MMP-7 induces N-cadherin cleavage and VSMC apoptosis 143 Figure 5 N-cadherin, MMP-7, and apoptosis in human IMA and atherosclerotic plaques. Representative western blot for N-cadherin (A) and cleaved caspase-3 (B) in IMA and coronary and carotid atherosclerotic plaques, n ¼ 3 per group. b-Tubulin is shown as loading control. Bar chart in (A) shows densitometric analysis of N-cadherin western blots. (C ) MMP activity in IMA and atherosclerotic plaques, n ¼ 15 and 20. * indicates a signiﬁcant differ- ence from IMA. To further study the involvement of MMP-7 in N-cadherin cleavage and VSMC apoptosis, we utilized a MMP-7 inhibitor. Apoptosis was induced in both human and mouse VSMCs using Fas-L and suspension culture, respectively, and in both cases the MMP-7 inhibitor signiﬁcantly reduced the amount of N-cadherin fragment and increased full-length N-cadherin (Figure 4A and B). Interestingly, the degree of inhibition of N-cadherin fragment formation observed with the MMP-7 inhibitor was comparable to that seen with BB-94 (Figure 4B), which suggests that MMP-7 is the primary MMP involved in N-cadherin cleavage. No further inhibition of N-cadherin cleavage was observed with co-treatment with the MMP-7 inhibitor and the plasmin inhibitor NALME (see Supplementary material online, Figure S1). Addition of a MMP-2 inhibitor had no signiﬁcant effect (data not shown). To ascertain whether reduced N-cadherin cleavage was associated with attenuated apoptosis in human VSMCs, cleaved caspase-3 was measured. As seen in the MMP-7 knockout mouse VSMCs, inhibition of MMP-7 in human VSMCs resulted in a signiﬁcant reduction in cleaved caspase-3 following addition of Fas-L (Figure 4C). Fas-L caused a signiﬁcant decline in pAkt, whereas signiﬁcantly elevated levels of pAkt were observed when VSMCs were treated with the MMP-7 inhibitor and Fas-L (Figure 4D). In contrast, active MMP-7 reduced pAkt levels (Figure 4D). 3.4 N-cadherin, apoptosis, and MMP-7 in atherosclerotic plaques Human carotid and coronary arterial plaques and IMAs were sub- jected to western blotting to examine N-cadherin and cleaved caspase-3 protein expression (Figure 5A and B). Full-length N-cadherin protein was seen in both atherosclerotic plaques and healthy arteries, however signiﬁcantly lower levels of full-length N-cadherin was detected in plaques compared with IMA (Figure 5A). Moreover, cleaved N-cadherin was only detected in the atherosclerotic arteries (Figure 5A). Cleaved caspase-3 was also signiﬁcantly higher in athero- sclerotic plaques compared with IMAs (86 + 33 vs. 4 + 2 O.D. mm2, Figure 5B). Additionally, MMP-7 activity was signiﬁcantly higher in atherosclerotic plaques than IMAs (Figure 5C). We assessed MMP activity using in situ zymography. We observed MMP activity in atherosclerotic plaques while little MMP activity was detected in IMAs (1681 + 518 vs. 37 + 12 FAU, Figure 6A and D). A signiﬁcant amount of the MMP activity was mediated by MMP-7 since MMP activity was signiﬁcantly reduced by 70% with the MMP-7 inhibitor (571 + 218 vs. 1681 + 518 FAU, Figure 6A and B). Apoptotic cells were co-located in areas of MMP activity in athero- sclerotic plaques, whereas MMP activity and apoptosis were low in IMAs (Figure 6C – E). Apoptosis was quantiﬁed in mouse brachiocephalic plaques using ISEL. The percentage of apoptotic plaque cells was signiﬁcantly lower in atherosclerotic plaques from ApoE2/2/MMP-72/2 mice than ApoE2/2/MMP-7þ/þ mice (0.05 + 0.01 vs. 0.12 + 0.03%, Figure 6F – H ). 4. Discussion This study demonstrates a link between MMP-7, N-cadherin cleavage, and VSMC apoptosis in atherosclerotic plaques. We have shown in vitro that during apoptosis, levels of active MMP-7 are elevated and that augmented levels of MMP-7 activity are associated with N-cadherin cleavage and increased apoptosis. Furthermore, we have shown increased MMP-7 activity, apoptosis, and N-cadherin cleavage in atherosclerotic plaques. Although VSMC apoptosis is rare in normal blood vessels, increased apoptosis is observed in unstable human atherosclerotic plaques4 and promotes plaque instability in mouse models of athero- sclerosis.5 – 7 We previously demonstrated that N-cadherin acts as