Sema3A/NRP1 binding is required for NEM recruitment, TGF-β1 upregulation, and Sema3A production.Figure 4Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.A, B Quantification of recruited NEM in sites of new angiogenesis by increasing VEGF doses, expressed as number of CD11b+ cells/mm2 of area (A) or /cm of vessel length (B). Data represent the mean ± SEM of individual images (n) acquired from three muscles/group. (A) Ctrl IgG2a, n = 11; Ctrl AbαNRP1A, n = 17; V Low IgG2a, n = 24; V Low AbαNRP1A, n = 28; V Med IgG2a, n = 25; V Med AbαNRP1A, n = 34; *P < 0.05, **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG2a versus Ctrl IgG2a P = 0.0068; V Low IgG2a versus V Low AbαNRP1A P = 0.0068; V Med IgG2a versus V Med AbαNRP1A P = 0.0460. (B) V Low IgG2a versus V Low AbαNRP1A P < 0.0001; V Low IgG2a versus V Med IgG2a P = 0.0193; V Med IgG2a versus V Med AbαNRP1A P = 0.0046. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.C Quantification of vessel length density generated by increasing VEGF doses after the different treatments. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: Ctrl IgG2a, n = 6; Ctrl AbαNRP1A, n = 6; V Low IgG2a, n = 19; V Low AbαNRP1A, n = 20; V Med IgG2a, n = 20; V Med AbαNRP1A, n = 25; *P < 0.05, **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG2a versus Ctrl IgG2a P < 0.0001; V Low AbαNRP1A versus Ctrl IgG2a P < 0.0001; V Low IgG2a versus V Med IgG2a P = 0.0014; V Low AbαNRP1A versus V Med AbαNRP1A P = 0.0179. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.D Tgfb1 gene expression in total skeletal muscles after the different treatments, expressed as fold-change versus the Ctrl IgG2a group. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; V Low IgG2a versus Ctrl IgG2a P = 0.0083; V Low IgG2a versus V Low AbαNRP1A P = 0.0315. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.E Immunohistochemistry for Sema3A protein on frozen muscle sections with H&amp;amp;E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 100 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 3; Ctrl IgG2a, n = 23; Ctrl AbαNRP1A, n = 12; V Low IgG2a, n = 53; V Low AbαNRP1A, n = 38; *P < 0.05 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG2a versus No stain P = 0.0007; V Low IgG2a versus Ctrl IgG2a P < 0.0001; V Low AbαNRP1A versus Ctrl IgG2a P = 0.0142; V Low IgG2a versus V Low AbαNRP1A P = 0.0001.