Sema3A restores NEM recruitment and accelerates vascular stabilization, without affecting the amount of induced angiogenesis.Figure 7A-C Mice, implanted with myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated on days 4 and 6 after cell implantation with intramuscular injections of Sema3A-Fc (0.1 or 10 mg/kg of average muscle tissue weight), or with control Fc protein. (A) Quantification of the number of NEM/cm of vessel length in sites of new angiogenesis after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 5 muscles/group: V Low Fc, n = 21; V Low 0.1 mg/kg, n = 22; V Low 10 mg/kg, n = 28; V Med Fc, n = 20; V Med 0.1 mg/kg, n = 30; V Med 10 mg/kg, n = 25; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low Fc versus V Low 10 mg/kg P = 0.0027; V Med Fc versus V Med 0.1 mg/kg P = 0.001; V Med Fc versus V Med 10 mg/kg P < 0.0001. (B) Quantification of vessel length density (VLD) in treated muscles after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 8; V Low 0.1 mg/kg, n = 20; V Low 10 mg/kg, n = 18; V Med Fc, n = 8; V Med 0.1 mg/kg, n = 23; and V Med 10 mg/kg, n = 27. Data were subjected to Kruskal-Wallis analysis with Dunn's multiple comparisons test, and no significant differences were detected. (C) Vascular stabilization rate was determined 2 weeks after cell implantation by measuring vessel length density after abrogation of VEGF signaling by VEGF-Trap. Treatment with 10 mg/kg Sema3A-Fc did not affect the stabilization of angiogenesis induced by low VEGF, but significantly increased the resistant fraction of vessels induced by medium VEGF. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 32; V Low Sema3A, n = 14; V Med Fc, n = 28; V Med Sema3A, n = 33; *P < 0.05 by one-way ANOVA with Bonferroni multiple comparisons test: V Med Fc versus V Med Sema3A P = 0.0458. D-F Mice received intramuscular injections of adenovirus expressing VEGF (AdVIC) or CD8 control (AdCD8) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A-Fc (10 mg/kg of average muscle tissue weight) or control Fc protein. (D) Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue), showing vessel density and morphology 1 week after injection of adenoviral vectors alone (no treatment) or after 3 weeks and with Fc or Sema3A-Fc treatment. Scale bar = 50 μm. (E) Quantification of vessel length density (VLD) on the same samples shows that treatment with Sema3A-Fc accelerated stabilization of vessels induced by transient and uncontrolled VEGF expression. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: Uninjected muscles, n = 72; AdCD8 1 week, n = 68; AdVIC 1 week, n = 53; AdVIC+Fc 3 weeks, n = 78; AdVIC+Sema3AFc 3 weeks, n = 91; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: AdCD8 1 week versus AdVIC+Fc 3 weeks P = 0.0037; all other comparisons indicated P < 0.0001. D-F Mice received intramuscular injections of adenovirus expressing VEGF (AdVIC) or CD8 control (AdCD8) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A-Fc (10 mg/kg of average muscle tissue weight) or control Fc protein. (F) Immunofluorescence staining of endothelial cells (CD31, in red) and NEM (CD11b, in green) on frozen sections of muscles 3 weeks after injection of adenoviral vectors and with Fc or Sema3A-Fc treatment. White arrows indicate NEM. Scale bar = 50 μm.