138 N-cadherin increased apoptosis of VSMCs. Additionally, we recently showed that a soluble form of N-cadherin, composed of the extra- cellular domain, acts as a mimetic reducing VSMC apoptosis, as well as features of atherosclerotic plaque instability in vivo.10 Studies in other cell types have suggested that cadherin cleavage by matrix-degrading metalloproteinases (MMPs) or a disintegrin and metalloproteases (ADAMs) occurs during apoptosis.11 – 14 Although we have previously observed that MMPs, speciﬁcally MMP-9 and MMP-12, cleave N-cadherin during VSMC proliferation,8,15 it was unknown whether MMP-dependent cleavage of N-cadherin modu- lates VSMC apoptosis. We therefore aimed to determine whether proteolytic cleavage of N-cadherin by MMPs leads to VSMC apoptosis and is therefore likely to contribute to plaque development and rupture. 2. Methods 2.1 Husbandry Knockout mice were kindly provided by Roger Lijnen (University of Leuven, Belgium) and then bred within the University of Bristol animal unit. C57BL/6J mice were obtained from Charles River and used as wild- type controls. Apolipoprotein E knockout mice (ApoE2/2) were crossed with MMP-7 knockout mice to generate ApoE2/2/MMP-72/2 as well as strain-matched controls (ApoE2/2/MMP-7þ/þ). These mice were placed on high-fat diet for 8 weeks and brachiocephalic arteries removed as pre- viously described.16 Housing, care, and all procedures were performed in accordance with the guidelines and regulations of the University of Bristol and the United Kingdom Home Ofﬁce. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.2 Cell culture The investigation conforms to the principles outlined in the Declaration of Helsinki. Human saphenous vein segments were obtained from consenting coronary artery bypass graft patients (Local ethics board REC#05/Q2007/ 77) and VSMCs grown from medial explants as described previously.8 Mouse VSMCs were grown from explants of mouse aorta removed from either MMP knockout or wild-type mice or C57BL/6J controls, as described previously.15 Each experiment was performed with VSMCs at passage 2 – 10 from at least three different individuals. Apoptosis was induced either by culturing VSMCs (5 104 cells/well in 24 well plates) in serum free media supplemented with 300 ng/mL human recombinant soluble Fas ligand (Fas-L), 50 ng/mL tumour necrosis factor-a (TNFa), 100 mM H2O2 or 50 mM BCl2 antagonist (HA-14, Cal- biochem), or by culturing VSMCs in suspension culture as described previously.17 MMP activity was inhibited by 1 mM BB-94 (Batimastat, a widely used and speciﬁc MMP inhibitor which inhibits many MMPs including MMPs 1, 2, 3, 7, 9, 12, 13 with Ki values of 0.15 – 2.3 nM,18 was a kind gift of Dr Clive Long, Pﬁzer), 2 mM MMP-2 inhibitor19 (Ki values of 1.7 mM for MMP-2, 444244, Calbiochem), 100 nM MMP-12 inhibitor20,21 (IC50 14 nM for MMP-12 and .270 nM for MMPs 2, 3, 8, 9, and 13, PF-356231, Biomol), or 10 nM MMP-7 inhibitor (IC50 10 nM for MMP-7, 444264, Calbiochem). g-Secretase, plasmin, and caspase activity were inhibited by 10 mM g-secretase inhibitor (565771, Calbiochem), 10 mM NALME22,23 (Sigma), and 20 mM pan caspase inhibitor (Z-VAD-FMK) inhibitor (550377, Calbiochem), respectively. DMSO was added to act as a vehicle control in all experiments. VSMCs were also treated with 60 nM active recombinant human MMP-7 (444270 Calbiochem) and 2 mg/mL cycloheximide (to prevent protein synthesis and thereby inhibit replenishment of full-length N-cadherin). H. Williams et al. 2.3 Atherosclerotic plaques Human carotid endarterectomy samples were obtained (local ethical approval #E3111) as described previously.24 Human right coronary artery plaques were removed from hearts collected for valve retrieval (local ethical approval REC#08/H0107/48). Surplus segments of internal mammary artery (IMA) were obtained from consenting patients under- going coronary artery bypass grafting (local ethical approval REC#04/ Q2007/6). Frozen samples were extracted in SDS lysis buffer or MMP-7 assay buffer (1 mM monothioglycerol, 50 mM Tris – HCl, pH 7.4) and ana- lysed by western blotting and MMP-7 activity assay, respectively, or frozen sections were cut for in situ zymography. 2.4 Cleavage of recombinant N-cadherin by MMP-7 As described previously,15 active MMP-7 (2 nM) was incubated with 2 nM puriﬁed soluble N-cadherin-Fc (SNC-Fc; consisting only of the extracellu- lar domain), for 30 min at 378C and then subjected to western blotting. 2.5 Western blotting Western blotting for N-cadherin, phosphorylated Akt (pAkt), total Akt, and b-tubulin was performed with equal protein concentrations, as described previously.8,9 The anti-N-cadherin antibody (610920, BD Trans- duction Laboratories) recognizes amino acids 802 – 819 in the C-terminus of N-cadherin. To detect SNC-Fc, western blotting for the Fc domain of mouse IgG was performed as described previously.15 Cleaved caspase-3 was detected using rabbit anti-cleaved caspase-3 antibody diluted 1:1000 (Cell Signalling No. 9661). Densitometric scanning was performed to quantify the optical density of detected bands (O.D. mm2) and nor- malized to b-tubulin values. 2.6 Measurement of apoptosis Apoptosis was assessed in vitro by measurement of cleaved caspase-3 levels using the Caspase-Glo luminescent assay (Promega) as described in the manufacturer’s instructions. Immunocytochemistry for cleaved caspase-3 was performed as described previously.10 Apoptosis was also measured in parafﬁn-wax embedded sections using in situ end labelling (ISEL) as described previously.25 The number of ISEL positive plaque cells was counted and expressed as a percentage of the total number of plaque cells. 2.7 MMP-7 activity assay Ten times concentrated conditioned media and tissue lysates were assessed for MMP-7 activity using a ﬂuorimetric assay. Samples and stan- dards were incubated with 0.14 mg/mL DQ-gelatin ﬂuorescent substrate (Invitrogen) with and without 10 nM MMP-7 inhibitor (444264, Calbio- chem) and ﬂuorescence read using a Fluostar Optima ﬂuorimeter from 0 to 90 min until peak ﬂuorescence was achieved. MMP-7 activity was calculated as that inhibited by the MMP-7 inhibitor. 2.8 In situ zymography MMP activity was detected as described previously.26 Sections were incu- bated with 10 nM MMP-7 inhibitor to identify MMP-7 activity. In some cases, immunohistochemistry for cleaved caspase-3 was performed as described previously10 after in situ zymography. MMP activity was quanti- ﬁed using image analysis and expressed as ﬂuorescent arbitrary units (FAU). 2.9 Statistics Experiments were carried out at least three times with VSMCs from different sources. Data were analysed by Student’s t-test or ANOVA and Student Newman – Keuls post-test and a signiﬁcant difference accepted when P , 0.05.