Ilomastat

At the day 0 of chondrogenic differentiation induction, hMSC-collagen microspheres were divided in inhibitor treatment groups. Microspheres were maintained in CM for 28 days; each group treated with protease inhibitors at an optimal concentration previously determined: E-64D (Aloxistatin, Cayman chemical, 20mM) and/or GM6001 (Ilomastat, Millipore, 25mM). Inhibitor stock solutions were prepared with DMSO at the concentration of 1mg/ml. One control group was treated with DMSO alone to check the effect of DMSO. The medium with inhibitors was freshly prepared and changed three times a week.

Protease activity in culture supernatants was determined by azocasein assay. Briefly, 350 µl reaction mixture containing 0.1 M Tris-HCl pH 8.0 and 1% azocasein (Sigma-Aldrich, cat. n. A2765) resuspended in 0.5% NaHCO₃ was added to 150 µl supernatant with/without MMPIs Ilomastat, Marimastat, Batimastat (Sigma-Aldrich, cat. n. M5939, M2699, SML0041) and incubated at 37°C for 20 minutes shaking. After addition of 1 ml 7% ice-cold perchloric acid, the solution was centrifuged. 150 µl 10 N sodium hydroxide were added to the clear supernatant and OD at 430 nm was measured. One protease unit was calculated as the amount of enzyme producing an increase of 1 OD unit per hour.

Serum starved ASM cells were suspended in a neutralized solution of bovine collagen-I (Sigma-Aldrich; C4243) and 10x DMEM (Sigma-Aldrich) and cast in 24-well tissue culture plates to create 7 mm-thick three dimensional (3D) collagen gels. Gels were allowed to set for 24 hours before being overlaid with DMEM. After 16 hours, gels released from wells and transferred to 6-well plates containing DMEM/F12 (Sigma-Aldrich) [36] (link). Contraction was stimulated with bradykinin (Sigma-Aldrich). Gel contraction was quantitated over 60 minutes using Genesnap camera (Genetools, Syngene, and Cambridgeshire, UK) with the reduction in initial gel area quantitated using Image J (http://rsb.info.nih.gov/ij). In experiments involving MMP inhibition, gels were treated with 10 µM ilomastat (Sigma-Aldrich) 1 hour prior to bradykinin stimulation.

Ilomastat (Sigma-Aldrich, St. Louis, MO), a broad spectrum MMPs inhibitor, was dissolved in a special solvent made by our institute [21 (link)]. For Ilomastat administration, mice were given 10 mg/kg of Ilomastat via intraperitoneal injection 2 h before irradiation referring to our previous study [1 (link)].

Protease-indipendent angiogenic properties and invasion were evaluated by in vitro capillary morphogenesis and 3D-Boyden chamber assays, as described above, with Matrigel coating in the presence of a physiological protease inhibitor cocktail, consisting of α2-antiplasmin (plasmin inhibitor; 5 μg/ml), Cystatin (cysteine protease inhibitor; 5 μM), PAI 1 (plasminogen activator inhibitor; 10 ng/ml), TIMP1, TIMP2 and TIMP3 (metallo-protease inhibitors; 0,5 μg/ml each one) purchased by Abcam. A completely artificial protease inhibitor cocktail (composed by Ilomastat, leupeptin, pepstatin A, E-64 and aprotinin; Sigma Aldrich) used in a previous study [13 (link)] was also used. Concentrations used were selected according to the manufacturer’s instructions and literature [16 (link), 17 (link)]. Protease inhibitor cocktails were added to un-polymerized Matrigel solution on the upper surface of the porous filter. To induce the amoeboid phenotype, cells were treated overnight with the protease inhibitor cocktails at the same concentrations used in the invasion assay. Cell viability upon protease inhibitors treatment was evaluated by Trypan blue dye (Sigma) exclusion assay.