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.

126 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Cytoskeleton 04/2015; 72(5). DOI:10.1002/cm.21221 · 3.12 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 10/2014; 9(10):e109441. DOI:10.1371/journal.pone.0109441 · 3.23 Impact Factor
  • Source
    • "While prior studies focused on an alternative angiogenic kinase VEGFR2, have suggested that endocytosis is also required for full activation of ERK [51]; other studies have revealed divergent observations [52]. In the present study, time course stimulation of EC with FGF2 showed that full activation of ERK requires endosomal signaling since ERK activation was blocked by DynK44A without effects on phosphorylation of FGFR1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Binding of angiogenic molecules with cognate receptor tyrosine kinases (RTK) is required for angiogenesis however the precise link between RTK binding, endocytosis, and signaling requires further investigation. Here, we use FGFR1 as a model to test the effects of the large GTPase and endocytosis regulatory molecule dynamin-2 on angiogenic signaling in context of distinct FGF ligands. In vitro, overexpression of dominant negative dynamin-2 (DynK44A) attenuates FGFR1 activation of Erk and tubulogenesis by FGF2. Furthermore, we identify FGF21, a non-classical, FGF ligand implicated in diverse human pathologies as an angiogenic molecule acting through FGFR1 and β-Klotho coreceptor. Disruption of FGFR1 activation of ERK by FGF21 is achieved by perturbation of the function of both dynamin-2 and Rab5 GTPase. In vivo, mice harboring endothelial selective overexpression of DynK44A, show impaired angiogenesis in response to FGF21. In conclusion, dynamin dependent endocytosis of FGFR1 is required for in vitro and in vivo angiogenesis in response to FGF2 and the non-classical FGF ligand, FGF21. These studies extend our understanding of the relationships between RTK binding, internalization, endosomal targeting, and angiogenic signaling.
    PLoS ONE 05/2014; 9(5):e98130. DOI:10.1371/journal.pone.0098130 · 3.23 Impact Factor
Show more