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 10⁶ 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 Figures S1 and S2 and Movies S1 and S2.

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.

EPAC/Rap-Mediated AJ Maturation and Actin Bundle Formation Depends on Raf-1 and Rok-α
(A–C) iMECs were seeded on chamber slides in the presence or absence of 007 (250 μM) and of Y-27632 (20 μM). After 2 hr, the localization of VEC (red) and pMLC (green) was analyzed by confocal microscopy. DAPI (blue) was used as a counterstain. In (B)–(C), VEC (red) was used to visualize the AJ; filamentous actin (green) was visualized by phalloidin staining. Note the strong increase in AJ maturation (A) and (C) and peripheral actin bundling induced by 007 in f/f, but not in Raf-1^Δ/Δ cells or in Y-27632-treated f/f cells. The graphs in (A) and (C) show the percentage of cells (mean ± SD) containing immature or mature junctions. Scale bars: 20 μm. See also Figure S3.

Raf-1 Affects VEC-Mediated AJ Formation
(A) Rok-α substrates are slightly hyperphosphorylated in continuously growing Raf-1^Δ/Δ pMECs. Expression of Raf-1, VEC, and Rok-α, and phosphorylation of Rok-α downstream targets were detected by immunoblotting.
(B and C) VEC fails to localize at cell-cell borders in sprouts formed by Raf-1^Δ/Δ pMECs in 3D cultures (B) and in matrigel plugs (C). VEC patterns (red) and nuclei (DAPI, blue) were analyzed by confocal microscopy.
(D) Delayed formation of VEC-containing AJ and mislocalization of activated myosin (pMLC) in Raf-1^Δ/Δ iMECs. iMECs were seeded on chamber slides in the presence of FGF-2 (50 ng/ml) and fixed at the indicated time points. The localization of VEC (red) and pMLC (green) was analyzed by confocal microscopy. Nuclei were stained with DAPI (blue). Note the consistent colocalization of VEC and pMLC in the f/f iMECs and the lack of colocalization in the Raf-1^Δ/Δ cells or in cells treated with the Rok inhibitor (20 μM Y-27632). The numbers below the pictures indicate the percentage of cells positive for junctional pMLC, ± SD.
(E) Rok inhibition does not rescue the sprouting defect of Raf-1^Δ/Δ pMECs. Y-27632 was added to the fibrin gels at indicated concentrations. Sprout formation was analyzed as in Figure 1A. Note the increased amount of individual cells scattered around the microcarrier in the f/f cultures treated with 50 μM Y-27632. A quantification of the results is shown in the right panel (minimum n = 25 microcarriers per genotype and treatment; error bars, SE of the mean). Blue traces have been added to two of the images lacking contrast to highlight the profile of the sprouts. Scale bars (B) and (C), 20 μm; (D), 10 μm; and (E), 200 μm. p < 0.001, according to Student's t tests. n.s., not significant.

The N-Terminal Domain of Raf-1 Is Sufficient to Restore Sprouting and Junctional VEC in Raf-1^Δ/Δ pMECs
(A) Reconstitution of Raf-1^Δ/Δ pMECs with the regulatory N-terminal domain restores the number and length of sprouts formed in 3D fibrin gels.
(B) Raf-1 CRD and RBD are essential for sprouting.
(C) The N-terminal domain of Raf-1, but not the CRD and RBD mutants, restore junctional VEC in Raf-1^Δ/Δ pMECs. f/f and Raf-1^Δ/Δ pMECs were transfected with GFP-tagged constructs and FACS sorted. Sprouting efficiency was assessed 3 days later. The results (number of sprouts/bead and sprout length) are plotted in the lower panels (n = number of microcarriers evaluated; error bars, SE of the mean). Scale bars: (A)–(B), 200 μm; (C), 20 μm. In (B), green traces have been added to three of the images lacking contrast to highlight the profile of the sprouts.

EPAC/Rap Activation Recruits the Raf-1/Rok-α Complex to VEC-Containing AJ
(A and B) iMECs were treated for 30 min with FGF-2 (50 ng/ml) or with the cAMP analog 007 (50 μM), a selective EPAC activator. Where indicated, cells were pretreated with GGTi-298 (10 μM; 1.5 hr) prior to stimulation to block Rap-dependent signaling. (A) VEC or (B) Raf-1 immunoprecipitates were prepared and the presence of VEC or Raf-1 and coimmunoprecipitating proteins were detected by immunoblotting.
(C) Rap1A/B is required for EPAC-mediated recruitment of Rok-α and Raf-1 to VEC. iMECS in which Rap1A and Rap1B were knocked down by siRNA interference, were stimulated with 007 (50 μM, 30 min). The presence of Rok-α and Raf-1 in VEC immunoprecipitates was detected by immunoblotting.
(D) Raf-1 and Rap1 are required for efficient Rok-α recruitment to VEC junctions. iMECs in which Rap1A and Rap1B were knocked down by siRNA interference, were seeded for 2 hr in the presence or absence of 007 (250 μM). Rok-α (red) and VEC (green) were visualized by confocal microscopy. DAPI (blue) was used as a counterstain. Note the absence of junctional Rok-α in Raf-1^Δ/Δ cells and in f/f cells treated with GGTi298.
(E) iMECs were treated and VEC immunoprecipitates were analyzed as in (A). In the right panel, 007-induced recruitment of Rok-α to VEC is plotted as fold Rok-α-VEC interaction in unstimulated cells (set as 1; mean ± SE of three experiments). The p value was calculated comparing f/f versus Raf-1^Δ/Δ cells.
(F) Small interfering RNA (siRNA)-mediated knock down of Rok-α, but not Rok-β abrogates 007-induced AJ maturation. siRNA-treated cells were plated for 2 hr in the presence of 007 (250 μM). VEC (green) and nuclei (DAPI, blue) were visualized by confocal microscopy. The graph shows the percentage of cells with mature AJ (mean ± SD). Knockdown efficiency is shown by immunoblotting. Scale bars: 20 μm. See also Figures S4 and S5.