Figure 1.

Vessel wall defects in smooth muscle cell-specific ephrin-B2 mutants. (A) Confocal images showing ephrin-B2 (green) and α-smooth muscle actin (α-SMA; red) immunostaining on sections of adult (30 wk) control and Efnb2^ΔSMC mutant aortae. (*) Vessel lumen. (B) Dilation affecting freshly isolated adult Efnb2^ΔSMC aortic arches (right) compared with control littermates (left). Arrows indicate vessel diameter. (C) Quantitation of relative aortic arch diameter of adult mice (>30 wk). P-value was calculated using two-tailed Student's t-test (n = 4). Error bars indicate SD. (D–F) 5-Ethynyl-2′-deoxyuridine (EdU) labeling (2-h pulse; red) of proliferating cells in control and mutant P8 aorta. (Green) α-SMA; (blue) nuclei (DAPI). (*) Vessel lumen. Quantitation of total α-SMA-positive cells (E) and EdU-labeled VSMCs (F). P-values were calculated using two-tailed Student's t-test (n = 3). Error bars indicate SD. (G) Western blot analysis of total and phosphorylated JNK (p-JNK), Erk1/2 (p-Erk1/2), and PDGFRβ (p-PDFGRβ) in control and Efnb2^ΔSMC aorta lysate, as indicated. Tubulin is shown as a loading control. Molecular weight markers (in kilodaltons) are indicated. (H,I) Strongly decreased levels of active Rac1 (GTP·Rac1) in Efnb2^ΔSMC aorta lysate relative to control (shown in H). (I) Tiam1 protein was nearly undetectable in mutant samples, whereas amounts of p27^kip1 were elevated. Tubulin is shown as a loading control. Molecular weight markers are indicated. (J) Quantitative analysis of band intensities in the Western blots shown in H and I and replicates. P-values were calculated using two-tailed Student's t-test (n = 3). Error bars indicate SD.