Palmitic Acid Impedes Extravillous Trophoblast Activity by Increasing MRP1 Expression and Function

Normal function of placental extravillous trophoblasts (EVTs), which are responsible for uteroplacental vascular remodeling, is critical for adequate delivery of oxygen and nutrients to the developing fetus and normal fetal programming. Proliferation and invasion of spiral arteries by EVTs depends upon adequate levels of folate. Multidrug resistance-associated protein 1 (MRP1), which is an efflux transporter, is known to remove folate from these cells. We hypothesized that palmitic acid increases MRP1-mediated folate removal from EVTs, thereby interfering with EVTs’ role in early placental vascular remodeling. HTR-8/SVneo and Swan-71 cells, first trimester human EVTs, were grown in the absence or presence of 0.5 mM and 0.7 mM palmitic acid, respectively, for 72 h. Palmitic acid increased ABCC1 gene expression and MRP1 protein expression in both cell lines. The rate of folate efflux from the cells into the media increased with a decrease in migration and invasion functions in the cultured cells. Treatment with N-acetylcysteine (NAC) prevented the palmitic acid-mediated upregulation of MRP1 and restored invasion and migration in the EVTs. Finally, in an ABCC1 knockout subline of Swan-71 cells, there was a significant increase in invasion and migration functions. The novel finding in this study that palmitic acid increases MRP1-mediated folate efflux provides a missing link that helps to explain how maternal consumption of saturated fatty acids compromises the in utero environment.


Introduction
Normal fetal growth and development depends upon normal structure and function of the placenta [1]. During the first trimester, invasive extravillous trophoblasts (EVTs) obstruct the maternal arterioles, preventing the premature onset of blood flow into the intervillous space. If this process is unsuccessful, an increase in oxygen levels leads to oxidative stress and injury to the placental villi, which in turn often results in obstetrical complications [1]. Aberrant EVT function, especially reduced migration and invasion into the uterus, may lead to preeclampsia [2].
ABC transporters are membrane proteins that utilize the energy released by the hydrolysis of ATP to transport substrates out of cells. [3][4][5][6]. In humans, 49 ABC transporters have been identified, comprising seven subfamilies designated ABC-A through ABC-G [3,[5][6][7]. In the placental chorionic villi, the transporters present in the apical membrane of the chorionic villi syncytiotrophoblasts are positioned to efflux substrates towards the maternal circulation while the transporters present in the basolateral membrane of villous trophoblasts efflux the substrates towards the fetal circulation [8,9]. ABC transporters are also present in the EVTs, which express ABCA1, ABCA5, and ABCC1/MRP1 [10,11]. Folic acid, which is required for normal migration and invasion of EVTs [12], is a major substrate of MRP1 [7].
In previous work, we have shown that cultured human EVTs incubated with palmitic acid have increased cell death, decreased total antioxidant capacity, and increased lipid Biomolecules 2022, 12, 1162 2 of 14 peroxidation [13]. We have also shown that consumption of a high fat diet leads to placental inflammatory vasculopathy in mice and programs metabolic disease in their offspring, both of which are resolved with NAC [14,15]. Human consumption of a high fat diet during pregnancy compromises the intrauterine environment and leads to oxidative stress, epigenetic changes, dyslipidemia, and inflammation [16,17], resulting in altered placental development and function [18,19]. These placental abnormalities are linked to pregnancy complications such as preeclampsia, intrauterine growth restriction (IUGR), gestational diabetes (GDM), preterm birth, and stillbirth [17,19]. Preeclampsia, IUGR, and GDM can, in turn, affect fetal programming [18,19]. Here, we report that palmitic acid increases MRP1-mediated folate efflux in first trimester EVTs, thus identifying a missing link that helps to explain how palmitic acid compromises the in utero environment.

Reagents
The HTR-8/SVneo cell line was provided by Dr. Charles Graham (Queens's University, ON, Canada). Swan-71 cell lines were provided by Dr. Gil Mor (Yale University, CT, USA). JEG-3 and BeWo cell lines were obtained from ATCC (Manassas, VA, USA). DMEM, RPMI, and fatty acid-free bovine serum albumin (BSA) were purchased from VWR International Inc. (Bridgeport, NJ, USA). EMEM and F-12K media were obtained from ATCC. Palmitic and linoleic acids were obtained from TCI Chemicals (Portland, OR, USA). FBS was purchased from Atlanta Biologicals (Flowery Branch, GA, USA). Monoclonal antibodies against ABCB1, ABCG2, ABCC1, ABCC7, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA). N-acetylcysteine (NAC), trypsin, and Alexa Fluor 488 conjugated goat antirabbit IgG secondary antibody were obtained from Thermo Fisher Scientific Inc. (Rockford, IL, USA). The [3H]-folic acid, diammonium salt (50 µCi) was a product of Moravek Biochemicals, Inc (Brea, CA, USA). Corning transwell with permeable polycarbonate membrane inserts was purchased from Thermo Fisher Scientific Inc. The plasmid for ABCC1 for the CRISPR/Cas9 study was purchased from VectorBuilder (Chicago, IL, USA). Mouse embryonic fibroblasts, 2-mercaptoethanol and sodium pyruvate were obtained from Thermo Fisher Scientific Inc. Mitomycin C, heparin sodium salt, and M2 medium were purchased from Sigma Aldrich. Recombinant human FGF-4 was obtained from Peprotech (Cranbury, NJ, USA). EDTA, tetrasodium tetrahydrate salt, and Matrigel matrix were purchased from MilliporeSigma (Burlington, MA, USA). The plasmid for ABCC1 gene knockout was built and purchased from VectorBuilder. In addition, the plasmid extraction kit was purchased from Thermo Fisher Scientific Inc.

Cell Lines and Cell Culture
Placental first trimester extravillous trophoblast cell lines HTR-8/SVneo and Swan-71 were used for in vitro studies. HTR-8/SVneo cells were derived from first trimester human extravillous trophoblasts by transfection with the gene encoding for Simian virus 40 large T antigen for immortalization. RPMI supplemented with 10% FBS and 1% penicillin streptomycin (PS) was used to culture HTR-8/SVneo cells. Swan-71 cells were derived from first trimester human extravillous trophoblasts by infection with human telomerase reverse transcriptase (hTERT) expressing virus. These cells were cultured in DMEM containing 10% FBS and 1% penicillin streptomycin (PS). JEG-3 and BeWo cells, which are human choriocarcinoma cell lines, were also used for in vitro studies. The JEG-3 cell line was cultured in EMEM with 10% FBS and 1% penicillin streptomycin (PS), and the BeWo cell line was grown in F-12K medium. All cells were incubated at 37 • C and 5% CO 2 . HTR-8/SVneo and Swan-71 cell lines represent placental extravillous trophoblast cells, whereas JEG-3 and BeWo cell lines represent placental syncytiotrophoblasts. For the CRISPR/Cas9 gene knockout experiment, cells were incubated in Opti-MEM medium during transfection. The ABCC1 knockout Swan-71 subline was cultured in DMEM with 10% FBS and was further supplemented with 0.6 mg/mL of G418.

Conjugation of Palmitic and Linoleic Acids with BSA
To increase the solubility of sodium palmitate and sodium linoleate in cell culture media, we conjugated sodium palmitate and sodium linoleate with fatty acid-free bovine serum albumin (BSA). Solutions of 200 mM sodium palmitate and sodium linoleate were prepared in 50% ethanol at 55 • C. These solutions were diluted to 50 mM using 10% fatty acid-free BSA and conjugation was carried out for 2 h at 40 • C in a shaking water bath. The final conjugate was sterile filtered and stored at −20 • C. Before use, the conjugated drugs were thawed in a water bath until a clear and homogenous solution of the drugs without any turbidity was obtained. The absence of turbidity was taken as an indication that the fatty acids were still conjugated to the BSA. The conjugated palmitic and linoleic acids are stable at −20 • C for 2 months.

Cell Viability Assay
MTT assay was carried out to determine the noncytotoxic concentrations of palmitic and linoleic acids in the HTR-8/SVneo, Swan-71, JEG-3, and BeWo cell lines. Cells were seeded at 5 × 10 3 in 96-well plates with a final volume of 180 uL per well and incubated at 37 • C in the presence of 5% CO 2 for 24 h. After 24 h, the cells were incubated with or without palmitic and linoleic acids for 72 h. MTT (2 mg/mL) was added to the cells, and the cells were further incubated for 4 h. Subsequently, the supernatant was aspirated and 100 uL of DMSO was added to each well. The absorbance was read at 570 nm in an Opsys microplate reader (Dynex Technologies, Chantilly, VA, USA). The viability of untreated cells was set as 100%. For each cell line, the concentration at which palmitic and linoleic acids showed 80% cell viability was selected for further studies. The MTT assays were performed in triplicate and each assay was run three times.

Immunoblotting Assay
Western blot was performed to determine the expression ABCB1, ABCG2, ABCC1, and ABCC7 in the four placental cell lines. Whole cell lysates were prepared by using RIPA buffer after treatment with or without palmitic and linoleic acids for 0, 24, 48, or 72 h. Protein content was determined using BCA assay and 40 ug protein aliquots were resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were left at 4 • C for overnight incubation with primary monoclonal antibodies. The membranes were then washed with TBST and incubated with HRP-conjugated secondary antibody for 1.5 h. Bands were detected using an ECL detection system (LI-COR Biosciences, Lincoln, NE, USA), followed by analysis using ImageJ software (Bethesda, MD, USA).

RT-qPCR Assay
Polymerase chain reaction (PCR) was carried out to determine if there was a change in mRNA levels of the ABCC1 gene in HTR/SVneo and Swan-71 cells after incubation with palmitic acid. Reverse transcription of total RNA into cDNA was carried out using SuperScriptII reverse transcriptase (Life Technologies, Inc., Carlsbad, CA, USA) using the manufacturer's instructions. Quantitative PCR was performed using SYBR Select Master Mix (Life Technologies, Inc.) in an AriaMx (Agilent Technologies, Santa Clara, CA, USA) real time qPCR system. The data were calculated by the comparative ∆∆CT method and presented as fold change compared to the control.

[3. H]-Folate Efflux Assay
An efflux assay was carried out to determine the effect of MRP1 upregulation on folate efflux. Cells were pretreated with palmitic acid for 0, 12, 24, 48, or 72 h and then incubated with [ 3 H]-folate for 6 h. Cells were washed with ice-cold PBS and lysed at different time points (0, 30, 60, and 120 min). Radioactivity of the lysates was measured with a Packard TRI-CARB 1900CA liquid scintillation analyzer.

Immunofluorescence Assay
An immunofluorescence assay was performed to determine the expression and localization of ABCC1 in HTR-8/SVneo and Swan-71 cell lines before and after treatment with palmitic acid. Cells were seeded at 1 × 10 4 cells per well in 24-well plates overnight. The cells were then treated with 0.5 mM or 0.7 mM palmitic acid, respectively, for 0, 24, 48, or 72 h. Cells were fixed using 4% formaldehyde and permeabilized using Triton X-100. Blocking was carried out with 6% BSA and was followed by incubation with monoclonal anti-MRP1 antibody. Subsequently, the cells were incubated with 1 mL Alexa fluor 488-conjugated goat antirabbit IgG per well for 2 h. The nuclei were counterstained by DAPI at room temperature for 15 min. Fluorescent images were taken using an EVOS FL autoimaging system (Life Technologies Corporation, Carlsbad, CA, USA).

Transwell Migration Assay
To test palmitic acid's effect on EVT migration, palmitic acid treated HTR-8/SVneo and Swan-71 cells (75 × 10 3 ) were added to each transwell in serum-free media, whereas media with 10% FBS was added to the lower chamber. Transwell insert pore size was 0.4 µm. After 24 h, the cells were fixed with 4% formaldehyde and the nonmigrated cells were removed from the transwell with a cotton swab. Each transwell was further incubated with 0.5% crystal violet. Brightfield images were taken using an EVOS FL autoimaging system from the underside of the transwell inserts. Cells were then trypsinized and counted with a hemocytometer.

Transwell Invasion Assay
For the invasion assay, the transwells were coated with Matrigel diluted in serum free media (1:25) overnight at room temperature. Subsequently, 75 × 10 3 cells were added to each transwell. The steps described above were then followed.

ABCC1 Gene Knockout by CRISPR/Cas9
The ABCC1 gene was knocked out using a CRISPR/Cas9 system to create an ABCC1 knockout subline of the Swan-71 cell line. The gRNA of the human ABCC1 targeting vector contains a specific 20 bp guide sequence: 5 -GTTGACAATCTCCCCGACCG-3 . Fugene6 transfection reagent (Promega, Madison, WI) was used to transfect the ABCC1 targeting vector into Swan-71 cells, following the manufacturer's instructions. The vector consists of a U6 promoter that regulates the transcription of guide RNA (gRNA), a CBh promoter that regulates the expression of Cas9 nuclease, and a CMV promoter that regulates the transcription of the neo gene responsible for resistance to G418. An aliquot of 1 × 10 3 cells in DMEM with 10% FBS was plated in a 100 mm petri dish and incubated overnight. The plasmid (8 g) was added to 376 µL of OPTI-MEM medium. This mixture was then incubated at room temperature for 30 min and added to the cell medium for a 48 h incubation at 37 o C and 5% CO 2 . The transfected cells were then rinsed with DPBS and incubated with selection medium containing 0.6 mg/mL G418 for 14 days. The limited dilution method was used to obtain three single colonies of transfected cells, which were developed for further studies. Expression of the MRP1 protein was measured using Western blotting to confirm the knockout of ABCC1. (Supplemental Figure S1). Untransfected HTR-8/SVneo cells were resistant to G418 and were therefore not suitable for CRISPR/Cas9.

Statistical Analysis
The experimental results are presented as the mean of three or more individual experiments. Deviations from the mean are presented as mean ± SD. ANOVA was used to determine statistical significance of p < 0.05.

Cytotoxicity of Linoleic and Palmitic Acids in JEG-3, BeWo, HTR-8, and Swan-71 Cells
Before investigating the role of ABC transporters in the placenta, the noncytotoxic concentrations of palmitic and linoleic acids in the four placental cell lines were determined using an MTT assay. As shown in Figure 1A, there was 80% survival with exposure to 0.5 mM palmitic acid in JEG-3, BeWo, and HTR-8/SVneo cells and 80% survival with exposure to 0.7 mM palmitic acid in Swan-71 cells. On treatment with linoleic acid, 80% cell survival was obtained at 1 mM in all four cell lines ( Figure 1B). Therefore, 1mM linoleic acid was used in all four cell lines, 0.5 mM palmitic acid was used for JEG-3, BeWo, and HTR-8/SVneo cells and 0.7 mM palmitic acid was used for experiments with Swan-71 cells.
The experimental results are presented as the mean of three or more individual experiments. Deviations from the mean are presented as mean ± SD. ANOVA was used to determine statistical significance of p < 0.05.

Cytotoxicity of Linoleic and Palmitic Acids in JEG-3, BeWo, HTR-8, and Swan-71 Cells
Before investigating the role of ABC transporters in the placenta, the noncytotoxic concentrations of palmitic and linoleic acids in the four placental cell lines were determined using an MTT assay. As shown in Figure 1A, there was 80% survival with exposure to 0.5 mM palmitic acid in JEG-3, BeWo, and HTR-8/SVneo cells and 80% survival with exposure to 0.7 mM palmitic acid in Swan-71 cells. On treatment with linoleic acid, 80% cell survival was obtained at 1 mM in all four cell lines ( Figure 1B). Therefore, 1mM linoleic acid was used in all four cell lines, 0.5 mM palmitic acid was used for JEG-3, BeWo, and HTR-8/SVneo cells and 0.7 mM palmitic acid was used for experiments with Swan-71 cells.

Expression of ABC Transporters in JEG-3, BeWo, HTR-8/SVneo, and Swan-71 Cells
The expression of the four most studied ABC transporters PgP, BCRP, MRP1, and MRP7, was evaluated in the four placental cell lines using Western blotting. There was no expression of PgP or MRP7 in any of the cell lines (Supplemental Figure S2B,D, respectively). BCRP was expressed in the BeWo cell line (Supplemental Figure S2A). Importantly, MRP1 was expressed in all four cell lines (Supplemental Figure S2C). Hence, the effect of palmitic and linoleic acids on MRP1 was studied in all four cell lines. The modulation of BCRP by both fatty acids was also studied in the BeWo cell line.

Palmitic Acid Increases MRP1 Expression in HTR-8/SVneo and Swan-71 Cells
The modulation of BCRP in BeWo cells and MRP1 in JEG-3, BeWo, HTR-8/SVneo, and Swan-71 cells by linoleic and palmitic acids was evaluated using Western blotting. Cells were treated with the above-mentioned concentrations of linoleic and palmitic acids and the expression of the ABC transporters was evaluated at different time points from 0-72 h. Treatment with linoleic acid did not alter the expression of BCRP in the BeWo cell line or the expression of MRP1 in any of the four cell lines (Supplemental Figure S2G-J). Treatment with palmitic acid did not increase BCRP expression in the BeWo cell line or MRP1 expression in the JEG-3 or BeWo choriocarcinoma cell lines (Supplemental Figure  S2E,F). Importantly, as shown in Figure 1C-E, there was an increase in the expression of MRP1 in HTR-8/SVneo and Swan-71 cell lines upon treatment with 0.5 mM and 1 mM palmitic acid, respectively, with expression directly related to time of exposure. Based on these results, these two cell lines, which represent first trimester extravillous trophoblast cells, were selected for further studies.

Palmitic Acid Increases ABCC1 Gene Expression in HTR-8/SVneo and Swan-71 Cells
To determine the effect of palmitic acid treatment on ABCC1 gene expression, qRT-PCR analysis was performed. As shown in Figure 1G, after treatment with palmitic acid for 72 h, there was a 2.5-fold upregulation in ABCC1 gene expression in HTR-8/SVneo cells compared to untreated control cells. Similarly, in Swan-71 cells, treatment with palmitic acid caused an approximately 3-fold increase in expression of the ABCC1 gene ( Figure 1H). These results indicate that, in addition to increasing in MRP1 protein expression, palmitic acid increases the expression of the ABCC1 gene.

Palmitic Acid Increases [ 3 H]-folate Efflux in HTR-8/SVneo Cells
The effect of palmitic acid on the function of MRP1 was studied using an efflux assay. As shown in Figure 2A, there was an increase in efflux activity in HTR-8/SVneo cells after palmitic acid treatment, with the time of exposure to palmitic acid directly related to [ 3 H]-folate efflux. The efflux activity was highest in cells treated with palmitic acid for the longest exposure time, which was 72 h.

Palmitic Acid Does Not Alter the Cellular Localization of MRP1
We further evaluated whether palmitic acid alters the cellular localization of MRP1 using an immunofluorescence assay. The MRP1 transporters are located on the cell membrane in both cell types. After a 72 h treatment with palmitic acid, there was no loss of membrane localization of MRP1 compared to controls in either HTR-8/SVneo or Swan-71 cells. However, an increase in MRP1 expression was seen, which further confirms our Western blot and qRT-PCR results.

Palmitic Acid Does Not Alter the Cellular Localization of MRP1
We further evaluated whether palmitic acid alters the cellular localization of MRP1 using an immunofluorescence assay. The MRP1 transporters are located on the cell membrane in both cell types. After a 72 h treatment with palmitic acid, there was no loss of membrane localization of MRP1 compared to controls in either HTR-8/SVneo or Swan-71 cells. However, an increase in MRP1 expression was seen, which further confirms our Western blot and qRT-PCR results.

Palmitic Acid Decreases Migration and Invasion in HTR-8/SVneo and Swan-71 Cells
To ascertain whether palmitic acid decreases the invasion and migration capacity of extravillous trophoblast cells, invasion and migration assays were carried out using the Boyden chamber method. Cells that traversed the transwell membrane were considered migrated or invaded cells. As shown in Figure 3A,B,E,F, there was a decrease in migration in both cell lines as the exposure time to palmitic acid increased. Migration was the lowest in cells treated with palmitic acid for 72 h, which was the longest exposure time.
For the invasion assays, cells were treated with palmitic acid for 0 to 72 h and the cells that passed through the Matrigel and the transwell membrane were considered invaded cells. As shown in Figure 3C,D,G,H, as the time of exposure to palmitic acid increased, there was a decrease in the invasion of HTR-8/SVneo and Swan-71 cells. The invasion was the lowest in the cells treated with palmitic acid for 72 h, which was the longest exposure time.

Figure 3. Effect of palmitic acid on migration and invasion in HTR8/SVneo and Swan-71 cells.
Migration (A) and invasion (C) in HTR8/SVneo cells after exposure to palmitic acid for 0 to 72 h and migration € and invasion (G) in Swan-71 cells after the same exposures. Images were captured from the underside of the transwell inserts. Quantification (B,D,F,H) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars indicate SD. * p < 0.05 compared to controls.

NAC Prevents Palmitic Acid-induced Upregulation of MRP1
To confirm that palmitic acid-induced upregulation of MRP1 was due to increased oxidative stress, HTR-8/SVneo and Swan-71 cells were treated with or without 1 mM NAC for 2 h before being exposed to palmitic acid. The expression of MRP1 was then evaluated in both cell lines using Western blotting. As shown in Figure 4A-D, the expression of MRP1 in NAC-and palmitic acid-treated cells was much lower compared to cells treated with palmitic acid alone and was comparable to the expression of MRP1 in control untreated cells. Interestingly, expression of MRP1 in cells treated with NAC alone was lower than in control untreated cells. These results indicate that the upregulation of MRP1 by palmitic acid is mediated by oxidative stress and is prevented by treating the cells with 1 mM NAC, a direct and indirect antioxidant. For the invasion assays, cells were treated with palmitic acid for 0 to 72 h and the cells that passed through the Matrigel and the transwell membrane were considered invaded cells. As shown in Figure 3C,D,G,H, as the time of exposure to palmitic acid increased, there was a decrease in the invasion of HTR-8/SVneo and Swan-71 cells. The invasion was the lowest in the cells treated with palmitic acid for 72 h, which was the longest exposure time.

NAC Prevents Palmitic Acid-induced Upregulation of MRP1
To confirm that palmitic acid-induced upregulation of MRP1 was due to increased oxidative stress, HTR-8/SVneo and Swan-71 cells were treated with or without 1 mM NAC for 2 h before being exposed to palmitic acid. The expression of MRP1 was then evaluated in both cell lines using Western blotting. As shown in Figure 4A-D, the expression of MRP1 in NAC-and palmitic acid-treated cells was much lower compared to cells treated with palmitic acid alone and was comparable to the expression of MRP1 in control untreated cells. Interestingly, expression of MRP1 in cells treated with NAC alone was lower than in control untreated cells. These results indicate that the upregulation of MRP1 by palmitic acid is mediated by oxidative stress and is prevented by treating the cells with 1 mM NAC, a direct and indirect antioxidant.

Figure 4. Effect of NAC on MRP1 expression and migration and invasion in palmitic acid-treated HTR-8/SVneo and Swan-71 cells. MRP1 expression in palmitic acid-treated HTR8/SVneo (A) and
Swan-71cells (B) with or without NAC pretreatment determined by Western blotting. Grayscale ratios were calculated with the help of ImageJ (C,D). Migration (top row) and invasion (bottom row) of palmitic acid-treated HTR8/SVneo cells (E) and Swan-71 cells (F) with or without NAC pretreatment. Images were captured from the underside of the transwell inserts. Quantification (G,H,I,J) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars represent SD. * p < 0.05 compared to controls.

NAC Increases Migration and Invasion in HTR-8/SVneo and Swan-71 Cells
To further test the effect of NAC on the function of extravillous trophoblast cells, additional migration and invasion experiments were carried out. We found that NAC increased migration in both HTR-8/SVneo and Swan-71 cells treated with palmitic acid, as shown in Figure S4E,F, respectively (top rows, quantification shown in Figure 4G,I, respectively). Similar results were observed with invasion assays where NAC restored invasion capacity in palmitic acid-treated HTR-8/SVneo and Swan-71 cell lines ( Figure 4E,F, respectively, bottom rows, quantification shown in Figure 4H,J, respectively). Invasion and migration in NAC-and palmitic acid-treated cells was comparable to invasion and migration in untreated cells in both cell lines, showing that, in addition to preventing the upregulation of MRP1, NAC also restores migration and invasion in EVTs.

MRP1 Inhibitor MK-571 Partially Restores Migration and Invasion in HTR8-SVneo and Swan-71 Cells
To confirm that the decrease in migration and invasion of the EVTs is MRP1 mediated, HTR9/SVneo and Swan-71 cells were treated with MK-571, an MRP1 inhibitor, for 2 h, before being exposed to palmitic acid. As shown in Figure 5A,E, respectively (quantifications in Figure 5B,F), we found that HTR-8/SVneo and Swan-71 cells treated with MK-571 and palmitic acid had increased migration compared to cells treated with palmitic acid alone, but the difference was not statistically significant. Similarly, cells pretreated with MK-571 had some improvement in invasive function as compared to cells treated Images were captured from the underside of the transwell inserts. Quantification (G-J) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars represent SD. * p < 0.05 compared to controls.

NAC Increases Migration and Invasion in HTR-8/SVneo and Swan-71 Cells
To further test the effect of NAC on the function of extravillous trophoblast cells, additional migration and invasion experiments were carried out. We found that NAC increased migration in both HTR-8/SVneo and Swan-71 cells treated with palmitic acid, as shown in Figure S4E,F, respectively (top rows, quantification shown in Figure 4G,I, respectively). Similar results were observed with invasion assays where NAC restored invasion capacity in palmitic acid-treated HTR-8/SVneo and Swan-71 cell lines ( Figure 4E,F, respectively, bottom rows, quantification shown in Figure 4H,J, respectively). Invasion and migration in NAC-and palmitic acid-treated cells was comparable to invasion and migration in untreated cells in both cell lines, showing that, in addition to preventing the upregulation of MRP1, NAC also restores migration and invasion in EVTs.

MRP1 Inhibitor MK-571 Partially Restores Migration and Invasion in HTR8-SVneo and Swan-71 Cells
To confirm that the decrease in migration and invasion of the EVTs is MRP1 mediated, HTR9/SVneo and Swan-71 cells were treated with MK-571, an MRP1 inhibitor, for 2 h, before being exposed to palmitic acid. As shown in Figure 5A,E, respectively (quantifications in Figure 5B,F), we found that HTR-8/SVneo and Swan-71 cells treated with MK-571 and palmitic acid had increased migration compared to cells treated with palmitic acid alone, but the difference was not statistically significant. Similarly, cells pretreated with MK-571 had some improvement in invasive function as compared to cells treated with palmitic acid alone, but the difference was not statistically significant (Figure 5C,D,G,H). The incomplete restoration of migration and invasion is likely due to weak inhibition of MRP1by MK-571.
Biomolecules 2022, 12, x FOR PEER REVIEW 10 of 15 with palmitic acid alone, but the difference was not statistically significant ( Figure  5C,D,G,H). The incomplete restoration of migration and invasion is likely due to weak inhibition of MRP1by MK-571.

Knockout of the ABCC1 Gene by CRISPR/Cas9 Completely Restores Migration and Invasion in Swan-71 Cells
As MK-571 only partially restored EVT migration and invasion, to evaluate the role of MRP1 upregulation more fully, we completely knocked out the ABCC1 gene in Swan-71 cells using CRISPR/Cas9 technology. With complete knockout of the ABCC1 gene, there was a significant increase in migration compared to wild-type Swan-71 cells treated with palmitic acid (Figure 6A,B). Similar results were observed in the invasion assay, where there was a significant increase in invasion in the ABCC1 gene knockout subline of Swan-71 cells treated with palmitic acid as compared to wild-type Swan-71 cells treated with palmitic acid (Figure 6C,D). These results indicate that MRP1 plays an important role in the migration and invasion of EVTs.

Knockout of the ABCC1 Gene by CRISPR/Cas9 Completely Restores Migration and Invasion in Swan-71 Cells
As MK-571 only partially restored EVT migration and invasion, to evaluate the role of MRP1 upregulation more fully, we completely knocked out the ABCC1 gene in Swan-71 cells using CRISPR/Cas9 technology. With complete knockout of the ABCC1 gene, there was a significant increase in migration compared to wild-type Swan-71 cells treated with palmitic acid (Figure 6A,B). Similar results were observed in the invasion assay, where there was a significant increase in invasion in the ABCC1 gene knockout subline of Swan-71 cells treated with palmitic acid as compared to wild-type Swan-71 cells treated with palmitic acid ( Figure 6C,D). These results indicate that MRP1 plays an important role in the migration and invasion of EVTs.
was a significant increase in migration compared to wild-type Swan-71 cells treated with palmitic acid (Figure 6A,B). Similar results were observed in the invasion assay, where there was a significant increase in invasion in the ABCC1 gene knockout subline of Swan-71 cells treated with palmitic acid as compared to wild-type Swan-71 cells treated with palmitic acid (Figure 6C,D). These results indicate that MRP1 plays an important role in the migration and invasion of EVTs. Figure 6. Effect of ABCC1 knockout on migration and invasion in Swan-71 cells exposed to palmitic acid. Migration (A) and invasion (C) in Swan-71 ABCC1 knockout and wild-type cells with or without exposure to palmitic acid. Images were captured from the underside of the transwell inserts. Quantification (B,D) was carried out by counting the migrated or invaded cells using a hemocytometer. Mean values were obtained from three independent experiments and error bars represent SD. * p < 0.05.

Discussion
There has been a significant rise in the frequency of obstetrical complications in the last decade [20]. Most pregnant women in the United States are overweight or obese when they begin their pregnancies [21] and placentas delivered by obese women have activated mitogen-activated protein kinase-Jun N-terminal kinase pathways and increased oxidative stress [21]. Increased consumption of Western diets high in fat has significantly increased the incidence of preterm birth, ectopic pregnancy, spontaneous abortions, and intrauterine growth restriction [21]. Maternal high fat diet also causes fetal programming, increasing the offspring's susceptibility for cardiometabolic dysfunction [22][23][24][25]. Noncommunicable maternal diseases (NCDs), which are driven by poor maternal nutrition [26,27], have been identified as a new focus area by The International Federation of Gynecology [28][29][30][31]. The adverse effects of maternal consumption of a high fat diet are largely related to saturated fatty acids, such as palmitic acid [32]. In this work, by using concentrations of fatty acids similar to those found in placentas of mothers consuming high fat diets [33,34], we elucidate one of the mechanisms whereby consumption of diets high in saturated fatty acids compromises the in utero environment.
Various ABC transporters have been identified in the placenta and their localization and function at the maternal-fetal interface across gestation has been studied [35]. It is well-known that alterations in the expression of these transporters during different stages of gestation or due to ABC transporter modulators can lead to changes in the transfer of substrates to and from the fetus [9,36,37]. However, there are limited reports about the function and presence of ABC transporters in extravillous trophoblasts [8,38].
While several reports have shown a clear association between maternal high fat diet and complications in pregnancy [16][17][18], the molecular mechanisms linking maternal high fat diet to undesired pregnancy outcomes and developmental changes in the fetus are poorly understood. We report, here, that the expression of MRP1, an ABC transporter, is increased in first trimester placental trophoblast HTR8/SVneo and Swan-71 cell lines treated with palmitic acid. In addition, we found that the expression of the ABCC1 gene is also increased by palmitic acid treatment in both cell lines. This effect was not seen in syncytiotrophoblast cell lines, suggesting that MRP1 upregulation by palmitic acid may be specific to EVTs. We also found that MRP1 was not found in the cytoplasm after palmitic acid treatment but remained localized to the EVT cell membrane, which is its normal cellular location [39], consistent with the increase in the number of MRP1 protein molecules affecting efflux function.
Others have reported that the expression and function of MRP1 increases in the presence of oxidative stress [40,41]. Oxidative stress is strongly linked to unfavorable obstetrical outcomes [42]. For example, accelerated placental aging is driven by oxidative stress and leads to preterm birth [42,43]. Our finding that NAC prevents upregulation of MRP1 and rescues migration and invasion in palmitic acid-exposed EVTs provides a connection between oxidative stress in the in utero milieu and EVT function. Saturated fatty acids such as palmitic acid have been shown to decrease total antioxidant capacity and increase lipid peroxidation in EVTs [10]. Oxidative stress, in turn, results in increased MRP1mediated folate efflux, depleting intracellular folate pools needed for normal EVT driven spiral artery remodeling. Furthermore, glutathione (GSH), the body's main antioxidant [44], is also a substrate of MRP1. Increases in MRP1 expression lead to increases in GSH efflux rates, locking together oxidative stress, MRP1 upregulation, and folate efflux in a feed forward loop.
Folic acid is prescribed to pregnant women to prevent fetal defects and to enhance migration and invasion functions of EVTs during placentation [1,12,45]. We show here, for the first time, that palmitic acid causes folate efflux from EVTs with a direct correlation between the rate of efflux and the time of exposure to this saturated fatty acid. In addition, palmitic acid treatment significantly decreases migration and invasion in both HTR8/SVneo and Swan-71 cells, linking a saturated fatty acid to impaired EVT function. We also found that pretreatment of palmitic acid-exposed EVTs with the antioxidant NAC significantly decreases MRP1 expression and restores EVT migration and invasion, suggesting that increased MRP1 expression is linked to impairment of EVT function. Consistent with MRP1 s role in impairing EVT function, inhibiting MRP1 pharmacologically with the weak inhibitor MK-571 partially restores migration and invasion in HTR-8/SVneo and Swan-71 cells. Furthermore, eliminating MRP1 completely, by knocking out the ABCC1 gene, restores migration and invasion significantly in Swan-71 cells.

Conclusions
EVTs play a very important role in the development of the placenta [46,47]. They migrate and invade spiral arteries, which is critical for remodeling [46,48,49]. Previous studies have shown that palmitic acid induces oxidative stress in cultured EVTs. Oxidative stress, in turn, is known to increase levels of MRP1 [13,41,50,51]. The novel finding in this study is that palmitic acid impairs EVT function by inducing MRP1-mediated folate efflux. Elucidation of pathways connecting maternal high fat diet consumption to fetal programming provides insights that can lead to future therapeutic approaches to safeguard the developmental origins of human health.