We focused our studies of developmental angiogenesis on the early postnatal mouse retina, which develops a stereotypical vascular pattern in a well-defined sequence of events (). Simultaneous vascular sprouting at the periphery and remodeling at the center (observable, for example, at postnatal day [P]5), allows the study of different aspects of vessel formation, maturation, and specialization in a single preparation. Retinas are ideal structures to visualize using whole-mount immunostaining and in-situ hybridization techniques, coupled with high resolution three-dimensional imaging by confocal laser scanning microscopy. We studied retinas from various mice between birth (P0) and P14. During this time, spreading of the inner vascular plexus proceeds from the optic disc to the peripheral margin. From approximately P6, vascular branches also extend from the inner plexus into the retina to form the outer plexuses (, P8, arrows).
Figure 1. Schematic presentation of retina development as a model system for investigation of angiogenic sprouting in the CNS. Corresponding top view micrographs of whole mount isolectin- labeled specimen are shown to the right. The top view displays the primary (more ...)
Characterization of the endothelial tip cell
High resolution imaging of isolectin B4–stained retinas revealed that the endothelial cells at the tips of vascular sprouts extended long filopodia (, a–d and f). In the retina, this was most evident at the edge of the expanding inner vascular plexus (, a and b), at sites of sprouting into and within the deeper retinal layers ( c), and at prospective fusion sites in the central, remodeling zone ( d, arrows). Endothelial filopodia were uniform in thickness (~100 nm) but of variable length, with the longest extending >100 μm. Staining of nuclei in combination with isolectin B4, vascular endothelial (VE) cadherin, and fibronectin (, a and e) revealed that the sprouting tip consisted of a single, highly polarized endothelial cell, hereafter referred to as the tip cell. The endothelial identity of this cell was further confirmed by staining for platelet–endothelial cell adhesion molecule (PECAM)-1, endomucin (unpublished data), and VEGF receptor (VEGFR)2 (see , d–f). Therefore, we consider the tip cell the leader in the phalanx of endothelial cells constituting a vascular sprout. Accordingly, trailing cells are referred to as stalk cells. Double staining for endothelial markers and nuclei revealed that the extension of long filopodia is largely restricted to the tip cells. Staining of the actin cytoskeleton by phalloidin conjugates highlighted the tip cells and in particular their leading edge and filopodia ( f). Tip cells were also distinguished from stalk cells by their strong expression of PDGF-B mRNA ( g) and VEGFR2 mRNA and protein (see , d–f), implying that tip cells have a gene expression profile that is distinct from the stalk cells. PDGF-B has been shown previously to play an essential role in the recruitment of pericytes to new vessels (Lindahl et al., 1997
). The VEGFR2 expression in tip cells is further discussed below. Vascular perfusion with labeled dextran demonstrated that the vascular lumen extends up to but not into the tip cells ( h). Together, these characteristics suggest that the tip cells are distinct and functionally specialized microvascular endothelial cells. Their morphology and localization in the sprout is schematically illustrated in i. Tip cells were not unique to the retina but were present in other parts of the developing mouse CNS harboring active angiogenesis (unpublished data).
Figure 2. Characterization of the endothelial tip cell. Isolectin staining is shown in green and nuclei staining is shown in blue. e, endothelium; m, macrophages/microglia. (a) High magnification confocal micrograph showing typical filopodia extension at (more ...)
Figure 5. Illustration of tip cell guidance toward VEGF sources and of VEGFR expression on endothelial filopodia. (a–c) Confocal laser scanning micrographs of VEGF-A in situ hybridization (black signal) combined with double labeling for isolectin and GFAP. (more ...)
To determine whether vascular sprouting in the CNS involved expansion of the endothelial population from the tip or the stalk, we performed double labeling of BrdU or the proliferation marker Ki-67, and isolectin B4. We did not observe proliferating tip cells in either 10 P5–P7 retinas labeled with Ki-67 or an additional 10 labeled with BrdU (, j–l). In contrast, proliferation was abundant in stalk cells and in the immature capillary plexus but occurred also in veins and to a lesser extent in arteries ( l; unpublished data). This suggests that tip cells are nonproliferative; proliferation in the spreading plexus occurs in the sprout stalks and further back in the remodeling plexus.
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 ( a and see 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 ( a). Endothelial proliferation was conspicuous in stalk cells ( 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.
Figure 3. Tip cell migration and stalk cell proliferation in the aortic ring sprouting model. (a) Selected sequence from a time-lapse movie focusing on a single sprout prelabeled for PECAM-1 (red). Note lamellipodia protrusions and continued migration of the leading (more ...)
Figure 6. Illustration of filopodia induction in hyaloid vessels of VEGF164tg. (a) Wild-type littermate showing normal smooth surface of the hyaloid vessels (arrows) lying on the inner surface of the retina. Filopodia are only present in the intraretinal vascular (more ...)
Tip cell filopodia extend on VEGF-producing astrocytes
The inner retinal vascular plexus develops in close association with a preexisting layer of astrocytes (Stone and Dreher, 1987
; Fruttiger, 2002
). We confirmed that the retinal vascular plexus initially forms super imposed on the astrocyte network that remodels after contact with the vasculature ( a). These two plexuses subsequently dissociate in the remodeling zone, indicating a transient importance of their close interaction in the peripheral sprouting region. All tip cells were closely attached to astrocytes and stretched most of their filopodia along the astrocyte cell bodies and processes ( b, filled arrowheads). Filopodia without apparent astrocyte contact were consistently shorter and undulating ( b, open arrowheads).
Figure 4. Astrocytes guide endothelial tip cell filopodia. (a) Over view micrograph, displaying overlap of vascular and astrocytic network. Note that the leading edge of the vascular plexus (arrows) is clearly visible in the astrocytic pattern (arrowhead). (b) (more ...)
These observations suggest that an astrocyte scaffold guides the extension of tip cell filopodia. Previous studies have implied that astrocytes are dominant in retinal vessel pattern formation (Fruttiger et al., 1996
). To analyze if changes in the architecture of the astrocyte plexus were dominant also with regard to the guidance of tip cell filopodia, we studied transgenic mice in which the growth factor PDGF-A was expressed under the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter (Fruttiger et al., 2000
). Since astrocytes strongly express the PDGF-A receptor (PDGFRα) (Fruttiger, 2002
), autocrine mitogenic stimulation results in super-numerous astrocytes intertwining into a dense network of radially oriented bundles ( h). The endothelial tip cells and their filopodia oriented along these abnormal astrocyte bundles (), leading to the formation of a super imposed multilayered vascular network ( f). PDGF-A knockout mice developed a sparser network of astrocytes and vessels, however, with retained association between tip cell filopodia and the astrocyte network (, i–k). Together, the effects of astrocyte hyper- or hypoplasia on retinal vascular patterning suggest that astrocytes provide the principal cues for guidance of endothelial tip cells and their filopodia.
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 (, a–c), 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).
VEGF-A stimulates tip cell filopodia
Since tip cell filopodia extended along VEGF-A mRNA-positive astrocytes, we asked whether ectopic VEGF-A could induce filopodial extension from endothelial cells. Support for this idea came from examination of the hyaloid vasculature in transgenic mice overexpressing VEGF from the lens-specific αA-crystallin promoter. (The generation and basic characterization of these mice will be reported elsewhere [unpublished data].) Hyaloid arteries extend from the optic disc and ramify around the lens supporting its early development, and ultimately these regress postnatally. The hyaloid arteries are normally straight vessels with smooth abluminal surfaces and few branch and fusion points; however, those in αA-crystallin–VEGF164 transgenics were densely covered with abluminal filopodia. These hyaloid vessels were also aberrantly fused into chaotic vascular networks (, a–c).These observations indicate that VEGF overexpression is sufficient to induce continuing filopodial extension, vascular sprouting, and hyperfusion in vessels in which these processes normally have ceased. However, these studies do not discriminate between the possibilities that filopodial extension is directly and dynamically regulated by VEGF or whether it is inherent to a tip cell phenotype induced by VEGF. To address these alternatives, we acutely deprived the growing retinal vasculature of VEGF-A by injecting a soluble VEGFR1 extracellular domain–Fc fusion protein (soluble Flt) into the eyes of P5 mice. Soluble Flt has a high affinity for VEGF-A and acts as a potent extracellular VEGF-A trap. 6 h after injection, most tip cell filopodia were completely retracted in the sprouting region (, d–g). To assess the dynamics of VEGF-A–regulated tip cell process extensions and migration, we employed the aortic ring assay. Filopodial retraction was widespread after addition of soluble Flt but not after treatment with other Fc fusion proteins (, h–k). Time-lapse videomicroscopy also showed that the protrusive activity of tip cell lamellipodia was impaired in conjunction with a block in tip cell migration and sprout elongation (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200302047/DC1
VEGF stimulates tip cell filopodia via VEGFR2
VEGF-A binds to and signals through two receptors, VEGFR1 (Flt-1) and -2 (Flk-1) (Ferrara, 1999
), which are coexpressed in angiogenic endothelium. In the retina, VEGFR2 in situ hybridization highlighted tip cells, but there was also significant signal in the stalk ( d). However, VEGFR1 mRNA expression was similar in both tip and stalk cells (Fruttiger, 2002
). VEGFR2 protein staining was prominent on tip cell filopodia ().
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 (), 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 (). 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 ( a). Immunolabeling showed that extracellular VEGF-A is distributed mainly along the astrocyte tracks in developing wild-type retinas ( 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 ( 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
Figure 7. VEGF gradients shaped by heparin-binding isoforms are necessary for directed tip cell filopodia extension. (a) RT-PCR analysis detects four VEGF isoforms in the retina at P5. The predominant form is VEGF 164 that binds to heparan sulfate proteoglycan. (more ...)
Normally, the tip cell filopodia extend directionally in a narrow plane defined by the astrocytic network (). Such filopodia also exist in VEGF120/120 mice, but they were fewer in number and shorter than in the wild-type retinas (). In addition, VEGF120/120 mutant tip cells extended filopodia also in several directions, i.e., upward (toward the inner limiting membrane (ILM), and backward (peripheral to central) (, a and b). Excessive filopodia extending from stalk cells were also seen. Filopodia that were not oriented centroperipherally and parallel to the astrocytic network were generally undulating and lacked clear spatial orientation ( b). In conclusion, the wide extracellular distribution of VEGF-A in 120/120 retinas correlated with loss of tip cell filopodial polarity.
Figure 8. Disturbance of VEGF gradients leads to misguidance of tip cell filopodia. Representative illustrations of the tip cell in wild-type (a), VEGF120/120 (b), and VEGF120tg (c) retinas. Left hand panels (a1–c1) show confocal images focused around the (more ...)
In addition to a change in distribution of extracellular VEGF-A, the remaining VEGF-A protein in VEGF120/120 mice lacks VEGF164 and 188–specific COOH-terminal sequences. This may affect signaling in ways that are critical for guidance. For example, VEGF164 (but not 120) binds to the axonal guidance receptor neuropilin-1, which enhances signaling via VEGFR2 in endothelial cells (Soker et al., 1998
). In an attempt to distinguish between the possibilities of isoform-specific signaling and extracellular VEGF gradients as alternative mechanisms of filopodial guidance, we compared the VEGF120/120 retinas with retinas from transgenic mice that overexpress specific VEGF isoforms and from mice injected intraocularly with VEGF164. When overexpressed from the lens-specific αA-crystallin promoter, each of the VEGF120, 164, or 188 isoforms led to abnormal filopodial guidance, including ectopic filopodia and a shortening of the remaining astrocyte-associated filopodia, similar to the situation in VEGF120/120 mice (; unpublished data). Direct injection of VEGF164 had a similar effect (). The most severe misguidance, which also resulted in entire tip cells extending across the ILM and into the vitreous, was seen in αA-crystallin–VEGF120 mice ( c), probably reflecting chronic exposure to high concentrations of VEGF (unpublished data).
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 αA-crystallin–VEGF mice and in response to direct intraocular injections of VEGF-A (unpublished data; see also ).
Figure 9. Receptor specificity. (a) Binding of VEGF-E to VEGFR–IgG fusion proteins. VEGFR–IgG fusion proteins were incubated with purified histidine-tagged growth factors and precipitated with protein A sepharose. After reducing SDS-PAGE and Western (more ...)
The observation that a disturbed VEGF-A gradient inhibits both filopodial extension and peripheral endothelial spreading in the retina suggests that directed tip cell migration depends on the ability of the tip cell to distinguish receptor signaling arising at the tips of filopodia from signals arising at the cell soma. To address the VEGFR dependence of this ability, we used a panel of VEGFR-specific ligands. Placenta growth factor (PlGF) and VEGF-B are selective ligands for VEGFR1 (Eriksson and Alitalo, 1999
). No endogenous mammalian VEGF is selective for VEGFR2; however, the orf virus–encoded VEGF-E protein shows selective high affinity binding to VEGFR2 (Ogawa et al., 1998
; Wise et al., 1999
). We produced recombinant VEGF-E protein and characterized its receptor-binding properties. This protein bound and activated the VEGFR2 protein selectively ( a). VEGF-E injection into the eye led to dramatic inhibition of peripheral plexus spreading () and to associated shortening of tip cell filopodia (). It also led to protrusion of short, ectopic filopodia from stalk cells (unpublished data). In contrast, injections of PlGF had no significant effect on spreading or filopodial extension, but it apparently had other biological effects, resulting in massive influx of macrophages (). A third VEGFR, VEGFR3, is mainly expressed in lymphatic endothelium in late embryogenesis and postnatal life but also plays a role in early blood vessel development (Dumont et al., 1998
). After intraocular injections of the VEGFR3-selective ligand VEGF-C156S (Joukov et al., 1998
), there was a lack of detectable effects on tip cell filopodial extensions in the developing retina (). Based on the selective and specific retinal responses to VEGF-E, we conclude that retinal tip cell migration depends on an intact gradient of VEGFR2 agonistic activity.
Although ectopic VEGFR2 agonists inhibited peripheral spreading, they increased proliferation widely in the retinal vascular plexus. This was observed in αA-crystallin–VEGF transgenics ( f) and after injection of VEGF-A ( g; unpublished data) or VEGF-E ( 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).