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path-finding is controlled by the growth cone, a lamellipodial structure present at the tip of growing axons which senses the gradients of chemoattractants and repellants.2 Recent findings3 revealed that a similar structure is present at the end of new capillaries, which are capped by specialized endothelial cells referred to as tip cells. Similar to growth cones, the tip cells probe their local environment by extending filopodia.
Though the morphological similarities between the vascular and nervous systems have been long noticed, their underlying reasons were only recently accounted for by invoking a mechanism of paracrine mutual guidance — each system secretes chemoattractants that act on its counterpart. For example, blood vessels secrete artemin4 and neurotrophin5 which recruit axons to grow alongside, while nerves secrete several VEGF isoforms6 that guide blood vessels to follow along the same paths.
Numerous secreted factors such as the above are chemoattractants and therefore can be functionally considered as guidance cues. This term has evolved, however, to define a narrower group of molecules which includes both secreted and membrane proteins, some of which convey repellant rather than attractant signals. This review will focus on the members of this group, currently consisting of the semaphorins, neuropilins, plexins, ephrins and the Eph receptors, netrins and UNC5s, and slits and roundabouts (Robo).
The signaling of all the four classes of guidance cues described here is not strictly repulsive but bifunctional: it can be modulated by other factors and conditions so as to result in either repulsion or attraction. Cell repulsion is brought about by several changes in cell morphology, adhesion and migration: the cellular cytoskeleton is altered, lamellipodia are retracted, the number of stress fibers increases, and the cell contracts and reduces the area of its contact with the substrate. The cell retracts from sources of soluble repellants, or dissociates from cells presenting repellants on their plasma membrane. This review will describe, therefore, signaling events relevant to these processes (Fig. 1).
2. Semaphorin Signaling
One of the earliest indications for the presence of neuronal receptors in the vascular system was the discovery that neuropilin-1 (Npn-1),
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Fig. 1. (A) Signaling of secreted sema3 via class A plexin and neuropilin. (B) Signaling of membrane-bound semaphorins via class B plexin.
previously known as a semaphorin receptor in neurons, is expressed by endothelial cells and functions as a specific receptor of VEGF165,7 in tandem with the established tyrosine kinase VEGF receptor VEGFR-2. Neuropilins are expressed as two isoforms and participate with several members of the plexin family as well as other membrane receptors in binding secreted class 3 semaphorins.
Semaphorins consist of more than 20 members in vertebrates (see Chapter 1), that can be grouped in several classes based on sequence homology: class 3 semaphorins are secreted, while semaphorins of classes 4, 5 and 6 are transmembrane proteins and semaphorin 7A is glycosylphosphatidylinositol (GPI)-anchored in the plasma membrane (Fig. 1). The secreted class 3 semaphorins are probably the most extensively studied group. The repellant nature of semaphorin signaling was
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first indicated by the capacity of semaphorin 3A (sema3A) to cause collapse of neuronal growth cones.8 Later studies revealed that this effect is not confined to neurons as both endothelial9 and epithelial10,11 cells contract in response to type 3 semaphorins.
2.1. Neuropilins
The role of Npn-1 in the development of the vascular system was first revealed upon inactivation of the mouse Npn-1 gene — an E12.5 embryonic lethal mutation12 which in addition to severe neural malformations produced a vascular phenotype consisting of defects in brain vasculature and morphological aberrations of the heart and the aorta.13 Since Npn-1 is a dual-ligand receptor, the phenotype could be attributed to defects in either VEGF165 or sema3 signaling (or both). This question was resolved by exploiting the fact that VEGF165 and sema3 bind to separate sites in Npn-1.14 Knock-in of Npn-1 in which the VEGF165 binding site was disabled replicated the Npn-1-null vascular phenotype, while impairment of the sema3 binding site resulted in viable offspring with an apparently normal vascular system.15 However, the vascular effects of the latter mutation could have been abrogated by redundancy between Npn-1 and Npn-2 signaling. Indeed, Npn-2 deletion together with knock-in of Npn-1 that does not bind sema3 produced cardiac outflow tract defects. Strikingly, similar defects were observed in sema3Cnull mice,16 suggesting that sema3C modulates cardiac development via both Npn-1 and Npn-2.
Unlike Npn-1 deletion, Npn-2 null mice are viable and do not exhibit obvious vascular defects. While the relative significance of VEGF versus sema3 binding to Npn-2 for vascular development was not dissected as in the case of Npn-1, a double knock-out of both Npn-1 and Npn- 2 caused embryonic lethality earlier than Npn-1 knock-out alone,17 including a defect in the formation of the vascular plexus of embryonic yolk sac. Further analysis of the Npn-2-null mouse revealed an overall absence of lymphatic small vessels and capillaries.18 The differences between the phenotypes of the Npn-1 and Npn-2-null mice reflect the mostly non-overlapping expression patterns of the two neuropilins. Npn-1 is expressed primarily in arteries and is absent from lymphatic
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vessels, where Npn-2 is the sole isoform.18 Npn-2 is also the dominant isoform in veins.
Despite their high degree of homology, the two neuropilins differ in their affinities to class 3 semaphorins. While sema3A binds preferentially to Npn-1, sema3C and 3F have higher affinity to Npn-2.19 With the exception of sema3E, sema3 signaling requires interaction with a heterodimer consisting of a plexin and a neuropilin molecule, wherein neuropilin provides the binding site.20 The neuropilins play no part, however, in transducing the effects of sema3 binding into the cell.21 The signaling downstream of the sema3 receptor complex is initiated by members of the plexin family, which unlike the neuropilins have a large cytoplasmic domain capable of binding several GTPases and possesses GTPase activating protein (GAP) catalytic activity towards R-Ras.22
2.2. Plexins
Several of the nine-member plexin family heterodimerize with either Npn-1, Npn-2, or both, forming together a receptor complex for secreted class 3 semaphorins.23 Similar to other components of guidance cue signaling pathways, the plexins were initially characterized as morphogens of the nervous system. While most plexins are expressed in endothelial cells, no specific functions in the vascular system were attributed to individual members of the plexin family. The first and currently still the only plexin associated with distinct roles in the vascular system is plexin D1 (plexD1), which has widespread expression in vascular endothelial cells of both the embryo and adult mouse.24,25 Similarly, plexD1 is expressed in the vasculature of the developing zebrafish, and its knock-down by anti-sense morpholinos resulted in defective branching and invasion of the interesegmental vessels into the somites, a region which these vessels normally avoid in the wild type (WT) zebrafish.26 This phenotype was replicated by knock-down of the zebrafish sema3a1 and sema3a2, orthologs of mammalian sema3A, indicating that these semaphorins, which are expressed in the somites, are the likely plexD1 ligands, though the extent of defective patterning was much lower than the one produced by plexD1 knock-down. Thus
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the confinement of the developing vasculature to the interesegmental space is the combined effect of a repellant secreted by the surrounding tissue — the somites, and a receptor of the repellant expressed on the surface of the vascular endothelial cells. PlexD1 loss of function was determined (ibid) to underlie also the excessive sprouting of blood vessels previously observed in the chemically induced out-of-bounds zebrafish mutant.27
The phenotype of the PlexD1 deletion in the mouse resembled plexD1 knock-down in the zebrafish. It produced unregulated growth of interesegmental blood vessels,25 and affected also the heart, which had a denser coronary vessel network and cardiac outflow tract defects. Interestingly, this phenotype resembled that of the sema3C-null mouse,16 as well as those of endothelial-specific deletion of Npn-1 and knock-in of mutant Npn-1 that does not bind semaphorin (Npn-1Sema−/−) in combination with Npn-2 knock-out.15 The co-expression of plexD1, Npn-1 and Npn-2 in the endothelium of the aorta, combined with the expression of sema3C in the outflow tract myocardium suggested that a mechanism analogous to the one in the zebrafish underlies the morphogenesis of the mouse heart. A somewhat different plexD1-dependent mechanism appears to operate in the vasculature, as unlike in the heart, the plexD1 ligand affecting the patterning of the intersomitic blood vessels is sema3E.11 The expression pattern of plexD1 and sema3E in the embryonic trunk are complementary to each other, as plexD1 is expressed in the intersomitic vasculature while sema3E is expressed in the somites. Surprisingly, and contrary to all other class 3 semaphorins, sema3E binding to plexD1 did not require a neuropilin co-receptor, since sema3E bound tissue sections of Npn-2-null and Npn-1Sema−/−- expressing embryos.11 The predominance of plexD1-sema3E signaling in the patterning of the vasculature is supported also by the above observation that the vascular patterning defects of sema3a1 and 3a2 knockdown were minor relative to plexD1 knock-down, and by the fact that such defects were not detected in all genetic backgrounds and were incompletely penetrant.28
The knowledge of the signaling downstream of the plexins is still fragmentary. Several plexin-binding proteins and their concomitant effectors are known, but the pathway leading from plexin to the activation
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of proteins that regulate actin dynamics is not fully mapped. In particular, there is still no data concerning the signaling downstream of plexD1, the plexin that appears to be the most relevant one to the morphogenesis of the cardiovascular system. In vitro experiments have shown that semaphorin treatment inhibits endothelial cell migration, adhesion, and in vitro tube formation.9,28,29 Similar to their effect on neurons, secreted semaphorins induce the collapse of endothelial cells30, a process that requires comprehensive re-arrangement of the actin cytoskeleton. Indeed, one of the major classes of effectors of semaphorin signaling via plexin are Rho-GTPases, proteins known to regulate actin dynamics. The signaling events downstream of the class A and class B plexins, representing receptors of secreted sema3 and of membrane-bound semaphorins, respectively, are probably the best studied ones. Based on these studies, it appears that the signaling pathway activated by the secreted semaphorins differs from the one activated by membrane-bound semaphorins. The differences are reflected primarily in the type of Rho-GTPases associated with each class of plexins.
A theme common to both plexin-A and plexin-B signaling is the antagonistic regulation of Rho-GTPases. Sema3 signaling requires the activities of Rac1 and Cdc42,32,33 two Rho-GTPases that induce growth of lamellipodia or filopodia, respectively. Rac1 activation results from the dissociation of the Rac1 GEF FARP2 from the plexA1 cytoplasmic domain and its subsequent activation.34 Class A plexins bind the Rholike GTPase Rnd1, and the Rho-GTPase RhoD.31,35 Rnd1, which binds also class B plexins,36 is constitutively active and is known to induce stress fiber disassembly and cause cell detachment and rounding.37 Similarly, Rnd1 binding triggers plexB1 GAP activity towards R-Ras, thus precipitating cell collapse.22 Both the binding to and activation of plexA1 by Rnd1 are antagonized by GTP-bound active RhoD, a GTPase supporting the motility of early endosomes along actin filaments.38 Though it is still unknown how RhoD blocks sema3Ainduced cell collapse, its association with membrane traffic suggests that it may promote the removal of the sema3A receptors from the plasma membrane.
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The downstream effectors of the GAP activity of class A and class B plexins, and the resulting deactivation of R-Ras are phosphatidylinositol 3-kinase (PI 3-K) and Akt, both of which are inactivated39,40 upon sema4D binding to plexA1 and plexB1. PI 3-K inhibition inhibits β1 integrin, and, subsequently, cell migration.40 These outcomes are similar to the effects of sema3A binding to plexA/Npn1 receptors.28
Class B plexins bind both Rac1 and RhoA. Rac1 binding is ligandand activation state-dependent, i.e. binding occurs upon sema4D interaction with plexB1,41 and is selective for GTP-bound Rac1.42 Experiments in Drosophila suggest that binding to plexB inactivates Rac,43 possibly by competing with Rac binding to its effector p21activated kinase (PAK). Conversely, binding to plexB upregulates RhoA activity in Drosophila.43 These coupled and opposite effects on Rac versus RhoA activity appear to be required for plexB-dependent morphogenesis of the Drosophila nervous system. While there is no evidence for RhoA binding to plexB in vertebrates, Rac1 does bind plexB, resulting in the inhibition of PAK.44 Since PAK promotes the formation of lamellipodia and the disassembly of stress fibers,45 its inhibition would result in the typical effects of semaphorin: retraction of lamellipodia and cell contraction.
Vertebrate class B plexins are linked in more than one way to RhoGTPase signaling, as their carboxy-termini bind to the Postsynaptic density 95, Disk large, Zona occludens-1 (PDZ) domains of the RhoA guanine exchange factors (GEF) PDZ-RhoGEF (PRG) and leukemia-associated Rho GEF (LARG).46−48 There is no consensus, however, over which plexB-dependent mechanisms regulate the activity of these RhoGEFs. Sema4D-induced neuronal growth cone collapse requires direct binding of PRG to plexB1,46,48 is dependent on PRG activity,48 and is enhanced by Rnd1 binding to plexB.36 Ligandinduced dimerization of PlexB activated RhoA, but failed to do so when the PDZ-binding motif of plexB was deleted.47 Dimerization of PRG and LARG has indeed been shown to stimulate their GTP exchange activity.49 Targeting of both plexB and PRG to the plasma membrane appeared to require interaction between the two proteins, and facilitated the plexB-dependent activation of its associated RhoGEF.48,50
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While semaphorin signaling via class B plexins clearly involves RhoGTPases, there is no evidence to indicate that the plexB-associated RhoA is activated by RhoGEFs bound to the carboxy-terminus of plexB. Moreover, the signaling downstream of the GTPases activated by semaphorin binding to plexins are only partially known. Actin depolymerization by cofillin is probably one of the sema3A effects involved in the collapse of the actin cytoskeleton, as cofillin is activated by LIMkinase dependent phosphorylation upon sema3A treatment.51
Similar to many plasma membrane receptors, plexins undergo tyrosine phosphorylation upon ligand binding. The non-receptor tyrosine kinase Fes is associated with plexA1, and is inhibited by Npn-1.52 The manner in which Npn-1 inhibits Fes is not known. It could conceivably be allosteric in nature, e.g. blockage of the Fes catalytic site by the cytoplasmic domain of Npn-1. Sema3A binding releases the Npn-1 inhibition and activates Fes, which phosphorylates the cytoplasmic domain of plexA1 as well as collapsin response mediator 2 (CRMP2), a tubulinbinding cytoplasmic protein53 and one of the first components of the semaphorin signaling pathway to be identified.54 While Fes activity was required for sema3A-induced cell collapse, the Fes phosphorylation site in plexA1 was not identified, nor was this phosphorylation shown to be required for sema3A signaling. In contrast, a CRMP2 phosphorylation site was identified at serine 522, and its replacement by alanine impaired sema3A signaling.55 Since CRMP2 promotes tubulin assembly,56 it is likely that its phosphorylation on serine 522 antagonizes this activity and contributes to the collapse of the cellular cytoskeleton. While the collapse of the actin cytoskeleton is mediated by Rho-GTPases, it appears that CRMP2 regulates a separate branch of semaphorin signaling whose downstream effector are the microtubules. Class A plexins bind also Src and the closely related kinase Fyn.52 Src and Fyn are activated upon sema3A binding and phosphorylate both plexA and Cdk5, a kinase expressed primarily in neurons but also in endothelial cells,57 which phosphorylates and inhibits PAK.58 Thus both plexA and plexB signaling appear to inactivate PAK, though via different pathways. Sema3A-dependent activation of Cdk5 resulted also in the phosphorylation of tau, which decreases its affinity to microtubules,59 consequently reducing microtubule stability.