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Blockade of Epha Receptor Tyrosine Kinase Activation Inhibits Vascular Endothelial Cell Growth Factor-Induced Angiogenesis

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Blockade of Epha Receptor Tyrosine Kinase Activation Inhibits Vascular Endothelial Cell Growth Factor-Induced Angiogenesis 2 Vol. 1, 2–11, November 2002 Molecular Cancer Research Blockade of EphA Receptor Tyrosine Kinase Activation Inhibits Vascular Endothelial Cell Growth Factor-Induced Angiogenesis Nikki Cheng,1 Dana M. Brantley,2 Hua Liu,3,4 Qin Lin,2 Miriam Enriquez,4 Nick Gale,5 George Yancopoulos,5 Douglas Pat Cerretti,3 Thomas O. Daniel,3,4 and Jin Chen1,2,6 Departments of 1Cancer Biology, 2Medicine, Division of Rheumatology, 4Division of Nephrology, and 6Cell Biology, Vanderbilt University School of Medicine, Nashville, TN; 3Immunex Corporation, Seattle, WA; and 5Regeneron Inc., Tarrytown, NY Abstract endothelial cell proliferation, migration, and assembly, as well Angiogenesis is a multistep process involving a diverse as recruitment of perivascular cells and extracellular matrix array of molecular signals. Ligands for receptor tyrosine remodeling. Three families of receptor tyrosine kinases (RTKs) kinases (RTKs) have emerged as critical mediators of have emerged as critical mediators of angiogenesis; these are the angiogenesis. Three families of ligands, vascular endo- vascular endothelial growth factor (VEGF), Tie, and Eph RTK thelial cell growth factors (VEGFs), angiopoietins, and families (1, 2). VEGF (VEGF-A/VEGF165) is a potent angio- ephrins, act via RTKs expressed in endothelial cells. genic factor in both embryonic development and in adult disease Recent evidence indicates that VEGF cooperates with states, such as cancer. VEGF and its RTKs, Flt-1/VEGFR1 and angiopoietins to regulate vascular remodeling and Flk-1/KDR/VEGFR2, are required for the development and angiogenesis in both embryogenesis and tumor remodeling of blood vessels during embryogenesis (3–8). neovascularization. However, the relationship between Moreover, VEGF signaling plays a crucial role in pathogenic VEGF and ephrins remains unclear. Here we show that angiogenesis, including the recruitment and maintenance of interaction between EphA RTKs and ephrinA ligands is tumor vasculature, and blocking VEGF function using soluble necessary for induction of maximal neovascularization receptors, neutralizing antibodies, or pharmacologic inhib- by VEGF. EphA2 RTK is activated by VEGF through itors significantly abrogates tumor angiogenesis and progres- induction of ephrinA1 ligand. A soluble EphA2-Fc sion (9–14). Recent evidence indicates that VEGF cooperates receptor inhibits VEGF-, but not basic fibroblast growth with other angiogenic factors, such as angiopoietins and their factor-induced endothelial cell survival, migration, RTK, Tie2, in regulating vascular remodeling and growth in sprouting, and corneal angiogenesis. As an indepen- tumors (15–17). However, the relationship between VEGF dent, but complementary approach, EphA2 antisense and ephrins remains unclear. oligonucleotides inhibited endothelial expression of The Eph family of RTKs and their ligands, originally EphA2 receptor and suppressed ephrinA1- and VEGF- identified as critical determinants of embryonic patterning and induced cell migration. Taken together, these data neuronal targeting (18), also regulates vascular development (1, indicate an essential role for EphA receptor activation in 2, 19). Targeted disruption of ephrinB2, EphB4,orEphB2/ VEGF-dependent angiogenesis and suggest a potential EphB3 results in embryonic lethality due to defects in primary new target for therapeutic intervention in pathogenic capillary network remodeling and subsequent patterning defects angiogenesis. in the embryonic vasculature (20–22), suggesting that Eph RTKs and their ligands are critical for vascular development during embryogenesis. The A class ligand, ephrinA1, has also Introduction been implicated in angiogenesis. EphrinA1 was originally Angiogenesis, the formation of new blood vessels from pre- identified as a tumor necrosis factor a (TNF-a)-inducible gene existing vasculature, is a multistep process involving a diverse in human umbilical vein endothelial cells (HUVECs) (23), and array of molecular signals. These include factors that stimulate is expressed in the developing vasculature during embryo- genesis (24). Moreover, ephrinA1 induces endothelial cell migration and capillary assembly in vitro, and angiogenesis in a Received 3/6/02; revised 6/17/02; accepted 6/24/02. The costs of publication of this article were defrayed in part by the payment of corneal pocket assay in vivo (25, 26), suggesting a role in page charges. This article must therefore be hereby marked advertisement in neovascularization of adult tissues. Indeed, expression of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ephrinA1 and its receptor EphA2 was observed in breast Grant support: NIH Grants HD36400 and DK47078; JDF grant I-2001-519; DOD grant BC010265; American Heart Association Grant 97300889N; ACS tumors and associated vasculature (27), and blocking EphA Institutional Research Grant IN-25-38 (to J. Chen); Vascular Biology Training receptor activation impaired tumor angiogenesis (28). These Grant T32-HL-07751-06 and American Heart Association Fellowship 0120147B studies indicate that Eph signaling is critical for normal blood (to D. Brantley); Cancer training Grant T-32 CA09592 (to N. Cheng); and a core facilities Grant 2P30CA68485 to the Vanderbilt-Ingram Cancer Center. vessel development as well as pathogenic angiogenesis. Requests for reprints: Jin Chen, Vanderbilt University School of Medicine, In this study, we provide evidence that Eph RTKs and their A-4323 MCN, 1161 21st Avenue South, Nashville, TN 37232. Phone: (615) 343-3819; Fax: (615) 343-7392. E-mail: [email protected] ligands are necessary for induction of maximal angiogenesis by Copyright D 2002 American Association for Cancer Research. VEGF. We show that ephrinA1 is a downstream target gene Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. Molecular Cancer Research 3 product induced by VEGF. Blocking EphA receptor activation inhibits VEGF-dependent endothelial cell migration, sprouting, and survival in vitro and angiogenesis in vivo. These effects are specific to VEGF, as blocking EphA class signaling has no impact of fibroblast growth factor (FGF)-mediated angio- genesis. As VEGF is a critical mediator of angiogenesis in pathogenic events such as cancer, our data suggest a potential new target for anti-angiogenic therapy. Results A Soluble EphA2-Fc Chimeric Receptor Inhibits VEGF-, but not FGF-induced Angiogenesis Given that both VEGF and ephrins regulate angiogenesis, and that VEGF is known to cooperate with other angiogenic factors to modulate blood vessel formation, we investigated whether EphA receptor activation is required for VEGF- mediated angiogenesis. To achieve this goal, we have utilized a soluble chimeric protein, EphA2-Fc, as a blocking reagent. This approach was taken because mutant mice deficient for individual Eph family members are either embryonic lethal or do not display overt phenotypes (20–22, 29). The EphA2-Fc soluble receptor fusion proteins consist of extracellular domain of the EphA2 receptor and the Fc portion of the human immunoglobulin IgG1. Because there is promiscuous binding between Eph receptors and ligands within the same subclass, this soluble receptor variant prevents multiple ephrinA class ligand interactions with endogenous receptors, effectively blocking signaling through class A Eph receptors. As shown in Fig. 1A, endothelial cells treated with ephrinA1 in the presence of excess EphA2-Fc showed a dramatic reduction in endogenous EphA2 receptor phosphorylation as compared to cells treated with ephrinA1 alone. However, EphA2-Fc does not affect the phosphorylation of VEGFR2 (Flk-1) (Fig. 1B), demonstrating the specificity of EphA2-Fc to class A ephrins. In the mouse cornea, soluble EphA2-Fc also markedly inhibited the angiogenic response induced by ephrinA1 (Fig. 1C), showing the efficacy of this reagent in blocking A class Eph receptor activation and neovascularization in vivo. We next investigated whether ephrin/Eph A class signals are required for VEGF-dependent angiogenesis. To do this, we used a well-established assay in which hydron pellets impregnated with either test or control proteins are implanted into mouse corneal pockets (30). Angiogenesis induced by exogenous factors in the normally avascular cornea was then documented and quantified. Consistent with previous observations, FIGURE 1. A soluble EphA2 receptor blocks endogenous EphA2 ephrinA1, VEGF, and basic fibroblast growth factor (bFGF) receptor signaling in vitro and ephrinA1-induced angiogenesis in vivo. A. EA926 endothelial cells were incubated with ephrinA1 in the presence or each induced corneal neovascularization (Figs. 1C and 2, A and absence of EphA2-Fc, and endogenous EphA2 was immunoprecipitated B) (16, 25, 31). To determine if VEGF function is dependent from cell lysates. EphrinA1-induced tyrosine phosphorylation of endoge- nous EphA2 receptor was inhibited by excess soluble EphA2-Fc. Uniform upon signaling by ephrinA class ligands, we implanted hydron loading was confirmed by immunoblotting with an anti-EphA2 antibody. B. pellets impregnated with VEGF or bFGF in the presence or HUVECs were stimulated with VEGF in the presence or absence of absence of EphA2-Fc. Soluble EphA2-Fc receptor itself does EphA2-Fc. Endogenous VEGF receptor was immunoprecipitated from cell lysates. VEGF-induced tyrosine phosphorylation of VEGR2 is not affected not induce angiogenesis in the cornea. Rather, the addition of by excess soluble EphA2-Fc. C. EphA2-Fc inhibits ephrinA1-induced
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