CL and mtMP activated ECs and disrupted their barrier function. HUVECs before (A,C) and after stimulation with CL (B, 25 μg/mL) or mtMP (D, 2.5 × 104/μL) for 2 hours at 37°C were stained for CD31 (PECAM1, green), VWF (red), and 4′,6-diamidino-2-phenylindole (blue) to mark cell–cell junction, endothelial cells, and the nucleus, respectively. Resting cells stained with VWF (E) and 4′,6-diamidino-2-phenylindole (F) served as controls (representative of 3-5 experiments; bar = 20 μm). (G) The conditioned media from cultured ECs before and after treated with CL or mtMPs were analyzed for VWF by immunoblot (top) and ELISA (bottom). (H) Confluent HUVECs on collagen-coated PET membrane were stimulated with CL or mtMPs in the presence and absence of fresh platelets for 3 hours at 37°C, followed by incubation with FITC-Dextran for 30 minutes at 37°C. Fluorescent dextran was detected in the bottom chambers (n = 3-4; 1-way ANOVA).
