The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth - PubMed (original) (raw)

The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth

Nathalie Kertesz et al. Blood. 2006.

Abstract

The receptor tyrosine kinase EphB4 and its ligand EphrinB2 play a crucial role in vascular development during embryogenesis. The soluble monomeric derivative of the extracellular domain of EphB4 (sEphB4) was designed as an antagonist of EphB4/EphrinB2 signaling. sEphB4 blocks activation of EphB4 and EphrinB2; suppresses endothelial cell migration, adhesion, and tube formation in vitro; and inhibits the angiogenic effects of various growth factors (VEGF and bFGF) in vivo. sEphB4 also inhibits tumor growth in murine tumor xenograft models. sEphB4 is thus a therapeutic candidate for vascular proliferative diseases and cancer.

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Figures

Figure 1.

Structure and biochemical properties. (A) Schematic illustration of truncated soluble proteins. (B) SDS-PAGE. Coomassie staining of EphB4-derived soluble proteins. (C) Binding of EphrinB2-AP fusion protein to EphB4 soluble proteins immobilized on Ni-NTA agarose beads. Results of 2 independent experiments are shown for each protein. Experiments were repeated 3 times (D) Tyrosine phosphorylation of EphB4 receptor in HUVECs in response to stimulation with EphrinB2-Fc (15 minutes) in the absence or presence of EphB4-derived soluble proteins. (E) Tyrosine phosphorylation of EphrinB2 by EphB4 in the presence of sEphB4. 293T cells transiently transfected with full-length EphrinB2 and EphB4 expression vector were cocultured (15 minutes) with or without sEphB4 for 24 hours (see “Monomeric sEphB4 antagonizes forward and reverse signaling” for details). Total amount of EphrinB2 by Western blot (bottom panel) and phosphorylation status of EphrinB2 after immunoprecipitation (IP) (top panel) are shown. Experiments were repeated twice.

Figure 2.

sEphB4 inhibits tubule formation on Matrigel in vitro. (A) HUVECs were cultured on standard Matrigel in growth factor-stimulated conditions with either sEphB4 or CF2 proteins for 24 hours. Shown are representative pictures from triplicate wells repeated twice. (B) Dose response. HUVECs were cultured as described in panel A with sEphB4 at indicated concentrations. (C) Quantitative analysis of data from panel B for tube length and the number of junctions in sEphB4-treated HUVECs (Bioquant Image Analysis; mean from triplicate wells in 2 repetition experiments). (D) sEphB4 inhibits invasion of HUVECs (top right) compared to cells treated with CF2 (bottom left) or no treatment (top left). Data are presented as number of invading cells ± SE from duplicate wells in 2 experiments (bottom right). (E) sEphB4 inhibits PDGF-stimulated attachment of HUAECs to Matrigel over 5 minutes and 15 minutes. Shown are number of attached cells as a percentage of control in triplicate wells; experiment was repeated twice. Photomicrographs in panels A, B, and D were taken using a Nikon Coolpix 5000 camera (Nikon, Tokyo, Japan) and a Carl Zeiss Invertoskop microscope (Zeiss, Goettingen, Germany) with a 4 ×/0.12 NA objective and 10 × eyepiece.

Figure 3.

sEphB4 inhibits angiogenesis in murine Matrigel and corneal micropocket assays. (A) Matrigel solution was injected subcutaneously into Balb/C nu/nu mice. After 6 days, plugs were removed and processed in paraffin. Individual sections were stained with Masson trichrome and representative photographs at × 20 magnification from triplicate plugs in 2 independent experiments are shown. Top left shows section of a Matrigel plug without any added proteins; top right, section of plug containing VEGF only; bottom left, section of plug with sEphB4 and VEGF; and bottom right, section of plug with CF2 and VEGF. Photomicrographs were taken with a Nikon Coolpix 5000 camera on a Nikon Eclipse E400 microscope with a 4 ×/0.13 NA objective and a 10 × eyepiece. (B) Quantitation of vascularized area averaged (± SEM) from all plugs (Scion Image software). (C) Immunofluorescent staining of dissected plugs (with or without sEphB4) with PECAM, EphB4, and EphrinB2 antibodies. (D) sEphB4 was added to Hydron polymer and sucralfate with or without bFGF and inserted into a micropocket in corneas of Balb/C mice in triplicate. Shown is neovascular response on day 4 after implantation: Hydron with bFGF (left), with no growth factor added (center), and growth factor and sEphB4 (right). (E) Quantitation of vascularization averaged (± SEM) over all corneas in each group.

Figure 4.

sEphB4 inhibits the tumor growth in a murine tumor xenograft model. (A) 1 × 104 MCF-7 and SCC-15 cells were grown in triplicate in the presence of increasing concentrations of sEphB4 for 72 hours. Cell viability was assessed by MTT assay. The experiment was repeated twice. (B) Mice (n = 6/group) were given implants with 5 × 106 SCC-15 or MCF-7 cells in a Matrigel preparation with PBS or sEphB4 (70 nM) and tumor volumes measured 3 times a week. Three weeks after implantation, tumors were harvested and weighed (right axis). Tumor volumes were significantly smaller in the sEphB4 arm, beginning on day 7 of measurement. The experiment was repeated twice. (C) Representative histology (hematoxylin and eosin) of SCC-15 tumors is shown. Tumor proliferation was assessed by immunohistochemical detection of Ki-67 protein and apoptosis by TUNEL. Microvasculature was assessed by CD31 immunohistochemistry. Number of cells staining positive was averaged over 5 random 40 × fields by a blinded observer and is shown at the bottom of each photomicrograph. (D) For assessing the effect of systemic administration of sEphB4, mice (n = 6/group) were given implants of tumor cells subcutaneously and treated with sEphB4 or CF2 (10 mg/kg/d) or an equal volume of PBS intravenously starting on day 4. Tumor volumes and weights were assessed as in panel B. Tumor volumes were significantly smaller in the sEphB4 arm compared to animals treated with PBS and CF2. (E) Histologic analysis of harvested MCF-7 tumors was performed as in panel C. All values are expressed as mean ± SEM. *P < .01 by Student t test. Bar in bottom right panel in E represents 100 μm in hematoxylin-eosin section, 60 μm in Ki-67 and apoptosis sections, and 75 μm in CD31 sections. Photomicrographs in panels C and E were taken using a Nikon Coolpix 5000 camera and a Nikon Eclipse E400 microscope with a 10 × eyepiece. Magnification was as follows: top row, 4 ×/0.13 NA; middle rows, 40 ×/0.75 NA; and bottom rows, 20 ×/0.5 NA objectives.

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The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth - PubMed (original) (raw)