EPH Receptors and Ephrins 39

artery-to-vein direction of sprouting angiogenesis has been observed in the avian yolk sac43 (Sec. 4.2).

2.4. Crosstalk with other angiogenic pathways

Activation of EphA2 by ephrin-A1 has been shown to mediate the angiogenic effects of tumor necrosis factor α (TNFα) both in vitro and in vivo (Fig. 3A). TNFα and other pro-inflammatory cytokines upregulate ephrin-A1 expression in endothelial cells, which in turn promotes

angiopoietin-2 EphA2

Fig. 3. Various factors that regulate Eph receptor and ephrin expression in endothelial cells. Thin black arrows and bars indicate upor down-regulation of expression levels, respectively. Thick gray arrows indicate increased angiogenic responses. Activated (tyrosine phosphorylated) EphB receptors mediate angiogenic responses through either attractive or repulsive effects depending on the conditions, the endothelial cell type, and the receptor involved (see text for details). P, tyrosine phosphorylation.

40 E. B. Pasquale

activation of EphA2 (as shown by increased tyrosine phosphorylation of this receptor) and capillary morphogenesis.5,18,44,45 Furthermore, activation of EphA2 is required for corneal neovascularization induced by TNFα.5 TNFα regulates ephrin-A1 expression in endothelial cells through the p38 MAP kinase and JNK,46 and it will be interesting to examine whether these MAP kinases in turn regulate the transcription factor HoxB3 (Sec. 2.1).

Consistent with a role in angiogenesis, ephrin-A1 is downregulated in human microvascular endothelial cells by treatment with the antiangiogenic factor endostatin.47 Ephrin-A1 is also an important mediator of the angiogenic effects of VEGF, which instead upregulates ephrin-A1 expression.19 Studies using EphA2 Fc to block the interaction between endogenous ephrin-A1 and EphA2, or EphA2 antisense oligonucleotides to reduce EphA2 expression, have shown that the ensuing stimulation of EphA forward signaling plays a role in some of the angiogenic activities of VEGF, such as microvascular endothelial cell survival, migration and sprouting in vitro as well as the formation of new blood vessels in vivo in corneal neovascularization assays and Matrigel assays.14,15,19,48 In contrast, endothelial cell proliferation induced by VEGF, and the angiogenic effects of basic fibroblast growth factor (FGF2), seem to be independent of ephrin-A1 and EphA2.5,19

There is also crosstalk between endostatin and VEGF and the EphB/ephrin-B signaling pathways (Fig. 3B). Endostatin downregulates ephrin-B1 and ephrin-B2 as well as EphB4 in human dermal microvascular endothelial cells.47 In contrast, VEGF upregulates ephrin-B2 in cultured endothelial cells49,50 and in vivo in arterial endothelial cells of the embryonic skin,49 in a subset of the blood vessels induced in corneal neovascularization assays,51 and in capillaries induced by VEGF transgenic expression in the mouse heart.52 A pathway responsible for ephrin-B2 expression likely involves Notch and TGFβ signaling.53 Indeed, TGFβx and activin-A can upregulate ephrin-B2 expression in mouse primary embryonic endothelial cells, similar to VEGF.49 Interestingly, loss-of-function studies in zebrafish embryos have shown that Notch signaling not only upregulates arterial markers like ephrin-B2 but also represses venous markers like EphB4.54

EPH Receptors and Ephrins 41

In turn, EphB receptor activation by ephrin-B2 Fc has been shown to attenuate VEGF-induced HUVE cell proliferation and migration.26,37 Other growth factors in addition to VEGF, as well as plating cells on a Matrigel substrate, have been reported to upregulate ephrin-B2 expression in endothelial cells.30,50 The other growth factors that have been shown to upregulate ephrin-B2 include VEGF-C, interleukin-6 and interleukin-8 in HUVE cells and hepatocyte growth factor and FGF2 in human aortic and dermal microvascular endothelial cells. Activation of EphB4 by ephrin-B2 Fc in the aortic endothelial cells in turn inhibits the angiogenic effects of FGF2. This effect involves upregulation of syndecan-1 expression and shedding of the ectodomain of this proteoglycan from the cell surface.55 The overproduced soluble syndecan-1 ectodomain inhibits FGF receptor signaling, likely by sequestering FGF2 away from its receptor. A further twist is that heparitinase, an enzyme that preferentially targets desulphated heparin, converts the soluble syndecan-1 ectodomain from an inhibitor to an activator of FGF2 binding to its receptor. Interestingly, enzymes with activity similar to heparitinase are present in inflamed tissue, where they would be predicted to modify the effects of ephrin-B2 on FGF receptor

signaling (Sec. 6.3).

Phorbol myristate acetate (PMA) also promotes the assembly of renal microvascular endothelial cells into capillary-like tubes, and this effect involves activation of EphB1 and EphB2 by endogenously expressed ephrin-B1.13 Ephrin-B1 levels are not changed by PMA treatment, however, suggesting another form of regulation that may involve ephrin-B1 clustering induced through phosphorylation by protein kinase C (PKC), a serine/threonine kinase that is activated by PMA.

The Tie2 receptor tyrosine kinase has also been shown to phosphorylate tyrosine residues in the cytoplasmic domain of ephrin-B1, at least in vitro, which may also modulate ephrin-B angiogenic activities.25 Ephrin-B2-EphB4 signaling in turn appears to increase the expression of Tie2 and its ligand, angiopoietin-1, because Tie2 and angiopoietin-1 are poorly expressed in ephrin-B2 knockout mice.39 Interestingly, the phenotype of the angiopoietin-1 and Tie2 knockout mice resembles that of ephrin-B2 and EphB4 knockout mice. This raises the intriguing possibility that ephrin-B2-EphB receptor signaling may mediate