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Address correspondence to Christer Betsholtz, Dept. of Medical Biochemistry, University of Göteborg, Medicinaregatan 9A, Box 440, SE 405 30 Göteborg, Sweden. Tel.: 46-31-7733460. Fax: 46-31-416108. E-mail: christer.betsholtz{at}medkem.gu.se
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Abstract |
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Key Words: VEGF; endothelial cell; filopodia; astrocyte; migration; proliferation
David Shima's present address is Eyetech Research Center, Eyetech Pharmaceuticals Inc., 42 Cummings Park, Woburn, MA 01801.
* Abbreviations used in this paper: CNS, central nervous system; GFAP, glial fibrillary acidic protein; ILM, inner limiting membrane; P, postnatal day; PECAM, plateletendothelial cell adhesion molecule; PlGF, placenta growth factor; VEGFR, vascular endothelial growth factor receptor.
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Introduction |
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The most pervasive vertebrate tubular organ, the vasculature, is first assembled from scattered precursor cells that shape blood islands, which fuse to create the first primitive plexus of vessels (Risau and Flamme, 1995). Subsequently, enlargement and remodeling of the plexus, involving sprouting, splitting, and regression of branches, shape hierarchical vascular patterns that allow directional blood flow. These patterns become precisely adapted to organ anatomy and physiology, hence they differ extensively between organs.
Principally, at least two different mechanisms may lead to organ-specific vascular patterns. First, the formation of a primary vascular network may be a random process followed by specific branch regression. This "vascular pruning" represents a major mechanism of vascular remodeling and is likely regulated at the level of endothelial cell survival, which depends on vascular endothelial growth factor (VEGF-A)* and unidentified signals from surrounding vascular smooth muscle cells or pericytes (Benjamin et al., 1999). Second, angiogenic sprouting and fusion may be a guided process, leading to specific primary vascular patterns. Such angiogenic guidance is mainly inferred by the seemingly nonrandom angiogenic sprouting in the developing central nervous system (CNS), for example, in the mammalian retina, where a vascular plexus initially forms superimposed on a preexisting astrocyte plexus (Stone and Dreher, 1987; Fruttiger et al., 1996).
Precision guidance of specialized cells is involved in the formation of other pervasive organ systems. Axonal guidance by attractive and repulsive forces is well established, and also the formation of the insect tracheal system, which is both structurally and functionally analogous to the vertebrate vasculature, relies on guidance of cells and subcellular processes along predefined tracks (for review see Zelzer and Shilo, 2000). Certain molecules with a role in axon and/or tracheal guidance have also been implicated in vascular morphogenesis (for review see Shima and Mailhos, 2000). For angiogenesis, however, the functions of these and other angiogenic modulators remain ill defined.
The concept of precision guidance requires a sensor that relays external signals into specific cell behavior. In axonal guidance, this is provided by a specialized tip structure, the growth cone. Also, the guidance of Drosophila tracheal branches depends on specialized sensor cells situated at the sprouting tips. These tip cells are unique in morphology and gene expression and appear to respond to guidance cues conferring positional information (Samakovlis et al., 1996). Both the growth cone and the tracheal tip cells use dynamic filopodia to sense guidance cues in their surroundings and to migrate (Kater and Rehder, 1995; Ribeiro et al., 2002). There is evidence from several studies that endothelial sprouts can also extend multiple filopodia at their distal tips (Bär and Wolff, 1972; Marin-Padilla, 1985 and earlier literature cited therein), indicating that growing vascular sprouts are endowed with specialized tip structures with potential functions in guidance and migration. These descriptions have received surprisingly little attention, and with few recent exceptions (Dorrell et al., 2002; Ruhrberg et al., 2002) they go unnoticed in today's concepts of vascular development. Importantly, the numerous pro- and antiangiogenic factors discovered during the past 15 yr have not been studied in relation to endothelial tip cells and their filopodia, and in particular, the possibility that endothelial tip cells may respond specifically to such factors has not been explored.
By analyzing mice lacking heparin-binding VEGF-A isoforms, we have recently provided evidence that the spatial distribution of secreted VEGF-A is critical for the balance between capillary branching and growth in vessel size (Ruhrberg et al., 2002). Here, we have used several genetic and pharmacological gain and loss of function approaches to show that different modes of VEGF-A distribution in the extracellular space independently guide tip cell migration and control proliferation in stalk cells. Collectively, our data explain how the pattern of cellular expression and extracellular distribution of a single growth factor shapes vascular patterns during angiogenic sprouting by regulating different events in defined subpopulations of endothelial cells.
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Results |
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The abundance of filopodia on tip cells is indicative of an active migratory phenotype. To study more directly the dynamics of tip cell behavior in real time, we used an organ culture model of vessel sprouting adapted from the rat aortic ring model (Nicosia and Ottinetti, 1990) in which all external stimuli such as TPA, VEGF, or bFGF were omitted. These modifications resulted in a system that is entirely self driven, characterized by rapid growth of sprouts, which form lumens and are enveloped by mural cells (unpublished data). The tips of the sprouts were composed of highly migratory cells with numerous filopodia- and lamellipodia-like processes (Fig. 3 a and see Fig. 6 i). Time-lapse recordings revealed that protrusion and retraction of lamellipodia from these tip cells was a highly dynamic process with single endothelial cells being retained at the tip of the sprout (Fig. 3 a). Endothelial proliferation was conspicuous in stalk cells (Fig. 3 c); however, we did not observe tip cell mitosis using Ki-67 or phospho-histone staining. Thus, with respect to the functional polarization in the sprout, angiogenesis in the aortic ring assay mimics retinal angiogenesis.
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Previous studies have shown that astrocytes express VEGF-A in CNS angiogenesis during developmental and pathological processes (Pierce et al., 1995; Stone et al., 1995; Provis et al., 1997). To map VEGF-A expression to specific cell types, we performed VEGF-A in situ hybridization in combination with isolectin/GFAP double staining. VEGF-A mRNA expression only occurred in GFAP-positive retinal astrocytes (Fig. 5, ac), with the strongest signals located in astrocytes at the leading edge and immediately ahead of the plexus. VEGF-A mRNA was also detected in astrocytes further back in the plexus surrounding veins and capillaries but not surrounding arteries (unpublished data).
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The intensity of VEGFR2 immunostaining on the filopodia suggested that this receptor is implicated in VEGF-mediated filopodia protrusion. To test this hypothesis, neutralizing VEGFR2-specific antibodies were injected into the eyes of P5 mice, and the retinas were removed for examination 6 h later. The tip cell filopodia were retracted, and plexus spreading was inhibited (Fig. 6, n and o), suggesting that VEGFR2-mediated signaling is necessary for tip cell filopodial extension. A neutralizing VEGFR1 antibody had no effect on tip cell morphology or their filopodia (Fig. 6, l and m). We conclude that the extension and maintenance of tip cell filopodia depends on VEGF-A signaling via VEGFR2.
Spatially restricted VEGF guides tip cell filopodia
We next asked if VEGF is directly involved in the guidance of filopodia along the astrocyte scaffold. Considering their length and display of VEGFR2 protein, the tip cell filopodia may be capable of sensing VEGF-A at considerable distance from the cell soma. However, it is also possible that VEGF-A stimulates random protrusion of filopodia and that other factors (e.g., extracellular matrix) subsequently guide the filopodia. Direct guidance by VEGF-A would require the existence of precisely shaped extracellular gradients or deposits of VEGF-A protein, which could be sensed by the filopodia. We showed recently that the heparin-binding isoforms VEGF164 and 188 are required for the establishment of steep extracellular VEGF gradients in the mouse embryonic hindbrain (Ruhrberg et al., 2002). Therefore, we asked if heparin-binding VEGF-A isoforms had a similar role in the retina. RT-PCR indicated that VEGF164 is the dominant isoform expressed in the developing retina followed by 120, 144, and 188 (Fig. 7 a). Immunolabeling showed that extracellular VEGF-A is distributed mainly along the astrocyte tracks in developing wild-type retinas (Fig. 7 b). In contrast, mouse mutants expressing only VEGF120 (120/120 mice) lacked a distinctive astrocytic association of extracellular VEGF; instead VEGF120 is distributed more diffusely in the retina (Fig. 7 c). This finding is consistent with the shallow gradient of VEGF protein around the hindbrain midline demonstrated previously in 120/120 mice (Ruhrberg et al., 2002).
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In summary, aberrantly oriented filopodia were seen in all situations in which disruption of a normal extracellular VEGF gradient occurred. However, aberrant filopodia orientation was not associated with the presence or absence of specific VEGF isoforms. Thus, a properly shaped extracellular pattern of VEGF-A distribution, rather than specific VEGF-A isoforms or concentrations, is necessary for the correct guidance of tip cell filopodia.
Tip cell migration depends on the distribution, whereas stalk cell proliferation depends on the concentration of VEGFR2 agonistic activity
In addition to the sensor role, filopodia are known to exert a motor function. Abnormal filopodial guidance would then correlate with altered or decreased tip cell migration. As a measure of directed tip cell migration, we assessed the peripheral spreading of the inner retinal plexus in the various situations of altered VEGF distribution. We observed slower spreading in 120/120 and A-crystallinVEGF mice and in response to direct intraocular injections of VEGF-A (unpublished data; see also Fig. 9, j and k).
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Although ectopic VEGFR2 agonists inhibited peripheral spreading, they increased proliferation widely in the retinal vascular plexus. This was observed in A-crystallinVEGF transgenics (Fig. 7 f) and after injection of VEGF-A (Fig. 7 g; unpublished data) or VEGF-E (Fig. 9 d), where the number of endothelial cells per vessel length, the size of the vessels, and the plexus density were increased. Together, these observations demonstrate that in the developing retina tip cell migration and stalk cell proliferation are independently controlled phenomena that depend on VEGF-A stimulation of VEGFR2. However, whereas tip cell migration depends on the extracellular VEGF-A distribution pattern (this study), stalk cell proliferation appears to depend on the actual VEGF-A concentration (this study; unpublished data).
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Discussion |
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The tip cell filopodia
Filopodia fulfill at least three different functions in a variety of cell types: intercellular communication, cell migration, and cell adhesion (Wood and Martin, 2002). Specialized filopodia may solely be devoted to cellcell communication, such as in the Drosophila wing imaginal disc where long, thin filopodial extensions (cytonemes) provide sites of contact and signaling between the disc anterior/posterior center and outlying cells (Ramirez-Weber and Kornberg, 2000). Neuronal growth cones use filopodia to probe their environment for guidance cues but also to migrate (Kater and Rehder, 1995; Tessier-Lavigne and Goodman, 1996). Filopodia and lamellipodia are regularly seen at the leading edge of migrating cells in culture, such as fibroblasts and keratinocytes, from which they reach out and adhere to the substrate, allowing the cell to pull itself forward.
In endothelial cells, filopodia may serve several of these functions. Our data indicate that in addition to tip cell filopodia sensing the VEGF gradient generated by retinal astrocytes, it is also probable that tip cells use the filopodia to attach to the astrocytes or astrocyte-derived matrix and to migrate. It is tempting to speculate that the latter may involve transient fibronectin deposition on astrocytes, which we have observed together with fibronectin-type integrin receptors localized at the tips of the endothelial filopodia (unpublished data)
The pleiotrophic actions of VEGF-A
Genetic ablation experiments of VEGF-A and VEGFR2 in mice (Shalaby et al., 1995; Carmeliet et al., 1996; Ferrara et al., 1996) revealed essential functions in the specification, differentiation, and assembly of angioblasts into primary vascular plexuses. VEGF also induces angiogenic sprouting (for review see Ferrara, 2000), vascular permeability (Senger et al., 1983), and recently proposed, the patterning of arteries (Lawson et al., 2002; Mukouyama et al., 2002; Stalmans et al., 2002). Thus, VEGF regulates several different processes critical for blood vessel formation and function; however, it is not clear how different cellular effects, like proliferation, migration, and survival, translate into higher order morphogenetic phenomena, such as vascular assembly and sprouting.
Our present results confirm that VEGF-A independently controls endothelial migration at the tip and proliferation in the stalk of the angiogenic sprout. Remarkably, these processes are functionally independent in that they affect distinct subpopulations of endothelial cells, and separable, since stalk cell proliferation can proceed in the absence of tip cell migration (this study), and vice versa (unpublished data). This is consistent with earlier studies, demonstrating separate migratory and proliferative activities during the process of angiogenesis (Ausprunk and Folkman, 1977; Sholley et al., 1984). As both functions are mediated by VEGFR2, the signals must be interpreted differently by the two endothelial subtypes. Moreover, the pattern of extracellular distribution of VEGF-A is interpreted differently; an uneven distribution in the form of steep soluble gradients or focal matrix depositions is a prerequisite for directional tip cell migration and filopodial extension. Proliferation apparently reflects the local concentration of VEGFR2 agonist.
Can the retinal angiogenic guidance paradigm be generalized?
Clearly, the tip-stalk paradigm is not restricted to the retina; tip cells with the described characteristics are found throughout the developing CNS (Ruhrberg et al., 2002; unpublished data) during angiogenesis in the aortic ring assay (this study) and in certain tumor models (unpublished data). The abnormal vessel profiles resulting from the manipulations of retinal VEGF gradients and levels are reminiscent of tumor angiogenesis and diabetic microangiopathy. This implicates abnormalities in the spatial regulation of VEGF/VEGFR signaling with the abnormal vessel phenotypes seen in these pathologies. We expect that our suggested model for VEGF-Adriven developmental angiogenesis will provide a helpful conceptual framework for the design and interpretation of future studies of pathological angiogenesis.
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Materials and methods |
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Whole mount immunohistochemistry and in situ hybridization
Eyes were fixed in 4% PFA in PBS at 4°C overnight and washed in PBS. Retinas were dissected, permeabilized in PBS, 1% BSA, and 0.5% Triton X-100 at 4°C overnight, rinsed in PBS, washed twice in PBlec (PBS, pH 6.8, 1% Triton-X100, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM MnCl2), and incubated in biotinylated isolectin B4 (Bandeiraea simplicifolia; L-2140; Sigma-Aldrich) 20 µg/ml in PBlec at 4°C overnight. After five washes in PBS, samples were incubated with streptavidin conjugates (Alexa 488, 568, or 633; Molecular Probes) diluted 1:100 in PBS, 0.5% BSA, and 0.25% Triton X-100 at 4°C for 6 h. TO-PRO 3 (1:1,000; Molecular Probes) served for nuclear staining. After washing and a brief postfixation in PFA, the retinas were either flat mounted using Mowiol/DABCO (Sigma-Aldrich) or processed for multiple labeling. The following antibodies were used: GFAP (1:75; Z 0334; Dako), VEGFR2 (1:50; 555307; BD PharMingen), VEGFR2 (1:50; AF644; R&D Systems), F4/80 (1:100; MCAP497; Serotec), fibronectin (1:200; A 0245; Dako), VE-cadherin (1:1, culture supernatant provided by Dietmar Vestweber, University of Muenster, Muenster, Germany), and Ki67 (1:200; NCL-Ki67p; Novo Castra). Alexa-488, 568, or 633 conjugated secondary antibodies (Molecular Probes). Rhodamine-phalloidin served for actin staining (1:40; Molecular Probes). VEGFR2 signal was amplified using the TSATM Fluorescein System (NEL701; NEN) according to instructions. Flat mounted retinas were analyzed by fluorescence microscopy using a Nikon E1000 microscope equipped with a digital camera (Nikon Coolpix 990) and by confocal laser scanning microscopy using a Leica LCS NT. Images were processed using Adobe Photoshop®.
For visualization of vascular lumina, FITC-conjugated Dextran (FD-2000S; Sigma-Aldrich) was warmed to 37°C and perfused through the heart of deeply anaesthetized mice (Avertin, intraperitoneally 10 µl/g body weight).
Mouse VEGF-A, PDGF-B, VEGFR1, and VEGFR2 cDNA fragments were used for whole mount in situ hybridization as described (Fruttiger, 2002). For double labeling, immunolabeling was performed after a 10-min postfixation in 4% PFA.
BrdU labeling was achieved by a 2-h BrdU pulse before fixation (100 µg Brd U/g body weight, intraperitoneally). For double labeling, isolectin labeling was followed by a 30 min 4% PFA fixation, three washes in PBS, a 1-h incubation in 6 M HCl and 0.1% Triton X-100, six washes in PBT, blocking, and anti-BrdU antibody (1:50, 347580; BD PharMingen) incubation.
Rat aortic ring assay
Serum-free aortic ring cultures were established according to the methodology of Bonanno et al. (2000) with modifications (unpublished data). Time-lapse microscopy was performed under low light fluorescence illumination using a Nikon Diaphot 200 inverted microscope and a Hamamatsu C474295 Orca1 CCD camera (Hamamatsu Photonic Systems). Aortic rings were maintained under normal culture conditions throughout the filming period. For vital immunostaining, cultures were incubated at 37°C for 30 min with a phycoerythrin-conjugated PECAM-1 antibody (clone TLD-1A2; BD PharMingen) diluted 1:100 in growth medium. After rinsing with growth medium a minimum of five times, the cultures were set up for filming, and images were captured every 10 or 15 min. Movies were created and analyzed using Kinetic AQM 2000 software (Kinetic Imaging).
For immunostaining, cultures were fixed in ice cold methanol for 10 min, rehydrated in a graded series of methanol/PBS. After a 10-min block in normal goat serum, PECAM-1 (phycoerythrin or biotin conjugate of the TLD-1A2 clone; 1:100 in PBS) and rabbitantiphospho-histone-H3 (1:200; Upstate Biotechnology) antibodies were added for 1 h at RT. After washing, labeling was detected using Alexa-488 streptavidin or goat antirabbit IgG (Molecular Probes). After rinsing with PBS, the areas of sprouting growth were cut out and mounted in SlowFade AntiFade (Molecular Probes). Imaging was performed on a Carl Zeiss MicroImaging Corp. LSM 510 confocal microscope, and quantification of sprout and lamellipodia lengths was performed using IPLab Spectrum software (Signal Analytics Corporation).
Intraocular injections
Pups were deeply anaesthetized by isofluran inhalation. Injections were performed using 10 µl gastight Hamilton syringes equipped with 33 gauge needles attached to a micromanipulator. Approximately 0.5 µl (1 µg/µl sterile filtered solution) was injected (variation due to reflux; delivery was checked by antibody staining). The following substances were used: Flt-1/Fc chimera (471-F1100; R&D Systems), neutralizing VEGFR1 antibody (AF471; R&D Systems), neutralizing VEGFR2 (AF644; R&D Systems), recombinant mouse VEGF-A 164 (493-MV/CF; R&D Systems), recombinant mouse PlGF-2 (465-PL/CF; R&D Systems), VEGF-E NZ2, VEGF-E NZ7, and VEGF-C156S (see Protein production).
Binding to soluble VEGFRIgG fusion proteins
100 ng of purified protein was incubated for 8 h at 4°C under gentle agitation with equimolar amounts of VEGFRIgG fusion proteins (Achen et al., 1998) in PBS supplemented with 0.5% BSA and 0.05% Tween-20 and 1 µg/ml heparin. Receptor-bound growth factors were precipitated with protein A sepharose, subjected to reducing SDS-PAGE, and after blotting visualized using pentahistidine antibody (QIAGEN) and the ECL detection system (Amersham Biosciences).
Protein production
Recombinant proteins were produced using the Bac-to-Bac system (Invitrogen). Nucleotides 658996 (VEGF-C; sequence data available from GenBank/EMBL/DDBJ under accession no. X94216), 235801 (VEGF; sequence data available from GenBank/EMBL/DDBJ under accession no. M27281), 329670 (VEGF-ENZ2; sequence data available from GenBank/EMBL/DDBJ under accession no. S67520), 387755 (VEGF-ENZ7; sequence data available from GenBank/EMBL/DDBJ under accession no. S67522), and 1121866 (human serum albumin; sequence data available from GenBank/EMBL/DDBJ under accession no. V00494) were cloned in-frame into a modified transfer vector (pFASTBAC1; Invitrogen) in between sequences coding for a melittin signal peptide and a hexahistidine tag. Human serum albumin was additionally hexahistidine tagged at the NH2 terminus of the mature protein in order to deploy the same stringent washing conditions in the purification as for the dimeric VEGFs. The VEGFR-3specific mutation in VEGF-C (C156S) has been described previously (Joukov et al., 1998). Conditioned serum-free medium (Sf900II; Invitrogen) of High Five cells was harvested 72 h postinfection and dialyzed against 30 mM sodium phosphate, 0.4 M sodium chloride, pH 6. The pH was adjusted to 8.0, and Ni2+NTA Superflow resin (QIAGEN) was added. Samples were agitated over night at +4°C, and the resin was then collected and applied to chromatography columns. The columns were washed with 30 mM sodium phosphate, 400 mM sodium chloride, 0.6 M glycerol, 20 mM imidazole at pH 8.0, and bound proteins were eluted with an imidazole step gradient. The eluate was dialyzed against PBS or 0.1% TFA and sterilized using Millex-GV filters (Millipore). The proteins were checked on silver-stained reducing SDS-PAGE gels and quantitated using the BCA protein assay (Pierce Chemical Co.).
Online supplemental material
Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200302047/DC1, illustrates the time-lapse analysis of tip cell migration and lamellipodia extension after VEGF sequestration in the aortic ring assay model. Quantitation of migration and lamellipodia length is presented.
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Acknowledgments |
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The study was supported by the Novo Nordisk Foundation, the Swedish Cancer Foundation, and the IngaBritt and Arne Lundberg Foundation to C. Betsholtz. H. Gerhardt is supported by a European Molecular Biology Organization postdoctoral fellowship and by the Swedish Cancer Foundation.
Submitted: 7 February 2003
Revised: 1 May 2003
Accepted: 1 May 2003
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