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First published online 6 May 2003
doi: 10.1242/jcs.00426

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Forward EphB4 signaling in endothelial cells controls cellular repulsion and segregation from ephrinB2 positive cells

Tim Füller^*, Thomas Korff^*, Adrienne Kilian, Gudrun Dandekar and Hellmut G. Augustin

Department of Vascular Biology and Angiogenesis Research, Institute of Molecular Oncology, Tumor Biology Center, Breisacher Strasse 117, 79106 Freiburg, Germany

View larger version (55K): [in a new window]   Fig. 2. Quantitative analysis of HUVEC adhesion to EphB4-Fc-and ephrinB2-Fc-coated adhesive and non-adhesive surfaces. (A) HUVECs were seeded on top of adhesive (left) or non-adhesive (right) plastic surfaces coated with ephrinB2-Fc (160 ng/mm2) or EphB4-Fc (160 ng/mm2). Adhesion of cells was assessed by digitally measuring the cell-covered area of the coated surface after 24 hours (representative pictures are shown below). HUVECs avoid adhesion to ephrinB2-Fc-coated adhesive plastic. In turn, EphB4-Fc promotes adhesion to non-adhesive plastic. (B) Dose dependency of ephrinB2-Fc-mediated anti-adhesive effects on HUVECs. EphrinB2-Fc (dark bars) inhibits HUVEC adhesion with an EC50 of 12.2 ng/mm2 and a hill slope of –1.5 (representative pictures are shown below). Clustering of ephrinB2-Fc (light bars) induced a left shift with an even steeper dose-response curve (EC50: 8.5 ng/mm2; hill slope: –2.4) (insert). The figure shows the mean±s.d. of one out of three experiments with similar results. The coverage of control coated adhesive plastic was set to 100%. ***P<0.001 compared with corresponding control. Bars, 200 µm.

View larger version (32K): [in a new window]   Fig. 4. Effect of EphB4-Fc and ephrinB2-Fc on lateral 2D migration of HUVECs (A) and VEGF-stimulated HUVEC chemotaxis (B). (A) EphB4-Fc (1 µg/ml) and VEGF (50 ng/ml) stimulate lateral EC migration (48 hours). In turn, ephrinB2-Fc (1 µg/ml) inhibits baseline as well as VEGF-induced lateral EC migration (mean±s.d.; analysis of four independent experiments performed in triplicates). (B) EphrinB2-Fc (2 µg/ml), but not EphB4-Fc (2 µg/ml), significantly inhibits VEGF- (20 ng/ml) mediated chemotaxis (4 hours) of HUVECs in a modified Boyden chamber assay (mean±s.d.; analysis of four independent experiments performed in triplicates). Control A: medium, upper and lower compartment; control B: medium, upper compartment, VEGF, lower compartment. **P<0.01 compared with baseline control; P<0.05 compared with VEGF induction.

View larger version (81K): [in a new window]   Fig. 1. Detection of EphB4 and ephrinB2 binding sites on cultured HUVECs by receptor body staining. Confluent HUVEC monolayers were fixed and incubated with EphB4-Fc (detecting ephrinB2) (B) or ephrinB2-Fc (detecting EphB2, EphB3 and EphB4) (C). Receptor body binding was visualized using a Cy3-coupled anti-Fc-antibody. Essentially, all HUVECs bind ephrinB2-Fc (C), whereas a subpopulation of cells binds EphB4-Fc (B), which is preferentially localized at intercellular contacts.

View larger version (69K): [in a new window]   Fig. 3. EphrinB2-Fc-induced detachment of ECs from 3D co-culture spheroids of HUVECs and HUASMCs (EC/SMC; left) and from explanted fragments of umbilical vein (right). (A,C,E) EC/SMC co-culture spheroids were treated for 24 hours with ephrinB2-Fc (2 µg/ml) or EphB4-Fc (2 µg/ml), fixed and whole mount stained for the EC marker CD31. Control spheroids (A) and EphB4-Fc-treated spheroids (E) have an intact surface monolayer of CD31+ ECs. EphrinB2-Fc (C) disintegrates the surface endothelial monolayer and induces EC detachment (arrows). (B,D,F) Explanted fragments of freshly isolated human umbilical cords were cultured for 24 hours in the presence of ephrinB2-Fc (2 µg/ml) or EphB4-Fc (2 µg/ml) after which they were fixed, paraffin embedded and stained for the EC marker CD34. Control (B) and EphB4-Fc-treated (F) umbilical veins have an intact monolayer of CD34+ ECs. By contrast, ephrinB2-Fc (D) induces detachment and denudation of umbilical veins. (G) Quantitation of umbilical vein denudation induced by ephrinB2-Fc. Umbilical vein integrity was assessed by automated image analysis quantitating the relative SMC surface area that is covered by CD34+ ECs. *P<0.05 compared with control.

View larger version (37K): [in a new window]   Fig. 5. Modulation of 3D sprouting angiogenesis (A) and endothelial alignment on Matrigel (B) by EphB4-Fc and ephrinB2-Fc. (A) Quantitative analysis of the cumulative sprout length (CSL, quantitated after 48 hours) originating from 10 collagen gel embedded HUVEC spheroids (one out of three independent experiments with similar results). Representatives of each experimental group are shown below the bar graph (bar, 100 µm). EphB4-Fc (1 µg/ml) as well as VEGF (50 ng/ml) induce capillary-like sprouting. By contrast, ephrinB2-Fc (1 µg/ml) inhibits baseline sprouting as well as VEGF-induced sprouting angiogenesis. (B) Quantitative cellular alignment analysis of ECs grown on Matrigel. HUVECs were grown on Matrigel for 24 hours after which the circumferential length of tube-like structures was quantitated by automated image analysis. EphrinB2-Fc significantly inhibits alignment of HUVECs. **P<0.01; ***P<0.01 (compared with baseline control); P<0.01 (compared with VEGF induction).

View larger version (49K): [in a new window]   Fig. 6. Effect of EphB4-Fc and ephrinB2-Fc on sprouting angiogenesis of mock and EphB4 (clone 4-8)-transfected PAECs. (A) PAEC spheroids were embedded in collagen gels and stimulated with ephrinB2-Fc or EphB4-Fc for 48 hours. PAECs have a high baseline sprouting that was set to 100%. The sprouting activity of EphB4-transfected PAECs was significantly inhibited upon treatment with ephrinB2-Fc (***P<0.001) but not upon EphB4-Fc treatment. By contrast, mock-transfected PAECs do not respond to either ephrinB2-Fc or EphB4-Fc treatment. (B) EphB4-overexpressing PAECs were stimulated with ephrinB2-Fc for 30 minutes and analyzed for EphB4 phosphorylation by immunoprecipitating EphB4 using ephrinB2-Fc. Blots were probed with an anti-EphB4 antibody (left) and reprobed with an anti-pTyr antibody (right). EphrinB2-Fc stimulation leads to prominent phosphorylation of EphB4 in PAECs overexpressing mEphB4.

View larger version (68K): [in a new window]   Fig. 7. Confrontation experiments of overexpressing cells EphB4- (clone 4-8), ephrinB2- (clone 2-6) and ephrinB2 (2-15). Equal numbers of cells were mixed and seeded at confluent cell density. Combinations of mock-transfected PAECs with either ephrinB2 (A), EphB4 (B) or ephrinB2 (C) results in complete intermingling of the two cell populations. By contrast, combinations of ephrinB2 (D) or ephrinB2 (E) with EphB4-overexpressing cells leads to segregation of the two cell populations as demonstrated by island formation of EphB4-expressing cells (red staining in A and C: EphB4-Fc receptor body staining for ephrinB2 expression; red staining in B, D and E: ephrinB2-Fc receptor body staining for EphB4). EphrinB2- or ephrinB2-mediated segregation of EphB4+ cells is associated with intense tyrosine phosphorylation of EphB4 (F). Biochemical analysis of confrontation experiments of mock-transfected cells with ephrinB4-transfected cells identified a weak phospho-EphB4 band. By contrast, co-culture of either ephrinB2 or ephrinB2 cells with EphB4 cells resulted in intense EphB4 tyrosine phosphorylation (F, arrowhead). In turn, analysis of ephrinB2 expression and phosphorylation identified abundant levels of full-length ephrinB2 and the truncated ephrinB2 (G, upper right arrowhead and arrow). Yet, a phospho-ephrinB2 band was only detectable in the confrontation experiments of ephrinB2/EphB4 co-cultures and in none of the other combinations (G, lower right arrowhead).

View larger version (58K): [in a new window]   Fig. 8. Proposed model of the functional consequences of EC ephrinB2 and EphB4 signaling during guided migration (A) and capillary network formation (B). The model is based on the functional data summarized in this manuscript and takes into account published data on the repulsive guidance of ECs and neural crest cells by surrounding cells (Helbling et al., 2000; Krull et al., 1997; Oike et al., 2002; Wang and Anderson, 1997). Similar to guided nerve cell outgrowth, forward EphB4 signals may direct ECs in a repulsive manner upon activation by surrounding cells avoiding areas where ephrinB2 is expressed (guided migration, A, stop signal). The opposite, promotion of EC migration may occur if ephrinB2-expressing angiogenic ECs are activated by EphB4 (A, go signal). Additionally, these effects may segregate ECs from each other to limit cellular intermingling and control arterio-venous positioning of cells (network formation, B). Propulsive and repulsive EC forces during capillary morphogenesis indicate an arterio-venous push and pull situation that supports an artery-to-vein model of invasive angiogenesis.

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