MMP-7 induces N-cadherin cleavage and VSMC apoptosis 141 Figure 3 MMP-7 promotes N-cadherin cleavage and apoptosis. Representative Western blots for full-length N-cadherin (A), and 35 kDa C- terminal fragment of N-cadherin (B) at 4 h after addition of 60 nM recombinant MMP-7. b-Tubulin is shown as loading control. Bar charts show den- sitometric analysis of western blots (A and B), data are shown as % of control and * indicates a signiﬁcant difference (P , 0.05) from control, n ¼ 6. Cleaved caspase-3 activity in human VSMCs treated with MMP-7 for 24 h (C ), * indicates a signiﬁcant difference (P , 0.05) from control, n ¼ 3. MMP-7 activity in human VSMC conditioned media with or without Fas-L for 4 h (D), * indicates a signiﬁcant difference (P , 0.05) from control, n ¼ 4. (E) Representative western blot for Fc domain of mouse IgG after 30 min incubation of 2 nM MMP-7 with 2 nM recombinant N-cadherin (SNC-Fc). Supplementary material online, Figure S1) or with a caspase inhibitor or g-secretase inhibitor (data not shown). 3.3 MMP-7 contributes to N-cadherin cleavage and apoptosis To identify which MMP was responsible for N-cadherin cleavage, we used aortic VSMCs isolated from a selection of MMP knockout mice. We ﬁrst ensured that we obtained the same effect in wild-type (C57BL/6J) mouse VSMCs as observed in human VSMCs. As seen in human VSMCs, we observed a signiﬁcant increase in N-cadherin fragment and a decrease in full-length N-cadherin in mouse VSMCs following Fas-L treatment, and this increase was inhibited by BB-94 (Figure 2B). Increased N-cadherin fragment and decreased full-length N-cadherin levels were also observed in VSMCs from MMP-3, MMP-9, and MMP-12 knockout mice after treatment with Fas-L (Figure 2C). However, this was not observed in VSMCs from MMP-7 knockout mice (Figure 2C and D). Importantly, we observed that basal levels of full-length N-cadherin in the various cell types used were comparable (wild-type controls: 15.3 + 3.1, MMP-32/2: 15.9 + 1.1, MMP-72/2: 14.5 + 1.6, MMP-92/2: 15.0 + 3.8, and MMP-122/2: 14.0 + 2.7, O.D. mm2, Figure 2C). Additionally, there was no difference in N-cadherin cleavage in the different wild-type VSMCs compared with the C57BL/6J control cells (see Supplemen- tary material online, Figure S2). Furthermore, supplementation with BB-94 did not signiﬁcantly affect the amount of N-cadherin fragment detected after Fas-L treatment of MMP-7 knockout VSMCs (Figure 2D). Cleaved caspase-3 activity was measured 8 h after induction of apoptosis with Fas-L using the Caspase-Glo assay. Cleaved caspase-3 was signiﬁcantly reduced in MMP-7 knockout VSMCs compared with control VSMCs (Figure 2E). In contrast, there was no statistical difference in cleaved caspase-3 in VSMCs from MMP-3, -9, and -12 knockouts compared with controls. Due to a large