144 H. Williams et al. Figure 6 MMP-7 and apoptosis in atherosclerotic plaques. In situ zymography of coronary atherosclerotic plaque in the absence (A) and presence (B) of 10 nM MMP-7 inhibitor. Green colour indicates MMP activity and nuclei are stained blue with DAPI. Dual detection of cleaved caspase-3 (red) and MMP activity (green) in coronary plaque (C ) and IMA (D). (E) High power image of marked area in (C), arrows indicate co-location of apoptosis and MMP activity. (F ) Quantiﬁcation of the percentage of apoptotic (ISEL positive) cells in atherosclerotic plaques from ApoE2/2/MMP-7þ/þ and ApoE2/2/MMP-72/2. * indicates a signiﬁcant difference from ApoE2/2/MMP-7þ/þ. ISEL staining of atherosclerotic plaques from ApoE2/2/ MMP-7þ/þ (G) and ApoE2/2/MMP-72/2 (H ) mice. ISEL positive apoptotic cells are green and nuclei are stained blue (DAPI). survival factor for VSMCs,9 and soluble N-cadherin reduces features of plaque instability.10 In this study, we examined whether proteolytic cleavage of N-cadherin occurs and contributes to VSMC apoptosis. We observed a time-dependent loss of full-length N-cadherin and appearance of a C-terminal fragment of N-cadherin ( 35 kDa) after induction of human VSMC apoptosis with various factors includ- ing Fas-L, suspension culture, or TNFa. The appearance of the 35 kDa fragment consisting of the intracellular and transmembrane portion of N-cadherin indicates extracellular cleavage. Previously a 38 kDa fragment N-cadherin was reported to be the result of MMP activity and was further processed by presenilin/g-secretase to 33 kDa.27 However, we were unable to determine any cleavage by presenilin/g-secretase. Additionally, we were unable to detect release of the extracellular domain into the conditioned media as seen previously for E-cadherin,28 presumably due to further proteo- lytic cleavage of the extracellular fragment of N-cadherin. Previous studies have highlighted cleavage of cadherins by various proteases including calpain,29 ADAMs,27 plasmin,30 presenilin-1/ g-secretase,14 and MMPs.13,28,31 – 34 During tumour regression, increased apoptosis was accompanied by elevated MMP activity and E-cadherin cleavage.32 Similarly, Herren et al.13 showed MMP-dependent cleavage of VE-cadherin during endothelial cell apoptosis. Wu and Huang31 identiﬁed that snake venom metallopro- teinases cause activation of MMP-2, cleavage of VE-cadherin, and