Progestin and thrombin regulate tissue factor expression in human term decidual cells - PubMed (original) (raw)

Progestin and thrombin regulate tissue factor expression in human term decidual cells

C J Lockwood et al. J Clin Endocrinol Metab. 2009 Jun.

Erratum in

Abstract

Context: Perivascular cell membrane-bound tissue factor (TF) initiates hemostasis via thrombin generation. The identity and potential regulation of TF-expressing cells at the human maternal-fetal interface that confers hemostatic protection during normal and preterm delivery is unclear.

Objectives: The objective of the study were to identify TF-expressing cells at the maternal-fetal interface in term and preterm decidual sections by immunohistochemistry and evaluate progestin, thrombin, TNF-alpha, and IL-1beta effects on TF expression by cultured human term decidual cells (DCs).

Interventions and main outcome measures: Serial placental sections were immunostained for TF. Leukocyte-free term DC monolayers were incubated with 10(-8) M estradiol (E2) or E2 plus 10(-7) M medroxyprogestrone acetate (MPA) +/- thrombin or TNF-alpha or IL-1beta. ELISA and Western blotting assessed TF in cell lysates. Quantitative real-time RT-PCR measured TF mRNA levels.

Results: Immunolocalized TF in DC membranes in preterm and term placental sections displayed higher Histologic Scores than villous mesenchymal cells (P < 0.05). TF was undetected in interstitial or extravillous trophoblasts. Compared with DCs incubated with E2, MPA and 2.5 U/ml thrombin each doubled TF levels (P < 0.05) and E2 + MPA + thrombin further doubled TF levels (P < 0.05), whereas TNF-alpha and IL-1beta were ineffective. Western blotting confirmed the ELISA results. Quantitative RT-PCR revealed corresponding changes in TF mRNA levels.

Conclusions: In human term placental sections, DC-expressed TF exceeds that of other cell types at the maternal-fetal interface and is localized at the cell membranes in which it can bind to factor VII and meet the hemostatic demands of labor and delivery via thrombin formation. Unlike the general concept that TF is constitutive in cells that highly express it, MPA and thrombin significantly enhanced TF expression in term DC monolayers.

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Figures

Figure 1

Figure 1

Immunohistochemical analysis of tissue factor expression at the decidual/placental interface. Serial sections of decidual basalis specimens were immunostained for TF and vimentin, as shown in an idiopathic preterm specimen (A, TF; B, vimentin). DCs (arrows), identified by positive vimentin staining, exhibited strong perimembranous TF staining. TF staining was virtually absent in interstitial trophoblasts (arrowheads). Similar results were seen in term specimens (C). Placental villi exhibited moderate TF staining in the mesoderm (M), in which the staining was primarily localized to perivascular adventitia (small arrows), as shown in a preterm specimen (D). Syncytiotrophoblasts (S) and cytotrophoblasts (C) exhibited no TF staining. Similar results were seen in term specimens (not shown). Negative control immunostaining using a nonspecific isotype-matched antibody revealed no positive signals (E). TF staining intensity (F); bars indicate HSCORE mean ±

sem

) among cell types was highest in DCs (*, vs. interstitial trophoblasts and the villous mesoderm of the same specimen group, P < 0.05), with moderate staining in the villous mesoderm (†, vs. interstitial trophoblasts of the same specimen group, P < 0.05). There were no significant differences in HSCOREs between specimen groups.

Figure 2

Figure 2

Effects of MPA and thrombin on TF expression in DCs. Term DCs were primed in E2 or E2 + MPA and then switched to DM with corresponding steroids ± 2.5 U/ml thrombin (Th) for 24 h. ELISAs were performed for TF in cell lysates with the results normalized to total cell protein: n = 10 (mean ±

sem

). *, vs. E2; **, vs. E2 + MPA and E2 + Th; P < 0.05.

Figure 3

Figure 3

Concentration-response effects of thrombin on TF expression in DCs. Term DCs were primed in E2 + MPA and then switched to DM with steroids ± thrombin (Th) at varying concentrations in units per milliliter for 24 h. ELISAs were performed for TF in cell lysates and measured by ELISA. Results are normalized to total cell protein (n = 3, mean ±

sem)

. *, vs. E2 + MPA; **, vs. + Th 0.25; P < 0.05.

Figure 4

Figure 4

Effects of thrombin, IL-1β, and TNF-α on TF expression in DCs. Term DCs were primed in E2 or E2 + MPA and then switched to DM with corresponding steroids ± 2.5 U/ml thrombin (Th) or 1 ng/ml of TNF-α or IL-1β for 24 h. ELISAs were performed for TF in cell lysates with the results normalized to total cell protein: n = 8 (mean ±

sem

). *, vs. E2 + MPA; P < 0.05.

Figure 5

Figure 5

Western blot of thrombin effects on TF output by DCs. Term DCs were primed in E2 or E2 + MPA and then switched to DM with corresponding steroids ± 2.5 U/ml thrombin (Th) for 24 h. Cell lysates were subjected to Western blotting after SDS-PAGE. See Patients and Methods for details.

Figure 6

Figure 6

Effects of MPA and thrombin on TF mRNA expression in DCs. Term DCs were primed in E2 or E2 + MPA and then switched to DM with corresponding steroids ± 2.5 U/ml thrombin (Th) for 6 h. TF mRNA was measured by quantitative real-time RT-PCR and normalized to β-actin mRNA; n = 9 (mean ±

sem

). *, vs. E2; P < 0.05.

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