Down-regulation of Placental Folate Transporters in Intrauterine Growth Restriction

Abstract

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). the main mechanism for cellular folate uptake at physiologic pH in mammalian cells, We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3^rd percentile, mean birth weight 1804 ± 110g, gestational age 35.7± 0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 10^th–90^th centile, mean birth weight 2493 ± 216 g, gestational age 33.9 ± 0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (−34%, p<0.05). IUGR MVM had a significantly lower folate uptake compared to the control group (−38%, p<0.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR.

1. Introduction

Periconceptional folate deficiency is a well-established risk factor for fetal structural malformations such as neural tube defects (NTD) [1] and low maternal folate intake and red cell folate levels are linked to restricted fetal growth [2–4]. In addition, altered fetal folate availability has been implicated in programming of adult disease [5, 6]. Folate is a water-soluble B vitamin regulating cellular function mediated by its involvement in the synthesis of nucleotides, which are needed for DNA synthesis and repair, and its function as a methyl donor, critical for DNA methylation.

Because folate is an essential micronutrient, folate requirements for fetal growth and development must be met by placental folate transfer, which is dependent on maternal folate intake/metabolism (determining maternal serum folate concentrations) and placental folate transport capacity. The mechanisms involved in maternal-fetal transport of folate are not well understood. Folates are small (Mw ~500), hydrophilic, anionic molecules that are transported across plasma membranes by specific transport mechanisms, including the Proton-Coupled Folate Transporter (PCFT), Folate Receptor-α (FR-α), and Reduced Folate Carrier (RFC) [7]. FR-α is anchored to the plasma membrane by glycophosphotidylinositol (GPI) [7], transports folate via receptor-mediated endocytosis/exocytosis and functions at a neutral to mildly acidic pH. PCFT (SLC46A1) mediates the co-transport of folate and protons, and has optimal activity at low pH [8]. RFC is an anionic exchanger, mediating the cellular uptake of folate in exchange for anions such as organic phosphates. RFC has been proposed to be the major route of delivery of folate to systemic tissues at physiologic pH. FR-α, PCFT, RFC have all been shown to be expressed and active in the human placenta [9–12].

The syncytiotrophoblast is the transporting epithelium of the human placenta and is believed to be the major barrier to transfer of amino acids, glucose, ions and folate. The syncytiotrophoblast has two polarized plasma membranes: the microvillous plasma membrane (MVM) directed towards maternal blood in the intervillous space and the basal plasma membrane (BM) facing the fetal capillary. Human IUGR is associated with a coordinated and complex regulation of placental nutrient and ion transport capacity. Whereas syncytiotrophoblast plasma membrane amino acid transport [13–17], Na⁺/K⁺-ATPase [18], Na⁺/H⁺ exchanger [19] and lactate transporter activity [20] is down-regulated in IUGR, MVM/BM glucose transporter activity and expression have been reported to be unchanged [21] and BM Ca²⁺-ATPase to be up-regulated in IUGR placentas [22].

Previous studies on placental folate transport in IUGR have generated conflicting results with findings suggesting increased [23], unchanged [24] or decreased placental folate transport capacity in this pregnancy complication [25]. Caviedes and co-workers recently reported down-regulation of gene and protein expression of folate transporters in term placental tissue in small-for-gestational age infants [25]. However, because folate transporters contribute to transplacental transport only when present in the syncytiotrophoblast plasma membranes and transport activity was not determined, the findings in the previous study may not be informative of placental folate transport capacity in IUGR. Hence, we tested the hypothesis that IUGR is associated with decreased folate transporter expression and transport activity in isolated syncytiotrophobast microvillous plasma membranes (MVM).

2. Subjects and Methods

2.1 Study subjects and tissue collection

The University of Western Ontario Health Sciences Research Ethics Board approved the collection of tissues for this study. Pregnant women attending St. Joseph’s Health Care Centre, London, Ontario, Canada, were enrolled following obtaining informed consent. The recruitment procedure and inclusion/exclusion criteria, has been described previously [15, 26]. In brief, women with singleton pregnancies delivered by either vaginal delivery or Cesarean section were included. Pregnancies complicated by chronic hypertensive disorders, pre-eclampsia, diabetes, thyroid disorders and congenital abnormalities were excluded. The gestational age was determined from last menstrual period and corrected by ultrasound in first trimester. Appropriate-for-gestational age control placentas were obtained from preterm deliveries (n = 12, range 27–36 weeks of gestation) and from uncomplicated term pregnancies (n = 7) with a birth weight between the 25^th to 75^th percentiles for corresponding gestational age. IUGR was defined as a birth weight less than the 3rd percentile for gestational age. The placentas of the IUGR group were obtained from preterm (n = 14, range 28–36 weeks of gestation) and term (n = 11) deliveries. Placentas were obtained immediately after delivery. After removing decidua basalis and chorionic plate, ten pieces of chorionic villus tissue were dissected from random locations representing the whole placenta and immediately snap frozen in liquid nitrogen. Prior to shipping samples to University of Colorado for analysis, samples and clinical information were de-identified.

2.2 Isolation of microvillous plasma membrane vesicles

Syncytiotrophoblast MVM was prepared according to established protocols [27] with some modifications [28]. In brief, after initial centrifugation steps (at 4°C, in 250 mM sucrose, 10 mM Hepes-Tris and protease inhibitors, pH 7.4), MVM were isolated from placental homogenates by Mg²⁺ precipitation. Samples were frozen in liquid nitrogen and stored in −80°C until use. The protein concentration was determined using BCA assay (Thermo Scientific, Waltham, MA), and the MVM enrichment was assessed by measuring alkaline phosphatase activity. MVM alkaline phosphatase activity in the control (26.1 ± 4.8-fold, n = 19) and IUGR (21.3 ± 3.0-fold, n = 25) groups was similar (P = 0.407).

2.3 Protein expression of folate transporters

Samples were diluted in urea buffer with DTT and phosphate-buffered saline to a final concentration of 1.0 µg/µL and heated to 95°C for 4 minutes. Proteins were then separated on a BioRad mini-Protean TGX any kD 15-well precast gel. Proteins were transferred by electrophoresis to nitrocellulose membranes at 30V for 18 hours at 4°C. The membranes were blocked for 20 hours at 4°C in TBS solution that contained 0.05% Tween and 5% dry milk. After being washed with TBS solution for 6 hours, membranes were incubated with commercial antibodies targeting FR-α (Santa Cruz Biotechnology, Inc) at 1:10,000 dilution or RFC (Santa Cruz Biotechnology, Inc) at 1,7000 dilution overnight at 4°C. Primary antibodies were validated by pre-incubation in antigen blocking peptide, which resulted in the disappearance or marked attenuation of bands at 40 kDa (FR-α), and 58kDa (RFC), respectively. After primary antibody incubation, membranes were washed with TBS solution for 3 hours and incubated with secondary donkey anti-goat IgG antibodies, (Santa Cruz Biotechnology, Inc) for 2 hours at room temperature at a dilution of 1:1000. Bands were visualized with enhanced chemiluminescence and blots were analyzed by using ImageJ software. Target protein expression was adjusted to loading by using β-actin (Sigma-Aldrich, St. Louis, MO) as a control. The mean value of controls was arbitrarily assigned a value of 1.0 for comparisons between groups.

2.4 Folate transport activity

Total MVM Methyltetrahydrofolic acid (MTHF) uptake was determined as reported previously [12]. In brief, MVM vesicles were preloaded by incubation in 300mmol/L mannitol, 1 mmol/L adenosine diphosphate (ADP), and 10mmol/L Hepes-Tris, pH 7.4 overnight at 4°C. Subsequently, MVM vesicles were pelleted and resuspended in a small volume of the same buffer (final protein concentration, approximately 5 – 10mg/mL). Membrane vesicles were kept on ice until immediately before transport measurements when samples were warmed to 37°C. At time zero, 30µL of vesicles were rapidly mixed (1:3) with the appropriate incubation buffer that contained [3', 5', 7, 9-³H]-MTHF (Moravek Biochemicals, Brea, CA) to a final concentration of 50 nmol/L. In initial time course studies, uptake of radiolabeled substrate was terminated by the addition of 2mL of ice-cold phosphate buffered saline solution (pH 7.4) after 10, 20 or 30 seconds. For subsequent folate uptake studies a 20 second time point was used. After stopping the reaction, vesicles were separated rapidly from the substrate medium by filtration on mixed ester filters (0.45µm pore size; Millipore Corporation, Bedford MA) and washed 4 × 2 mL with phosphate buffered saline solution. In all uptake experiments, each condition was studied in triplicate for each membrane vesicle preparation. Filters were dissolved in 2 mL liquid scintillation fluid and counted; uptakes were expressed in picomoles folate per milligram of protein over time. Non-mediated uptake/nonspecific binding was determined in the presence of 1.5 mmol/L unlabeled MTHF. The ³H-MTHF uptakes could not be performed in 3 of the control and 3 of the IUGR due to inadequate amount of MVM samples.

2.5 Data presentation and statistics

The number of experiments (n) represents the number of different placentas studied. In the MTHF uptake experiments, each condition was studied in triplicate, and data were averaged to represent a value for each placenta. Data are presented as mean ± SEM. Statistical significance of differences between control and IUGR groups were assessed using Student’s t test (GraphPad Prism). A P value < 0.05 was considered significant.

3. Results

3.1 Characteristics of study subjects

Selected clinical data for the control and IUGR groups are provided in Table 1. There were no statistical differences between the two groups with regard to maternal age, body mass index (BMI) or gestational age. Birth weight was 28 % lower (P < 0.01) and placental weight was reduced by 36% (P < 0.001) in the IUGR compared with the controls. Uterine artery and/or umbilical artery Doppler blood flow measurements were available in half of the IUGR pregnancies and persistent uterine artery notching and/or an umbilical artery pulsatility index > the 95^th centile were present in all but one of these cases.

Table 1.

Selected characteristics of study subjects

	Control (n = 19)	IUGR (n = 25)
Maternal age (years)	25.9 ± 1.29	28.7 ± 1.23
Body mass index (kg/m2)#	28.3 ± 2.6	26.8 ± 2.0
Gestational age (weeks)	33.9 ± 0.95	35.7 ± 0.61
Birth weight (g)	2493 ± 236	1804 ± 110*
Birth weight percentile+	55.9 ± 4.6	2.4 ± 0.3***
Placental weight (g)	566 ± 42.0	394 ± 18.4**
Fetal sex (M/F)	7/12	8/17
Mode of delivery (C/V)	6/13	15/10

Data are presented as means ± S.E.M.

^#n=10 (Control) and 18 (IUGR);

⁺by corresponding gestational age;

^*P<0.05;

^**P<0.01;

^***P<0.001, Student’s t test.

Abbreviations: C, caesarean section; F, female; M, male; n, numbers; V, vaginal delivery

3.2 Placental folate transporter protein expression

Folate transporter expression was studied in MVM. The expression of FR-α was not significantly different between the two groups (Figure 1A and 1B), whereas MVM protein expression of RFC was significantly decreased in the IUGR group compared with controls (−34%, p <0.05, Figure 1C and 1D).

Figure 1. MVM protein expression of folate transporters.

(A) Representative Western blots for FR-α. (B) Histogram summarizing the Western blot data of FR-α. (C) Representative Western blots for RFC. (D) Histogram summarizing the Western blot data of RFC. *P<0.05; Mean ± S.E.M.; unpaired Student’s t test. n=19 (Control) and 25 (IUGR). Abbreviations: C, control; I, IUGR.

3.3. ³H-MTHF uptake in MVM

The time course of uptake of ³H-MTHF was assessed in triplicate in three samples at 10, 20, and 30 seconds (Figure 2). Binding/uptake of MTHF increased over time at pH 7.4 and no plateau was noted after 30 seconds. Based on these data, 20 seconds was chosen for subsequent experiments to compare MTHF binding/uptakes between the two groups. MVM isolated from IUGR placentas had a significantly lower MTHF uptake compared to the control group (−38%, p < 0.05, Figure 3A). This relationship remained significant between the term IUGR and control groups (−56%, p < 0.05, Figure 3B). There was a significant positive correlation between MVM RFC expression and folate transport activity (r=0.47, p< 0.0028). We further analyzed the association of MVM MTHF uptake and gestational age, however, MTHF uptake in MVM did not correlate with gestational age in either the control or IUGR groups (Figure 4A and 4B).

Figure 2. Time dependence of ³H-MTHF uptake in MVM.

MTHF uptake measured at a pH of 7.4. Each point represents the mean of three determinations.

Figure 3. MVM ³H-MTHF uptake.

(A) MTHF uptake measured at a pH of 7.4 in control and IUGR MVM. (B) MTHF uptake in term and preterm pregnancies. Data is presented as mean ± SEM, unpaired Student’s t-test. n=16 (Control) and 22 (IUGR).

Figure 4. Relationship between MVM ³H-MTHF uptake and gestational age.

Relationship between MVM ³H-MTHF uptake and gestational age in control (A) and IUGR (B) groups. MTHF uptake was measured at a pH of 7.4.

4. Discussion

The novel finding in this report is that IUGR is associated with a decreased folate transport activity in the syncytiotrophoblast microvillous plasma membrane, which is believed to be the rate-limiting step in transplacental folate transfer. In addition, MVM protein expression of RFC, the main mechanism for cellular folate uptake at physiologic pH in mammalian cells, was decreased in IUGR. These observations suggest that a decreased MVM folate transport in IUGR is due to the lower MVM abundance of RFC, a conclusion that is supported by the positive correlation between MVM RFC expression and folate transport activity. These findings add to the body of work demonstrating that placental insufficiency is associated with an array of specific changes in placental signaling and nutrient transporters. This work will further our understanding of the mechanisms underpinning fetal growth restriction and may have implications for the management of IUGR pregnancies.

Our findings that IUGR is associated with decreased folate transporter expression and transport activity in isolated syncytiotrophobast microvillous plasma membranes extend, and are in general agreement with, the study of Caviedes and collaborators reporting down-regulation of gene and protein expression of folate transporters in term placental tissue in small-for-gestational age infants [25]. In contrast, Keating and coworkers reported increased uptake of folic acid into cultured primary human trophoblast cells isolated from IUGR placentas [23]. The reasons for these discrepant findings remain to be fully established but may be due to that trophoblast cells do not fully maintain their in vivo phenotype when cultured for 3–4 days and/or that folic acid, rather than methyltetrahydrofolic acid (the predominant form of folate taken up from the circulation), was used as a tracer in the previous study [23]. Furthermore, Bisseling et al. used ex vivo dually perfused isolated cotyledons and found no change in maternal-to-fetal folate transfer or MVM RFC expression in IUGR [29]. It is possible that the relatively low number of IUGR placentas used for perfusion experiment (n=6) and RFC expression studies (n=4) may have contributed to the lack of differences between AGA and IUGR placentas in the study of Bisseling and coworkers [29].

Although the literature is not entirely consistent [3], several studies demonstrate a strong positive relationship between maternal red blood cell folate and birth weight [2–4]. Furthermore, a meta analysis of 8 randomized controlled trials investigating the effect of folate supplementation on fetal growth clearly shows a dose-response relationship between folate intake and birth weight [2]. However, the mechanisms linking folate availability to fetal growth remain to be established. We recently reported that folate deficiency in pregnant mice causes inhibition of placental mTOR signaling, down regulation of placental nutrient transport and IUGR [30], consistent with the model that impaired placental function links folate deficiency to restricted fetal growth. Thus, decreased MVM folate transport capacity in IUGR as demonstrated in the present study may directly contribute to restricted fetal growth by inhibiting placental functions regulated by mTOR signaling, including amino acid transport, mitochondrial function and protein synthesis [31]. Moreover, syncytiotrophoblast mTOR signaling regulates the plasma membrane trafficking of FR-α and RFC and it possible that placental mTOR inhibition in IUGR [15, 32] represents the mechanism of decreased MVM folate transport expression and activity in this pregnancy complication.

The decreased MVM folate transport capacity in IUGR observed in the current study may also limit fetal folate availability. Consistent with this hypothesis, Lindblad and co-workers reported that umbilical folate levels were markedly reduced in South Asian women with pregnancies complicated by IUGR [33]. Decreased fetal folate availability in IUGR may contribute to the decreased fetal growth but also program the fetus for future disease. Exposures during early life modulate the risk of developing type-2 diabetes, coronary heart disease, and hypertension later in life, a concept known as the developmental origins of health and disease (DOHaD) or fetal programming [6]. Reduced folate availability has been implicated in fetal programming [34], which may in part be mediated by the link between folate deficiency and fetal growth restriction [35] and in part due altered methylation of critical genes. Reflecting the clinical relevance of the role of folate availability and fetal programming, folic acid supplementation during pregnancy in a generally undernourished population was shown to reduce the prevalence of metabolic syndrome (MS) in children at 6 to 8 years of age [36]. Moreover, recently Wang and coworkers reported a strong association between maternal plasma folate concentrations and the metabolic health of the children in a US population [37]. In experimental studies, folate supplementation in pregnant rats modified growth patterns and the metabolic response to fasting in adult offspring [38], providing further evidence that fetal folate availability during pregnancy affects metabolic health in offspring.

The major strength of the current study is the use of microvillous plasma membranes isolated from the syncytiotrophoblast, the transporting epithelium of the human placenta, allowing the generation of information on folate transport capacity specifically in the membrane that is believed to be the rate limiting step of transplacental folate transport. The acquisition of data on both folate transporter expression and activity represents another strength and the parallel decrease in both MVM RFC protein expression and folate transport activity in IUGR provides additional confidence in our findings. Limitations of our study include the lack of detailed information on maternal folate status and/or intake and that the syncytiotrophoblast basal plasma membrane was not studied, which would have allowed us to obtain a more complete picture of placental folate transport capacity in IUGR. Using birth weight centiles as the primary definition for restricted fetal growth identifies small-for gestational age infants rather than IUGR per se. However, we used <3^rd percentile as our cutoff, which is likely to predominantly identify infants with growth restriction due to limitations in oxygen and nutrient delivery. Moreover, we have previously reported that placental mTOR signaling is inhibited and MVM System A amino acid transport activity and transporter isoform expression are decreased in this cohort of IUGR infants [15], demonstrating placental insufficiency at the molecular level. In addition, uterine or umbilical blood flow was compromised in the subgroup of IUGR cases in which uterine and/or umbilical Doppler flow measurements were available. Thus, it is likely that our IUGR group represents true IUGR and that the number of constitutionally small infants is small or negligible. Our samples were collected from both caesarean sections and vaginal deliveries and it is possible that MVM folate transport is affected by labor. However, we have previously reported that the route of delivery does not affect various placental transport functions including MVM and BM glucose transport activity [21], MVM sodium-proton exchanger activity [19] and MVM triglyceride hydrolase activity [39], suggesting that the mode of delivery does not influence MVM folate transport activity in the current study.

Based on solid scientific evidence, current recommendations for folate supplementation in reproductive age and pregnant women emphasizes the importance of supplementation 2–3 months prior to conception and during the first 12 weeks of pregnancy, in particular in high risk women, which includes women with a history of neural tube defect in previous pregnancy [40]. In contrast, the evidence for folate supplementation beyond the first trimester is less compelling. Given the findings in the current study, it may be speculated that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR. This could be beneficial by increasing fetal folate availability despite the placenta transport defect and may also stimulate placental mTOR signaling, thereby improving placental function.