142 H. Williams et al. Figure 4 MMP-7 inhibitor (MMP-7i) retards N-cadherin cleavage and VSMC apoptosis. (A) Representative western blot (n ¼ 3) for full length and 35 kDa C-terminal fragment of N-cadherin protein in human VSMCs 4 h after the addition of Fas-L with or without 10 nM MMP-7 inhibitor (MMP-7i). (B) Representative western blot (n ¼ 3) for full length and 35 kDa C-terminal fragment of N-cadherin protein in mouse wild-type VSMCs grown in suspension (Susp) to induce apoptosis and treated with 10 nM MMP-7 inhibitor. Bar charts (A and B) show densitometric analysis of western blots, data are shown as percent of control and * indicates a signiﬁcant difference (P , 0.05) from Fas-L or suspension culture alone. (C ) Cleaved caspase-3 activity in human VSMCs treated with Fas-L with or without 10 nM MMP-7 inhibitor for 8 h, * indicates a signiﬁcant difference (P , 0.05) from Fas-L alone, n ¼ 4. (D) Representative western blots (n ¼ 3) for pAkt, and total Akt. Bar chart shows densitometric analysis of western blots, data are shown as percent of control and * and $ indicate a signiﬁcant difference (P , 0.05) from control and Fas-L, respectively. b-Tubulin is shown as loading control. All control samples were supplemented with DMSO to act as a vehicle control. variation in the response of the MMP-12 knockout VSMCs, we deter- mined the effect of a MMP-12 inhibitor on cleaved caspase-3 activity. No difference in cleaved caspase-3 activity was observed [699316 + 288179 vs. 690540 + 258043 luminescent arbitrary units (LAU)]. We investigated whether addition of a human recombinant MMP-7 affected N-cadherin cleavage and apoptosis. Addition of recombinant MMP-7 caused a signiﬁcant increase in cleavage of N-cadherin, as shown by a reduction in full-length N-cadherin (Figure 3A) and pro- duction of the 35 kDa fragment (Figure 3B). Cycloheximide (which was added to all conditions to inhibit replenishment of N-cadherin by protein synthesis) alone had no signiﬁcant effect on N-cadherin levels compared with control (data not shown). Increased N-cadherin cleavage was associated with signiﬁcantly augmented apoptosis (Figure 3C). To establish whether MMP-7 activity is increased during Fas-L induced apoptosis, we measured MMP-7 in the conditioned media of Fas-L-treated VSMCs. MMP-7 activity was signiﬁcantly increased in Fas-L treated VSMCs (Figure 3D), conﬁrming that MMP-7 activity is elevated during VSMC apoptosis. To establish whether the cleavage of N-cadherin by MMP-7 is direct or indirect, recombinant N-cadherin was incubated with active MMP-7. We observed that cleavage of N-cadherin occurred in the absence of cells and other proteases and therefore is direct (Figure 3E).