Mca plgl dpa ar nh2

Metalloproteinase activity of HUVECs was determined using the fluorogenic peptide substrate Mca-PLGL-Dpa-AR-NH₂ (ES001; R&D Systems). HUVEC monolayers, at control conditions and at different times after wounding, were washed and incubated with 3 µM fluorogenic peptide diluted in DMSO and TNC buffer (50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂ and 0.002 % NaN₃; pH, 7.5) during 1 h at 37 °C. The reaction was stopped by adding stop solution 10X (100 nM EDTA + 0.02 % NaN₃). The fluorescence signal was read with a Varioskan flash spectral scanning multimode reader (Thermo Scientific).

Recombinant human MMP-12 (R&D Systems, Minneapolis, MN, USA) activity was determined with a quenched fluorogenic peptide Mca-PLGL-Dpa-AR-NH2 (R&D Systems, Minneapolis, MN, USA), as previously described [57 (link)]. Briefly, MMP-12 was activated by incubation with an assay buffer (50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35; pH 7.5) at 37 °C for 30 h. The fluorescence intensity was measured with a Varioskan flash spectral scanning multimode reader (Thermo Scientific, Waltham, MA, USA). When necessary, a mixture of 0.4 ng/mL of activated rhMMP-12 with 6 µM of fluorogenic peptide was incubated with 30 µM endoglin peptides or the MMP-12 inhibitor MMP-408. Endoglin-related peptides P583 (TSKGLVLP), P447 (SLSFQLGLYL), and P230 (GPRTVTVK) with N-terminal acetylation and C-terminal amidation [57 (link)] were synthesized and purified to >90% purity (Biomedal SL, Sevilla, Spain).

Human recombinant MMP9 (hrMMP9; R&D Systems) was activated by adding 1 mM (final concentration) ρ-aminophenylmercuric acetate (APMA) and incubating for 24 h at 37 °C. HrMMP9 activity was confirmed by measuring cleavage of the fluorogenic peptide, Mca-PLGL-Dpa-AR-NH₂ (R&D Systems), according to the manufacturer’s instructions. HDF-depleted ECM was incubated with 10 µg/mL of active hrMMP9, whereas DCN (ab167743, Abcam, Cambridge, UK) and VTN (ab94369, Abcam) was incubated at a 1:2 protein to active hrMMP9 ratio, diluted in MMP buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl₂ and 0.05% (w/v) Brij-35, pH 7.5) at 37 °C with agitation, for 24 h. For HDF ECM assay, the buffer was removed and dialyzed for 4 h against ddH₂O (Thermo Fisher), and samples freeze-dried overnight. DCN and VTN samples were run on 4–12% NuPAGE Novex Bis-Tris Gels (1 µg of protein per well) using standard SDS–PAGE procedures. Nondigested control bands and digested bands were excised using a scalpel blade. ECM or DCN and VTN incubated with MMP buffer alone served as negative protease controls, respectively.