MMP-7 induces N-cadherin cleavage and VSMC apoptosis apoptosis of endothelial cells. Furthermore, N-cadherin cleavage during activated hepatic stellate cell apoptosis is promoted by MMP-235 and inhibited by the endogenous inhibitor of MMPs (TIMP-1).12 In our study, combined treatment with Fas-L and a global non-speciﬁc MMP inhibitor signiﬁcantly reduced N-cadherin cleavage, while plasmin, caspase, and g-secretase inhibitors had no effect, indicating that MMP activity is involved in N-cadherin cleavage. Using VSMCs from MMP knockout mice, we identiﬁed a role for MMP-7 in the cleavage of N-cadherin during VSMC apoptosis. In support of this, addition of recombinant active MMP-7 caused loss of full-length N-cadherin, appearance of the N-cadherin fragment, and increased apoptosis in human VSMCs while a MMP-7 inhibitor reduced Fas-L-induced N-cadherin cleavage and apoptosis in human VSMCs. Incubation of recombinant N-cadherin with MMP-7 revealed that cleavage of N-cadherin was direct and did not require the pres- ence of other proteases. Interestingly, we also showed that as a result of Fas-L treatment, MMP-7 activity is augmented in human VSMCs. We observed that as a result of N-cadherin cleavage, pAkt levels were reduced, conﬁrming our previous ﬁnding that N-cadherin acts as a survival signal via activation of Akt.9 Together this data clearly indicates that MMP-7 contributes to the cleavage of N-cadherin and the induction of VSMC apoptosis. Although previous studies have shown that MMP-7 cleaves E-cadherin,28,34 this is the ﬁrst demon- stration that MMP-7 can cleave N-cadherin. Recently, we observed that MMP-9- and MMP-12-dependent cleavage of N-cadherin occurs during VSMC proliferation.15 Interestingly, in this study, we observed no involvement of MMP-9 and MMP-12 in N-cadherin cleavage during VSMC apoptosis. This indicates that different MMPs are responsible for N-cadherin cleavage during these different cellular processes of proliferation and apoptosis. Polymorphisms of MMP-7 have been associated with coronary artery disease.36 Moreover although MMP-7 is absent in control arteries, it is present in atherosclerotic plaques.37 More speciﬁcally, MMP-7 was expressed speciﬁcally in lipid-laden macrophages con- ﬁned to the sites susceptible to rupture; the border between the acel- lular lipid cores and the ﬁbrous areas.37 They proposed therefore that MMP-7, speciﬁcally expressed in atherosclerotic lesions, could cleave structural proteoglycans including versican, potentially leading to sep- aration of caps and shoulders from lipid cores and plaque instability. Additionally, ApoE38 and apolipoprotein C-II39 are substrates for MMP-7 which will affect atherosclerotic plaque formation and stab- ility. We observed elevated MMP-7 activity in atherosclerotic plaques compared with controls, which correlated and co-located with increased apoptosis and N-cadherin cleavage. We therefore suggest that in addition to matrix and apolipoprotein cleavage, MMP-7 further contributes to plaque instability by induction of apop- tosis of VSMCs as a result of N-cadherin cleavage. MMP-7 may also exert a pro-apoptotic effect through other proteolytic mechanisms. For example, it has been shown that MMP-7 can cleave Fas-L, releas- ing soluble Fas-L and thus facilitating Fas-induced apoptosis.40 Indeed, in our study, we have utilized this well characterized death pathway as an apoptotic stimulus. Moreover, as stated earlier, MMP-7 has numer- ous other substrates which are present in atherosclerotic plaques and which cleavage of may inﬂuence cell survival. Deletion of MMP-7 in ApoE knockout mice led to a 78% increase in VSMC content of the plaque.16 We demonstrated in this study that MMP-7 deﬁciency attenuated apoptosis, indicating that MMP-7 plays a pro-apoptotic role in atherosclerotic plaques in vivo. Despite the increased numbers of VSMCs in atherosclerotic plaques of MMP-7 145 knockout mice however, there was no effect on plaque growth or stability.16 MMP-7 gene deletion may have other effects on other cell types, apart from VSMCs, within the plaques which are beneﬁcial for plaque stability or may have other effects on arterial physiology that counteract the beneﬁcial changes in VSMC content. For example, MMP-7 has been implicated in the maintenance of arterial tone, and therefore MMP-7 knockout mice may suffer increased tensile force across the ﬁbrous cap as a result of reduced medial tone.41 Hence, the greater stability conferred on the lesion by increased VSMCs may be negated by increased strain on the ﬁbrous cap. In summary, we have shown for the ﬁrst time that MMP-7 activity is up-regulated during apoptosis of VSMCs. Moreover, we have observed that MMP-7 causes cleavage of N-cadherin which is associ- ated with increased VSMC apoptotic rates. We propose that MMP-7 may play a key role in atherosclerotic plaque development and rupture via N-cadherin cleavage and induction of VSMC apoptosis. Supplementary material Supplementary material is available at Cardiovascular Research online. Conﬂict of interest: none declared. Funding This work was supported by the British Heart Foundation (PG/05/103). Funding to pay the open access publication charge was provided by the British Heart Foundation (PG/05/103). References 1. Virmani R, Burke AP, Farb A, Kolodgie FD. Pathology of the vulnerable plaque. J Am Coll Cardiol 2006;47:C13–C18. 2. Geng Y, Libby P. Evidence for apoptosis in advanced human atheroma. Co-localisation with interleukin-1 b converting enzyme. Am J Pathol 1995;147:251–266. 3. Dhume A, Soundararajan K, Hunter W, Agrawal D. Comparison of vascular smooth muscle cell apoptosis and ﬁbrous cap morphology in symptomatic and asymptomatic carotid artery disease. Ann Vasc Surg 2003;17:1–8. 4. Bauriedel G, Hutter R, Welsh U, Bach R, Sievert H, Luderitz B. Role of smooth muscle cell death in advanced coronary primary lesions: implications for plaque stab- ility. Cardiovasc Res 1999;41:480–488. 5. von der Thusen JH, van Vlijmen BJM, Hoeben RC, Kockx MM, Havekes LM, van Berkel TJC et al. Induction of atherosclerosis plaque rupture in apolipoprotein E2/2 mice after adenovirus-mediated transfer of p53. Circulation 2002;105: 2064– 2070. 6. Zadelaar A, von der Thusen J, Boesten L, Hoeben R, Kockx M, Versnel M et al. Increased vulnerability of pre-existing atherosclerosis in ApoE knockout mice follow- ing adenovirus-metiated Fas ligand gene transfer. Atherosclerosis 2005;183:244– 250. 7. Clarke M, Figg N, Maguire J, Davenport A, Goddard M, Littlewood TD et al. Apop- tosis of vascular smooth muscle cells induces features of plaque vulnerability in ather- osclerosis. Nature Med 2006;12:1075– 1080. 8. Uglow EB, Slater S, Sala-Newby GB, Aguilera-Garcia CM, Angelini GD, Newby AC et al. Dismantling of cadherin-mediated cell-cell contacts modulates smooth muscle proliferation. Circ Res 2003;92:1314–1321. 9. Koutsouki E, Beeching CA, Slater SC, Blaschuk OW, Sala-Newby GB, George SJ. N-cadherin-dependent cell-cell contacts promote human saphenous vein smooth muscle cell survival. Arterioscler Thromb Vasc Biol 2005;25:982 –988. 10. Lyon CA, Johnson JL, Williams H, Sala-Newby GB, George SJ. Soluble N-cadherin over-expression reduces features of atherosclerotic plaque instability. Arterioscler Thromb Vasc Biol 2009;29:195–201. 11. Makrigiannakis A, Coukos G, ChristoﬁdouSolomidou M, Gour BJ, Radice GL, Blaschuk O et al. N-cadherin-mediated human granulosa cell adhesion prevents apoptosis - A role in follicular atresia and luteolysis? Am J Pathol 1999;154:1391–1406. 12. Murphy F, Waung J, Collins J, Arthur MJP, Nagase H, Mann D et al. N-cadherin clea- vage during activated hepatic stellate cell apoptosis is inhibited by tissue inhibitor of metalloproteinase-1. Comp Hepatol 2004;3:S8. 13. Herren B, Levkau B, Raines EW, Ross R. Cleavage of b-catenin and plakoglobin and shedding of VE-cadherin during endothelial apoptosis: evidence for a role for caspases and metalloproteinases. Mol Biol Cell 1998;9:1589–1601.