Fig. 4

Involvement of RhoA, Rac1, αV integrin and CD44 on capillary morphogenesis after Marimastat treatment. A Western blotting of total and GTP-loaded forms of small Rho-GTPases RhoA and Rac1 in control conditions and after ECFC treatment with Marimastat. RhoA-GTP and Rac1-GTP, GTP-loaded forms of small Rho GTP-ases; RhoA and Rac, total un-loaded forms of small Rho GTP-ases, used as a reference loading control. Numbers on the left refer to molecular weights expressed in kDa. Histograms report band densitometry. Results are the mean of 5 different experiments performed in duplicate and are shown as mean value ± SD. *p < 0.05 significantly different from control. B In vitro angiogenesis of ECFCs in the presence of Rho Activator II (5 µg/ml) and Rho inhibitor I (1 µg/ml) was measured by capillary morphogenesis after 6 h in mesenchymal conditions (CTRL) and amoeboid conditions induced by Marimastat. Representative microphotographs (×10) of capillary-like structures are shown. Capillary network was quantified by Angiogenesis Analyzer Image J tool. Histograms represent the mean number of number of meshes, total length, and total branching length, and total meshes area respectively. Data are representative of measures obtained from at least nine fields. C The left panel shows western blotting analysis of integrin alphaV and CD44 after siITGAV and siCD44 treatment. LIPO: treatment of cells with the transfection reagent alone, lipofectamine. After silencing, ECFCs were subjected to capillary morphogenesis The assay was performed as usually and results are shown as described in B. Results are the mean of 5 different experiments performed in duplicate and are shown as mean value ± SD. *p < 0.05; **p < 0.001; ***p < 0.0001 significantly different from control or the experimental point indicated