EphB4 controls blood vascular morphogenesis during postnatal angiogenesis

Abstract

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.

Figure 1

Ephrin and Eph receptor expression analysis in experimental and human glioma. (A) RT–PCR expression analysis of SF126 human glioma xenografts following transplantation into nude mice. In order to discriminate between tumor cell-derived (human origin) and blood vessel-derived (mouse origin) expression of individual Ephs and ephrins in glioma xenografts, species-specific primers were designed. (B) RT–PCR expression analysis for ephrinB2 and EphB4 on human surgical specimens of normal brain, low-grade glioma and high-grade glioma. (C) Summary of results of RT–PCR expression analysis on human astrocytoma. In situ hybridization of normal brain (D, E) and high-grade glioma (F–H) for ephrinB2 and EphB4 using DIG-labeled cRNA-probes. Higher magnification (H) shows strong signal for EphB4 mRNA in tumor vessels (arrowhead) and tumor cells adjacent to necrosis (arrow denotes the margin of necrotic area). Cells are denoted glial cells and neurons on the basis of morphological critereria. G=glial cell, N=neuron, V=vessel, NE=necrosis. Counterstain with methyl green. Bar scale as indicated.

Figure 2

Generation of retrovirus packaging cell lines to manipulate EphB4 signaling in vivo. (A, B) Stable virus-producing clones of Phoenix E producer cells were established. Western blot analysis of infected NIH 3T3 cells detected EphB4wt (A) and EphB4dn (B) at the predicted molecular weights of approx. 120 and 70 kDa, respectively. (C) Immunofluorescence staining of virally transfected NIH 3T3 cells using ephrinB2-Fc chimeras. Scale bar=10 μm. (D) SF126 cells and NIH 3T3 cells were in vitro incubated with virus-containing cell supernatants. PCR analysis for the presence of the neomycin-resistance gene (upper panel) and Western blot analysis for EphB4 protein (lower panel). (E) Western blot analysis of virally transfected NIH 3T3 cells for EphB4 and PTyr under baseline conditions (upper panel) and after stimulation with ephrinB2-Fc chimera (lower panel). (F) Schematic summary of experimental approach and manipulation of EphB4 signaling by endothelial overexpression of EphB4wt and EphB4dn.

Figure 3

Interference with endothelial EphB4 signaling in vivo alters tumor blood vessel morphology. RT–PCR expression analysis (A) and Western blot analysis (B) of SF126 xenografts following s.c. coimplantation with virus-producing Phoenix E cells. (C) PCR amplification utilizing primers located in the SV40 promoter of the vector and the untruncated part of the EphB4 cDNA was used to test for the presence of cross-contaminating viruses. PCR analysis for the presence of the neomycin-resistance gene served as a control. (D) Immunohistochemical staining for EphB4 and CD31 on consecutive cryo-fixed sections. Counterstain with hematoxylin. Scale bar=100 μm. (E) Immunofluorescent staining for EphB4 and ephrinB2 on consequent zinc-fixed sections of e-EphB4 wt tumors. Arrows indicate EphB4/ephrinB2 double-positive blood vessels. Scale bar=20 μm. (F) Immunohistochemical staining for CD31. Counterstain with hematoxylin. Scale bar=100 μm.

Figure 4

Endothelial EphB4 regulates vascular morphogenesis and permeability, independently of its tyrosine kinase activity. Intravital fluorescence videomicroscopy of SF126 cells that were coimplanted with virus-producing Phoenix E cells on days 7 (A–C) and 14 (D–F) after implantation. Scale bar=100 μm. Quantitative measurements of tumor blood vessel surface (G), functional tumor blood vessel density (H), and tumor blood vessel diameter (I). Values are represented as means±s.d. Number of animals per experimental group: e-pLXSN n=5; e-EphB4wt n=5; e-EphB4dn n=6. ^*P<0.05 versus e-pLXSN.

Figure 5

Manipulation of endothelial EphB4 signaling results in circumferential blood vessel growth and a reduced tumor blood vessel permeability. Consecutive cryofixed sections of SF126 glioma xenografts were immunohistochemically stained for CD31 (A–C) and the proliferation-associated antigen Ki-67 using a murine-specific antibody (Tec3) (D–F). Scale bar=100 μm. (G) Measurement of endothelial cell proliferation index in SF126 xenografts. Values are represented as means±s.d. Number of animals per experimental group: e-pLXSN n=3; e-EphB4wt n=3; e-EphB4dn n=3. ^*P<0.05 versus e-pLXSN. (H) Intravital fluorescence videomicroscopy of e-EphB4wt tumor on day 21 after implantation. Arrows point at sites of impaired interconnection of angiogenic sprouts and failed formation of a functional vascular network. Scale bar=100 μm. (I) Measurement of vascular branching points. Values are represented as means±s.d. Number of animals per experimental group: e-pLXSN n=5; e-EphB4wt n=5, e-EphB4dn n=6. ^*P<0.05 versus e-pLXSN. (J) Measurement of tumor blood vessel permeability by calculating the permeability index for individual blood vessels. Therefore, the ratio of extravascular to intravascular fluorescence intensities was determined for multiple tumor blood vessels. Values are represented as means±s.d. Number of animals per experimental group: e-pLXSN n=5; e-EphB4wt n=5, e-EphB4dn n=6. ^*P<0.05 versus e-pLXSN. (K–M) Double-immunofluorescent staining of cryo-fixed sections of SF126 xenografts (day 21 after implantation) for desmin (green) and CD31 (red) in order to assess pericyte/endothelial cell association. Scale bar=20 μm. (N) RT–PCR expresion analysis of SF126 xenografts for Ang-1, Ang-2, and Tie2 following s.c. coimplantation with virus-producing Phoenix E cells. (O) Western blot analysis for mTie2 (anti-Tie2) and PTyr (anti-PTyr) after immunoprecipitation of mTie2 from SF126 xenografts following s.c. coimplantation with virus-producing Phoenix E cells.

Figure 6

Manipulation of endothelial EphB4 signaling confers reduced tumor blood vessel permeability via the Tie2/Ang axis. (A) In situ hybridization of SF126-e-pLXSN, e-EphB4 wt and e-EphB4 dn tumors for Ang-1 mRNA (upper panel) and Ang-2 (lower panel), demonstrating localization of the increased Ang-1 signal to perivascular mural cells or pericytes, whereas Ang-2 m-RNA expression was restricted to the endothelial lining. Scale bar=20 μm. (B) Endothelial–pericytic assembly in SF126 e-pLXSN tumors (top left) compared with SF126 e-EphB4 wt tumors revealed by transmission electron microscopy. Upper panel left: Small tumor microvessel of an e-pLXSN tumor rich on endothelial nuclei (E) and loosely covered by periendothelial cells (arrows). Arrowheads point to the broad intercellular space at the endothelial cell/pericyte interface as a consequence of loose association of pericytes to endothelial cells. Upper panel right: Cross section of a large tumor blood vessel demonstrating a regular endothelium (E), which is tightly associated to pericytes (P). Arrows indicate strands of pericytic cytoplasm closely attached to the endothelium. A common basal lamina (BL) for the endothelium and pericytes is well-established and the intercellular space at the endothelial cell/pericyte interface is reduced to a minimum. Lower panel left: Small tumor blood vessel containing numerous mitochondria (black arrows) and profiles of free ribosomes and granular endoplasmic reticulum (black arrowheads), referring to activated endothelium. White arrows indicate tight endothelial cell to endothelial cell contacts and white arrowheads tight endothelial cell to pericyte contacts. Lower panel right: Newly formed, capillary-sized blood vessel containing abundant vesicles indicating enhanced transcytosis. Even these capillaries are tightly covered by pericytes (arrows). Er=Erythrocytes. Scale as indicated.

Figure 7

The effects of endothelial EphB4 expression on tumor blood vessels are reproducible with other tumor xenografts. (A–C) Intravital fluorescence videomicroscopy of SF188 cells that were coimplanted with virus-producing Phoenix E cells, day 14 after implantation. Scale bar=100 μm. (D–F) Intravital fluorescence videomicroscopy of SF767 cells that were coimplanted with virus-producing Phoenix E cells, day 14 after implantation. (G) Measurement of tumor blood vessel permeability on SF767 e-pLXSN, e-EphB4 wt, and e-EphB4 dn tumors by calculating the permeability index. Values are represented as means±s.d. Number of animals per experimental group: e-pLXSN n=4; e-EphB4wt n=5; e-EphB4dn n=5. ^*P<0.05 versus e-pLXSN. Scale bar=100 μm.

Figure 8

Effects of EphB4 signaling on vascular morphology are not limited to tumor angiogenesis. (A–C) Intravital fluorescence videomicroscopy of virus-producing Phoenix E cells that were implanted alone, day 14 after implantation. Scale bar=100 μm. Quantitative measurements of functional density (D), diameter (E), and permeability (F) of newly formed blood vessels revascularizing the implanted Phoenix E cells. Values are represented as means±s.d. Number of animals per experimental group: e-pLXSN n=5; e-EphB4wt n=5; e-EphB4dn n=5. ^*P<0.05 versus e-pLXSN. (G, H) Retinal digest preparations on day p15, that is, 10 days following intravitreal injection of Phoenix E cells. Scale bar=100 μm, or as indicated in the insets of higher magnification. Quantitative measurement of retinal blood vessel diameters (I) and vascular branching points (J). Values are represented as means±s.d. Number of animals per experimental group: e-pLXSN n=5; e-EphB4wt n=5. ^*P<0.05 versus e-pLXSN.