Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

Chapter 11

EPH RECEPTOR TYROSINE KINASES: MODULATORS OF ANGIOGENESIS

Jin Chen,1,2,3 Dana Brantley-Siders,1 and John S. Penn4

1Department of Medicine, Division of Rheumatology, 2Department of Cancer Biology, 3Department of Cell and Developmental Biology, and 4Department of Ophthalmology and Visual Sciences, Vanderbilt University, Vanderbilt University School of Medicine, Nashville, Tennessee

1.INTRODUCTION

Retinal neovascularization is the critical pathological component of a number of blinding conditions such as diabetes mellitus, retinopathy of prematurity, and age-related macular degeneration and is the leading cause of irreversible vision loss in developed countries.1 Although the present treatment, retinal laser photocoagulation, is partially effective, this procedure can destroy postmitotic retinal neurons and permanently affect visual function.2 During the last several years, a number of therapeutic agents have been developed, aiming at pharmacological inhibition of retinal angiogenesis.

Angiogenesis, or the outgrowth of new sprouts from pre-existing vessels, involves a complex cascade of events (Reviewed by 3,4). First, the wall of the intact vessel loosens, reducing endothelial cell-smooth muscle cell

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J.S. Penn (ed.), Retinal and Choroidal Angiogenesis, 203–219.

© Springer Science+Business Media B.V. 2008

interaction, and endothelial cells are activated by angiogenic factors in response to hypoxia, ischemia, or developmental cues. The endothelial cells then invade the surrounding tissue through matrix degradation, proliferate and migrate toward the angiogenic stimulus, and coalesce into tubular structures. Finally, maturation of the new vessel is accomplished through recruitment of perivascular supporting cells and deposition of extracellular matrix. Each of these events is tightly regulated at the molecular level. In general, there is an upregulation of pro-angiogenic factors such as growth factors, integrins, and proteinases, and a downregulation of angiogenic inhibitors.5,6

Among the diverse array of molecules involved in angiogenesis, receptor tyrosine kinases (RTKs) have emerged as critical mediators of neovascularization.3,7 Vascular endothelial cell growth factor A (VEGF-A, hereafter referred to as VEGF), a RTK ligand, plays an essential role in hypoxia-induced proliferative retinopathy. VEGF expression is associated both temporally and spatially with the development of retinopathy in vivo.8-11 Suppression of VEGF activity by monoclonal antibodies, soluble receptor

chimeric proteins, or antisense oligonucleotides inhibits angiogenesis in the retina.12,13 More recently, the angiopoietins and their Tie-2 receptors have

been implicated in proliferative retinopathy.14,15 This review will focus on the youngest family of essential vascular RTKs, the Eph receptors, and their corresponding ligands.

The present review aims at summarizing our current understanding of Eph/ephrin function in vascular remodeling during embryogenesis and neovascularization in adult tissues. Much of the data on post-natal angiogenesis has been obtained from tumor studies. Therefore, this review will provide an overview of Eph molecule function in tumor neovascularization and discuss the potential role of this family of RTKs in retinal angiogenesis.

2.EPH RTKS AND EPHRIN LIGANDS

Eph receptors are unique RTKs that play critical roles in embryonic development and human diseases. First discovered in a human cDNA library screen for homologous sequences to the viral oncogene vfps, Eph receptors

comprise the largest class of RTKs, and they display many unique features. The Eph family consists of at least 15 receptors and 9 ligands (Figure 1).16,17

The Eph receptors have been divided into two subclasses, A and B, according to sequence similarity and affinity to their ligands. In general, Eph class A (EphA) receptors bind to glycosylphosphatidyl inositol (GPI)- anchored ephrin ligands (ephrin-A), while Eph class B receptors (EphB)

(SAM) domain, and a PDZ binding motif (PSD-95 post synaptic density protein, Discs large, Zona occludens tight junction protein).19,20 The juxtamembrane region, kinase domain, and SAM domain contain several tyrosine residues, phosphorylation of which may create docking sites for interactions with signaling proteins containing SH2/SH3 (Src-Homology- 2/3) or PTB domains. The SAM domain has been implicated in mediating protein-protein interactions via the formation of homoand hetero-typic oligomers. The PDZ binding motif binds to PDZ domain-containing proteins, which are thought to serve as scaffolds for the assembly of multiprotein signaling complexes at the membrane.

Of the 9 known ligands, 6 are attached to the cell surface by a GPI linkage (class A) and 3 by transmembrane domains (class B). Analyses of amino acid sequences of ephrin ligands indicate that each ligand consists of a signal peptide at the amino-terminus, followed by a conserved receptorbinding region containing several cysteine residues and a spacer region. At the C-terminus, the A class ligands contain a hydrophobic region comprising the GPI linkage. In contrast, ephrin-B ligands contain a transmembrane domain and a cytoplasmic domain that, like the Eph receptors themselves, contains a PDZ-binding motif and conserved tyrosine residues that may be phosphorylated and serve as docking sites for proteins containing SH2/SH3 or PTB domains. These structural motifs control ligand attachment, receptor and ligand clustering, and regulate the binding of ephrins to specific Eph receptors to elicit distinct biological responses (Figure 1).

Compared to other RTKs, Eph receptor signaling is unique due to the bidirectional signals activated by both receptor and ligand. Therefore, both the cell containing the receptor and the adjacent cell containing the ligand receive signals upon receptor-ligand binding. In the unbound state, the Eph tyrosine kinase domain is distorted by the helical conformation of the juxtamembrane region that renders the kinase domain inactive. Cell-cell contact allows Eph receptors to bind to their ligands and trigger a series of events that lead to receptor activation. The high-affinity binding of Eph-ephrin heterodimers causes them to cluster together on the cell surface.21 This heterodimeric conformation is thought to allow the trans- phosphorylation of the juxtamembrane domain, altering its inhibitory helical structure and allowing the kinase domain to be activated by phosphorylation of the activation loop. The newly phosphorylated tyrosines are then able to interact with various downstream signaling effectors. Eph receptor forward signaling and ephrin ligand reverse signaling will not be reviewed in detail here, but interested readers are referred to the excellent body of recent literature.22,23

3.ROLE OF EPH/EPHRIN IN VASCULAR REMODELING DURING EMBRYONIC DEVELOPMENT

Many Eph RTK family members are expressed in the vasculature during embryogenesis. In mice, Xenopus, and chick, ephrin-B2 is expressed in endothelial cells in the extraembryonic yolk sac primary capillary plexus, in large arteries within the embryo, and in the endocardium of the developing heart.24-28 The principal receptor for ephrin-B2, EphB4, displays a reciprocal

expression pattern in embryonic veins in the yolk sac, larger veins including the arterial cardinal vein and vitelline vein, and also in endocardium.24,26,28,29

These expression patterns provided the first evidence for a molecular distinction between arterial and venous endothelium.

Although the Eph/ephrin system was originally identified in the nervous system, genetic studies using knockout mice have firmly established the role of these molecules in vascular development. Targeted disruption of either ephrin-B2 or EphB4 results in embryonic lethality at E11 or E9.5-10, respectively, due to strikingly similar defects in angiogenic remodeling of both arteries and veins as well as patterning defects in cardiac myocardium.28,29 Although vasculogenesis occurs normally in homozygous null embryos, with the formation of primitive capillary network structures, these networks fail to remodel and branch into more complex vascular networks. Similar angiogenic remodeling defects within intersomitic vessels are produced by overexpression of dominant-negative EphB4 in Xenopus embryos.26 Interestingly, overexpression of both full length ephrin-B ligands and their corresponding cytoplasmic domain deletion mutants recapitulates this phenotype, suggesting that remodeling of intersomitic vessels in the Xenopus trunk occurs through forward signaling rather than reverse.26 Reverse signaling through ephrin-B2, however, can affect angiogenesis in mice, as demonstrated by replacement of the endogenous ephrin-B2 gene with a cytoplasmic deletion mutant. Much like null mutants, ephrin-B2 C/ C “knockin” mutants display defects in remodeling of vessels in the yolk sac and in the embryo, as well as recapitulating heart defects.30 These data suggest that ephrins and Eph receptors have more complex functions in mammals than in lower vertebrates.

Although vasculogenesis appears to occur normally in EphB4-deficient mice,29 a recent report suggests that EphB4 may still modulate differentiation of hemangioblasts in cooperation with other pro-angiogenic factors. Wang et al. reported that EphB4-deficient embryoid bodies display delayed expression of the hemangioblast marker VEGFR-2/Flk-1, as well as defective vascular morphogenesis, in response to VEGF and bFGF in vitro.31 These data suggest that EphB RTKs and ephrin signaling may

modulate vasculogenesis and might explain why EphB4-deficient embryos die sooner than ephrin-B2-null mutants.28,29 Thus, ephrins might regulate

sensitivity to early vascular developmental cues in addition to exerting direct effects on angiogenic remodeling of the embryonic vasculature.

In addition to its expression in arterial endothelium, ephrin-B2 has been detected in mesenchyme surrounding some blood vessels, and it begins to be

expressed in smooth muscle cells and pericytes surrounding vessels as development proceeds.26,29,32,33 Thus, mesenchymal expression of ephrin-B2

may also affect morphogenesis of EphB RTK-expressing endothelium. This hypothesis is supported by experiments in which ephrin-B2 is differentially overexpressed either ubiquitously or in endothelial cells specifically. Defective patterning of intersomitic vessels and defective outgrowth of venous vessels in the head region were observed in transgenic embryos overexpressing ephrin-B2 ubiquitously, but not in endothelial-specific, Tie-2 promoter-driven ephrin-B2 transgenics.34 The embryos ubiquitously overexpressing ephrin-B2 also display neonatal lethality due to aortic aneurysms that result from lack of vascular smooth muscle cell recruitment, and smooth muscle cell outgrowth and migration were impaired in ascending aortic explants relative to wild-type control littermates.34 Again, these defects were not observed in Tie-2p-ephrin-B2 transgenics overexpressing ephrin-B2 in endothelium. Tissue-specific deletion of ephrin-B2 in endothelium and endocardium, however, was sufficient to recapitulate angiogenic remodeling defects observed in conventional knockout animals.35 Since the full complement of vascular defects was produced by deletion of ephrin-B2 in endothelium, although mesenchymal expression remained intact, the authors argued that mesenchymal ephrin-B2 is not sufficient for vessel remodeling. Ephrin-B2 might, however, be necessary for proper remodeling, as demonstrated by several in vitro studies.

Zhang et al. showed that mesenchymal expression of ephrin-B2 enhances differentiation of paraaortic splanchnopleuric mesoderm and endothelial precursor-enriched cell populations within this tissue into endothelium, whereas overexpression of EphB4 was inhibitory.36 Differentiation induced by mesenchymal ephrin-B2 was accompanied by morphogenesis into cordlike tubules and enhanced smooth muscle cell recruitment, demonstrating the importance of mesenchymal ephrin-B2 in vascular morphogenesis and maturation. Inhibition of vascular morphogenesis by mesenchymal EphB4 may be due to the anti-adhesive, repulsive effects of this receptor. Füller et al. recently reported that treatment with soluble ephrin-B2-Fc, a soluble form of the ephrin-B2 ectodomain, inhibited attachment of EphB4-positive endothelial cells, as well as induced detachment of three-dimensional spheroids and delamination of endothelial cells from umbilical vein explants.37 Treatment with EphB4-Fc had the opposite effect, inducing

migration and sprouting of endothelial cells. Similar anti-adhesive, antimigratory effects were reported in primary mouse microvascular endothelial cells in response to ephrin-B2-Fc.38 In addition, EphB4-positive endothelial cells co-cultured with endothelial cells expressing either full-length or a cytoplasmic deletion mutant of ephrin-B2 resulted in segregation of these cells. These data suggest that forward signaling through EphB4 restricts interaction with ephrin-B2 expressing cells, which could assist separation of arterial and venous domains in vascular morphogenesis.37

Ephrin-B2 and EphB4 are not the only Eph family members that regulate embryonic vessel patterning. Ephrin-A1 is expressed in the developing vasculature and promotes angiogenesis in vitro and in vivo.39-44 Though no data are yet available concerning the role of ephrin-A1 in embryonic angiogenesis, this ligand and its principal receptor, EphA2, are known to regulate post-natal angiogenesis as discussed in the next section. Ephrin-B1 is also expressed in the embryonic vasculature, in both arteries and veins, as is EphB3. In addition, EphB2 is expressed in vascular-associated mesenchyme.24 Although targeted disruption of EphB2 or EphB3 alone produced no discernable vascular phenotype, approximately 30% of double mutants die at E11 due to vascular remodeling defects in the head, heart, and intersomitic regions of the embryo, demonstrating that these EphB RTKs also participate in developmental angiogenesis.24 Although ephrin-B1 expression cannot compensate for the loss of ephrin-B2 in null mutants, in vitro studies have demonstrated that this ligand can induce angiogenic responses in cultured endothelial cells,24,45-47 as can reverse signaling through ephrin-B1.48 These data suggest that the ephrin-B1 ligand might also be necessary, though not sufficient, for vascular remodeling during embryogenesis. The vascular phenotypes of Eph/ephrin knockout and transgenic animals are summarized in Table 1.

Impairment in endothelial cell migration and assembly and tumor angiogenesis

4.ROLE OF EPH/EPHRIN IN POST-NATAL ANGIOGENESIS

Eph RTKs and ephrins also regulate post-natal angiogenic remodeling. Expression of ephrin-B2 persists in adult arterial endothelium and vascular smooth muscle cells surrounding arteries, and EphB4 expression persists in adult venous endothelium, suggesting that this ligand-receptor pair may regulate boundary maintenance and/or vascular remodeling in mature tissues.32,33 Indeed, in cultured endothelial cells, soluble ephrin-B2 facilitates adhesion and migration, processes critical for angiogenic remodeling.49

Eph RTKs and ephrins can induce post-natal vascular remodeling. For example, soluble ephrin-A1,40,43 ephrin-B2,50 and the ectodomain of EphB1

[48] induce corneal angiogenesis in adult mice, demonstrating that these mature endothelial cells have the capacity to respond to ephrin and Eph RTK signals. In addition, ephrin-B2 and ephrin-A1 can also induce an angiogenic response from subcutaneous vessels in vivo.50,51 Matrigel plugs harboring soluble ephrin-B2 induced an angiogenic response from subcutaneous host vessels when implanted into mice.50 Similarly, surgical sponges impregnated with soluble ephrin-A1 and implanted in the subcutaneous dorsal flank of wild-type mice induced sprouting of adjacent subcutaneous vessels and infiltration of new vessel sprouts into the sponges. When ephrin-A1- containing sponges were introduced into EphA2-deficient mice, this angiogenic response was greatly diminished, suggesting that efficient vascular remodeling in response to ephrin-A1 requires EphA2 RTK.51 Lung

microvascular endothelial cells isolated from adult mice can also respond to ephrin-A1, which induces assembly and migration in vitro.39,51 These

processes are dependent upon expression of EphA2 RTK, since endothelial cells isolated from EphA2-deficient mice display impaired angiogenic responses to ephrin-A1 and these responses are rescued upon restoration of EphA2 expression.51

The female reproductive system is a major site for physiological angiogenesis. Ephrin-A1 expression has been observed in normal human endometrial epithelial cells.52 Given the pro-angiogenic activity of this ligand, it is possible that ephrin-A1 could contribute to normal endometrial angiogenesis. Ephrin-A1 expression is downregulated in biopsies from endometriosis tissues relative to normal endometrial tissue,53 however, it is unclear how this molecule might regulate pathogenic angiogenesis in this disease. B class Eph RTKs and ephrins may play a more direct role in endometrial disease, as EphB4 and ephrin-B2 overexpression was recently reported in human endometrial hyperplasias and carcinomas.54,55 Since B class ephrins, such as ephrin-B2, are expressed in vascular beds of mature tissues and in tumor xenografts,32 it is possible that EphB4 overexpression could affect neovascularization of endometrial cancer. Further investigation of this hypothesis could facilitate development of new therapies for endometrial cancer, since high microvascular density correlates with advanced disease and a poor patient survival rate.56

Cyclic vascular remodeling also occurs in the ovary during formation of the corpus luteum, a transient structure that provides progesterone to establish and sustain pregnancy. After ovulation, granulosa and theca cells of the developing follicle and steroid-producing cells in the corpus luteum produce pro-angiogenic factors that promote vascularization of the corpus luteum.57 Ephrin-B2 expression has been observed in murine corpus luteum

vessels.32 Moreover, a recent report correlates expression of B class Eph RTKs and ephrins with corpus luteum formation in humans. EphB1, B2, and B4, as well as ephrin-B1 and -B2 mRNA expression was detected in human ovary samples.58 Interestingly, ephrin-B1 expression was localized to theca and granulosa cells during the window of corpus luteum formation and neovascularization. Given the pro-angiogenic functions of this molecule in vitro, it would be interesting to determine if ephrin-B1 regulates angiogenesis in the ovary, particularly since overexpression of ephrin-B1 has also been detected in human ovarian carcinomas.59

5.ROLE OF EPH/EPHRIN

IN TUMOR ANGIOGENESIS

Although it is now clear that Eph receptors and ephrin ligands play a critical role in vascular development during embryogenesis, the function of these molecules in pathological angiogenesis has just begun to be investigated. A survey of expression patterns of Eph molecules in tumor vasculature reveals that the ephrin-A1 and EphA2 ligand-receptor pair is consistently expressed in tumor-associated endothelium in a variety of tumors, including tumor xenografts (MDA-MB-435 human breast cancer and KS1767 human Kaposi’s sarcoma) and human tumor specimens (lung anaplastic adenocarcinoma and squamous carcinoma, gastric cancer, colon carcinoma, and kidney clear cell carcinoma).60 Expression patterns of ephrin-A1 and EphA2 in two murine tumor models that are angiogenesis-dependent, the RIP-Tag islet carcinoma transgenic model and the 4T1 transplantable metastatic mammary carcinoma model, have also been determined.39 EphrinA1 ligand was expressed predominantly in tumor tissue, and EphA2 receptor expression was mainly associated with the tumor vasculature. In addition, a soluble EphA2 receptor inhibited tumor neovascularization in a dorsal vascular window assay. These data suggest a role of ephrin-A/EphA molecules in promoting angiogenesis in tumors.

The first functional evidence that Eph receptors regulate tumor angiogenesis came from studies using soluble EphA-Fc receptors. A soluble EphA2-Fc or EphA3-Fc receptor that blocks endogenous EphA receptor signaling inhibited tumor growth and angiogenesis in murine 4T1 malignant mammary carcinomas.39 In addition, local or systemic delivery of soluble EphA-Fc receptors also inhibited angiogenic islet formation and reduced tumor volume in multi-stage pancreatic carcinomas in RIP-Tag transgenic mice,61 indicating a possible relevance for EphA targeting in clinical cancer therapeutics. Because soluble EphA receptors can interact with multiple ephrin ligands and block multiple EphA receptor signaling pathways, the