Figure 1.

Elastase activity in first-trimester cytotrophoblasts. A: First-trimester cytotrophoblasts (CTBs) were cultured with Congo Red-labeled elastin (1 mg/ml) for 48 hours and stained with an antibody to cytokeratin-7 (green). Serial images at 2-μm intervals along the z axis were captured by confocal microscopy; inset depicts intracellular elastin fragments. Scale bars = 50 μm (n = 3). B: CTBs were isolated from placentas of fewer than 9 weeks gestation or greater than 9-weeks gestation. Cells were cultured with a vehicle control (DMSO), the broad-spectrum caspase inhibitor zVADfmk (50 μmol/L), the broad-spectrum MMP inhibitor NNGH (50 μmol/L), the broad-spectrum cathepsin inhibitor Z-FG-NHO-BzOMe (10 μmol/L), or (C) with all three inhibitors for 18 hours. B and C: Intracellular elastase activity was determined using a cell-permeable elastase substrate. Median + range (n = 3). B: *P < 0.05; Kruskal-Wallis test. D and E: Membrane-associated elastase activity in SGHPL4 (D) and first-trimester CTBs (E) was assessed using the substrate N-succinyl-(L-alanine)₃-p-nitroanilide. Cell supernatants were assayed for elastase activity in the presence of vehicle control (DMSO), zVADfmk (50 μmol/L), NNGH (50 μmol/L), Z-FG-NHO-BzOMe (10 μmol/L), or the uPA inhibitor uPA-STOP (10 μmol/L). Median, (n = 3); *P < 0.05; Kruskal-Wallis test.