Gestational age-dependent gene expression profiling of ATP-binding cassette transporters in the healthy human placenta.

The ATP-binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal-fetal interface. We and others have demonstrated a gestational age-dependent expression pattern of two ABC transporters, P-glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational-age dependent manner in normal human pregnancy. Using the TaqMan® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.

P-Glycoprotein (P-gp)/ABCB1 plays a functional role in extravillous trophoblast (EVT) invasion and is decreased in the pre-eclamptic placenta.

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/SVneo 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.

Chorioamnionitis Induces a Specific Signature of Placental ABC Transporters Associated with an Increase of miR-331-5p in the Human Preterm Placenta.

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.

Impact of bacterial and viral challenge on multidrug resistance in first- and third-trimester human placenta.

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.

TGF-ß1 regulation of multidrug resistance P-glycoprotein in the developing male blood-brain barrier.

P-glycoprotein (P-gp), an efflux transporter encoded by the abcb1 gene, protects the developing fetal brain. Levels of P-gp in endothelial cells of the blood-brain barrier (BBB) increase dramatically during the period of peak brain growth. This is coincident with increased release of TGF-ß1 by astrocytes and neurons. Although TGF-ß1 has been shown to modulate P-gp activity in a number of cell types, little is known about how TGF-ß1 regulates brain protection. In the present study, we hypothesized that TGF-ß1 increases abcb1 expression and P-gp activity in fetal and postnatal BBB in an age-dependent manner. We found TGF-ß1 to potently regulate abcb1 mRNA and P-gp function. TGF-ß1 increased P-gp function in brain endothelial cells (BECs) derived from fetal and postnatal male guinea pigs. These effects were more pronounced earlier in gestation when compared with BECs derived postnatally. To investigate the signaling pathways involved, BECs derived at gestational day 50 and postnatal day 14 were exposed to ALK1 and ALK5 inhibitors and agonists. Through inhibition of ALK5, we demonstrated that ALK5 is required for the TGF-ß1 effects on P-gp function. Activation of ALK1, by the agonist BMP-9, produced similar results to TGF-ß1 on P-gp function. However, TGF-ß1 signaling through the ALK1 pathway is age-dependent as dorsomorphin, an ALK1 inhibitor, attenuated TGF-ß1-mediated effects in BECs derived at postnatal day 14 but not in those derived at gestational day 50. In conclusion, TGF-ß1 regulates P-gp at the fetal and neonatal BBB and both ALK5 and ALK1 pathways are implicated in the regulation of P-gp function. Aberrations in TGF-ß1 levels at the developing BBB may lead to substantial changes in fetal brain exposure to P-gp substrates, triggering consequences for brain development.

Prenatal endotoxemia and placental drug transport in the mouse: placental size-specific effects.

Lipopolysaccharide (LPS) in high doses inhibits placental multidrug resistance P-glycoprotein (P-gp--Abcb1a/b) and breast cancer resistance protein (BCRP--Abcg2). This potentially impairs fetal protection against harmful factors in the maternal circulation. However, it is unknown whether LPS exposure, at doses that mimic sub-lethal clinical infection, alters placental multidrug resistance. We hypothesized that sub-lethal (fetal) LPS exposure reduces placental P-gp activity. Acute LPS (n = 19;150 µg/kg; ip) or vehicle (n = 19) were given to C57BL/6 mice at E15.5 and E17.5. Placentas and fetal-units were collected 4 and 24 h following injection. Chronic LPS (n = 6; 5 µg/kg/day; ip) or vehicle (n = 5) were administered from E11.5-15.5 and tissues were collected 4 h after final treatment. P-gp activity was assessed by [³H]digoxin accumulation. Placental Abcb1a/b, Abcg2, interleukin-6 (Il-6), Tnf-α, Il-10 and toll-like receptor-4 (Tlr-4) mRNA were measured by qPCR. Maternal plasma IL-6 was determined. At E15.5, maternal IL-6 was elevated 4 h after single (p<0.001) and chronic (p<0.05) LPS, but levels had returned to baseline by 24 h. Placental Il-6 mRNA was also increased after acute and chronic LPS treatments (p<0.05), whereas Abcb1a/b and Abcg2 mRNA were unaffected. However, fetal [³H]digoxin accumulation was increased (p<0.05) 4 h after acute LPS, and maternal [³H]digoxin myocardial accumulation was increased (p<0.05) in mice exposed to chronic LPS treatments. There was a negative correlation between fetal [³H]digoxin accumulation and placental size (p<0.0001). Acute and chronic sub-lethal LPS exposure resulted in a robust inflammatory response in the maternal systemic circulation and placenta. Acute infection decreased placental P-gp activity in a time- and gestational age-dependent manner. Chronic LPS decreased P-gp activity in the maternal myocardium and there was a trend for fetuses with smaller placentas to accumulate more P-gp substrate than their larger counterparts. Collectively, we demonstrate that acute sub-lethal LPS exposure during pregnancy impairs fetal protection against potentially harmful xenobiotics in the maternal circulation.