Inhibitory effects of caspase inhibitors on the activity of matrix metalloproteinase-2

Matrix metalloproteinase (MMP)-2, a zinc-dependent endopeptidase, plays a detrimental role in several diseases including ischemia and reperfusion (I/R) injury of the heart. Caspases are a group of cysteine-dependent, aspartate-directed proteases which regulate cellular Apoptosis. Interestingly, protective effects of Caspase inhibitors independent of Apoptosis have been shown in I/R injury of the heart. The cardioprotective actions of both these classes of Protease Inhibitors led us to hypothesize that Caspase inhibitors may also reduce MMP-2 activity. Five known Caspase inhibitors (Z-IE(OMe)TD(OMe)-fmk, Ac-DEVD-CHO, Ac-LEHD-cmk, Z-VAD-fmk and Ac-YVAD-cmk) were tested for their possible inhibitory effects on MMP-2 activity in comparison to the MMP inhibitors ONO-4817 and ARP-100 (which themselves were unable to inhibit Caspase-3 activity). MMP-2 activity was assessed by an in vitro troponin I (TnI) proteolysis assay and a quantitative kinetic fluorescence assay using a fluorogenic peptide substrate (OmniMMP). TnI proteolysis was also measured by western blot in neonatal cardiomyocytes subjected to hypoxia-reoxygenation injury. Using human recombinant MMP-2 and TnI as its substrate, the Caspase inhibitors, in comparison with ONO-4817, significantly inhibited MMP-2-mediated TnI degradation in a concentration-dependent manner. The kinetic assay using OmniMMP revealed that these Caspase inhibitors blocked MMP-2 activity in a concentration-dependent manner with similar IC50 values. TnI degradation in neonatal cardiomyocytes was enhanced following hypoxia-reoxygenation and this was blocked by ARP-100 and Ac-LEHD-cmk. Inhibition of MMP-2 activity is an additional pharmacological action which contributes to the protective effects of some Caspase inhibitors.

Caspase inhibitors; Caspases; Hypoxia-reoxygenation; I/R; MMPs; Matrix metalloproteinase-2; TnI; Troponin I; ischemia and reperfusion; matrix metalloproteinases; troponin I.