Fig. 2

Evaluation of the effect of groups of protease inhibitors and Marimastat on in vitro invasion and angiogenesis. A Boyden chamber invasion assay through a thick Matrigel coating, in the presence of groups of the following protease inhibitors: TIMP1, TIMP2, TIMP3, alpha2-antiplasmin, PAI-1 and cystatin, and with all inhibitors mixed together (MIX) added to the Matrigel solution before polymerization. Histogram refers to quantification of Matrigel invasion assay obtained by counting the total number of migrated cells/filter. B In vitro angiogenesis measured by capillary morphogenesis at 24 h in the presence and in the absence of the same groups of inhibitors tested in A. Here it is shown the histogram representing the mean number of master junctions. Angiogenesis Analyzer Image J tool was used for the quantification of capillary network. Quantification was performed at 24 h after seeding and data are representative of measures obtained from at least nine photographic fields for each condition. C Cell viability was tested after Marimastat cell treatment for 6 (similar results not shown) and 24 h and evaluated by Trypan blue dye exclusion assay. The columns of histograms show in white the percentage of live cells and in black the percentage of dead cells. DMSO is to be considered the real control since it was used as Marimastat solvent. To be noted that in the next figures CTRL is to be considered as DMSO. D Histogram shows the collagenolytic activity of endothelial cells under mesenchymal (CTRL) and amoeboid conditions (TIMP1, TIMP2, TIMP3 or Marimastat), expressed as % collagen degradation with respect to the positive control obtained by addition of exogenous collagenase. Ctrl−: collagenolytic activity in the absence of cells and exogenous collagen; Ctrl+: collagenolytic activity in the absence of cells but in the presence of exogenous collagenase; CTRL: ECFCs; TIMP1-2-3 or Marimastat: collagenolytic activity of ECFCs in the presence protease inhibitors indicated. E Boyden chamber invasion assay through a thick Matrigel coating, in the presence of VEGF, here used as positive control, and Marimastat. The assay was performed as described in A. F Capillary network was quantified by Angiogenesis Analyzer Image J tool. Histograms represent the mean number of number of nodes, number of meshes, total length, and total branching length, respectively, at 24 h. Representative microphotographs (×10) of capillary-like structures at 6 and 24 h are shown. Data are representative of measures obtained from at least nine fields. G Effect of Marimastat on cell proliferation was quantified by AlamarBlue® assay and fluorescence was measured at 530 nm/590 nm. n = 3 independent samples