FIGURE 4.

α-Defensins stimulate expression of cathepsin activity by endothelial cells and macrophages and increase endothelial permeability. A, confluent monolayers of BAECs were incubated for 120 min at 37 °C with ¹²⁵I-LDL alone (Saline) or with α-defensins (α-def, 50–150 nm), α-defensin and rRAP (20 nm), anti-LRP antibody (20 nm) ± GB111-NH₂, or CA-074, and permeability was assessed. The results are expressed as the percent increase in radioactivity (cpm) in the bottom chamber relative to saline-treated cells. The mean ± S.D. and p values are shown. B, bovine aortic endothelial cells grown to confluence in 48-well culture plates (150,000 cells/well) were incubated with or without α-defensins (50 or 150 nm) and with or without rRAP or anti-LRP antibodies for 6 h. Z-Arg-Arg-AMC was added to the supernatants with or without GB111-NH₂ or CA-074. The results are expressed in arbitrary fluorescence units (FU). The mean ± S.D. and p values are shown. C, RAW cells were incubated in saline buffer or saline supplemented with α-defensins (50 or 150 nm) with or without rRAP or anti-LRP antibodies for 6 h. Z-Arg-Arg-AMC was added to the supernatants alone or in the presence of GB111-NH₂, and fluorescence was recorded over the ensuing 35 min as described in B. The mean ± S.D. and p values are shown. D, aortas from 4-month-old WT or Def^+/+ mice fed a regular chow diet were incubated with the cathepsin catalytic site probe GB123, with or without the inhibitor GB111-NH₂, to measure specific binding (25). Images were obtained with an IVIS Kinetic system using 640/695 excitation and emission filters, respectively (25). Representative sections (n = 5 animals) are shown. E, cathepsin expression was calculated as described in Methods. Vessels from 5–8 animals in each group were studied. The mean ± S.D. and p values are shown.