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. 2021 Dec 20:12:771818.
doi: 10.3389/fphar.2021.771818. eCollection 2021.

Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

Esha Ganguly  1 , Ananth Kumar Kammala  1 , Meagan Benson  1 , Lauren S Richardson  1   2 , Arum Han  2 , Ramkumar Menon  1
Affiliations

Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

Esha Ganguly et al. Front Pharmacol. .

Abstract

Current intervention strategies have not been successful in reducing the risks of adverse pregnancy complications nor maternal and fetal morbidities associated with pregnancy complications. Improving pregnancy and neonatal outcomes requires a better understanding of drug transport mechanisms at the feto-maternal interfaces, specifically the placenta and fetal membrane (FM). The role of several solute carrier uptake transporter proteins (TPs), such as the organic anion transporting polypeptide 2B1 (OATP2B1) in transporting drug across the placenta, is well-established. However, the mechanistic role of FMs in this drug transport has not yet been elucidated. We hypothesize that human FMs express OATP2B1 and functions as an alternate gatekeeper for drug transport at the feto-maternal interface. We determined the expression of OATP2B1 in term, not-in-labor, FM tissues and human FM cells [amnion epithelial cell (AEC), chorion trophoblast cell (CTC), and mesenchymal cells] using western blot analyses and their localization using immunohistochemistry. Changes in OATP2B1 expression was determined for up to 48 h after stimulation with cigarette smoke extract (CSE), an inducer of oxidative stress. The functional role of OATP2B1 was determined by flow cytometry using a zombie violet dye substrate assay. After OATP2B1 gene silencing, its functional relevance in drug transport through the feto-maternal interface was tested using a recently developed feto-maternal interface organ-on-a-chip (OOC) system that contained both FM and maternal decidual cells. Propagation of a drug (Rosuvastatin, that can be transported by OATP2B1) within the feto-maternal interface OOC system was determined by mass spectrometry. FMs express OATP2B1 in the CTC and AEC layers. In FM explants, OATP2B1 expression was not impacted by oxidative stress. Uptake of the zombie violet dye within AECs and CTCs showed OATP2B1 is functionally active. Silencing OATP2B1 in CTCs reduced Rosuvastatin propagation from the decidua to the fetal AEC layer within the feto-maternal interface-OOC model. Our data suggest that TPs in FMs may function as a drug transport system at the feto-maternal interface, a function that was previously thought to be performed exclusively by the placenta. This new knowledge will help improve drug delivery testing during pregnancy and contribute to designing drug delivery strategies to treat adverse pregnancy outcomes.

Keywords: drug transport; human fetal membranes; organ-on-a-chip; pregnancy; transporter protein.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Localization and expression of OATP2B1 transporter protein in fetal membrane tissues and human placenta. (A) Bright field microscopy showing localization of OATP2B1 (brown) in human fetal membrane AECs and CTCs (left image) at a magnification of 20x (main image) and 100x (inset image) and placental syncytiotrophoblast at a magnification of 20x (main and inset image (right image). 20x and 100x images were taken in different regions of the tissue. Images are representative of three biological replicates. Scale bar = 30 µM. (B) Western blot analysis and quantification of OATP2B1 in human fetal membranes and placenta (maternal and fetal side). β-Actin is used as a loading control. Representative western blots are shown. n = 6 biological replicates. Error bars represent mean ±SEM.
FIGURE 2
FIGURE 2
Expression of OATP2B1 transporter protein in human fetal membrane cells. (A) Cell surface expression of OATP2B1 in fetal membrane cells (AECs, AMCs, CTCs) and maternal decidual cells (DECs) using primary antibody conjugated with CFR647. Representative histograms of stained samples are shown. The mean fluorescence of these histograms was calculated using the FlowJo Software. (B) Western blot analysis and quantification of OATP2B1 in fetal membrane AECs, AMC, CTCs, and maternal DECs. Actin was used as a loading control. Representative blots are shown. Error bars represent mean ± SEM. n = 4 biological replicates.
FIGURE 3
FIGURE 3
Functional validation of OATP2B1 in fetal membrane and placental cells. (A) Functional activity of OATP2B1 in fetal membrane (AECs and CTCs) and placental (BeWo) cells was determined by flow cytometry. Representative histograms of zombie violet dye uptake are shown. (B) The mean fluorescence intensity of these histograms was calculated using the FlowJo software. n = 4 biological replicates. Error bars represent mean ± SEM.
FIGURE 4
FIGURE 4
Expression of OATP2B1 in fetal membrane and placenta explants upon stimulation with CSE-induced oxidative stress. (A) Western blot analysis and quantification of OATP2B1 in CSE-treated fetal membrane explants shows no significant effect on OATP2B1 expression. (B) Western blot analysis and quantification of OATP2B1 in CSE-treated placental explants shows a time-dependent reduction in OATP2B1 expression (***p < 0.001). β-Actin was used as a loading control. Representative blots are shown. n = 5 biological replicates. Error bars represent mean ± SEM.
FIGURE 5
FIGURE 5
Effect of OATP2B1 silencing on zombie violet dye uptake assay in placental BeWo cells and fetal membrane CTCs. (A) Western blot analysis and quantification of OATP2B1 in siRNA-treated BeWo cells and CTCs to determine the efficiency of knockdown. OATP2B1 was silenced in BeWo cells (70%) and CTCs (80%) using short-interfering RNA (siRNA). Non-targeted scrambled siRNA is used as control siRNA. β-Actin was used as a loading control. Representative blots are shown. Error bars represent mean ± SEM. (B) OATP2B1 siRNA-treated BeWo cells and CTCs were subjected to the zombie violet dye uptake assay. The data shows that the uptake of zombie violet dye (as indicated by the percent change in mean fluorescence intensity compared to the unstained controls) was reduced to a greater extent in OATP2B1 siRNA-treated CTCs compared to the siRNA treated BeWo cells. Representative histograms of the dye uptake determined by flow cytometry analysis are shown. The percent change in mean fluorescence intensity (MFI) in OATP2B1 knockdown CTCs and BeWo cells (**p < 0.01) compared to their respective controls was calculated using the FlowJo software. n = 4 biological replicates. Error bars represent mean ± SEM.
FIGURE 6
FIGURE 6
Role of OATP2B1 in rosuvastatin transport across the feto-maternal interface-OOC. (A) Schematic showing the four concentric cell culture chambers of the feto-maternal interface-OOC used to determine the role of OATP2B1 in transporting drug across the different fetal membrane chambers [AECs (blue, chamber 1), AMCs (pink, chamber 2), CTCs (yellow, chamber 3), and maternal DECs (green, chamber 4)]. Rosuvastatin (200 ng/ml) was introduced in the decidual chamber of the feto-maternal interface-OOC with either control mock siRNA or OATP2B1-knockdowned CTCs. Following incubation for 4 h, media was collected from different chambers of feto-maternal interface-OOC for mass spectrometry analysis. (B) Under control siRNA conditions, 18.45 and 1.73% of rosuvastatin propagated from the maternal DEC to the CTC chamber as well as to the AEC chamber, respectively. OATP2B1 silencing in OATP2B1 resulted in only 2.57% rosuvastatin propagation from the maternal DEC chamber to the CTC chamber, and only 0.32% of rosuvastatin propagation from the DEC chamber to the AEC chamber, and then zero to the CTC chamber. Data is represented as mean ± SEM. Rosuvastatin propagation across the feto-maternal interface -OOC was confirmed from six independent experiments.
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