Figure 5
Figure 5. EGFL7 stimulates angiogenic sprouting in vitro and in vivo in an αvβ3 integrin-dependent manner. (A) HUVEC spheroids embedded in collagen gels were treated with EGFL7 and/or VEGF. (B) Prior to spheroid formation, HUVECs were transfected with scrambled (scr) siRNA or siRNA directed against EGFL7 (siEGFL7). Spheroids embedded in the collagen gels were left to sprout in the absence or presence of VEGF. (C) HUVECs were infected with adenovirus encoding for DsRed or EGFL7 and subsequently transfected with scr or siEGFL7 and used for spheroid formation assays. (D) HUVEC spheroids embedded in collagen gels were treated with EGFL7, VEGF as a positive control, the specific αvβ3 inhibitor cRGDfV along with EGFL7, the inhibitor control peptide cRADfV along with EGFL7, or with each of the compounds alone. (E) HUVECs were transfected with DsRed, wild-type EGFL7, EGFL7 ΔRGD, or EGFL7 RAD and subjected to spheroid assays. The mean cumulative sprout length per spheroid was calculated based on the analysis of at least 10 spheroids per experimental group. Representative images of spheroid assay and statistical analysis are shown (mean ± SEM, n = 3, *P < .05). Bar represents 100 μm. (F) CAM assays were employed to study whether or not EGFL7 affects vessel formation in ovo. Water, EGFL7, EGF as a positive control, EGFL7 plus cRGDfV, EGFL7 plus cRADfV, or each of the compounds alone were applied on the CAMs, which were subsequently photographed and analyzed with regard to vessel formation. Each experimental group contained at least 6 CAMs and 1 out of 3 representative experiments is displayed. Representative images of CAMs and statistical analysis are shown (mean ± SEM, n = 3, *P < .005). (G) In order to determine the proangiogenic effect of EGFL7 in vivo, 3 groups of nude mice were repetitively injected with either control, recombinant EGFL7 or VEGF as a positive control. The upper panel shows representative examples of ears of each group after 2 weeks of treatment. The lower panel displays the quantification of the cumulative vessel length per ear area (mm/cm2) in all groups upon treatment (mean ± SEM, n = 6,*P < .01).

EGFL7 stimulates angiogenic sprouting in vitro and in vivo in an αvβ3 integrin-dependent manner. (A) HUVEC spheroids embedded in collagen gels were treated with EGFL7 and/or VEGF. (B) Prior to spheroid formation, HUVECs were transfected with scrambled (scr) siRNA or siRNA directed against EGFL7 (siEGFL7). Spheroids embedded in the collagen gels were left to sprout in the absence or presence of VEGF. (C) HUVECs were infected with adenovirus encoding for DsRed or EGFL7 and subsequently transfected with scr or siEGFL7 and used for spheroid formation assays. (D) HUVEC spheroids embedded in collagen gels were treated with EGFL7, VEGF as a positive control, the specific αvβ3 inhibitor cRGDfV along with EGFL7, the inhibitor control peptide cRADfV along with EGFL7, or with each of the compounds alone. (E) HUVECs were transfected with DsRed, wild-type EGFL7, EGFL7 ΔRGD, or EGFL7 RAD and subjected to spheroid assays. The mean cumulative sprout length per spheroid was calculated based on the analysis of at least 10 spheroids per experimental group. Representative images of spheroid assay and statistical analysis are shown (mean ± SEM, n = 3, *P < .05). Bar represents 100 μm. (F) CAM assays were employed to study whether or not EGFL7 affects vessel formation in ovo. Water, EGFL7, EGF as a positive control, EGFL7 plus cRGDfV, EGFL7 plus cRADfV, or each of the compounds alone were applied on the CAMs, which were subsequently photographed and analyzed with regard to vessel formation. Each experimental group contained at least 6 CAMs and 1 out of 3 representative experiments is displayed. Representative images of CAMs and statistical analysis are shown (mean ± SEM, n = 3, *P < .005). (G) In order to determine the proangiogenic effect of EGFL7 in vivo, 3 groups of nude mice were repetitively injected with either control, recombinant EGFL7 or VEGF as a positive control. The upper panel shows representative examples of ears of each group after 2 weeks of treatment. The lower panel displays the quantification of the cumulative vessel length per ear area (mm/cm2) in all groups upon treatment (mean ± SEM, n = 6,*P < .01).

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