Journal: Developmental Cell

Article Title: Angiogenic Sprouting Requires the Fine Tuning of Endothelial Cell Cohesion by the Raf-1/Rok-α Complex

doi: 10.1016/j.devcel.2011.11.012

Figure Lengend Snippet: Raf-1 Is Required for In Vitro Sprouting in 3D Fibrin Gels and In Vivo for Pathological Angiogenesis (A) pMECs isolated from Raf-1 Δ/ΔEC animals show dramatically reduced sprouting in 3D fibrin gels. pMECs were allowed to adhere to microcarriers and embedded in fibrin gels containing FGF-2 and VEGF (200 ng/ml each). The number of sprouts/bead and the length of sprouts were microscopically assessed after 3 days in culture. The results are plotted in the right panel. n = number of microcarriers evaluated; error bars, SD of the mean. (B) Sprout formation monitored by time lapse microscopy. Still images taken at different time points during sprouting are shown. In Raf-1 Δ/Δ , pMEC cultures single-tip cells break off from the developing sprouts, migrate as single cells (black arrow), and eventually undergo apoptosis, as indicated by membrane blebbing (green arrow). (C) Raf-1 Δ/Δ pMECs exhibit a mesenchymal phenotype during sprouting in fibrin gels. Cultures were stained with Rhodamin-phalloidin to visualize F-actin structures. (D and E) Raf-1 Δ/ΔEC mice fail to vascularize subcutaneous matrigel plugs. Matrigel, containing FGF-2 and VEGF (1 μg each), was subcutaneously injected into f/f and Raf-1 Δ/ΔEC mice. Ten days later, plugs were fixed and stained with anti-VEC antibodies (D) or with CD31/DAPI to visualize ECs. In (D), dashed lines indicate the outer margin of the plug. In (E), a quantification of CD31-positive vessels/field is shown in the right panel. Four plugs/genotype were analyzed; error bars, SD of the mean. (F and G) Raf-1 ablation impairs xenograft growth and vascularization. The graph shows tumor volume and mass assessed 14 days after subcutaneous implantation of 10 6 Lewis lung carcinoma cells (LLC-1) into f/f and Raf-1 Δ/ΔEC mice. In (G), CD31 immunohistochemistry was used to visualize ECs and vessels invading the tumors. A quantification of the CD31-positive vessels/field is shown in the right panel; error bars, SD of the mean. Scale bars: (A), (D), and (E), 200 μm; (C) and (G), 100 μm. p values are according to Student's t tests. See also and .

Article Snippet: For VEC staining, samples were fixed (1% PFA in 250 mM HEPES [pH 7.4], 15 min at RT), washed (TBST, 0.2% Tween-20, 15 min) prior to blocking (0.1% gelatin in PBST, 0.2% Tween, 1 hr at 4°C), and incubated with primary rat anti-mouse VEC antibodies (BD PharMingen; 1:100 in blocking solution, 1 hr at 37°C).

Techniques: In Vitro, In Vivo, Isolation, Time-lapse Microscopy, Staining, Injection, Immunohistochemistry

Journal: Developmental Cell

Article Title: Angiogenic Sprouting Requires the Fine Tuning of Endothelial Cell Cohesion by the Raf-1/Rok-α Complex

doi: 10.1016/j.devcel.2011.11.012

Figure Lengend Snippet: Raf-1 Is Required for the Recruitment of Rok-α to AJ and for Junctional Myosin Activation (A–C) Growth factors and increasing cell densities induce the recruitment of Raf-1 and Rok-α to VEC. iMECs were treated with FGF-2 or VEGF (both 50 ng/ml) for 30 min (A) or with FGF-2 (50 ng/ml) for the indicated times (B) or seeded at low (LD) or high density (HD) (C). VEC immunoprecipitates were prepared and the presence of VEC and coimmunoprecipitating proteins was detected by immunoblotting. In (A), the right panel shows growth factor-induced recruitment of Raf-1 to VEC plotted as fold Raf-1-VEC interaction in unstimulated cells (set as 1; mean ± SE of four experiments). (D) Raf-1 is required for growth factor-stimulated recruitment of Rok-α to VEC. iMECs were treated with FGF-2 or VEGF as in (A). The right panel shows FGF-2 induced recruitment of Rok-α to VEC plotted as fold Rok-α–VEC interaction in unstimulated cells (set as 1; mean ± SE of three experiments). The p value was calculated by comparing f/f versus Raf-1 Δ/Δ cells. (E) pMLC is not associated with AJ in Raf-1 Δ/Δ iMECs. iMECs were stimulated with FGF-2 (50 ng/ml) for 30 min. pMLC (green) and DAPI (blue) were visualized by confocal microscopy. The percentage of cells positive for junctional pMLC (mean ± SD) is shown in the lower panel. (F) Rok-α does not colocalize with VEC in Raf-1 Δ/Δ iMECs. Rok-α (red) and VEC (green) were visualized by confocal microscopy. DAPI was used as a counterstain. Scale bar: 20 μm. (G) pMLC fails to localize at cell-cell borders in sprouts formed in matrigel plugs implanted in Raf-1 Δ/ΔEC mice. Scale bar: 20 μm. In (A), IgG represents control immunoprecipitate using an unrelated, isotype-matched antibody, instead of the VEC antibody. In (A)–(D), input = whole cell lysate. See also Figure S3 .

Article Snippet: For VEC staining, samples were fixed (1% PFA in 250 mM HEPES [pH 7.4], 15 min at RT), washed (TBST, 0.2% Tween-20, 15 min) prior to blocking (0.1% gelatin in PBST, 0.2% Tween, 1 hr at 4°C), and incubated with primary rat anti-mouse VEC antibodies (BD PharMingen; 1:100 in blocking solution, 1 hr at 37°C).

Techniques: Activation Assay, Western Blot, Confocal Microscopy