Expression of ADAMTS13 in Normal and Abnormal Placentae and Its Potential Role in Angiogenesis and Placenta Development

Abstract

Objective: ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 repeats, member 13) is primarily synthesized in liver. The biosynthesis of ADAMTS13 and its physiological role in placenta are not known.

Approach and results: We used real-time polymerase chain reaction, immunohistochemistry, and Western blotting analyses, as well as proteolytic cleavage of FRETS (fluorescent resonance energy transfers)-VWF73, to determine ADAMTS13 expression in placenta and trophoblasts obtained from individuals with normal pregnancy and patients with severe preeclampsia. We also determined the role of ADAMTS13 in extravillous trophoblasts using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, wound scratch assay, transwell migration assay, tube formation assay, and tissue outgrowth assays. We showed that full-length and proteolytically active ADAMTS13 was expressed in normal human placenta, primarily in the trophoblasts and villous core fetal vessel endothelium during pregnancy. Placental expression of ADAMTS13 mRNA, protein, and proteolytic activity was at the highest levels during the first trimester and significantly reduced at the term of gestation. Additionally, significantly reduced levels of placental ADAMTS13 expression was detected under hypoxic conditions and in patients with preeclampsia. In addition, recombinant ADAMTS13 protease stimulated proliferation, migration, invasion, and network formation of trophoblastic cells in culture. Finally, knockdown of ADAMTS13 expression attenuated the ability of tube formation in trophoblast (HTR-8/SVNEO) cells and the extravillous trophoblast outgrowth in placental explants.

Conclusions: Our results demonstrate for the first time the expression of ADAMTS13 mRNA and protein in normal and abnormal placental tissues and its role in promoting angiogenesis and trophoblastic cell development. The findings support the potential role of the ADAMTS13-von Willebrand factor pathway in normal pregnancy and pathogenesis of preeclampsia.

Keywords: ADAMTS13; placenta; preeclampsia; protein synthesis; trophoblast.

Fig. 1. ADAMTS13 expression in human placenta

A. Real-time PCR detected the relative abundance of ADAMTS13 mRNA/GAPDH in human placental villous tissues from first (1^st) (n=10), second (2^nd) (n=3), and third (3^rd) (n=10) trimester of pregnancy. Human liver expressing normal ADAMTS13 mRNA (Liver) was used as a positive control and blank CHO cell mRNA was used as a negative control. B and C. ADAMTS13 protein in the placental villous tissue lysate by Western blotting with a rabbit anti-ADAMTS13 IgG. D and E. ADAMTS13 protein and activity detected by Western blotting and FRETS-VWF73, respectively, in the conditioned medium of placental villous tissues. All results were presented as the mean ± SEM. * indicates a p value < 0.05. n.s. indicates a p value >0.05.

Fig. 2. Cellular expression of ADAMTS13 protein in human placentae and deciduae throughout pregnancy

Immunoreactivity of anti-ADAMTS13 IgG in fixed trophoblasts (Aa-c) and extravillous trophoblasts in deciduae (Ba-b). CK7 was used as a marker for trophoblasts (Ad-f). HLA-G was used for a maker for extravillous trophoblasts (Bc-d) and CD34 for villous core blood vessel endothelium (Ag-i). Negative controls containing only the secondary antibody were all negative (Aj-l & Be-f). Bar=50 μm. Arrows in Aa-c indicate villous core blood vessel endothelium. Arrowheads in Aa-c indicate stroma cells. CT, cytotrophoblast; ST, syncytiotrophoblast; EVT, extravillous trophoblasts.

Fig. 3. ADAMTS13 expression is down-regulated in placentase from patients with severe preeclampsia

A. Relative ratios of ADAMTS13 to β-actin mRNA in patients with severe preeclampsia (PE, n=22) and in normotensive pregnany (Control, n=25) placentae. B. ADAMTS13 protein detected by Western blotting in placental villous tissues from patients with severe preeclampsia (n=22) and normotensive pregnancy (n=25). C. Quantification of the relative aboundance of ADAMTS13 protein in severe preeclamptic placentae and control placentae is shown. D and E. ADAMTS13 mRNA by real-time PCR and ADAMTS13 protein by Western blotting in placental explant cultured under normal and hypoxic conditions. F. Quantification of the immunoreactivity of a full-length ADAMTS13 is shown. All results are presented as the mean ± SEM (n=10). * indicates the p valule <0.05.

Fig. 4. Expression of ADAMTS13 in primary placental trophoblasts and HTR-8/SVNEO cells

A. Relative ratios of ADAMTS13 to GAPDH mRNA levels in placental trophoblasts. Human liver ADAMTS13 mRNA was used as a positive control and blank CHO cell mRNA was used as a negative control. B. Western blotting demonstrates a full-length ADAMTS13 protein in the cell lysate (Lys) and conditioned medium (Med) of primary trophoblast (TB) and HTR-8/SVNEO cells. Recombiannt ADAMTS13 (rA13) expressed in CHO cells transiently transfected with ADAMTS13 plasmid was used as a positive control. C. ADAMTS13 activity (mean ± SEM) in the conditioned media of primary trophoblasts (TB) and HTR-8/SVNEO cells is shown. D. Immunofluoreceent staining of primary trophoblasts (TB) and HTR-8/SVNEO cells with monoclonal anti-CK7 (10μg/ml) and rabbit anti-ADAMTS13 IgG (20μg/ml) as indicated in the photographs (Bar=50μm). CK7 is a marker for placental trophoblasts.

Fig. 5. ADAMTS13 promotes trophoblast cell proliferation, migration, invasion and network formation

A. Proliferation of HRT-8/SVNEO trophoblasts after treatment for 48 hours with a buffer or VEGF₁₆₅ (5 nM) or recombiannt ADAMTS13 (0-8 nM). Each experiment was performed in 6 replicates. ANOVA determined the statistical signfiacance of the difference among the groups. B and C. Migration of HTR-8/SVNEO cells after incubation with VEGF₁₆₅ (5 nM) and recombinant ADAMTS13 (8 nM) for 0 and 24 hours as indicated by a wound scratch assay (Bar=100μm). D and E. The effect of 24 hours treatment of VEGF₁₆₅ (5 nM) and recombinant ADAMTS13 (8 nM), respectively, on the invasion of HTR-8/SVNEO cells (Bar= 20 μm) by a transwell assay. F and G. The formaiton of tube network in HTR-8/SVNEO cells treated without and with recombinant ADAMTS13 (8 nM), respectively, for 8 hours (Bar=100μm) assessed by the Matrigel assay. The figure shows the representative images of three independent experiments and data are expressed as % of control. ** and *** indicate p values <0.01 and <0.001, respectively.

Fig. 6. Silencing of ADAMTS13 expression inhibits the network formation in HTR-8/SVNEO cells

A, B, and C. Real-time PCR, Western blotting, and quantitation of ADAMTS13 protein demonstrate the signficant reduction of ADAMTS13 mRNA and protein in HTR-8/SVNEO transfected with siRNA specific for ADAMTS13 (50 nM) compared with those with control siRNA (50 nM). D, E, and F. the network formation assessed by matrigel assay in HTR-8/SVNEO cells transfected with siRNA to ADAMTS13 (si-A13, 50 nM) or control siRNA(si-Ctrl, 50 nM) for 48 hours (Bar=100μm). The figure shows the representative images of six independent experiments and data are expressed as % of control. ** and *** indicate the p values of <0.01 and < 0.001, respectively.

Fig. 7. Silencing of ADAMTS13 suppresses the outgrowth of EVTs from human placental explant in culture

A. Whole mount immunofluorescent assay indicating the silencing efficiency of 72 hours of treatment with ADAMTS13 siRNA (100 nM) in the outgrowing EVTs (Bar=100μm). The antibodies used for immunofluorescence staining are indicated on the images. B. Representative bright field images show the outgrowth of the placental explants after 72 hours of cululture treated with 100 nM of siRNA specific for ADAMTS13 (si-A13) and control siRNA (si-Ctrl). The outgrowth area and distance of the explants under different treatments are indicated by the dashed lines and white arrows. Quantiation of the outgrowth distance (C) and the average surface area of EVT outgrowth (D) is shown. The figure shows the representative images of nine independent experiments and data are expressed as % of control.** indicates the p valule <0.01.