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2. Effects on Vascular Cell Behavior and Signaling Pathways

Eph receptor forward signaling and ephrin reverse signaling can dramatically influence the behavior of endothelial cells and vascular smooth muscle cells both in vitro and in angiogenesis assays in vivo. The signaling pathways regulated by Eph receptors and ephrins in vascular cells are beginning to be elucidated through in vitro studies, but the importance of these pathways for physiological angiogenesis in vivo remains to be verified. Ephrin extracellular domains fused to the Fc portion of human IgG1 have been extensively used to activate Eph receptor signaling pathways in angiogenesis assays because they are soluble and dimeric, and can be multimerized by anti-Fc antibodies. Thus, they can be used to induce the Eph receptor dimerization and further clustering that are important for proper signaling.1,13 Similarly, Eph receptor ectodomains fused to Fc have been used to stimulate ephrin reverse signaling. Eph receptor and ephrin Fc fusion proteins, however, can also function as inhibitors because they disrupt endogenous Eph receptorephrin interactions. For example, EphA receptor Fc fusion proteins have been used to inhibit EphA forward signaling, which has established the importance of EphA receptors in postnatal angiogenesis14,15 (Sec. 6).

2.1. Ephrin-A1 and EphA2

Ephrin-A1 and EphA2 are the main ephrin and Eph receptor of the A class that have thus far been implicated in endothelial cell function (Fig. 2A). In a widely used in vitro angiogenesis model, endothelial cells plated onto reconstituted basement membrane proteins (Matrigel) respond by forming capillary tube-like structures. Interestingly, one of the consequences of plating human umbilical vein endothelial (HUVE) cells on Matrigel is the upregulation of ephrin-A1.16 The effect of ephrin-A1 in capillary morphogenesis on Matrigel was confirmed by showing that exogenously added ephrin-A1 Fc promotes the assembly of capillary structures in HUVE cells and mouse pulmonary microvascular endothelial cells, both of which express high levels of the EphA2 receptor.5,17 Furthermore, reducing expression of the transcription factor Homeobox B3 (HoxB3) with antisense oligonucleotides decreases

EPH Receptors and Ephrins 33

ephrin-A1 expression and impairs capillary morphogenesis in dermal microvascular endothelial cells.18 Given that treatment with ephrin-A1 Fc restores capillary-like tube formation in the HoxB3-deficient cells, these data suggest that the HoxB3-dependent expression of ephrin-A1 is important for endothelial capillary morphogenesis driven by extracellular matrix proteins. Not all endothelial cells may respond to ephrin-A1 Fc, however. Human renal microvascular endothelial cells, for example, reportedly do not form capillary-like tubes in response to ephrin-A1 Fc.17 The reason for this lack of responsiveness to ephrin-A1 Fc remains mysterious, since these cells express EphA2 and can form capillary-like tubes when treated with ephrin-B1 Fc (Sec. 2.3).

Consistent with a role as an angiogenic factor, ephrin-A1 Fc also promotes endothelial cell migration. Ephrin-A1 Fc acts as a chemoattractant for bovine adrenal capillary endothelial cells and microvascular endothelial cells in transwell migration assays and promotes the movement of cells into a “wound” devoid of cells in a confluent endothelial cell monolayer.5,14,19,20 Ephrin-A1 Fc also induces endothelial cell sprouting in an in vitro capillary sprouting assay.19 In this assay, microvascular endothelial cells are cultured on collagen-coated beads embedded in fibrin gels and form capillary sprouts that extend out from the beads into the fibrin matrix.

Additional studies have shown that ephrin-A1 Fc also promotes the formation of blood vessels in a variety of in vivo angiogenesis assays. These assays include: (i) corneal neovascularization assays, where hydron pellets impregnated with ephrin-A1 Fc induce the formation of blood vessels when implanted in a micropocket in the normally avascular rodent cornea;5,19 (ii) Matrigel assays, where Matrigel injected under the mouse skin forms plugs that promote the assembly of endothelial cells into blood vessels when supplemented with ephrin-A1 Fc; and (iii) assays in which surgical sponges impregnated with ephrinA1 Fc and implanted in the dorsal flank of mice attract an increased number of host blood vessels compared to control sponges.20

Ephrin-A1 appears to have similar pro-angiogenic effects when it is endogenously expressed in endothelial cells and as an exogenous Fc fusion protein. Hence, the angiogenic effects of ephrin-A1 can be mainly attributed to its stimulation of EphA receptor forward signaling because

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ephrin-A1 Fc lacks the ability to mediate reverse signals. Indeed, mutants of the EphA2 receptor that inhibit EphA receptor forward signaling in a dominant negative manner block the in vitro angiogenic effects of ephrin-A1 Fc, while a constitutively active EphA2 mutant enhances angiogenic responses.20,21 Furthermore, an EphA antagonist such as EphA2 Fc strongly inhibits capillary formation in an in vitro rat aortic ring explant assay and in an in vivo Matrigel assay.15 Since EphA2 Fc can also activate ephrin-A1 reverse signaling (Sec. 2), this experiment corroborates the idea that ephrin-A1 reverse signaling may not promote angiogenesis.

The requirement for EphA2 signaling in endothelial cell migration and vascular assembly has been confirmed by the impaired angiogenic responses to ephrin-A1 Fc in microvascular endothelial cells isolated from EphA2 knockout mice as well as in endothelial cells in which EphA2 expression was downregulated with antisense oligonucleotides.19,20 A signaling pathway involving phosphatidylinositol (PI) 3 kinase and the Rho family GTPase, Rac1, has been implicated in the effects of EphA2 on microvascular endothelial cell migration in vitro.20,22 Although EphA2 is not expressed in the embryonic vasculature, this receptor has been confirmed as a key player in postnatal angiogenesis in vivo because EphA2 knockout mice exhibit a diminished angiogenic response to surgical sponges impregnated with ephrin-A1 Fc.20 Furthermore, microvascular endothelial cells from EphA2 knockout mice fail to elongate and assemble into capillaries in Matrigel plugs implanted into wild-type recipient mice. The EphA2-deficient cells also have impaired survival in the Matrigel plugs, but appear to proliferate normally, which is consistent with the lack of in vitro effects of ephrinA1 Fc on endothelial cell proliferation.19

2.2. Ephrin-A1 and EphA4

Endothelial ephrin-A1 likely coordinates different aspects of angiogenesis by activating EphA receptors not only in endothelial cells, but also in the surrounding vascular smooth muscle cells (Fig. 2A). In cultured smooth muscle cells, ephrin-A1 Fc causes a repulsive response involving increased assembly and contractility of actin stress fibers and