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The ephrins and Eph receptors in angiogenesis

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Abstract

Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, vascular development and adult neovascularization. Engagement of Eph receptors by ephrin ligands mediates critical steps of angiogenesis, including juxtacrine cell–cell contacts, cell adhesion to extracellular matrix, and cell migration. Recent evidence from in vitro angiogenesis assays and analysis of mice deficient for one or more members of the Eph family establishes the role of Eph signaling in sprouting angiogenesis and blood vessel remodeling during vascular development. Furthermore, elevated expression of Eph receptors and ephrin ligands is associated with tumors and associated tumor vasculature, suggesting that Eph receptors and their ephrin ligands also play critical roles in tumor angiogenesis and tumor growth. This review will focus on the relevance of Eph receptor signaling in embryonic and adult neovascularization, and possible contributions to tumor growth and metastasis.

Introduction

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a multi-step process involving a diverse array of molecular signals. These include factors that stimulate endothelial cell proliferation, migration, and assembly, as well as recruitment of perivascular cells and extracellular matrix remodeling. Endothelial cell receptor tyrosine kinases (RTK) have been recognized as critical mediators of angiogenesis. These are the vascular endothelial growth factor (VEGF) receptor, Tie, and Eph RTKs [27], [63], [71]. The functions of both VEGF/VEGF receptor and angiopoietins/Tie-2 receptor families in vascular development and angiogenesis are well studied, and reviewed elsewhere [10], [18], [22], [23], [27], [38], [52], [63], [71]. The Eph receptor tyrosine kinase family, however, represents a new class of RTKs, and its role in angiogenesis is just beginning to emerge.

First discovered in a human cDNA library screen for homologous sequences to the viral oncogene vfps [31], Eph receptors are a unique class of RTK. First, unlike other families of RTKs, which bind to soluble ligands, Eph receptors interact with cell surface-bound ephrin ligands. Ephrins attach to the cell membrane either through a glycosylphosphatidyl inositol (GPI) anchor or a transmembrane domain. Second, distinct from other RTK ligands, ephrins do not promote cell proliferation; rather, they mediate cellular repulsion, adhesion, cell attachment to extracellular matrices, and cell migration in various cell types. Finally, these receptor–ligand interactions activate signaling pathways in a bi-directional fashion, through both the Eph receptors and ephrin ligands (also referred to as counter receptors).

Eph receptor tyrosine kinases and their ephrin ligands mediate many important biological processes in both invertebrates and vertebrates [33]. Their functions are best studied in the nervous system, where Eph and ephrin molecules are involved in patterning the developing hindbrain rhombomeres, axon pathfinding, and guiding neural crest cell migration [24]. Recently, a series of publications has demonstrated the essential role of Eph/ephrin in vascular development during embryogenesis and in adult angiogenesis. This review will focus mainly on the contributions of Eph and ephrin signaling to the process of developmental and pathological blood vessel formation.

Section snippets

Structure of Eph RTKs and ephrin ligands

The Eph family of receptor tyrosine kinases is the largest known family of RTKs identified, consisting of at least 14 receptors and 8 ligands [13], [26]. Homologues of Eph receptors and ephrin ligands have been identified in vertebrate and invertebrate species, such as mice, Xenopus laevis, zebrafish and Caenorabhditis elegans [13]. For clarity, the Eph receptors have been divided in two subclasses, classes A and B, depending on the type of interaction with the ephrin ligands. In general, Eph

Bi-directional signaling between Eph receptors and ephrin ligands

Eph/ephrin signaling is complex in several respects. First, within each subclass, receptor–ligand binding is promiscuous. With few exceptions, one ligand is able to bind to multiple receptors; and conversely, one receptor can bind to multiple ligands within the same subclass [26]. Thus, the specificity of ligand–receptor interaction apparently comes from the cell-specific localization of these molecules in vivo. Another unique aspect of the ligand–receptor interaction in the Eph family is that

Regulation of Eph receptor/ligand expression and activities

Regulation of Eph ligand and receptor expression and activity occurs at many levels. Such regulation includes induction of Eph/ephrin transcription, re-distribution of Eph/ephrin molecules within the cell membrane upon cell stimulation, and internalization of receptor–ligand complex. Interestingly, many of the known modulators of Eph/ephrin signaling are known regulators of angiogenesis.

Regulation of cellular proliferation

Unlike many angiogenic or growth factors such as VEGF, EGF and bFGF, ephrins are not known to directly promote cellular proliferation or transformation. However, over-expression of EphA1 in fibroblasts and EphA2 in normal breast epithelial cells promotes colony growth in soft agar and tumor formation in nude mice, indicating the ability of EphA2 receptor to transform normal cells [45], [73]. Further evidence of Eph/ephrin involvement in cell proliferation came from the ability of Eph/ephrin

Function in vascular development during embryogenesis

The assembly of endothelial cells and supporting mesenchyme into mature and functional blood vessels is a multi-step process involving a diverse array of molecular signals. Ligands for receptor tyrosine kinases (RTK) such VEGFs and angiopoietins are well known as critical mediators of blood vessel formation during embryogenesis. Recent studies from in vitro angiogenesis assays and in vivo homozygous null mice now firmly establish an essential role of Eph receptors and their ephrin ligands in

Effects on tumor cells

Elevated expression and activity of Eph receptors have been correlated with the growth of solid tumors. EphA1, the first Eph receptor identified, was found to be expressed in erythropoeitin producing hepatoma cells [31], and in breast, liver, lung, and colon carcinoma [46]. Over-expression of EphA1 in NIH3T3 cells led to the foci formation in soft agar and promoted tumor formation in nude mice [45]. EphA2 has also been found to be upregulated in a number of human tumors including melanoma,

Perspectives

Eph receptors and ephrin ligands mediate cell–cell interaction in many important biological processes. While their functions in the nervous system are well studied, the work on ephrin/Eph functions in the vasculature is only just beginning. Many important questions remain to be addressed. What are the signaling mechanisms downstream of Eph/ephrins that govern interactions between endothelial cells, endothelial and perivascular supporting cells, and endothelial and tumor cells? Do they regulate

Acknowledgements

We thank Drs. Ann Richmond, Lynn Matrisian, Charles Lin, Mark Boothby, and Rebecca Muraoka for critical reading of the manuscript. This work was supported by National Institutes of Heath grant HD36400 and DK47078, American Heart Association grant 97300889N, and an ACS Institutional Research Grant IN-25-38 to J. Chen, Vascular Biology Training Grant T32-HL-07751-06 and American Heart Association Fellowship 0120147B to D. Brantley, and Cancer training grant T-32 CA09592 to N. Cheng. This work was

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