Fonseca et. al. Enzymatic, physicochemical and biological properties of MMP-sensitive alginate hydro

Lower than 005 3 results 31 pvglig cleavage by dierent

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Unformatted text preview: cubated with FRET-PVGLIG (Fig. 1), different rates of enzymatic cleavage were observed: it was rapidly cleaved, albeit at different rates, by the two gelatinases (MMP-2 and MMP-9), by collagenase 3 (MMP-13) and by type-I transmembrane MMP-14 (MT1-MMP), but only minimally cleaved by collagenases MMP-1 and MMP-8. The kinetic parameters for FRET-PVGLIG cleavage are depicted in Table 1. The PVGLIG sequence showed to be a good 3286 | Soft Matter, 2013, 9, 3283–3292 Paper Fig. 1 Fluorescence-based measurements of FRET peptide digestion in the presence of different MMPs. While the commercial FRET-PLGL substrate (inset) was rapidly cleaved by all the tested enzymes, the FRET-PVGLIG peptide was cleaved at different rates by MMP-2, MMP-9, MMP-13 and MMP-14, and only minimally cleaved by MMP-1 and MMP-8. Table 1 Kinetic parameters for the cleavage of FRET-PVGLIG by different MMPs MMP kcat (sÀ1) Km (mM) kcat/Km (MÀ1 sÀ1) MMP-1 MMP-2 MMP-8 MMP-9 MMP-13 MMP-14 0.02 Æ 0.00 0.89 Æ 0.02 0.08 Æ 0.00 2.10 Æ 0.05 0.58 Æ 0.08 1.48 Æ 0.07 9.4 Æ 0.3 5.3 Æ 0.2 25.9 Æ 0.8 4.3 Æ 0.2 4.7 Æ 1.0 5.8 Æ 0.5 2.5 Â 103 1.7 Â 105 3.0 Â 103 4.9 Â 105 1.2 Â 105 2.6 Â 105 substrate for MMP-2, MMP-9, MMP-13 and MMP-14, presenting high kcat/Km ratios on the order of 105 MÀ1 sÀ1.21 3.2 hMSC expression of MMP-2 and MMP-14 under basal and osteogenic conditions Zymography analysis of hMSC CM from days 1 to 21 revealed the presence of signicant amounts of MMP-2 zymogens (proMMP-2: 72 kDa28), but not the active form (64 kDa28). The levels detected in undifferentiated hMSC cultures (BM) were always higher than those from osteogenic (OM) cultures (Fig. 2A). No other secreted MMPs were detected, namely MMP9 (proMMP-9: 92 kDa, MMP-9: 83 kDa28) or MMP-13 (proMMP13: 53 kDa, MMP-13: 48 kDa29). As a membrane-type enzyme, MMP-14 could not be detected with this technique. The soluble FRET-PVGLIG (Fig. 2B) was cleaved by hMSCsecreted proteases present in CM collected at days 1, 7, 14 and 21, while the enzymatic digestion of the scrambled FRETGIVGPL peptide was only minimal (Fig. 2B, inset). hMSC osteogenic differentiation upon induction was conrmed by ALP and vK positive stainings (Fig. 2C), and by the gradual increase in ALP activity along the 21 days of culture in OM. ALP activity under OM conditions peaked around the 1st and 3rd weeks of culture and was always higher than under basal conditions, as expected (Fig. 2D). The qRT-PCR analysis of MMP-2 and MMP-14 mRNA levels showed that the two MMPs were continually expressed throughout the culture (Fig. 3). An apparent down-regulation of MMP-2 expression (Fig. 3A) occurred under osteogenic This journal is ª The Royal Society of Chemistry 2013 View Article Online Paper Soft Matter Downloaded by State University of New York at Buffalo on 16/04/2013 03:56:51. Published on 08 February 2013 on http://pubs.rsc.org | doi:10.1039/C3SM27560D Fig. 3 qRT-PCR analysis of endogenous MMPs gene expression in basal and osteogenic medium in 2D MSC cultur...
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