Fig. 5
AD0157 inhibits in vivo VEGF-C-induced lymphangiogenesis and blocks the lymphangiogenic process ex vivo. a Representative pictures of gelatin sponges soaked with either rhVEGF-C (1 μg/mL) alone (positive control) or rhVEGF-C + AD0157 at different concentrations and implanted between the mouse ear skin layers. Lymphatic and blood vasculatures were examined by LYVE-1 (green) and CD31 (red) immunostainings, respectively. Dapi staining was used to detect cell nuclei (blue). Scale bars represent 1500 and 500 μm on higher magnification. A total of 10 mice were analyzed in each experimental condition. b The graphs represent the computerized quantification of the lymphatic density (left panel), the normalized frequency of lymphatic vessels from the sponge edge to its center (middle panel), and the blood density (right panel). Arrows indicate the maximal distance of LEC migration (Lmax). c Ex vivo lymphatic sprouting from mouse thoracic duct explants embedded in a type I collagen gel and cultured in the absence (control) or presence of AD0157 for 7 days. Scale bars represent 500 μm. d Graphs represent the number of microvessel intersections (Ni) quantified on binarized images using a grid of concentric rings (left graph) and the LEC density at a distance (d) = 0.25 mm from the ring border (right graph). A total of 10 lymphatic rings collected from five different mice were analyzed in each experimental condition. e Microvessel outgrowth in rat mesenteric windows incubated in medium containing either rhVEGF-C (200 ng/mL) alone or in combination with AD0157, ranging from 0.5 to 2.5 μM, for 5 days. Scale bars represent 200 μm. f Lymphatic density and number of branchings in rat mesenteric windows exposed or not to AD0157. A total of 10 mesenteric windows harvested from five different rats were evaluated in each experimental condition. Values are expressed as mean ± s.e.m., and the Wilcoxon-Mann-Whitney significance tests were used to compare the differences between control and AD0157 treatment. *p < 0.05, **p < 0.01, ***p < 0.001