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Fig. 3. NO/cGMP increase mesoangioblast migration and myogenic differentiation in vitro. (A-C) Mesoangioblast migration on gelatin-coated membranes. (A) Comparison of the chemoattractant properties of VEGF (10 ng/ml), TGFß (100 ng/ml), TNF (10 ng/ml), HGF (10 ng/ml) or bFGF (10 ng/ml) measured in a 6-hour migration assay (n=5). (B) Effect of pretreatment of mesoangioblasts for 12 hours without (NT) or with DETA-NO (20 µM), IMN (50 µM), 8-Br-cGMP (1 mM) or DETA-NO plus ODQ (1 µM). Migration is expressed as the percentage of that observed in untreated controls (NT) (n=5). (C) Representative images of the transmigrated mesoangioblasts after staining with crystal violet. (D,E) Mesoangioblast transmigration through an H5V endothelial cell monolayer. In these experiments, LacZ-expressing mesoangioblasts, pretreated with or without DETA-NO, IMN, 8-Br-cGMP and ODQ as above were used. Transmigration induced by TNF, VEGF, HGF (D) or L6E9 myoblasts and myotubes (E) was measured after 6 hours. The migration index was calculated as described in the Materials and Methods (n=4). (F,G) Mesoangioblasts pretreated for 12 hours without (NT) or with DETA-NO (20 µM), IMN (50 µM), 8-Br-cGMP (1 mM) or DETA-NO plus ODQ (1 µM) were co-cultured for 5 days in differentiating conditions together with rat L6E9 myoblasts (co-fusion conditions, F) or preformed L6E9 myotubes (post-fusion conditions, G). The graphs show the fusion index, i.e. the number of murine mesoangioblast-derived nuclei in myosin-expressing cells with more than two nuclei vs. the total number of rat and murine nuclei measured (n=5). Double and triple asterisks and crosses, P<0.01 and P<0.001, respectively vs. NT (asterisks) and DETA-NO-treated cells (crosses). Error bars, s.e.m. Bar in C, 200 µm.

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