MMP-7 induces N-cadherin cleavage and VSMC apoptosis 139 Figure 1 N-cadherin cleavage occurs during VSMC apoptosis. Representative western blot for full-length (FL, 130 kDa) and 35 kDa C-terminal fragment of N-cadherin protein in human VSMCs after induction of apoptosis with Fas-L at various time-points (A), full-length N-cadherin at 4 h (B), and 35 kDa C-terminal fragment of N-cadherin after 4 h (C ). Bar charts show densitometric analysis of western blots (B and C) and * indicates a signiﬁcant difference (P , 0.05) from control, n ¼ 5. (D) Representative western blot for full-length (130 kDa) and 35 kDa C-terminal fragment of N-cadherin protein in human VSMCs after induction of apoptosis with 50 mM Bcl2 antagonist (Bcl2 antag) for 18 h, 100 mM H2O2, or 50 ng/mL TNFa for 22 h, and suspension culture for 24 h. b-Tubulin is shown as loading control. 3. Results 3.1 N-cadherin is cleaved during VSMC apoptosis Treatment with Fas-L resulted in a time-dependent cleavage of full- length N-cadherin in human VSMCs (Figure 1A), which reached signiﬁ- cance after 4 h of treatment (Figure 1B). The appearance of 35 kDa fragment of N-cadherin was also observed after 4 h (Figure 1A and C). Other inducers of apoptosis (Bcl2 antagonist, TNFa, H2O2, or sus- pension culture) also caused cleavage of full-length N-cadherin and the appearance of the 35 kDa N-cadherin fragment (Figure 1D), conﬁrming that this was not a unique effect of Fas-L-induced apoptosis. Using western blotting, we were unable to detect release of the extracellular domain of N-cadherin into the conditioned media (data not shown). 3.2 MMPs contribute to N-cadherin cleavage Addition of the MMP inhibitor BB-94 (Figure 2A) signiﬁcantly reduced the amount of N-cadherin fragment produced in response to Fas-L and increased full-length N-cadherin correspondingly, indicating that MMPs are involved in N-cadherin cleavage. In contrast, N-cadherin cleavage was not retarded by treatment of human VSMCs with a plasmin inhibitor (NALME) with or without BB-94 (see