Syncytialization alters the extracellular matrix and barrier function of placental trophoblasts

Abstract

The human placenta is of vital importance for proper nutrient and waste exchange, immune regulation, and overall fetal health and growth. Specifically, the extracellular matrix (ECM) of placental syncytiotrophoblasts, which extends outward from the placental chorionic villi into maternal blood, acts on a molecular level to regulate and maintain this barrier. Importantly, placental barrier dysfunction has been linked to diseases of pregnancy such as preeclampsia and intrauterine growth restriction. To help facilitate our understanding of the interface and develop therapeutics to repair or prevent dysfunction of the placental barrier, in vitro models of the placental ECM would be of great value. In this study, we aimed to characterize the ECM of an in vitro model of the placental barrier using syncytialized BeWo choriocarcinoma cells. Syncytialization caused a marked change in syndecans, integral proteoglycans of the ECM, which matched observations of in vivo placental ECM. Syndecan-1 expression increased greatly and predominated the other variants. Barrier function of the ECM, as measured by electric cell-substrate impedance sensing (ECIS), increased significantly during and after syncytialization, whereas the ability of THP-1 monocytes to adhere to syncytialized BeWos was greatly reduced compared with nonsyncytialized controls. Furthermore, ECIS measurements indicated that ECM degradation with matrix metalloproteinase-9 (MMP-9), but not heparanase, decreased barrier function. This decrease in ECIS-measured barrier function was not associated with any changes in THP-1 adherence to syncytialized BeWos treated with heparanase or MMP-9. Thus, syncytialization of BeWos provides a physiologically accurate placental ECM with a barrier function matching that seen in vivo.

Keywords: barrier; extracellular matrix; placenta; syncytialize; trophoblast.

Figure 1.

Representative phase contrast imaging of BeWo trophoblasts treated with DMSO as a control for 48 h (A) and of BeWo trophoblasts treated with 25 µM forskolin for 48 h (B). Representative confocal imaging of BeWo trophoblasts treated with DMSO as a control for 48 h (C) and of BeWo trophoblasts treated with 25 µM forskolin for 48 h (D). In C and D, red = cell membrane and blue = nuclei. E: relative migration of BeWos after 48 h of treatment with 25 µM forskolin or DMSO as a control. N = 6 for migration assay groups. Statistical analysis performed using unpaired Student’s t test. Significance is designated by a bar connecting groups (***P < 0.001).

Figure 2.

Digital droplet PCR of syndecan-1 (SDC-1) (A), syndecan-2 (SDC-2) (B), syndecan-3 (SDC-3) (C), and syndecan-4 (SDC-4) (D) expression levels, expressed as copies of gene per nanogram (ng) of RNA, in BeWos treated with DMSO or 25 µM forskolin for 48 h. N = 6 for all groups. Statistical analysis performed using unpaired Student’s t test. Significance is designated by a bar connecting groups (***P < 0.001).

Figure 3.

Syndecan-1 protein levels in the cell lysates of BeWos treated with DMSO or 25 µM forskolin for 48 h (A), as measured by ELISA. Syndecan-4 protein levels in the cell lysates of BeWos treated with DMSO or 25 µM forskolin for 48 h (B), as measured by ELISA. Immunofluorescent imaging for syndecan-1 (green) in cells treated with DMSO (C) and forskolin (D). Lysate protein levels were normalized to lysate total protein levels and are expressed in picograms (pg) of syndecan-1 or syndecan-4 per microgram (µg) of total protein. N = 6 for all groups. Statistical analysis performed using unpaired Student’s t test. Significance is designated by a bar connecting groups ( ***P < 0.001).

Figure 4.

A: normalized impedance of BeWos treated with DMSO or 25 µM forskolin for 48 h, as well as media alone. Impedance was continuously measured at 4,000 Hz frequency. N = 3 for each group. B: area under the curve (AUC) of 48 h of impedance measurement. N = 3 for each group. C: THP-1 adhesion to BeWos treated with DMSO or 25 µM forskolin for 48 h, expressed as the percentage of total THP-1s added to each well. N = 5 for each group. Statistical analysis performed using unpaired Student’s t test. Significance is designated by a bar connecting groups (*P < 0.05, **P < 0.01).

Figure 5.

Syndecan-1 protein levels in the cell lysates (A) and media (B) of BeWos first treated with 25 µM forskolin for 48 h and then treated with forskolin, forskolin (For) + heparanase (HPSE), For + MMP-9, or For + HSPE + MMP-9 (Combo) for 24 h, as measured by ELISA. Syndecan-4 protein levels in the cell lysates (C) and media (D) of BeWos first treated with 25 µM forskolin for 48 h and then treated with forskolin, forskolin (For) + heparanase (HPSE), For + MMP-9, or For + HSPE + MMP-9 (Combo), as measured by ELISA. Lysate protein levels were normalized to lysate total protein levels and are expressed in picograms (pg) of syndecan-1 or syndecan-4 per microgram (µg) of total protein. N = 6 for all groups. Statistical analysis performed using one-way ANOVA with post hoc Dunnett’s multiple-comparisons test. Significance is designated by a bar connecting groups (*P < 0.05, **P < 0.01, ***P < 0.001). MMP-9, matrix metalloproteinase-9.

Figure 6.

A: normalized impedance of BeWos first treated with 25 µM forskolin for 48 h and then treated with forskolin, forskolin (For) + heparanase (HPSE), For + MMP-9, or For + HSPE + MMP-9 (Combo) for 24 h, as well as media alone. Continuous impedance measurements throughout the 24 h of treatment with enzymes is shown, which was measured at 4,000 Hz frequency. N = 3 for each group. B: area under the curve (AUC) of 24 h of impedance measurement. N = 3 for each group. C: THP-1 adhesion to BeWos first treated with 25 µM forskolin for 48 h and then treated with forskolin, forskolin (For) + heparanase (HPSE), For + MMP-9, or For + HSPE + MMP-9 (Combo) for 1 h, expressed as the percentage of total THP-1s added to each well. MMP-9, matrix metalloproteinase-9. Significance is designated by a bar connecting groups (**P < 0.01).