Figure 5.

Defective PDZ-dependent signaling of ephrinB2 results in impaired migration and proliferation of kidney MVECs and compromises internalization and phosphorylation of VEGFR2. (A) Phase contrast and immunofluorescence images of primary kidney MVECs. Sorted and cultured MVECs express CD31 (marker for ECs) but not E-cadherin (marker for epithelial cell) and CD11b (marker for monocyte/macrophage). (B) Migration assay in response to mouse VEGF stimulation (10 ng/ml) using kidney MVECs isolated from WT, ephrinB2 5Y (5Y), and ephrinB2 ΔV (dV) mice. Migration is assessed by the percentage of wound closure. Creation of wound in monolayer of cells denotes time zero. n=3 per group. *P<0.05 versus WT; **P<0.01 versus WT; ^#P<0.05 versus 5Y. (C) Proliferation assay in response to mouse VEGF stimulation (10 ng/ml) using kidney MVECs assessed by BrdU incorporation for 24 h. n=3 per group. **P<0.01. (D) The left panel shows representative fluorescence images of individual kidney MVECs which indicate the distribution of VEGFR2 in response to VEGFA (100 ng/ml) for 30 minutes. The right panel indicates the proportion of internalized (red) compared with surface (green) VEGFR2 based on fluorescence intensities. n≥20 per condition. **P<0.01. (E) The left panel shows representative kidney MVECs stained for p-VEGFR2 at tyrosine 1175 (red) in control conditions or with VEGF (100 ng/ml) stimulation for 30 minutes. The right panel indicates the proportion of phosphorylated compared with total VEGFR2 based on fluorescence intensities. The ratio of p-VEGFR2 to total VEGFR2 is normalized to the values of WT group. n=20 per condition. ** P<0.01. NS, no significant difference. Scale bar, 20 μm in A; 10 μm in D and E.