No abstract

The ABC transporters P-glycoprotein (P-gp, official gene symbol ABCB1) and breast cancer resistance protein (BCRP, official gene symbol ABCG2) protect the conceptus from exposure to toxins and xenobiotics present in the maternal circulation. Viral or bacterial challenges alter expression of placental multidrug transporters in rodents. We hypothesized that exposure to lipopolysaccharide (LPS, bacterial antigen) and polyinosinic-polycytidylic acid (poly(I:C), viral antigen) would decrease P-gp and BCRP in the human placenta. Placental explants from first and third trimesters were challenged with 0.1 to 10 μg/mL LPS or 1 to 50 μg/mL poly(I:C) for 4 or 24 hours; mRNA levels, protein expression, and localization were assessed by quantitative real-time PCR, Western blot analysis, and immunohistochemistry, respectively. Toll-like receptor (TLR)-3 and TLR-4 mRNA expression increased from the first to third trimester (P < 0.01), and the receptors localized to cytotrophoblasts in the first trimester and to syncytiotrophoblasts in the third trimester. LPS exposure in first-trimester explants decreased (P < 0.001) ABCB1 and ABCG2 mRNA and protein levels. In contrast, poly(I:C) decreased (P < 0.05) ABCB1, TLR-3, and TLR-4 mRNA levels in the third trimester but not first trimester. LPS and poly(I:C) treatments increased (P < 0.01) IL-8 and chemokine ligand 2. Results suggest that bacterial infections likely alter exposure of the conceptus to toxins and drugs during early pregnancy, whereas viral infections may disrupt fetal protection in later stages of pregnancy.

Background/Aims: The ATP-binding cassette (ABC) transporters mediate drug biodisposition and immunological responses in the placental barrier. In vitro infective challenges alter expression of specific placental ABC transporters. We hypothesized that chorioamnionitis induces a distinct pattern of ABC transporter expression. Methods: Gene expression of 50 ABC transporters was assessed using TaqMan® Human ABC Transporter Array, in preterm human placentas without (PTD; n=6) or with histological chorioamnionitis (PTDC; n=6). Validation was performed using qPCR, immunohistochemistry and Western blot. MicroRNAs known to regulate P-glycoprotein (P-gp) were examined by qPCR. Results: Up-regulation of ABCB9, ABCC2 and ABCF2 mRNA was detected in chorioamnionitis (p<0.05), whereas placental ABCB1 (P-gp; p=0.051) and ABCG2 (breast cancer resistance protein-BCRP) mRNA levels (p=0.055) approached near significant up-regulation. In most cases, the magnitude of the effect significantly correlated to the severity of inflammation. Upon validation, increased placental ABCB1 and ABCG2 mRNA levels (p<0.05) were observed. At the level of immunohistochemistry, while BCRP was increased (p<0.05), P-gp staining intensity was significantly decreased (p<0.05) in PTDC. miR-331-5p, involved in P-gp suppression, was upregulated in PTDC (p<0.01) and correlated to the grade of chorioamnionitis (p<0.01). Conclusions: Alterations in the expression of ABC transporters will likely lead to modified transport of clinically relevant compounds at the inflamed placenta. A better understanding of the potential role of these transporters in the events surrounding PTD may also enable new strategies to be developed for prevention and treatment of PTD.

Dysregulation of trophoblast differentiation is implicated in the placental pathologies of intrauterine growth restriction and pre‐eclampsia. P‐glycoprotein (P‐gp encoded by ABCB1) is an ATP‐binding cassette transporter present in the syncytiotrophoblast layer of the placenta where it acts as a molecular sieve. In this study, we show that P‐gp is also expressed in the proliferating cytotrophoblast (CT), the syncytiotrophoblast (ST) and the extravillous trophoblast (EVT), suggesting our hypothesis of a functional role for P‐gp in placental development. Silencing of ABCB1, via siRNA duplex, results in dramatically reduced invasion and migration, and increased tube formation and fusion in the EVT‐like HTR8/SV neo cell line. In both EVT and CT explant differentiation experiments, silencing of ABCB1 leads to induction of the fusion markers human hCG, ERVW‐1 and GJA1 and terminal differentiation of both trophoblast subtypes. Moreover, P‐gp protein levels are decreased in both the villous and the EVT of severe early‐onset pre‐eclamptic placentas. We conclude that, in addition to its role as a syncytial transporter, P‐gp is a key factor in the maintenance of both CT and EVT lineages and that its decrease in severe pre‐eclampsia may contribute to the syncytial and EVT placental pathologies associated with this disease.

During placental development, cytotrophoblast progenitor cells differentiate into the syncytiotrophoblast and invasive extravillous trophoblasts (EVTs). Some EVTs further differentiate into endovascular trophoblasts (enEVTs) which exhibit endothelial-like properties. Abnormal placental development, including insufficient enEVT-mediated remodeling of the uterine spiral arteries, is thought to be a precipitating factor in the onset of preeclampsia (PE), a pregnancy-related hypertensive disorder. Several members of the transforming growth factor-β (TGF-β) superfamily, such as TGF-βs, Nodal, and Activin have been reported to either promote or inhibit the invasive EVT pathway. These ligands signal through serine/threonine receptor complexes to activate downstream signaling mediators, Smad2 and Smad3. In this study, we determined Smad2 and Smad3 expression pattern in placenta and their effects on trophoblast invasion and differentiation. Total Smad2/3 levels were relatively constant across gestation while the ratio of active phosphorylated forms to their total levels varied with gestational stages, with a higher pSmad2/total Smad2 in later gestation and a higher pSmad3/total Smad3 in early gestation. Immunofluorescent staining revealed that pSmad3 was localized in nuclei of EVTs in anchoring villi. On the other hand, pSmad2 was mostly absent in this invasive EVT population. In addition, pSmad3/total Smad3, but not pSmad2/total Smad2, was significantly lower in both early onset and late onset PE cases, as compared to gestational age-matched controls. Functional studies carried out using a first trimester trophoblast cell line, HTR-8/SVneo, and first trimester human placental explants showed that Smad2 and Smad3 had differential roles in the invasive pathway. Specifically, siRNA-mediated knockdown of Smad2 resulted in an increase in trophoblast invasion and an upregulation of mRNA levels of enEVT markers while the opposite was observed with Smad3 knockdown. In addition, Smad2 siRNA accelerated the EVT outgrowth in first trimester placental explants while the Smad3 siRNA reduced the outgrowth of EVTs when compared to the control. Furthermore, knockdown of Smad2 enhanced, whereas overexpression of Smad2 suppressed, the ability of trophoblasts to form Brkić et al. Differential Role of Smad2 and Smad3 in Placenta endothelial-like networks. Conversely, Smad3 had opposite effects as Smad2 on network formation. These findings suggest that Smad2 and Smad3 have opposite functions in the acquisition of an enEVT-like phenotype and defects in Smad3 activation are associated with PE.