Membrane-associated MMP-12 mediates elastolysis in first-trimester CTBs. First-trimester CTBs cultured for 48 hours on Matrigel-coated coverslips were immunostained with control IgG (A) or an antibody to MMP-12 (B) (catalytic domain; green); nuclei were counterstained with propidium iodide (red). Scale bar = 50 μm. Pictures are representative of three independent CTB isolations. C: Membrane-associated elastase activity in first-trimester CTBs 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), the broad-spectrum MMP inhibitor NNGH (50 μmol/L), or the specific MMP-12 inhibitor 470.1 (100 μmol/L). Median, (n = 6); *P < 0.05; ***P < 0.001; Kruskal–Wallis test.

MMP-12 expression in cultured VSMCs and excised human spiral arties. Serial sections of first-trimester placenta (n = 5) and decidua basalis (n = 8) were immunostained with control IgG (A), an antibody to cytokeratin-7 (CK-7; B, C) or HLA-G (I) to detect trophoblasts, an antibody to α-smooth muscle actin (α-SMA) to detect VSMCs (G and H), or an antibody to MMP-12 (carboxyl-terminal) (D–F, J–L). Sections were counterstained with hematoxylin. Scale bar = 50 μm. Serial sections: (A and D), (B and E), (C and F), (G and J), (H and K), (I and L). EC indicates endothelial cells; vCTB, villous cytotrophoblasts; VSMC, vascular smooth muscle cell; eEVT, endovascular extravillous trophoblasts.

Elastin uptake in HASMCs is regulated by MMP and NO. HASMCs were cultured with elastin fragments (1 mg/ml) in the presence of vehicle control (DMSO), the broad-spectrum MMP inhibitor NNGH (50 μmol/L), or L-NAME (5 mmol/L) for 24 hours. Cultures were monitored by time-lapse video-microscopy, and the time point at which an elastin fragment appeared within a cell was recorded. Forty-five cells were scored for each treatment. A: Selected images taken at hourly intervals, showing accumulation of elastin fragments within a cell. Arrows denote elastin fragments. Scale bar = 10 μm. B: Time course of elastin uptake. Mean ± SEM (n = 4). C: Percentage of HASMC-containing elastin fragments at 6, 12, and 24 hours. Black bars represent control cells, gray bars represent NNGH-treated cells, and white bars represent L-NAME-treated cells. Median + range (n = 4); *P < 0.05, **P < 0.01, ***P < 0.001; two-way analysis of variance.

Elastin uptake and MMP expression in first-trimester trophoblasts. SGHPL-4 were cultured with elastin fragments (1 mg/ml) (closed diamonds) in the presence of vehicle control (DMSO), the broad-spectrum MMP inhibitor NNGH (50 μmol/L; closed triangles), or L-NAME (5 mmol/L; open squares) for 24 hours. Cultures were monitored by time-lapse video-microscopy, and the time point at which an elastin fragment appeared within a cell was recorded; 45 cells were scored for each treatment. A: Selected images taken at hourly intervals, showing accumulation of elastin fragments within a cell. Arrows denote elastin fragments. Scale bar = 10 μm. B: Time course of elastin uptake. Mean ± SEM (n = 4). C: Percentage of SGHPL-4–containing elastin fragments at 6, 12, and 24 hours. Black bars represent control cells, gray bars represent NNGH-treated cells, and white bars represent L-NAME–treated cells. Median + range (n = 4); *P < 0.05, **P < 0.01; two-way analysis of variance.

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.

HASMCs exhibit elastase activity attributable to serine proteases and MMP. A: HASMCs were cultured on uncoated plastic dishes (control), Matrigel-coated dishes, or with first-trimester CTB-conditioned medium (50% [vol/vol]), or elastin fibers (1 mg/ml) for 48 hours. Intracellular elastase activity was quantified using a cell-permeable elastase substrate and flow cytometry. Median (n = 3); *P < 0.05; Kruskal-Wallis test. B: HASMCs were cultured on plates coated with elastin fragments for 48 hours and were incubated with vehicle control (DMSO), the broad-spectrum caspase inhibitor zVADfmk (50 μmol/L), the MMP inhibitor NNGH (50 μmol/L), or the cathepsin inhibitor Z-FG-NHO-BzOMe (10 μmol/L) for a further six hours. Intracellular elastase activity was determined as above. Median (n = 4). C: HASMCs were cultured on elastin coated flasks for 48 hours then incubated with vehicle control (DMSO), the serine protease inhibitor PMSF (10 mg/ml; 6 hours), the NOS inhibitor L-NAME (5 mmol/L; 24 hours), or the iNOS inhibitor 1400W (5 μmol/L; 24 hours). Intracellular elastase activity was determined as above. Median (n = 4); *P < 0.05; Kruskal-Wallis test. D and E: Membrane-associated elastase activity in HASMCs 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), the caspase inhibitor zVADfmk (50 μmol/L), the MMP inhibitor NNGH (50 μmol/L), the uPA inhibitor uPA-STOP (10 μmol/L), or the cathepsin inhibitor Z-FG-NHO-BzOMe (10 μmol/L). D: HASMCs were cultured in uncoated flasks (white bars) or in flasks coated with elastin fragments (black bars) for 48 hours. Median + range (n = 4); *P < 0.05, **P < 0.01; Kruskal–Wallis test. E: HASMCs were cultured on elastin coated flasks, in the absence or presence of the NOS inhibitor L-NAME (5 mmol/L; 24 hours) or the iNOS inhibitor 1400W (5 μmol/L; 24 hours). Control supernatants were analyzed in the presence of vehicle control (DMSO) or the serine protease inhibitor PMSF (10 mg/ml). Median (n = 4); *P < 0.05; Kruskal–Wallis test.

MMP-12 expression in cultured HASMCs and excised human spiral arties. A and B: Cultured HASMCs were immunostained with control IgG (A) or an antibody to the catalytic domain of MMP-12 (B) (green). Nuclei were counterstained with propidium iodide (red). Scale bar = 50 μm. Intact human spiral arteries (C) and arteries denuded of endothelium (D) were fixed immediately and stained with an antibody to CD31 (green). Nuclei were counterstained with propidium iodide (red). Scale bar = 100 μm. C: Arrows denote endothelial cells. Note autofluorescence of elastin. E–H: Spiral arteries were denuded of endothelium and were fixed immediately (E) or perfused with unconditioned first-trimester CTB medium (1:1 DMEM: Ham's F12; control) (F), CTB-conditioned medium (diluted 1:1 with DMEM: Ham's F12) (G), or rhTRAIL (0.5 μg/ml in DMEM: Ham's F12) (H) and cultured for 24 hours. Sections were stained with an antibody to the catalytic domain of MMP-12 (green). Scale bar = 100 μm. Nuclei were counterstained with propidium iodide (red). Images are representative of six independent experiments; however, MMP-12–positive VSMCs were only observed in seven or eight (∼30%) of the 24 serial tissue sections examined from each vessel. I: HASMC supernatants were assayed for membrane-associated elastase activity in the presence of vehicle control (DMSO), the broad-spectrum MMP inhibitor NNGH (50 μmol/L), or the specific MMP-12 inhibitor 470.1 (100 μmol/L). Median (n = 6); *P < 0.05; ***P < 0.001; Kruskal–Wallis test.