1. EphB4 regulates VEGF‐induced phosphorylation of endothelial ERK1/2 downstream of VEGF‐R2 activation
EphB4 regulates VEGF‐induced phosphorylation of endothelial ERK1/2 downstream of VEGF‐R2 activation HDMEC were treated in vitro with VEGF alone or together with ephrinB2‐Fc or the VEGF‐R2 small molecule inhibitor axitinib as a positive control. A, BVEGF‐R2 internalization was quantified by FACS. Mean ± SEM; n = 6 independent samples/group; **P < 0.01 and ***P < 0.001 vs. not stimulated control, #P < 0.05, ###P < 0.001 (one‐way ANOVA with Bonferroni multiple comparisons test).CVEGF‐R2 phosphorylation at tyrosine Y1175 (C) was quantified by immunocytochemistry. Mean ± SEM; n = 4 independent samples/group; *P < 0.05 and **P < 0.01 vs. not stimulated control (one‐way ANOVA with Bonferroni multiple comparisons test).DExpression of VEGF‐R2 target genes Igfpb3 and Esm1 was quantified by real‐time qRT–PCR. Mean ± SEM; n = 8 independent samples/group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. not stimulated control, ###P < 0.001 (one‐way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic‐transformation).E–HMuscles were harvested 3 and 4 days after implantation of V‐low or V‐low sEphB4 clones, or V‐high cells while treating animals systemically with ephrinB2‐Fc or control Fc proteins. Frozen sections were immunostained for phosphorylated ERK1/2 (p‐ERK1/2) and the endothelial junctional protein VE‐cadherin (VE‐CAD) (E, G), and the percentage of p‐ERK1/2‐positive endothelial cells was quantified (F, H). EphB4 inhibition and stimulation, respectively, increased and decreased ERK1/2 activation downstream of VEGF‐R2. *in IF panels = lumens of vascular enlargements. Scale bar = 20 μm. Mean ± SEM; n = 4 independent samples/group; *P < 0.05 and ***P < 0.001 (one‐way ANOVA with Bonferroni multiple comparisons test).
Elena Groppa et al. EMBO Rep. 2018;embr
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