VASCULAR DEVELOPMENT
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Novartis Foundation Symposium 283
VASCULAR DEVELOPMENT
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Contents
Symposium on Vascular development, held at the Novartis Foundation, London, 13–15 June 2006 Editors: Derek J. Chadwick (Organizer) and Jamie Goode This symposium is based on a proposal made by Eckhard Lammert and Adam Wilkins Christer Betsholtz Chair’s introduction
1
Maria Grazia Lampugnani and Elisabetta Dejana The control of endothelial cell functions by adherens junctions 4 Discussion 13 Maike Schmidt, Ann De Mazière, Tanya Smyczek, Alane Gray, Leon Parker, Ellen Filvaroff, Dorothy French, Suzanne van Dijk, Judith Klumperman and Weilan Ye The role of Eg fl7 in vascular morphogenesis 18 Discussion 28 Max Levin, Andrew J. Ewald, Martin McMahon, Zena Werb and Keith Mostov A model of intussusceptive angiogenesis 37 Discussion 42 Tomáš Kucˇera, Jan Eglinger, Boris Strilic´ and Eckhard Lammert Vascular lumen formation from a cell biological perspective 46 Discussion 56 Christopher J. Drake, Paul A. Fleming and W. Scott Argraves The genetics of vasculogenesis 61 Discussion 71 Steven Suchting, Catarina Freitas, Ferdinand le Noble, Rui Benedito, Christiane Bréant, Antonio Duarte and Anne Eichmann Negative regulators of vessel patterning 77 Discussion 80 v
vi
CONTENTS
Taija Mäkinen and Kari Alitalo Lymphangiogenesis in development and disease 87 Discussion 98 Irene Noguera-Troise, Christopher Daly, Nicholas J. Papadopoulos, Sandra Coetzee, Pat Boland, Nicholas W. Gale, Hsin Chieh Lin, George D. Yancopoulos and Gavin Thurston Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis 106 Discussion 121 Georg Breier, Alexander H. Licht, Anke Nicolaus, Anne Klotzsche, Ben Wielockx and Zuzana Kirsnerova HIF in vascular development and tumour angiogenesis 126 Discussion 133 Karina Yaniv, Sumio Isogai, Daniel Castranova, Louis Dye, Jiro Hitomi and Brant M. Weinstein Imaging the developing lymphatic system using the zebrafish 139 Discussion 148 Frances High and Jonathan A. Epstein Signalling pathways regulating cardiac neural crest migration and differentiation 152 Discussion 161 Ralf H. Adams Investigation of the angiogenic programme with tissue-specific and inducible genetic approaches in mice 165 Discussion 171 Gary K. Owens Molecular control of vascular smooth muscle cell differentiation and phenotypic plasticity 174 Discussion 191 Andrea Lundkvist, Sunyoung Lee, Luisa Iruela-Arispe, Christer Betsholtz and Holger Gerhardt Growth factor gradients in vascular patterning 194 Discussion 201 Deborah A. Freedman, Yasushige Kashima and Kenneth S. Zaret Endothelial cell promotion of early liver and pancreas development 207 Discussion 216
CONTENTS
vii
Jörg Wilting, Kerstin Buttler, Jochen Rössler, Susanne Norgall, Lothar Schweigerer, Herbert A. Weich and Maria Papoutsi Embryonic development and malformation of lymphatic vessels 220 Discussion 227 Joaquim Miguel Vieira, Quenten Schwarz and Christiana Ruhrberg Role of the neuropilin ligands VEGF164 and SEMA3A in neuronal and vascular patterning in the mouse 230 Discussion 235 Final discussion 238 Tracheal tube development in Drosophila Closing remarks 240 Contributor index 242 Subject index 244
238
Participants
Ralf H. Adams Vascular Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Hellmut G. Augustin Department of Vascular Biology & Angiogenesis Research, Tumour Biology Center, Breisacher Str 117, Freiburg, D-79106, Germany Christer Betsholtz (Chair) Laboratory of Vascular Biology, Division of Matrix Biology, House A3, Plan 4, Department of Medical Biochemistry and Biophysics, Scheeles vag 2, Karolinska Institutet, SE-171 77 Stockholm, Sweden Georg Breier Institute of Pathology, Technische Universität Dresden, Fetscherstr 74, 01307 Dresden, Germany Jamie Davies Genes & Development IDG, Anatomy Building, University of Edinburgh, Teviot Place, Edinburgh EH10 5HF, UK Elisabetta Dejana FIRC Institute of Molecular Oncology, Via Adamello 16, I-20139 Milan, Italy Christopher J. Drake Cardiovascular Developmental Biology Center, Department of Cell Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA Anne Eichmann Institut National de la Santé et de la Recherche Medicale U833, Collège de France, 11 Place Marcelin Berthelot, 75005 Paris, France Jonathan A. Epstein Department of Cell and Developmental Biology, Cardiovascular Institute, University of Pennsylvania, 954 BRB II, 421 Curie Boulevard, Philadelphia, PA 19104, USA Deborah A. Freedman Cell and Developmental Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA viii
PARTICIPANTS
ix
Holger Gerhardt Vascular Biology Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Frances High University of Pennsylvania, BRB II/III Room 949, 421 Curie Boulevard, Philadelphia, PA 19104, USA Jan Kitajewski Department of Pathology, Irving Cancer Research Center 217B, Columbia University Medical Center, 1130 St Nicholas Avenue, New York, NY 10032, USA Eckhard Lammert Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 Dresden, Germany Keith E Mostov Department of Anatomy; Program in Cell Biology; and Department of Biochemistry and Biophysics, University of California School of Medicine, Genentech Hall, Room N212B, Mail Code 2140, 600 16th Street, San Francisco, CA 94158-2517, USA Taija Mäkinen Lymphatic Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Gary K. Owens Department of Molecular Physiology & Biological Physics, University of Virginia School of Medicine, 415 Lane Road, PO Box 801394, Room 1322 Medical Research Building 5, Charlottesville, VA 22908, USA Christiana Ruhrberg Institute of Ophthalmology, University College London, 11–43 Bath Street, London EC1V 9EL, UK Masabumi Shibuya Division of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo, 108-8639, Japan Claire L. Shovlin BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK Anne E. Uv Institute of Biomedicine, University of Göteborg, Medicinaregatan 9A, Box 440, 40530 Göteborg, Sweden Neil A. Vargesson Section of Molecular and Cellular Medicine, Division of Biomedical Sciences, Faculty of Natural Sciences, Imperial College London,
x
PARTICIPANTS
Workspace D, 1st Floor, Sir Alexander Fleming Bldg, South Kensington, London SW7 2AZ, UK Brant M. Weinstein Section on Vertebrate Organogenesis, Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Building 6B, Room 309, 6 Center Drive, Bethesda, MD 20892, USA Adam Wilkins BioEssays, 10/11 Tredgold Lane, Napier Street, Cambridge CB1 1HN, UK Jörg Wilting Zentrum für Kinderheilkunde und Jugendmedizin, Pädiatrie I, Forschungslabor, Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany George D. Yancopoulos Regeneron Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, NY 10591, USA Weilan Ye Tumor Biology and Angiogenesis Department, Genentech Inc., Mail Stop 230A, Building 15, Room 152015, 1 DNA Way, San Francisco, CA 94080, USA
Chair’s introduction Christer Betsholtz Laboratory of Vascular Biolog y, Division of Matrix Biolog y, Department of Medical Biochemistry and Biophysics, Scheeles vag 2, Karolinska Instituet, SE-171 77 Stockholm, Sweden
The overall theme of this symposium is vascular development, a subject that has progressed quite rapidly over recent years. It is also a broad subject: often it is described as encompassing just two steps, vasculogenesis and angiogenesis, but it is clear that this division might be somewhat artificial, and moreover, that multiple, complex phenomena are encompassed by these terms. Vasculogenesis involves the differentiation of vascular cells from non-vascular cells (mesodermal or mesenchymal), the assembly of these cells into vessels, and sometimes the incorporation of circulating precursors (bone marrow-derived endothelial progenitor cells) into the vasculature. Angiogenesis is even more complicated, involving a number of different morphogenetic phenomena in the vasculature, such as sprouting of new shoots, circumferential growth, the proliferation and enlargement of vessels, splitting of vessels (intussusception) and remodelling. This last term is often used in the literature as a description of the transition of an immature plexus to a refi ned hierarchically organized pattern of vasculature. Branch regression is also included in remodelling. The cellular and molecular processes collected under the simple terms ‘angiogenesis’, ‘vasculogenesis’ and ‘remodelling’ are extremely complicated and we therefore need to diversify our nomenclature in order to defi ne and focus our studies, and know what we are discussing. Just focusing on one of these processes, sprouting in angiogenesis, it becomes immediately clear that even this process is quite complicated. Hypoxia acting via hypoxia-responsive transcriptional machinery leads to the induction of signalling molecules that set off an angiogenic response. This response involves (1) the destabilization and degradation of vascular wall components (the matrix and the dissociation of mural cells that stabilize the vasculature), followed by (2) activation of the endothelial cells, their migration and proliferation to form a sprout, (3) the branching of the sprout and connection of the branches into a communicating and functional vessel network, and finally (4) the stabilization of the newly formed vessels by basement membrane deposition and maturation, and by the recruitment and incorporation of new mural cells. 1
2
BETSHOLTZ
We are beginning to appreciate that there is functional subspecialization among the vascular cells. This is relatively recent. An example is what occurs at the tip of the vascular sprout, where the leading cell, the tip cell, distinguishes itself morphologically and genetically from the trailing lumen-forming endothelial cells, the stalk cells. There are also mural cells: those associated with the sprouts and subsequent microvessels are referred to as pericytes. These cells are associated with the sprout from the onset of sprouting. Functional subspecialization among the vascular cells, and even within the endothelial compartment, is perhaps something that has been underappreciated so far. It may underlie the unequal cellular responses to the extracellular cues stimulating sprouting, as well as different types of intracellular signalling programmes in the different cell types. The cells are also connected to each other by cell adhesion molecules and junctional complexes; there is likely to be intense cell–cell communication via cell adhesion molecule-associated signalling mechanisms and juxtacrine or paracrine signalling. One of the emerging themes in vascular development is that the processes that are being uncovered at the growing vessel tips follow the same developmental principles that operate in other organogenesis processes. We have observed attractive signalling, repulsive signalling, cell–cell signalling leading to the establishment of cell boundaries, and we have noticed quite striking similarities to what has been observed for the development of the insect trachea. Remarkably, these processes of tubular sprouting are also similar to a process of subcellular sprout extension, namely neurite extension and guidance. Vascular development is interesting in its own regard, but one of the major driving forces for scientific progress in this area is the therapeutic promise. There are diseases which could benefit from the enhanced formation of new blood vessels if we learn how to stimulate their formation, such as infarction, fractures, thrombosis and even male baldness. There are other diseases where blood vessel formation is an intrinsic part of the pathological problem. Cancer is the most obvious example, but there is also clinical success with anti-angiogenic treatment in the area of ocular diseases. What is happening in the therapeutic area? Currently, there is just one validated target for anti-angiogenic therapies, and there are no clinically validated proangiogenic therapies. The anti-angiogenic target is vascular endothelial growth factor (VEGF), for which there are currently a few inhibitors in clinical use. Those of us who have been in the angiogenesis field for a while have, however, witnessed a long list of failures in human clinical trials of candidate anti-angiogenic molecules. Endostatin and angiostatin are well known examples, but there are many more that haven’t made it into the clinics. It’s reasonable to ask why molecules that act potently as anti-angiogenic factors in vitro or in animal models, fail in human clinical trials. One of the answers may be that the complexity of vascular development is enormous, and that we are still suffering from a quite profound
CHAIR’S INTRODUCTION
3
lack of insight into the molecular mechanisms of angiogenesis. For example we know very little about the molecular targets of some of the anti-angiogenic drugs that were entered into clinical trials some years ago. In this meeting we will discuss recent data on the guidance of endothelial cells in angiogenic sprouting and vessel remodelling. We will further discuss cell–cell communication at endothelial cell junctions, polarization of endothelial cells and lumenization of the vessel. Mural cells were in the shadows for a long time but they have recently been highlighted: a couple of papers will look at these. We have a second vascular system to consider—the lymphatics. I am glad that there are representatives of the field of lymphangiogenesis present at this symposium. We are also realizing that the developing vasculature is not just responding to surrounding cues, but also delivering developmentally important cues to the organs that it invades (in addition to oxygen and nutrients). Thus, vessels appear to fulfi l inductive functions during organogenesis. Reciprocal inductive signalling is a well known principle in epithelial–mesenchymal interactions, and it appears to also take place between blood vessels and surrounding cell types. Technology is important for studying the vasculature, and experimental genetics has become a major technology for advancing this field. Two participants at this symposium will discuss new developments that push the limits of mouse genetics. Finally, the symposium will have a translational component, as we will discuss vascular drug targets, new and old.
The control of endothelial cell functions by adherens junctions Maria Grazia Lampugnani* and Elisabetta Dejana*†1 IFOM, FIRC Institute of Molecular Oncolog y, * Mario Negri Institute for Pharmacological Research and † Department of Biomolecular Sciences and Biotechnologies, Faculty of Sciences, University of Milan, Milan, Italy
Abstract. Cell to cell junctions are important regulators of endothelial responses both in quiescent and angiogenic vessels. Endothelial cells express tight and adherens junctional structures. Although different in their specific molecular composition, these junctional complexes present a relatively similar general arrangement. Both types of junctions are formed by transmembrane adhesive proteins that bind homophilically to identical proteins on an adjacent cell and start a sequence of signalling events. Signal transmission is mediated by interaction with cytoplasmic and transmembrane partners. Adherens junctions are ubiquitous along the vascular tree. In these structures adhesion is mediated by VE-cadherin and its intracellular partners. In vitro and in vivo data show that VE-cadherin is required for endothelial integrity in quiescent vessels and for the correct organization of new vessels. VE-cadherin regulates endothelial functions through different mechanisms that include: (i) direct activation of signalling molecules such as PI3 kinase and Rac, to sustain survival and organization of the actin cytoskeleton; (ii) regulation of gene transcription, possibly modulating the nuclear level of transcription co-factors such as β -catenin and p120; (iii) formation of complexes with growth factor receptors, such as the type 2 receptor of VEGF (VEGFR2) and modulation of their signalling properties. 2007 Vascular development. Wiley, Chichester (Novartis Foundation Symposium 283) p 4–17
Cell–cell contacts control critical endothelial functions both in quiescent conditions, and in activated situations, such as inflammation and angiogenesis. Junctional proteins restrain cell migration, inhibit proliferation and apoptosis, and contribute to the maintenance of apical–basal polarity. In general, therefore, junctional signals should counteract angiogenesis and should be inhibited when vessels are induced to proliferate. Indeed, angiogenesis is accompanied by increased vessel permeability (Eliceiri et al 1999) and reduction of endothelial barrier function accompanies most inflammatory situations (Weis et al 2004a). 1
This paper was presented at the symposium by Elisabetta Dejana, to whom correspondence should be addressed. 4
ENDOTHELIAL CELL TO CELL JUNCTIONS AND VE-CADHERIN
5
A somehow intuitive consequence of these observations is that cell to cell junctions need to be weakened to allow the vessels to grow and be strengthened to maintain the endothelium in a quiescent state. An important aspect to define is the molecular mechanism through which this regulation takes place. Molecular architecture of endothelial junctions Various junctional structures have been identified at endothelial cell to cell contacts. Tight junctions, located in the most apical position toward the vessel lumen, exert a strong control on solute permeability and cell trafficking. These structures are present only in endothelia that need to maintain a strict control of permeability, such as brain microcirculation or large vessel endothelia. Adherens junctions on the other hand are ubiquitous along the vascular tree (Liebner et al 2006). Although the molecular composition of the different types of junctions varies, in general they are formed by both transmembrane and cytoplasmic components. At junctions, dimeric adhesive proteins bind to other identical dimers present on a nearby cell. The result is the lateral clustering of the adhesive molecules at cell to cell contacts, forming a zipper-like structure along the cell periphery (Dejana 2004). The recognition/adhesive information is delivered inside the cell by cytoplasmic and transmembrane partners. Junctions therefore behave as true signalling complexes (Liebner et al 2006). VE-cadherin and adherens junctions The major transmembrane component of endothelial adherens junctions is vascular endothelial (VE-) cadherin. This member of the cadherin family of adhesive receptors is expressed specifically in endothelial cells from the time of the earliest endothelial differentiation in the embryo. The extracellular domain of this singlepass transmembrane protein comprises five homologous repeats. The minimal functional unit of VE-cadherin is a homodimer that recognizes as a ligand, in a calcium-dependent way, an identical dimer present on a nearby cell. This starts a process of clustering of VE-cadherin molecules at the apposing cell membranes, which is sustained by both cis- and trans-interactions between cadherin molecules of the same cell and on the opposing cell, respectively (Gumbiner 2005). From the junction, signals are transmitted with the cooperation of interacting partners such as the intracellular molecules collectively known as catenins: α -, β -, γ -catenin/ plakoglobin and p120. β -catenin, plakoglobin and p120 bind strongly to defined sequences of the VE-cadherin cytoplasmic domain (Table 1), while α -catenin binds indirectly through the bridge of β - or γ -catenin. Other cytoplasmic and transmembrane proteins form complexes with VE-cadherin and contribute to the modulation of its adhesive and signalling activities (Table 1).
TABLE 1
VE-cadherin interactors 6
Transmembrane
Region of VE-cadherin involved in the binding
VEGFR2
n.d., β -catenin required
Dep1
n.d., β -catenin required
VE-PTP
membrane-proximal extracellular domain EC5
Cytoplasmic β -catenin
Induced
+ (in vivo)
+ (in vitro) +
↑
+
n.d.
↓
+
↑
+
↓
+
↑
Tiam Csk Src
n.d., β -catenin required phosphotyrosine 685 n.d.
+
SHP2
n.d., β -catenin required
+
Shc
cytoplasmic domain (aa703–784 in human VE-cadherin)
+
+
+
↑ ↑
Functional consequence Regulation of VEGFR2 internalization, mitogenic and antiapoptotic activities (1,2) endothelial barrier-function (3,4) Down-regulation of VEGFR2, Tyr-phosphorylation, internalization and mitogenic response (1,2) Reduced phosphorylation of VEGFR2 in response to VEGF, increased VE-cadherin-mediated barrier integrity, defective vasculogenesis (5,6) Association with VE-cadherin required to control vessel morphogenesis (7) Association with VE-cadherin contributes to junction stabilization (8) Involved in the maintenance of endothelial barrier function (9) Protection from apoptosis activated by VE-cadherin clustering (7) Regulation of actin and focal contact organization (10) Involved in density-dependent inhibition of cell growth (11) Mediates VEGF-induced VE-cadherin phosphorylation in vivo and in vitro (12) Down-regulation of catenin’s tyrosine phosphorylation. Target of thrombin (13) Possibly involved in the regulation of response to VEGF (14)
(1) Lampugnani et al 2003, (2) Lampugnani et al 2006, (3) Weis et al 2004a, (4) Weis et al 2004b, (5) Nawroth et al 2002, (6) Baumer et al 2006, (7) Carmeliet et al 1999, (8) Lampugnani et al 1995, (9) Iyer et al 2004, (10) Lampugnani et al 2002, (11) Baumeister et al 2005, (12) Lambeng et al 2005, (13) Ukropec et al 2000, (14) Zanetti et al 2002. Some of the reported interactions are direct and the molecular domains of VE–cadherin involved have been identified, while others remain to be determined (n.d., not determined). These interactions can be constitutive or induced by cell stimulation or clustering of VE–cadherin at cell to cell contacts as in confluent cultures. VE–cadherin partners may transfer intracellular signals and influence cell behaviour in a way dependent on cell confluency.
LAMPUGNANI & DEJANA
p85/PI3K
p120
↑
+
cytoplasmic domain (aa703–784 in human VE-cadherin) cytoplasmic domain (aa703–784 in human VE-cadherin) cytoplasmic domain (aa621–702 in human VE-cadherin) n.d., β -catenin required
γ -catenin
Modulated by cell confluence
Constitutive
ENDOTHELIAL CELL TO CELL JUNCTIONS AND VE-CADHERIN
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VE-cadherin and endothelial functions Data collected in several in vitro and in vivo models indicate that VE-cadherin expression and clustering strongly modulate endothelial cell behaviour (Carmeliet et al 1999, Corada et al 1999, 2001, 2002, Liao et al 2002, Weis et al 2004a, Crosby et al 2005). The involvement of VE-cadherin in the organization of new vascular structures is clearly indicated by the effect of homozygous null mutation of the VE-cadherin gene. In the mouse embryo this mutation induces a lethal phenotype at around 9.5 dpc due to major defects in the organization of the vascular tree (Carmeliet et al 1999). Among the various examples that illustrate the requirement of VE-cadherin for the formation of a new endothelial network, we report in Fig. 1 an ex vivo model of organ culture. The still avascular allantois of an 8.0 dpc mouse embryo can be explanted and maintained in culture. After about 18 h endothelial cells differentiate and form a vascular network (Fig. 1 a–e) (Drake & Fleming 2000). If the allantois is explanted from embryos with homozygous null mutation of the VE-cadherin gene, endothelial cells differentiate (see PECAM1and Fli1-positive cells in Fig. 1) (Crosby et al 2005), but they fail to organize in a three-dimensional structure (Fig. 1 f–l). Beyond illustrating the biological role of VE-cadherin, this experimental model is also useful to screen for the antiangiogenic activity of antibodies to VE-cadherin (Crosby et al 2005). Conversely, the importance of VE-cadherin in preservation of vascular integrity is underlined by the study of the effects of a blocking monoclonal antibody, BV13, directed to the EC1 region of the extracellular domain of the protein (Corada et al 2002). In vivo administration of this antibody strongly increases vascular permeability in several organs of adult mice (Corada et al 1999). Consistently, a monoclonal antibody, BV9, directed to the extracellular domain of human VE-cadherin enhances the permeability of monolayers of human endothelial cells (Corada et al 2001). While these data support the concept that VE-cadherin is a key element in the control of endothelial integrity, they also imply that targeting VE-cadherin for therapeutic intervention may produce severe systemic effects on quiescent vasculature. However, another monoclonal antibody, E4G10, directed to a different epitope (May et al 2005) was found to target VE-cadherin specifically at angiogenic sites, leaving resting vessels unperturbed (Liao et al 2002). VE-cadherin is in a different conformational state in resting versus growing endothelia and E4G10 recognizes an epitope of VE-cadherin that is accessible only in activated angiogenic vessels, while it is masked in quiescent endothelia. VE-cadherin signalling pathways VE-cadherin may modulate endothelial behaviour through different pathways (see Table 1). We have described the association to VE-cadherin of the p85 subunit of
8
LAMPUGNANI & DEJANA
VE positive
a
8.0 dpc mouse embryo
VE null
f
b
d
c
e
g
i
h
l
FIG. 1. VE-cadherin null allantois explants fail to undergo normal vascular morphogenesis in culture. Allantoises (small panel on the left) explanted from 8.0 dpc embryos can be maintained in culture for 16–24 hours. In this time frame endothelial cells differentiate, (see PECAM1 [green fluorescence, not visible in this grey scale reproduction, but please see colour figure at http://www.novartisfound.org.uk/dejana_figure1.jpg] and Fli1 [red fluorescence] positive cells) and organize in a vascular like network (panel a). The allantois from a homozygous null VE-cadherin mutant embryo contains a comparable number of PECAM1 (green fluorescence) and Fli1 (red fluorescence) positive endothelial cells. However, the cells fail to form stable vascular structures and only form sparse aggregates (f ). Small panels on the right (b–e, wildtype, g-l, VE-null) show independent magnifications (b,c and g,h 20×, and d,e and i,l 40×, respectively) of the 10 × magnification fields on the left (a,f ).
phosphatidylinositol 3 (PI3) kinase in confluent cells. This results in activation of Akt and enhanced resistance to apoptosis (Carmeliet et al 1999). The C-terminal truncated VE-cadherin, unable to bind β -catenin, does not recruit p85 and does not protect cells from apoptosis. VE-cadherin, again only when clustered in stable junctions, induces recruitment at cell–cell contacts of Tiam, a Rac-specific guanosine exchange factor. The effect is activation of Rac, inhibition of Rho and reorganization of actin cytoskeleton and adhesion plaques (Lampugnani et al 2002). Tyrosine kinases and phosphatases have been reported to form complexes with VE-cadherin. Src kinase is constitutively associated to VE-cadherin and mediates VEGF-induced VE-cadherin phosphorylation (Lambeng et al 2005). This may contribute to the maintenance of VEGF-stimulated angiogenic processes.
ENDOTHELIAL CELL TO CELL JUNCTIONS AND VE-CADHERIN
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C-terminal src kinase, csk, binds via its SH2 domain to the phosphorylated tyrosine 685 of VE-cadherin (Baumeister et al 2005). The functional consequence of such an interaction is inhibition of cell proliferation. Through its most membraneproximal extracellular domain, VE-cadherin associates with the receptor-typevascular endothelial protein tyrosine phosphatase (VE-PTP) (Nawroth et al 2002). This reduces tyrosine phosphorylation of VE-cadherin, reduces cell layer permeability, and most importantly, influences a correct vasculogenesis in the embryo (Baumer et al 2006, Nawroth et al 2002). The non-receptor tyrosine phosphatase SHP2 associates with VE-cadherin via β -catenin (Ukropec et al 2000). Thrombin induces phosphorylation of SHP2 and release of SHP2 from the complex. This is accompanied by tyrosine phosphorylation of the VE-cadherin-associated catenins ( β -, γ -, p120) and may be part of the mechanism through which thrombin increases endothelial permeability. The transmembrane phosphatase Dep-1 (density enhanced phosphatase 1) is recruited by VE-cadherin and modulates the responses to VEGFR-2 (see below, Lampugnani et al 2003). It was also found that VE-cadherin regulates gene transcription. This has been observed by comparison of the transcriptome of endothelial cells genetically ablated of VE-cadherin and that of the same cell line gene transduced to express VE-cadherin. 85 and 66 genes have been found up-regulated and down-regulated, respectively, by the presence of VE-cadherin clustered at cell to cell contacts. One reasonable mechanism through which VE-cadherin can exert its control on gene transcription is through sequestration at junctions of transcriptional co-factors, such as β -catenin and p120 (Park et al 2005). This would reduce the nuclear level of these factors and transcriptional regulation of their target genes. While this mechanism may be operating for genes presenting in the promoter regions at least one putative β -catenin/Tcf-Lef-1 binding domain, the precise molecular mechanism of such an effect is still unclear. In addition, the pathways through which VE-cadherin can control genes not under putative β -catenin control remain to be defined. VE-cadherin can also actively cross-talk with receptors for growth factors. This has been analysed in detail for VEGFR2, a classical modulator of endothelial function (Lampugnani et al 2003). When VE-cadherin is clustered at cell–cell contacts, as is the case in mature quiescent vessels and in confluent cultures, the two proteins can form a complex and, as a consequence, receptor signalling is re-directed from mitogenesis to survival (see below and Fig. 2). The complex between VE-cadherin and VEGFR2 observed in vivo is constitutive and transiently disrupted by treatment with VEGF (Weis et al 2004b). While in cell culture VEGF induces the formation of the complex (Zanetti et al 2002). A possible explanation of such discrepancy is the effect of blood flow as a modulator of endothelial functions. Although the molecular organization of the complex between VE-cadherin and VEGFR2 remains to be defined in detail, we have shown that β -catenin is required
10
LAMPUGNANI & DEJANA
Confluent VE-positive cells
** Akt
Survival
Sparse or VE-null cells
P42/44 MAP
kinases
Proliferation
FIG. 2. VE-cadherin clustering at cell to cell contacts directs VEGFR2 signalling. In confluent cells, as in mature quiescent vessels, VE-cadherin clustered at cell–cell contacts can form a complex with VEGFR2. The complex forms in response to VEGF stimulation which induces tyrosine phosphorylation of the receptor (represented as ** in the figure) (Shibuya & ClaessonWelsh 2006). β -catenin is indispensable for the complex to form (Lampugnani et al 2003) and the transmembrane tyrosine-phosphatase Dep1 is recruited in the complex. Association with VE-cadherin reduces VEGFR2 internalization and allows its dephosphorylation by Dep1 (Lampugnani et al 2003). The fi nal outcome is that the internalized receptor is poorly phosphorylated and transfers a weak mitogenic signal from intracellular compartments (Lampugnani et al 2006). In the absence of VE-cadherin, VEGFR2 is highly phosphorylated in response to VEGF (Lampugnani et al 2003), is efficiently internalized and can signal more effectively from intracellular compartments (Lampugnani et al 2006).
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for its formation. The biochemical consequence of its interaction with VEcadherin is that VEGFR2 is less tyrosine-phophorylated following stimulation with VEGF. This results in reduced activation of MAPK and reduced proliferation (Lampugnani et al 2003). Somewhat surprisingly, under the same experimental conditions, the anti-apoptotic response to VEGF is enhanced (Carmeliet et al 1999). The inhibition of tyrosine phosphorylation of VEGFR2 is due to the recruitment of the complex of the transmembrane phosphatase density-enhanced phosphatase 1 (Dep1). This transmembrane phosphatase has been reported to preferentially dephosphorylate particular tyrosine residues of Met receptor (Palka et al 2003) and PDGFβ receptor (Kovalenko et al 2000). If a similar site selectivity also operates for VEGFR2 it may represent the basis of the decreased proliferation and enhanced anti-apoptosis signalled by VEGFR2 in confluent VE-cadherin positive cells (Lampugnani et al 2003). Dep1 could preferentially dephosphorylate the tyrosines involved in recruiting mitogenic mediators (such as PLCγ ) (Takahashi et al 2001), leaving intact the phosphotyrosines that sustain the antiapoptotic signal (Shibuya & Claesson-Welsh 2006). A further consequence of the formation of a complex between VE-cadherin and VEGFR2 is that the internalization of the activated receptor is inhibited. If VE-cadherin is not expressed or not clustered at cell–cell contacts VEGFR2 can enter intracellular compartments to a greater extent and in a highly phosphorylated form (Lampugnani et al 2006). In this active state, VEGFR2 can activate PLCγ, which can sustain mitogenesis from intracellular compartments. This process is strongly inhibited in VE-cadherin positive cells in which the receptor is retained more effectively at the membrane and, due to Dep1 activity, is actively dephosphorylated. As a consequence, the mitogenic signal from the internalized receptor is much weaker (Lampugnani et al 2006). The implications of these results are that: (i) signalling from VEGFR2 is under a junctional control; (ii) the formation of a complex between VE-cadherin and VEGFR2 limits its mitogenic signalling; (iii) internalization is a way for the receptor to escape inactivation and signal more effectively and for a longer time; and (iv) any system that modulates the recruitment of VEGFR2 to the complex with VE-cadherin may effectively modulate the activity of this receptor. Conclusions It appears that endothelial cell–cell junctions are the association sites of a complex array of transmembrane and cytoplasmic molecules. These molecules functionally interact and the result of such cross-talk is a fine tuning of the endothelial behaviour. Cell–cell junctions represent possible targets of therapeutic intervention since modulation of junction adhesion and signalling may strongly influence endothelial growth, apoptosis and permeability.
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Acknowledgements This work was supported by the Associazione Italiana per la Ricerca sul Cancro, the European Community (QLRT-2001-02059, Integrated Project Contract No LSHG-CT-2004-503573; NoE MAIN 502935; NoE EVGN 503254).
References Baumeister U, Funke R, Ebnet K, Vorschmitt H, Koch S, Vestweber D 2005 Association of Csk to VE-cadherin and inhibition of cell proliferation. EMBO J 24:1686–1695 Baumer S, Keller L, Holtmann A et al 2006 Vascular endothelial cell-specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development. Blood 107:4754– 4762 Carmeliet P, Lampugnani MG, Moons L et al 1999 Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98:147–157 Corada M, Mariotti M, Thurston G et al 1999 Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo. Proc Natl Acad Sci USA 96:9815–9820 Corada M, Liao F, Lindgren M et al 2001 Monoclonal antibodies directed to different regions of vascular endothelial cadherin extracellular domain affect adhesion and clustering of the protein and modulate endothelial permeability. Blood 97:1679–1684 Corada M, Zanetta L, Orsenigo F et al 2002 A monoclonal antibody to vascular endothelialcadherin inhibits tumor angiogenesis without side effects on endothelial permeability. Blood 100:905–911 Crosby CV, Fleming PA, Argraves WS et al 2005 VE-cadherin is not required for the formation of nascent blood vessels but acts to prevent their disassembly. Blood 105:2771–2776 Dejana E 2004 Endothelial cell-cell junctions: happy together. Nat Rev Mol Cell Biol 5:261–270 Drake CJ, Fleming PA 2000 Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95:1671–1679 Eliceiri BP, Paul R, Schwartzberg PL, Hood JD, Leng J, Cheresh DA 1999 Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. Mol Cell 4:915–924 Gumbiner BM 2005 Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol 6:622–634 Kovalenko M, Denner K, Sandstrom J 2000 Site-selective dephosphorylation of the plateletderived growth factor beta-receptor by the receptor-like protein-tyrosine phosphatase DEP1. J Biol Chem 275:16219–16226 Lambeng N, Wallez Y, Rampon C et al 2005 Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues. Circ Res 96:384–391 Lampugnani MG, Corada M, Caveda L et al 1995 The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol 129:203–217 Lampugnani MG, Zanetti A, Breviario F et al 2002 VE-cadherin regulates endothelial actin activating Rac and increasing membrane association of Tiam. Mol Biol Cell 13:1175–1189 Lampugnani MG, Zanetti A, Corada M et al 2003 Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148. J Cell Biol 161:793–804 Lampugnani MG, Orsenigo F, Gagliani MC, Tacchetti C, Dejana E 2006 Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J Cell Biol 174:593–604
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Liao F, Doody JF, Overholser J et al 2002 Selective targeting of angiogenic tumor vasculature by vascular endothelial-cadherin antibody inhibits tumor growth without affecting vascular permeability. Cancer Res 62:2567–2575 Liebner S, Cavallaro U, Dejana E 2006 The multiple languages of endothelial cell-to-cell communication. Arterioscler Thromb Vasc Biol 26:1431–1438 May C, Doody JF, Abdullah R et al 2005 Identification of a transiently exposed VE-cadherin epitope that allows for specific targeting of an antibody to the tumor neovasculature. Blood 105:4337–4344 Nawroth R, Poell G, Ranft A et al 2002 VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts. EMBO J 21:4885–4895 Palka HL, Park M, Tonks NK 2003 Hepatocyte growth factor receptor tyrosine kinase met is a substrate of the receptor protein-tyrosine phosphatase DEP-1. J Biol Chem 278:5728– 5735 Park JI, Kim SW, Lyons JP et al 2005 Kaiso/p120-catenin and TCF/beta-catenin complexes coordinately regulate canonical Wnt gene targets. Dev Cell 8:843–854 Shibuya M, Claesson-Welsh L 2006 Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res 312:549–560 Takahashi T, Yamaguchi S, Chida K, Shibuya M 2001 A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J 20:2768–2778 Ukropec JA, Hollinger MK, Salva SM, Woolkalis MJ 2000 SHP2 association with VE-cadherin complexes in human endothelial cells is regulated by thrombin. J Biol Chem 275:5983– 5986 Weis S, Cui J, Barnes L, Cheresh D 2004a Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. J Cell Biol 167:223–229 Weis S, Shintani S, Weber A et al 2004b Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction. J Clin Invest 113:885–894 Zanetti A, Lampugnani MG, Balconi G et al 2002 Vascular endothelial growth factor induces SHC association with vascular endothelial cadherin: a potential feedback mechanism to control vascular endothelial growth factor receptor-2 signaling. Arterioscler Thromb Vasc Biol 22:617–622
DISCUSSION Lammert: Perhaps connecting the sprouts with the cadherins would be an interesting avenue: can we understand how stalk cells proliferate, weakening their junctions, and how this can be explained by VE-cadherin being upstream of VEGF receptor, for example? Betsholtz: That’s a good suggestion. It is an interesting possibility that the junctions are structurally different at the sprout sites. Dejana: One would expect that confluent cells remain relatively insensitive to VEGF activation. However, it is probably the local concentration of VEGF that makes the difference. There might be a concentration threshold, and cells would start to migrate once this is passed. VEGF can induce phosphorylation of VEcadherin and reduce the strength of adhesion at junctions. At the right concentration of VEGF, junctions would become weaker and endothelial cells would release themselves from inhibition of growth, and then start to migrate.
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Davies: Most of the things you talked about were global responses of a cell to the loss of VE-cadherin expression, such as internalization of receptor and so forth. Do you think everything will be global, or do you have any evidence that part of a cell can develop differently to another part? For example, if one part of a cell has lost expression, it can go into its lamellipodium-forming mode and forge forward, whereas the other side of the cell knows that it is still connected and just needs to pull its neighbours with it. A cell at one position has neighbours on one side but not on the other. One part of the membrane would be responding differently. Dejana: Let’s imagine a wound in the vessel wall. The cells at the front are released from contact inhibition of growth, and would migrate much more effectively. Cells at the migrating front present part of their membrane free, i.e. not in contact with neighbouring cells. VEGF receptors, present in that area, maintain a high response to migration and proliferation. In contrast, receptors at the back where the cells are still in contact with other cells are less responsive. This phenomenon may dictate the direction of movement. Betsholtz: Jamie Davies, it sounds to me that you are describing the tip cell, which is partially detached at one side and reacts to VEGF with a migratory response. But it still remains attached to the vasculature at the trailing end of the cell. Davies: It could be the tip cell, or even, if this is true of other cadherins, such as N-cadherin, could be modulated by loss of connection with pericytes. Gerhardt: We have recently looked at the distribution of VE-cadherin in the growing sprout. In the retinal model, the tip cell seems to have weakened junctions at its contact point with the stalk cell. It also relocates a lot of the VE-cadherin to the sprouting front, to the tips of the fi lopodia (unpublished results). We know that the response to stimulation of VEGFR-2 also differs between the tip cells and the stalk cell. The question I find interesting in the in vivo situation is why the tip cell doesn’t respond with proliferation. If you inject VEGF into the retina, almost the entire vascular plexus starts to proliferate. All the cells grow except the tip cells. But these cells retain stable VE-cadherin contacts. If you want to make an analogy to cell culture, I would consider these cells as being confluent. They start to proliferate, not migrate. Dejana: The concentration of VE-cadherin at junctions doesn’t tell the whole story. The most important parameter is the state of phosphorylation of these proteins. When we add histamine or other permeability increasing agents, we don’t see cell retraction if we don’t use very high concentrations: we just see phosphorylation of VE-cadherin at the junctions. The overall organization of junctions may change depending on the functional state of the cells. Maybe junctions of the cells at the tip allow their migration but not proliferation. Drake: We have compared tip cells with angioblasts. What we have concluded is the following. Angioblasts do not express VE-cadherin. In the tip cell, VEcadherin is only expressed at the site where the tip cell and stalk cell share a
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common cell-cell junction. Physically, tip cells appear to adhere tightly to the extracellular matrix. I think cell matrix adhesion is what is inhibiting VE-cadherin expression in both angioblasts and tip cells. When one moves back to the stalk cell, which expresses VE-cadherin, the cells are engaged in cell–cell versus cell– matrix interactions. Dejana: Our view is more concentrated on VE-cadherin. Certainly, interaction with the matrix is important. There are a series of signals from integrin to VEcadherin and vice versa. VEGFR2, can also interact with integrins, there might be a better interaction of this receptor with integrins while the cells are growing and migrating, while the receptor would interact with cadherins better when the cells are stabilized. Augustin: What is the relevance of your model in the context of sprouting angiogenesis? I think we would all agree that the tip cells have altered junctional complexes, and that subconfluent cells reflect some of the properties of an activated endothelial cell. Yet, I am not sure whether the two are the same. We have done a lot of transcriptomic profi ling of confluent and subconfluent cells. Essentially, we can validate 100% of the genes expressed by subconfluent endothelial cells in the carotid denudation model, whereas we find that only few of the genes expressed by subconfluent cells are actually involved in angiogenesis. Lateral cell migration as it occurs in vivo following a carotid denudation is distinctly different in terms of function and phenotype versus the three-dimensional sprouting angiogenesis in a complex matrix, where we see very distinct matrix-dependent gene expression profi les. Dejana: It might be different, because cell migration is more related to wound repair. In sprouting cells junctions may undergo very subtle modifications. These two models might have something in common but be quite different in nature. Augustin: You have determined the VE-cadherin-dependent transcriptome of endothelial cells. If you were to validate those genes you presented as candidates, in which model would they validate? Dejana: The general hypothesis is that VE-cadherin induces stabilization signals. Therefore the genes up- or down-regulated by this molecule should also be up- or down-regulated in confluent, stabilized monolayers. Owens: I have a question about cell autonomous functions of the cadherins. If you make a chimeric knockout mouse where half of the cells lack the VEcadherins, do they populate blood vessels or undergo apoptosis? Dejana: We haven’t looked at that, and I’m not aware of anyone else who has. However, VE-cadherin is a way through which cells communicate, if you do not have VE-cadherin you may interrupt communication. So, VE-cadherin null cells may die either because they do not express the molecule (cell autonomous) or because they cannot get in touch with the other endothelial cells.
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Owens: It would be interesting to know if VE-cadherin null embryonic stem cells are incapable of differentiating into endothelial cells within chimeric knockout mice, but capable of forming other cell lineages. In addition, such experiments may provide insights about the importance of intercellular interactions via cadherins in conferring information to cells about who their neighbours are and where they are at. You spoke extensively about homotypic EC–EC interactions. What is known about heterotypic cell–cadherin interactions and vascular development? I recall seeing ultrastructural evidence that adherens-type junctions between mural cells (smooth muscle cells and pericytes) and endothelial cells are highly abundant during development, but tend to become much rarer later on. Dejana: In vivo in the brain there is a contribution from N-cadherin, which is expressed on astrocytes, pericytes and smooth muscle cells. The interesting concept is that N-cadherin can interact with the fibroblast growth factor (FGF) receptor. This type of interaction may have a different meaning in terms of intracellular signalling. It may be important for the guidance of the endothelium, and FGF has been implicated in this role. It might also be important in the remodelling and stabilization of the vessels. This is still speculation because there aren’t many molecular data. With regard to your other point, you are saying that even in the absence of VE-cadherin there may be other mechanisms that determine the endothelial phenotype. We can’t exclude this. What we know so far is that in the absence of VE-cadherin, or when the junctions are broken using blocking antibodies, or when we use RNAi for VE-cadherin, we could validate most of the genes that we found to be modulated by the absence of this molecule. In vivo if we give blocking antibodies to VE-cadherin, we kill the animal in 24 h. There is a recent paper (Luo et al 2005) on the knockout of N-cadherin specifically in endothelial cells. The embryo died within 10.5 d of development. They claim this is due to the fact that in the absence of N-cadherin there is down-regulation of VE cadherin. Gerhardt: The data that we generated on N-cadherin were from the chicken system. In that system we found that N-cadherin was distributed on the abluminal side of the endothelium in contact with the pericytes (Gerhardt et al 2000). Blocking antibodies lead to a detachment of the pericytes, particularly in the brain parenchyma. Interestingly, we found that the intracellular components that associated with N- and VE-cadherin are different (Liebner et al 2000). It appeared that the junction between endothelium and pericyte only has β -catenin, and we couldn’t find the prominent association with γ -catenin, at this time indicating that it was a very loose type of junction. Now it might be good to look at other associations that might be important. Dejana: A nice idea is that N-cadherin may be important for the guidance of vessels. Gerhardt: We haven’t been able to study this in detail.
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Drake: In studying de novo blood vessel formation, with Elisabetta Dejana, we looked at the VE-cadherin nulls; the null embryos were able to form lumenized blood vessels. There was a recent paper that was a follow-up of an earlier Ncadherin knockout which showed that like the VE-cadherin nulls, the N-cadherin nulls also were able to form lumenized blood vessels. Whatever is directing the polarity of endothelial cells required for lumen formation is, therefore, an open question. References Luo Y, Radice GL 2005 N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis. J Cell Biol 169:29–34 Gerhardt H, Wolburg H, Redies C 2000 N-cadherin mediates pericytic–endothelial interaction during brain angiogenesis in the chicken. Dev Dyn 218:472–479 Liebner S, Gerhardt H, Wolburg H 2000 Differential expression of endothelial β -catenin and plakoglobin during development and maturation of the blood–brain and blood–retina barrier in the chicken. Dev Dyn 217:86–98
The role of Eg fl7 in vascular morphogenesis Maike Schmidt*, Ann De Mazière¶, Tanya Smyczek*, Alane Gray†, Leon Parker¶, Ellen Filvaroff‡, Dorothy French§, Suzanne van Dijk¶, Judith Klumperman¶ and Weilan Ye1* * Tumor Biolog y and Angiogenesis Department and † Molecular Biolog y Department, ‡ Molecular Oncolog y Department, § Patholog y Department, Genentech Inc. 1 DNA Way, South San Francisco, CA 94080, USA and ¶ Cell Microscopy Center, Department of Cell Biolog y and Institute for Biomembranes, University Medical Center, Utrecht, 3584C, Utrecht, The Netherlands
Abstract. EGFL7 was identified by a number of groups as a putative secreted factor produced by the vascular endothelial cells (ECs). In a recent publication, we showed that EGFL7 regulates midline angioblast migration in zebrafish embryos—a key step in vascular tubulogenesis. In this study, we further characterized the zebrafish vasculature in the Eg fl7 knockdown embryos at the ultrastructural level, and found that malformation of axial vessels is indeed due to the accumulation of angioblasts and aberrant connection among themselves, but not abnormal interaction between ECs and other cell types. Using in vitro biochemical assays, we demonstrated that EGFL7 is tightly associated with the extracellular matrix (ECM), and it supports EC migration either as a single factor or in combination with other ECM molecules. In order to evaluate if the biological function of EGFL7 is evolutionarily conserved, we generated Eg fl7 knockout mice and analysed vascular development in a number of tissues. We found that vascular coverage of a given tissue is reduced or delayed, and vascular morphogenesis is defective in the Eg fl7 mutant mice. Taken together, we conclude that EGFL7 provides a proper microenvironment for endothelial cell migration, thereby enabling accurate patterning. Our study indicates that the molecular composition of the ECM influences vascular morphogenesis. 2007 Vascular development. Wiley, Chichester (Novartis Foundation Symposium 282) p 18–36
Actively growing tissues such as embryos and tumours require adequate blood supply. They satisfy this need by producing pro-angiogenic factors, which promote new blood vessel formation from existing vessels (angiogenesis), or from progenitor cells (vasculogenesis) (Carmeliet & Jain 2000, Folkman 1997, Hanahan 1997). Although the initiation of new vessel formation is often triggered by mitogenic 1
This paper was presented at the symposium by Weilan Ye, to whom correspondence should be addressed. 18
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factors that act directly on endothelial cells (ECs) or their progenitors, the construction of a functional vascular network is a complex biological process, which involves all or many of the following steps: (a) EC and supporting cell differentiation and proliferation; (b) EC and supporting cell migration; (c) EC coalescence into cord-like structures; (d) transition from cord to tube (tubulogenesis) (Hogan & Kolodziej 2002); (e) formation of secondary vessels from existing ones (angiogenesis) (Burri & Djonov 2002, de Waal & Leenders 2005); (f) transition from a primitive vascular plexus to a complex network (remodeling) (Carmeliet & TessierLavigne 2005) and (g) recruitment of perivascular supporting cells (Jain 2003, Betsholtz et al 2005). To date, a significant number of molecules have been shown to regulate vasculogenesis and angiogenesis (Alva & Iruela-Arispe 2004, Coultas et al 2005, Davis & Yancopoulos 1999, Ferrara et al 2003, Klagsbrun & Eichmann 2005, Yancopoulos et al 1998). What we learn from studying these factors not only greatly enhances our understanding of vascular biology, but also leads to the development of therapeutic strategies to treat angiogenesis disorders (Ferrara et al 2004, Ferrara & Kerbel 2005, Folkman 2003). However, our knowledge regarding the complex vascular development process is still fragmented; likewise, our means to leverage the angiogenesis process toward medical benefits are limited. Advancements in these two areas should be facilitated by the discovery and characterization of additional angiogenesis regulators. In an effort to identify novel molecules involved in the regulation of vascular development, we performed a whole-mount in situ hybridization screen to identify factors that are expressed in and around the mouse embryonic vasculature. Out of ∼900 genes examined, about two dozen are expressed in the vasculature. One of the vascular-associated genes is Eg fl7, which encodes a ∼30 kDa putative secreted factor with an N-terminal EMI domain, two centrally located EGF-like domains, and a leucine- and valine-rich C-terminus that has some similarities to a coiled-coil domain. We and other groups found that Eg fl7 is specifically expressed in vascular endothelial cells, and its expression is up-regulated during active vascular growth (Campagnolo et al 2005, Fitch et al 2004, Parker et al 2004, Soncin et al 2003). When Eg fl7 is knocked down using a morpholino antisense oligo in zebrafish embryos, we found that midline angioblast migration along the dorsal ventral axis is blocked, and tubulogenesis of axial vessels does not occur (Parker et al 2004). These findings suggest that EGFL7 plays an important role in regulating vascular development. EGFL7 is required for proper positioning of endothelial cells To further understand the mechanism by which EGFL7 influences axial vessel morphogenesis in the zebrafish embryos, we carried out ultrastructural analysis on cross-sections of zebrafish embryos at the 30 somite stage using electron
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microscopy. We asked if the lack of EC migration along the dorsal–ventral (D–V) axis is due to altered interaction between ECs and the surrounding cells, and we found that ECs do not form abnormal connections with their surrounding cells in the Eg fl7 knockdown (KD) embryos (Fig. 1). Consistent with what we observed using deconvolution microscopy, we saw that ECs accumulate in the space between the hypocord and the gut tube in the KD embryos, whereas ECs in the control oligo injected embryos have readily separated along the D–V axis and assembled into the aorta and posterior cardinal vein at this age. Strikingly, aberrant EC–EC connections are frequently observed in the KD embryos, as adherent junctions are seen across the space where vascular lumen would normally form (Fig. 1 C and D). The abnormal connections are quantified and summarized in Table 1. These
FIG. 1. Ultrastructural analysis of the zebrafish axial vessels. Overview of the vascular area transversely sectioned immediately posterior to the yolk ball-yolk ball extension transition in control (A) and Eg fl7 knockdown (B) zebrafish embryos. In the knockdown embryo, the lumen of the axial vein is obliterated (C) by a cell junction (D, arrow). A, dorsal aorta; V, axial vein; N, notochord; H, hypochordal cell(s); E, endoderm; S, somite; K, pronephric duct. Scale bar: (A, B) 10 µm; (C) 2 µm; (D) 0.5 µm.
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TABLE 1 Quantitative electron microscopic analysis of vascular defects in the Eg fl7 knockdown (KD) embryos
Hypochord–endoderm distance ( µm) Number of sections with cell junctions crossing aorta and/or vein lumen Number of cell junctions crossing aorta and/or vein lumen Number of junctional interfaces in aorta and vein endothelium Number of junctional interfaces in aorta endothelium Number of junctional interfaces in vein endothelium Number of hypochord-aorta appositions Number of endoderm-vein appositions
Wild-type (n = 13)
Eg fl7 KD (n = 12)
P
43.0 ± 4.2 0 out of 13
28.0 ± 4.3 5 out of 12
