mm in control and PRP-stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti-VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up-regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose- and time-dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up-regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short-term activation should be considered when the controlled release system of PRP components is designed. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · Apr 2015 · Cytoskeleton
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    • "Unexpectedly, we observed that the percentages of Flk1þ cells were significantly decreased at both 48 and 96 h in 1% DMSOtreated P19 cells as determined by flow cytometry (Fig. 5C, D). Recent studies have demonstrated the importance of Flk1 endocytosis for its biological function and signaling capability, suggesting that many key events of VEGFR signaling are regulated by endosomal receptor trafficking (for review, Eichmann and Simons, 2012; Nakayama et al., 2013). In this context, we hypothesized that Flk1, a receptor tyrosine kinase could be internalized and transported to various intracellular compartments in response to VEGF activation triggered by 1% DMSO. "
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    ABSTRACT: Dimethyl sulfoxide (DMSO) is widely used to induce multilineage differentiation of embryonic and adult progenitor cells. To date, little is known about the mechanisms underlying DMSO-induced mesodermal specification. In this study, we investigated the signaling pathways and lineage-determining genes involved in DMSO-induced mesodermal specification in P19 cells. Wnt/β-catenin and TGF-β superfamily signaling pathways such as BMP, TGF-β and GDF1 signaling were significantly activated during DMSO-induced mesodermal specification. In contrast, Nodal/Cripto signaling pathway molecules, required for endoderm specification, were severely downregulated. DMSO significantly upregulated the expression of cardiac mesoderm markers but inhibited the expression of endodermal and hematopoietic lineage markers. Among the DMSO-activated cell lineage markers, the expression of Mixl1 and Flk1 was dramatically upregulated at both the transcript and protein levels, and the populations of Mixl1+, Flk1+ and Mixl1+/Flk1+ cells also increased significantly. DMSO modulated cell cycle molecules and induced cell apoptosis, resulting in significant cell death during EB formation of P19 cells. An inhibitor of Flk1, SU5416 significantly blocked expressions of TGF-β superfamily members, mesodermal cell lineage markers and cell cycle molecules but it did not affect Wnt molecules. These results demonstrate that Mixl1 and Flk1 play roles as key downstream or interacting effectors of Wnt/TGF-β signaling pathway during DMSO-induced mesodermal specification in P19 cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Dec 2014 · Journal of Cellular Physiology
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    • "Although cell surface interaction between SCF and KIT has been extensively studied [31]–[33], the precise mechanisms of their endocytosis and intracellular transport have not been clarified. Meanwhile, CME has been reported to be involved in the endocytosis and/or intracellular transport of some receptors such as epidermal growth factors (EGFs) [29], [34], vascular endothelial growth factor (VEGF) [35], [36] and Notch [37], [38]. So, in this study, we analyzed the roles of CALM in the endocytosis and transport of KIT. "
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    ABSTRACT: CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage-Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM-/- mice. Also, colony forming activity was impaired in CALM-/- LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM-/- LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM-/- murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM-/- MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM-/- MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM-/- MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM-/- MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM-/- KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.
    Full-text · Article · Oct 2014 · PLoS ONE
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