Cardiovascular Research (2010) 87, 137–146 doi:10.1093/cvr/cvq042 MMP-7 mediates cleavage of N-cadherin and promotes smooth muscle cell apoptosis Helen Williams , Jason L. Johnson , Christopher L. Jackson , Stephen J. White , and Sarah J. George * Bristol Heart Institute, University of Bristol, Bristol Royal Inﬁrmary, Research Floor Level 7, Upper Maudlin St., Bristol BS2 8HW, UK Received 22 July 2009; revised 26 January 2010; accepted 29 January 2010; online publish-ahead-of-print 5 February 2010 Time for primary review: 34 days Aims Vascular smooth muscle cell (VSMC) apoptosis can lead to thinning of the ﬁbrous cap and plaque instability. We previously showed that cell – cell contacts mediated by N-cadherin reduce VSMC apoptosis. This study aimed to determine whether matrix-degrading metalloproteinase (MMP)-dependent N-cadherin cleavage causes VSMC apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods Induction of human VSMC apoptosis using different approaches, including 200 ng/mL Fas ligand (Fas-L) and culture in and results suspension, caused N-cadherin cleavage and resulted in the appearance of a C-terminal fragment of N-cadherin ( 35 kDa). Appearance of this fragment during apoptosis was inhibited by 47% with the broad-spectrum MMP inhibi- tor BB-94. We observed retarded cleavage of N-cadherin after treatment with Fas-L in aortic mouse VSMCs lacking MMP-7. Furthermore, VSMC apoptosis, measured by quantiﬁcation of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L. Addition of recombinant active MMP-7 increased the amount of N-cadherin fragment by 82% and augmented apoptosis by 53%. The involve- ment of MMP-7 was corroborated using human cells, where a MMP-7 selective inhibitor reduced the amount of fragment formed by 51%. Importantly, we observed that treatment with Fas-L increased levels of active MMP-7 by 80%. Finally, we observed signiﬁcantly increased cleavage of N-cadherin, MMP-7 activity, and apoptosis in human atherosclerotic plaques compared with control arteries, and a signiﬁcant reduction in apoptosis in atherosclerotic plaques from MMP-7 knockout mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion This study demonstrates that MMP-7 is involved in the cleavage of N-cadherin and modulates VSMC apoptosis, and may therefore contribute to plaque development and rupture. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Keywords Vascular smooth muscle † Apoptosis † Atherosclerosis † Matrix-degrading metalloproteinase-7 † N-cadherin 1. Introduction Rupture of atherosclerotic plaques is the major cause of myocardial infarction and stroke.1 Vascular smooth muscle cells (VSMCs) form a protective ﬁbrous cap over atherosclerotic plaques. VSMC apopto- sis in the ﬁbrous cap2 and subsequent weakening of the cap3 is thought to be an important regulator of plaque stability. In normal blood vessels VSMC apoptosis is rare, however increased apoptosis is observed in unstable human atherosclerotic plaques.4 Recently, mouse models of atherosclerosis have provided direct evidence that VSMC apoptosis causes plaque instability.5 – 7 Consequently, reducing VSMC apoptosis is an attractive strategy for attenuating plaque instability. A greater understanding of the mechanisms underlying apoptosis of VSMCs in the ﬁbrous cap may aid the development of new therapies for reducing plaque instability. We have previously demonstrated that N-cadherin, a predominant cell – cell junction protein in VSMCs,8 provides a vital pro-survival signal to VSMCs via PI3-kinase-dependent activation of Akt.9 Over- expression of N-cadherin reduced apoptosis of VSMCs in suspension culture, while conversely, over-expression of dominant negative * Corresponding author. Tel: þ44 117 3423154; fax: þ44 117 3423581, Email: s.j.george@bris.ac.uk Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: journals.permissions@oxfordjournals.org. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non- commercial purposes provided that the original authorship is properly and fully attributed; the Journal, Learned Society and Oxford University Press are attributed as the original place of publication with correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org.