EVL regulates VEGF receptor‐2 internalization and signaling in developmental angiogenesis
2021; Springer Nature; Volume: 22; Issue: 2 Linguagem: Inglês
10.15252/embr.201948961
ISSN1469-3178
AutoresJoana Zink, Maike Frye, Timo Frömel, Claudia Carlantoni, David John, Danny Schreier, Andreas Weigert, Hebatullah Laban, Gabriela Salinas, Heike Stingl, Lea Günther, Rüdiger Popp, Jiong Hu, Benoît Vanhollebeke, Hannes Schmidt, Amparo Acker‐Palmer, Thomas Renné, Ingrid Fleming, Peter M. Benz,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle29 January 2021Open Access Transparent process EVL regulates VEGF receptor-2 internalization and signaling in developmental angiogenesis Joana Zink Joana Zink Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, GermanyThese authors contributed equally to this work Search for more papers by this author Maike Frye Corresponding Author Maike Frye [email protected] orcid.org/0000-0002-6257-7636 Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, GermanyThese authors contributed equally to this work Search for more papers by this author Timo Frömel Timo Frömel Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Claudia Carlantoni Claudia Carlantoni Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author David John David John German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Insitute for Cardiovascular Regeneration, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Danny Schreier Danny Schreier Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Andreas Weigert Andreas Weigert Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Hebatullah Laban Hebatullah Laban Department of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Gabriela Salinas Gabriela Salinas NGS-Integrative Genomics Core Unit (NIG), Institute of Human Genetics, University Medical Center Göttingen (UMG), Göttingen, Germany Search for more papers by this author Heike Stingl Heike Stingl Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Lea Günther Lea Günther Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Rüdiger Popp Rüdiger Popp Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Jiong Hu Jiong Hu Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Benoit Vanhollebeke Benoit Vanhollebeke orcid.org/0000-0002-0353-365X Laboratory of Neurovascular Signaling, ULB Neuroscience Institute Department of Molecular Biology, University of Brussels, Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Brussels, Belgium Search for more papers by this author Hannes Schmidt Hannes Schmidt Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Amparo Acker-Palmer Amparo Acker-Palmer orcid.org/0000-0002-8107-927X Institute of Cell Biology and Neuroscience and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Thomas Renné Thomas Renné Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Ingrid Fleming Ingrid Fleming orcid.org/0000-0003-1881-3635 Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Peter M Benz Corresponding Author Peter M Benz [email protected] orcid.org/0000-0002-3994-8937 Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Joana Zink Joana Zink Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, GermanyThese authors contributed equally to this work Search for more papers by this author Maike Frye Corresponding Author Maike Frye [email protected] orcid.org/0000-0002-6257-7636 Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, GermanyThese authors contributed equally to this work Search for more papers by this author Timo Frömel Timo Frömel Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Claudia Carlantoni Claudia Carlantoni Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author David John David John German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Insitute for Cardiovascular Regeneration, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Danny Schreier Danny Schreier Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Andreas Weigert Andreas Weigert Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University, Frankfurt am Main, Germany Search for more papers by this author Hebatullah Laban Hebatullah Laban Department of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Gabriela Salinas Gabriela Salinas NGS-Integrative Genomics Core Unit (NIG), Institute of Human Genetics, University Medical Center Göttingen (UMG), Göttingen, Germany Search for more papers by this author Heike Stingl Heike Stingl Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Lea Günther Lea Günther Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Rüdiger Popp Rüdiger Popp Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Jiong Hu Jiong Hu Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Benoit Vanhollebeke Benoit Vanhollebeke orcid.org/0000-0002-0353-365X Laboratory of Neurovascular Signaling, ULB Neuroscience Institute Department of Molecular Biology, University of Brussels, Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Brussels, Belgium Search for more papers by this author Hannes Schmidt Hannes Schmidt Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Amparo Acker-Palmer Amparo Acker-Palmer orcid.org/0000-0002-8107-927X Institute of Cell Biology and Neuroscience and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Thomas Renné Thomas Renné Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Ingrid Fleming Ingrid Fleming orcid.org/0000-0003-1881-3635 Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Peter M Benz Corresponding Author Peter M Benz [email protected] orcid.org/0000-0002-3994-8937 Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany Search for more papers by this author Author Information Joana Zink1,2, Maike Frye *,3, Timo Frömel1,2, Claudia Carlantoni3, David John2,4, Danny Schreier3, Andreas Weigert5, Hebatullah Laban6, Gabriela Salinas7, Heike Stingl1,2, Lea Günther1,2, Rüdiger Popp1,2, Jiong Hu1,2, Benoit Vanhollebeke8, Hannes Schmidt9, Amparo Acker-Palmer10, Thomas Renné3, Ingrid Fleming1,2 and Peter M Benz *,1,2,† 1Centre for Molecular Medicine, Institute for Vascular Signalling, Goethe University, Frankfurt am Main, Germany 2German Centre of Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany 3Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4Insitute for Cardiovascular Regeneration, Goethe University, Frankfurt am Main, Germany 5Institute of Biochemistry I-Pathobiochemistry, Faculty of Medicine, Goethe-University, Frankfurt am Main, Germany 6Department of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany 7NGS-Integrative Genomics Core Unit (NIG), Institute of Human Genetics, University Medical Center Göttingen (UMG), Göttingen, Germany 8Laboratory of Neurovascular Signaling, ULB Neuroscience Institute Department of Molecular Biology, University of Brussels, Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Brussels, Belgium 9Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany 10Institute of Cell Biology and Neuroscience and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt am Main, Germany †Present address: Department of CardioMetabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany *Corresponding author. Tel: +49 40 471057004; E-mail: [email protected] *Corresponding author. Tel: +49 69 6301 6052; E-mail: [email protected] EMBO Reports (2021)22:e48961https://doi.org/10.15252/embr.201948961 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling. SYNOPSIS This study reveals that the Ena/VASP protein family member EVL is a regulator of endothelial cell proliferation and sprouting in the postnatal retina. Genetic deletion of EVL (but not its homolog VASP) impairs the radial sprouting of the retinal vascular plexus and reduces endothelial cell proliferation, tip cell density and filopodia formation in the retina. EVL depletion impairs VEGF-induced endothelial cell proliferation and sprouting in vitro and ex vivo. EVL depletion reduces the internalization and phosphorylation of VEGF receptor-2 and its downstream signaling via the MAPK/ERK pathway. Introduction The development of the vertebrate vasculature requires the concerted action of growth factors and guidance cues that target endothelial cells. In the angiogenic vasculature, the highly motile and invasive endothelial tip cells form actin-rich lamellipodia and filopodia, which probe the environment for guidance cues, such as vascular endothelial growth factor (VEGF), and thereby determine the direction of growth (Gerhardt et al, 2003; Carmeliet et al, 2009). VEGF binding triggers homo- and heterodimerization of endothelial VEGF receptors (primarily VEGFR2 and VEGFR3), which induces receptor tyrosine phosphorylation and the activation of downstream signaling pathways that control proliferation, migration, and sprouting (Carmeliet & Jain, 2011; Simons et al, 2016). Extracellular regulated kinase (ERK)1/2 is a prominent downstream target of VEGFR2 signaling and the phosphorylation of VEGFR2 on Y1173 (murine sequence; Y1175 human sequence) is crucial for ERK1/2 activation. Indeed, a non-phosphorylatable Y1173F VEGFR2 mutant has the same effect as VEGFR2 gene ablation, namely early embryonic lethality due to severe vascular defects (Takahashi et al, 2001; Sakurai et al, 2005). Given its crucial role in vascular development, the biological activity of VEGFR2 is tightly regulated, i.e., at the level of receptor expression, as well as by the presence of co-receptors and auxiliary proteins, and the activity of signal terminating tyrosine phosphatases, e.g., CD148, PTP1b, and VE-PTP (Lampugnani et al, 2006; Lanahan et al, 2010; Hayashi et al, 2013; Simons et al, 2016). Although the surface expression of VEGFR2 is prerequisite for ligand binding, the endocytosis of the receptor is essential to activate many, if not all of the downstream signaling pathways, including ERK1/2 (Simons et al, 2016). A prominent example for this mechanism is ephrin-B2, which controls VEGF receptor internalization and is necessary for VEGF-induced tip cell filopodia extension and vascular sprouting (Sawamiphak et al, 2010; Wang et al, 2010). However, downstream mechanisms and proteins involved in VEGF receptor endocytosis and signaling have not been well defined. Like axonal growth cones, the principal role of endothelial tip cells is to navigate, a process that requires the correct probing of microenvironmental cues and their translation into directed cell migration. Therefore, it is not surprising that molecular guidance cues are largely conserved between axonal growth cones and endothelial tip cells and both form filopodia to explore their local environment (Soker et al, 1998; Wang et al, 1998; Serini et al, 2003; Wang et al, 2003; Banu et al, 2006; Adams & Eichmann, 2010; Dent et al, 2011; Fischer et al, 2018). Despite their pivotal role in tip cell navigation, little is known about the processes regulating filopodia assembly in endothelial cells (Carmeliet et al, 2009; Fischer et al, 2018). Studies in axonal growth cones have highlighted the potential importance of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family for filopodia formation (Drees & Gertler, 2008). The Ena/VASP proteins are important mediators of cytoskeleton control, linking kinase signaling pathways to actin assembly and are localized at sites of high actin turnover, including cell–cell contacts, focal adhesions, the leading edge of lamellipodia, and the tips of filopodia, where they promote actin polymerization and regulate the geometry of F-actin networks (Krause et al, 2003; Sechi & Wehland, 2004; Benz et al, 2009). In axonal growth cones and fibroblasts, these proteins bundle actin fibers and antagonize the capping of elongating filaments, thereby promoting filopodia formation and cell motility (Lebrand et al, 2004; Schirenbeck et al, 2006; Applewhite et al, 2007; Dent et al, 2007; Drees & Gertler, 2008; Barzik et al, 2014; Winkelman et al, 2014). In mammals, the Ena/VASP family of proteins consists of mammalian enabled (Mena), VASP, and Ena-VASP-like protein (EVL). In endothelial cells, Ena/VASP proteins are required for intercellular adhesion and Ena/VASP gene disruption impairs vessel integrity and endothelial barrier function in vivo (Furman et al, 2007; Benz et al, 2008). However, a potential role of Ena/VASP proteins in tip cell filopodia formation and angiogenesis is not known. Given the structural and molecular similarities between axonal growth cones and endothelial tip cells, this study set out to investigate the role of Ena/VASP proteins in angiogenic sprouting in vitro and in vivo in the postnatal murine retina. Results Differential expression of Ena/VASP protein family members in postnatal retinal endothelial cells To address potential roles of Ena/VASP proteins in angiogenic sprouting, expression of VASP, EVL and Mena was assessed in endothelial cells isolated from the postnatal murine retina. CD31 and CD34 double-positive endothelial cells were isolated by FACS from retinas from wild-type mice on postnatal day 5 (P5), and subjected to RNA-Seq (Fig EV1). Analysis of the transcriptomes confirmed high expression levels of multiple endothelial biomarkers, including von Willebrand factor (vWF), tyrosine-protein kinase receptor Tie2 (TEK), VE cadherin (CDH5), endoglin (ENG), CD146 (MCAM) and VEGFR2 (KDR). Endothelial selective sorting was confirmed by expression analysis of marker genes for astrocytes, immune cells, Müller glial cells, neurons and retina pigment epithelial cells, which were either very low or undetectable in the isolated cells (Fig 1A). Notably, while P5 retinal endothelial cells expressed VASP and EVL, Mena RNA was hardly detectable (Fig 1B). Consistent with expression data, confocal microscopy confirmed VASP and EVL protein expression in endothelial cells of the postnatal mouse retina (Fig 1C and D). No Mena was detectable in these cells, arguing against a significant role of Mena in retinal endothelial cells. However, our antibodies readily detected Mena in other retinal cells, including neurons (Fig 1E). Click here to expand this figure. Figure EV1. Overview of retinal endothelial cell isolation for RNA sequencing P5 retinas from wild-type mice were digested with collagenase and dispase. CD31 and CD34 double-positive endothelial cells were isolated by FACS, pooled into two samples, and analyzed by RNA-Seq on an Illumina HiSeq 4000 sequencer generating 50 bp single-end reads (ca. 30–40 Mio reads/sample). Download figure Download PowerPoint Figure 1. Expression of Ena/VASP proteins in postnatal retinal endothelial cells A, B. RNA sequencing of CD31 and CD34 double-positive retinal endothelial cells from P5 wild-type mice. (A) RNA levels (FPKM; fragments per kilobase million) of marker genes of endothelial cells (green; CD31 (gene name PECAM1), CD34, von Willebrand Factor (vWF), tyrosine-protein kinase receptor Tie2 (TEK), VE-cadherin (CDH5), endoglin (ENG), CD146 (MCAM) and VEGFR2 (KDR)), astrocytes (blue, GFAP), immune cells (red; T cells (CD3E), B-cells (CD19), all leukocytes (CD45, PTPRC), and monocytes/macrophages (F4/80, EMR1)), Müller glial cells (orange; aquaporin 4 (AQP4)), neurons (yellow; retinal ganglion cells (RNA binding fox-1 homolog 3, RBFOX3), amacrine cells (parvalbumin, PVALB), bipolar cells (PKC-α, PRKCA), horizontal cell (calbindin, CALB1), photoreceptors (rods, CD73 (NT5E); cones, transducing (GNAT1)), and retinal pigment epithelial cells (magenta; retinal pigment epithelium-specific 65 kDa protein (RPE65). (B) RNA levels of VASP (red), EVL (green), and Mena (blue) in the CD31 and CD34 double-positive P5 retinal endothelial cells. Error bars represent SEM; n = 18 animals (36 retinas) from six independent litters; four independent experiments. ***P < 0.001, one-way ANOVA with Bonferroni's multi comparison test. C–E. Staining of Ena/VASP proteins in blood vessels of P5 mouse retinas. P5 wild-type mouse retinas were fixed and stained with isolectin B4 (IB4, green) to visualize endothelial cells and VASP- (C), EVL- (D), or Mena-specific (E) antibodies (red). Yellow color in the merged images indicates the expression of VASP and EVL proteins in endothelial cells. No Mena protein expression was detected in P5 retinal endothelial cells (E). Representative images from three independent experiments are shown. Scale bars, 10 µm. Download figure Download PowerPoint EVL expression in endothelial cells Based on the transcriptomic and immunofluorescent analyses, we focused on the in vivo roles of VASP and EVL for vascular sprouting. "Knockout first" mutant EVL mice were obtained from the European Mouse Mutant Archive (Skarnes et al, 2011) and used to generate global EVL-deficient (EVL−/−) mice (Fig EV2A). Alternative splicing generates two EVL isoforms, a short (393 amino acids) and a 21 amino acids longer variant (EVL-I, (Lambrechts et al, 2000)). Western blotting using a novel polyclonal antibody raised against the murine EVL protein (Fig EV2B and C) detected both the EVL and EVL-I isoforms in lung and spleen lysates from adult wild-type mice and in the P5 brain and retina. However, only the short EVL isoform was detected in adult wild-type brain and retina and neither of the EVL isoforms were detectable in tissues derived from EVL−/− mice (Fig EV2D). Consistent with the Western blot analysis, flow cytometry of CD31+CD34+ cells from P5 mouse retinas revealed strong EVL expression in wild-type but not EVL−/− endothelial cells (Fig 2A). VASP is highly expressed in endothelial cells and required for endothelial barrier function in vivo (Furman et al, 2007; Benz et al, 2008; Benz et al, 2009; Kraft et al, 2010), but little is known about EVL function in endothelial cells. We analyzed EVL expression in endothelial cells from different human and mouse vascular beds. Both EVL isoforms were detected in the human endothelium-derived cell line EA.hy926, but only the short EVL protein isoform was detected in primary human dermal lymphatic endothelial cells (HDLEC) (Fig EV2C). Murine brain (MBEC) and lung (MLEC) endothelial cells isolated from wild-type mice also expressed both EVL protein isoforms but as expected no EVL protein signal was detectable in corresponding endothelial cells from EVL−/− mice (Fig 2B and C). Similar to the subcellular distribution of VASP in endothelial cells (Benz et al, 2009), EVL was concentrated at actin stress fibers, focal adhesions, filopodia, and the leading edge of lamellipodia in migrating MLEC (Figs 2D and EV3A). Click here to expand this figure. Figure EV2. Targeted disruption of the mouse EVL gene Generation of global and tissue-specific EVL-deficient mice. In the knockout first alleles, a trapping element consisting of a splice acceptor (SA), the promoterless lacZ gene, a polyadenylation signal (pA), and a neomycin resistance (neo) is inserted in intron 3 of the EVL gene. Splicing (dashed line) from EVL exon 3 (gray box) to the splice acceptor of the trapping cassette induces the global disruption of the EVL gene (EVL−/−). FLP-mediated recombination of the FRT sites (green rectangles) deletes the trapping elements and generates conditional alleles (EVLfl/fl) with loxP sites flanking the critical EVL exons 4–6. Recombination of loxP sites (red triangles) by the tamoxifen-inducible, pdgfb-driven Cre (Pdgfb-iCre/ERT2) deletes EVL exons 4–6, creates a frameshift mutation, and thus generates endothelial-specific EVL-deficient mice (EVLΔEC). Characterization of EVL-specific antibodies. HEK293 cells were transfected with EVL, VASP, or Mena (CMV-EVL, -VASP, -Mena) or MOCK (CMV-expression plasmid without insert) and analyzed by Western blotting with EVL-specific antibodies (left panel). Expression of VASP and Mena in the corresponding lysates was confirmed by Western blotting with anti-VASP (middle) or anti-Mena (right) antibodies, respectively. Primary human dermal lymphatic endothelial cells (HDLEC), human endothelium-derived cells (EA.hy926), and human monocytic cells (THP-1) were lysed and analyzed by Western blotting with EVL-specific antibodies (upper panel) or actin-specific antibodies (lower panel). Western blot analysis of EVL expression in lung, spleen, brain, and retina from adult wild-type (WT), P5 wild-type (WT P5), and adult EVL−/− (−/−) mice. EVL-specific antibodies detected the short EVL (60 kDa) and the long EVL-I (65 kDa) protein isoforms. MOCK and EVL-transfected HEK cells were used as positive or negative controls, respectively. Actin was used as loading control. Download figure Download PowerPoint Figure 2. EVL expression in mouse endothelial cells A. Wild-type and EVL−/− P5 retinas were digested, labeled with CD31-, CD34-, and EVL-specific antibodies, and analyzed by flow cytometry. Endothelial cells were defined as CD31/CD34-double-positive cells (gate indicated in the left panel) and analyzed for EVL expression (middle panel). Mean fluorescence intensity (MFI) of EVL in wild-type (magenta) and EVL−/− (black) cells is shown in the right panel. WT: n = 3, EVL−/−: n = 2; two retinas per animal. Error bars represent SEM. B, C. EVL protein expression in sparse/migrating wild-type (WT) and EVL−/− mouse brain endothelial cells (MBEC) and mouse lung endothelial cells (MLEC). Actin was used as loading control. Western blots are representative of three independent experiments. D. MLEC from wild-type (upper panel) and EVL−/− (lower panel) mice stimulated with 10 ng/ml VEGF were stained for actin (cyan) and EVL (magenta). Asterisks indicate focal adhesions, white arrows indicate filopodia, and white arrowheads indicate the leading edge of lamellipodia. Representative images from four independent experiments are shown. Scale bar, 20 µm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Subcellular localization of EVL and postnatal angiogenesis in VASP−/− mice A. EVL localizes to focal adhesions in endothelial cells. MLEC from wild-type mice were stained for phospho-paxilin (green) as a marker for focal adhesions, EVL (red) and actin (blue). White arrows indicate integrin-based focal adhesions at the tips of actin stress fibers. Representative images from three independent experiments are shown. Scale bar, 10 µm. B, C. Postnatal retinal angiogenesis in VASP−/− mice. (B) Isolectin B4-stained vasculature in whole mount retinas of wild-type (WT) and global VASP−/− mice on postnatal days 3 and 5 (P3, P5) assessed by confocal microscopy. Scale bars 200 µm. (C) Analysis of the radial vascular outgrowth relative to retinal radius and normalized to wild-type littermates. Error bars represent SEM; no significant difference was observed between the two genotypes at P3 (P > 0.999) or P5 (P > 0.999) (one-way ANOVA with Bonferroni's multi-comparison test). Download figure Download PowerPoint Impact of individual and combined VASP/EVL deletion on postnatal retinal angiogenesis To study the roles of VASP and EVL for sprouting angiogenesis in vivo, we compared the postnatal development of the retinal vasculature in VASP−/− (Hauser et al, 1999) and EVL−/− mice. The radial extension of the vascular plexus from the optic nerve to the periphery at P3 and P5 was significantly delayed in EVL−/− mice. The magnitude of the delay inversely correlated with the vascularization of the retina; e.g., the delay was most pronounced in P3 retina, smaller in the P5 retina and vanished in the mostly normoxic P7 retina (Fig 3A and B), suggesting a role of EVL particularly in the early stages of hypoxia/VEGF-driven retinal angiogenesis. In contrast, VASP deficiency had no significant impact on the rate of the vascular sprouting in the postnatal retina (Fig EV3), and mice with combined EVL/VASP deficiency were not statistically different from EVL−/− animals (Fig 3C). Figure 3. Delayed postnatal retinal angiogenesis in EVL−/− mice Isolectin B4 stained vasculature in whole mount retinas of wild-type (WT) and global EVL−/− mice on postnatal days 3, 5, and 7 (P3, P5, P7) assessed by confoca
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