Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion

Cecilia Y Cheung; Debra F Anderson; Marion Rouzaire; Loïc Blanchon; Vincent Sapin; Robert A Brace

doi:10.1177/1933719118765979

. 2018 Mar 27;26(10):1351–1359. doi: 10.1177/1933719118765979

Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion

Cecilia Y Cheung ^1,^2,^✉, Debra F Anderson ², Marion Rouzaire ³, Loïc Blanchon ³, Vincent Sapin ³, Robert A Brace ^1,²

PMCID: PMC6949972 PMID: 29587617

Abstract

Vascular endothelial growth factor (VEGF) has been proposed as an important regulator of amniotic fluid absorption across the amnion into the fetal vasculature on the surface of the placenta. However, the activators of VEGF expression and action in the amnion have not been identified. Using the pregnant sheep model, we aimed to investigate the presence of the retinoic acid (RA) pathway in ovine amnion and to determine its effect on VEGF expression. Further, we explored relationships between RA receptors and VEGF and tested the hypothesis that RA modulates intramembranous absorption (IMA) through induction of amnion VEGF in sheep fetuses subjected to altered IMA rates. Our study showed that RA receptor isoforms were expressed in sheep amnion, and RA response elements (RAREs) were identified in ovine RARβ and VEGF gene promoters. In ovine amnion cells, RA treatment upregulated RARβ messenger RNA (mRNA) and increased VEGF transcript levels. In sheep fetuses, increases in IMA rate was associated with elevated VEGF mRNA levels in the amnion but not in the chorion. Further, RARβ mRNA was positively correlated with VEGF mRNA levels in the amnion and not chorion. We conclude that an RA pathway is present in ovine fetal membranes and that RA is capable of inducing VEGF. The finding of a positive relationship between amnion VEGF and RARβ during altered IMA rate suggests that the retinoid pathway may play a role through VEGF in regulating intramembranous transport across the amnion.

Keywords: RAR and RXR, sheep fetal membranes, intramembranous transport, amniotic fluid

Introduction

During pregnancy, the amnion is a multifunctional tissue originating from the embryonic ectodermal layer. The amnion together with the underlying chorion encompasses the accumulated amniotic fluid serving as a protective enclosure to facilitate growth and maturation of the developing fetus. Early studies described the amniotic membrane as epithelial with transport characteristics.^1–3 Subsequently, a role for the placental amnion in the regulation of amniotic fluid volume (AFV) has been proposed.⁴ In humans, the amnion is partitioned into 3 regions with distinct morphology and gene expression patterns⁵: placental amnion, reflected amnion juxtaposed to the membranous chorion, and amnion overlaying the umbilical cord.⁶ Recent studies have established the concept that AFV is regulated by vesicular transport pathways within the placental amnion⁷ to modulate uptake and transcytosis of amniotic fluid from the amniotic cavity into the fetoplacental circulation within the placental plate. This transport process is referred to as intramembranous absorption (IMA).^8,9

Vascular endothelial growth factor (VEGF) has been shown to stimulate vesicular transcytosis by postreceptor activation of the protein tyrosine kinase c-Src signaling¹⁰ to initiate caveolar-mediated uptake and transport of macromolecules such as albumin across vascular endothelial cells.¹¹ In humans, VEGF is expressed in the amniotic epithelium, and VEGF receptors are present in endothelium of placental blood vessels.^12,13 In animal models including sheep, VEGF is similarly localized in fetal membranes and its receptors in intramembranous blood vessel endothelium.^14,15 We previously have shown that VEGF activated caveolin-1 phosphorylation in ovine amnion cells,¹⁰ consistent with a role in mediating transport by caveolar vesicles. Further, conditions that increase amniotic fluid absorption were associated with upregulations of VEGF gene expression in the sheep amnion.^16,17 Together, these findings suggest that VEGF may be an important mediator of amniotic fluid transport across the amnion and thus regulate AFV.¹⁸ However, the activators of VEGF to initiate transamnion transport pathways have not been described.

Retinoic acid (RA), the active derivative of vitamin A, is an important regulator of embryonic development through modulation of cell growth and differentiation.^19,20 These effects are mediated by the RA receptors RARα, β, and γ and RXRα, β, and γ that belong to a class of ligand-activated nuclear transcription factors. Their action in transcriptional regulation of target genes is dependent on coordinated assembly of RAR–RXR heterodimers that bind to RA response element (RARE) at direct repeats predominantly spaced by 5 nucleotides (DR5) sequences in the promoter region of target genes.²¹ The RAR and RXR transcription factors are expressed in the placenta and regulate expression of placental genes²² including placental lactogen and epidermal growth factor receptor.²³ In studies of retinal pigment cells²⁴ and pancreatic islet cells,²⁵ RA has been shown to modulate vascular angiogenesis and VEGF gene expression. More recently, the retinoid pathway has been described in human amniotic membranes and may be metabolically active to modulate amnion functions as has been shown for the AQP3 water channel.^26,27 Therefore, the possibility exists that the retinoid pathway in the amnion may be involved in regulating the VEGF-mediated intramembranous transport pathway.

In the present study, we used the ovine model to test the hypotheses that an RA pathway is present and functional in ovine amnion and that RA induces VEGF expression presumably through its receptors. Further, we tested whether modifications in IMA rate would be associated with changes in VEGF gene expression and RA levels in the amnion. Because the sheep amnio–chorionic interface is vascularized with fetal microvessels that arise from placental cotyledons,²⁸ the chorion was studied in addition to the amnion as potential target for RA action. Specifically, our objectives were to (1) investigate the presence of RA receptors in ovine amnion and chorion, (2) determine whether ovine VEGF is a target gene inducible by RA similar to its effect on RARβ, a well-recognized RA inducible gene, (3) analyze the gene expression profiles of RARβ and VEGF in the amnion and chorion of fetuses during experimentally modified IMA rates, and (4) explore relationships between IMA rate, RARs, and VEGF expression under conditions of altered IMA rate and AFV. In this study, we utilized changes in target gene expression as an indirect measure of gene activity. Determination of RA levels in the amnion and chorion of experimental animals were not performed because of the short half-life due to rapid degradation.

Materials and Method

Amnion Cell Culture and Experimentation

These studies were approved by our Institutional Animal Care and Use Committee and were conducted in accordance with guidelines of the National Research Council’s Guide for the Care and Use of Laboratory Animals. Amniotic membranes were collected from 4 near-term ovine fetuses for preparation of primary cultures of amnion cells using procedures described in our previous studies.^10,29 Following digestion with 0.625% trypsin, amnion cells were plated onto T-75 flasks (Corning Life Sciences, Union City, California) in Dulbecco Modified Eagle Medium: Nutrient Mixture F12 (DMEM/F12; Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1× antibiotic/antimycotic (Thermo Fisher Scientific). The cells were maintained at 37°C in a humidified atmosphere of air supplemented with 5% CO₂. At the second subculture, the cells were harvested and plated onto 6-well plates (Falcon; Thermo Fisher Scientific) for experimentation. At 70% to 80% confluence, cells were serum withdrawn for 24 hours and then treated with all trans-retinoic acid (atRA) at 1 µmol/L (Sigma-Aldrich, St Louis, Missouri) dissolved in dimethyl sulfoxide (DMSO). The concentration of RA used in this study was a well-accepted dose for optimal activation of RA nuclear receptors and routinely used in our previous studies.³⁰ For blockade experiments, cells were pretreated for 1 hour with cycloheximide (1 µg/mL; Sigma-Aldrich) to inhibit protein synthesis followed by testing with atRA in the presence of cycloheximide. Experiments were terminated at 6, 12, or 24 hours, and cells were collected for RNA extraction using an RNeasy kit (Qiagen, Inc, Valencia, California).

Amniotic Fluid Transport Studies in Sheep Fetuses

We utilized amnion and chorion tissues from 16 late gestation fetal sheep from recent studies.³¹ The fetuses were implanted with chronic catheters and subjected to 2-day experimental protocols under 4 conditions (n = 4 per group): (1) control, (2) continuous fetal urine drainage, (3) urine drainage with isovolumic fluid replacement (lactated Ringer’s solution), and (4) continuous intra-amniotic infusion (2 L/d of lactated Ringer’s solution). During the experiment, urine flow rate, lung liquid secretion rate, and swallowed volume were continuously monitored. Amniotic fluid volume was determined at the beginning and end of each experiment, and the average IMA rate during the 2-day experimental period was calculated from the change in AFV and the time integrated amniotic inflows and outflows.^9,32–34 At completion of the experiment, amnion and chorion tissues were collected immediately and placed in RNAlater (InVitrogen, Thermo Fisher Scientific) for RNA extraction using an RNeasy kit.

Real-Time RT-qPCR

The VEGF messenger RNA (mRNA) levels were determined by real-time Quantitative Reverse Transcription - Polymerase Chain Reaction (RT-PCR) as previously described.^31,35 Total RNA (2 µg) was reverse transcribed using MultiScribe reverse transcriptase. Sample complementary DNA (25 ng) was amplified using custom-designed (Primer Express Software v3.0. Applied Biosystems, Thermo Fisher Scientific) ovine-specific primers and probes for VEGF₁₆₄ (Table 1) in a TaqMan Gene Expression Assay system (Applied Biosystems, Grand Island, NY). The temperature profile was: initial 2-step hold at 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The amplified sequence was validated by sequencing and alignment to the consensus ovine VEGF sequence. Two endogenous references, 18S ribosomal RNA and ovine ribosomal protein lateral stalk subunit P0 (RPLO), were used as housekeeping genes. Standard curves were incorporated into the assays for VEGF and each of the 2 endogenous references. All samples were quantified in triplicates and normalized to the geometric mean of the housekeeping genes. For the quantification of RARα, β, γ and RXRα, β, γ, and retinaldehyde dehydrogenase 2 (RALDH2), 1 µg RNA was reverse transcribed with a Superscript III First-Strand Synthesis System (Invitrogen, Thermo Fisher Scientific) for RT-PCR. The ovine-specific primer sequences of RARs and RXRs used for amplification are detailed in Table 1. Quantitative RT-PCR was performed using sensifast SYBR (Bioline, Paris, France) and LightCycler480 SYBR Green-I Master (Roche, Lyon, France) with the temperature profile: 95°C for 10 minutes, followed by 45 cycles of 10 seconds at 95°C, 10 seconds at 61°C, and 15 seconds at 72°C. Each sample was quantified in triplicate and normalized to the housekeeping genes ribosomal protein lateral stalk subunit P0( RPLP0) and ribosomal protein S17 ( RPS17). The relative quantity of target mRNA was determined by the comparative cycle threshold (C_T) method after PCR efficiency correction and normalization to the geometric mean of the C_T values for the 2 housekeeping genes (ΔC_T). For comparison of expression levels, fold change of target mRNA was calculated as the 2^−ΔΔC _T value using the control group as the calibrator.³⁶

Table 1.

Custom Designed Ovine Primers and Probe.

Gene	Primer/Probe	Nucleotide Sequence	Amplicon Size
VEGF₁₆₄	Forward	5′-GGCGAGGCAGCTTGAGTTAA-3′	75 bp
	Reverse	5′-CACCGCCTCGGCTTGTC-3′
	Probe	5′-6FAMCGAACGTACTTGCAGATGMGBNFQ-3′
RARα	Forward	5′-AAATCCTCAGGCTACCACTATG-3′	152 bp
RARα	Reverse	5′-CAGTACTGGAACGGTTCCG-3′	152 bp
RARβ	Forward	5′-TTTACACTTGTCACCGAGATAAG-3′	192 bp
RARβ	Reverse	5′-CAGCTCTGCCGTCATCTCGT-3′	192 bp
RARγ	Forward	5′-GAGGGTCTCCACCTTTCGAG-3′	177 bp
RARγ	Reverse	5′-ACACGTAGCACGGCTTGTAGA-3′	177 bp
RXRα	Forward	5′-GGCTTGATGTCCTCGCTGCT-3′	166 bp
RXRα	Reverse	5′-TTAGCTCGCCCATCAATGGCA-3′	166 bp
RXRβ	Forward	5′-TGTCAGTACTGCCGCTACCAG-3′	139 bp
RXRβ	Reverse	5′-TCCTGTCCACAGGCATCTCC-3′	139 bp
RXRγ	Forward	5′-GTCGGGATAACAAAGACTGCC-3′	171 bp
RXRγ	Reverse	5′-AATCCTCTCCACGGGCATATC-3′	171 bp
RALDH2	Forward	5′-CCTCCAGGTGTGGTCAATATTA-3′	162 bp
RALDH2	Reverse	5′-CAGGGTCACTCTCTTCAGGTT-3′	162 bp

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Abbreviations: RAR, retinoic acid receptor; RALDH2, retinaldehyde dehydrogenase 2; VEGF, vascular endothelial growth factor.

In Silico Analysis

Potential regulatory sequences of RARE were identified by in silico screening of promoters using the V$RXRF matrix bundled with Genomatix software (matrix family library version 6.3).

Statistical Analysis

Data are presented as mean ± standard error. One-factor analysis of variance (ANOVA) was used to compare mRNA levels among groups and 2-factor ANOVA among groups over time. If the null hypothesis was rejected, Fisher least significant difference was used for post hoc testing. The Kruskall-Wallis test was used when the data were nonnormally distributed. Relationships between IMA rate, AFV, VEGF mRNA, and RAR mRNA levels were determined by least squares bivariate regression. Pearson correlation coefficient (r) was used to express the degree of linearity between 2 variables. Data were logarithmically transformed as needed to normalize variances prior to analysis. P < .05 was considered significant.

Results

Retinoic Acid Pathway Components in Ovine Fetal Membranes

Retinoic acid receptor isoforms were identified in ovine fetal membranes by RT-PCR and gel electrophoresis. Expression of RARα, RARβ, and RARγ as well as RXRα and RXRβ was readily detected in both amnion and chorion, whereas RXRγ was detectable but the level was low at the limit of detectability (Figure 1). For all receptor isoforms, expression levels in the amnion appeared less abundant than in the chorion (Figure 1). To determine the functionality of RARs in the amnion, the ability of RA to stimulate RARβ, a well-known retinoid inducible gene, was tested in ovine amnion cells treated with atRA (1 µmol/L). RARβ mRNA levels significantly increased at 6, 12, and 24 hours of treatment (P < .01; Figure 2A) when compared to controls treated with DMSO alone. Following cycloheximide pretreatment (1 µg/mL), RARβ mRNA levels were similarly elevated in response to atRA at all 3 time points (P < .01) without attenuation relative to atRA treatment alone (Figure 2B). The presence of DR5 sequences in the RARE of the sheep RARβ promoter was demonstrated by in silico analysis, which permitted the identification of 2 copies of the RARE-DR5 sequences proximal to the transcription start site of the RARβ gene (data not shown).

Figure 1. — Identification of retinoic acid receptors (RAR) α, β, γ and RXRα, β, γ by gel electrophoretic separation of RT-PCR products from ovine amnion (A) and chorion (Ch). M indicates molecular marker; NC, negative control, that is, sample buffer without complementary DNA.

Figure 2. — Retinoic acid induction of retinoic acid receptor β (RARβ) in ovine amnion cells. A, Amnion cells were treated with *all trans-*retinoic acid (atRA) at 1 µM in dimethyl sulfoxide (DMSO) for 6, 12, or 24 hours. Control cells were treated with DMSO alone. *, P < .001 compared to DMSO at the same time point. B, Amnion cells were treated with atRA (1 µmol/L) in the presence of cycloheximide (Chx, 1 µg/mL). Control cells were treated with DMSO + cycloheximide. *, P < .001 compared to DMSO + cycloheximide at the same time point. The results were analyzed statistically by 2-factor analysis of variance (ANOVA) for treatment over time.

The upregulation of RARβ levels by atRA in amnion cells was associated with parallel stimulation of VEGF. All trans-retinoic acid at 1 µmol/L increased VEGF mRNA levels at 6 hours compared to DMSO controls and continued to increase significantly at 12 and 24 hours (Figure 3A). Pretreatment with cycloheximide (1 µg/mL) similarly did not suppress the VEGF response to atRA over the 24-hour period (3B). In silico analysis of the ovine VEGF gene promoter demonstrated the presence of 1 copy of RARE-DR5 sequence situated at ∼2500 base pairs upstream from the transcription start site (data not shown).

Figure 3. — Retinoic acid stimulation of vascular endothelial growth factor (VEGF₁₆₄) expression in ovine amnion cells. A, Amnion cells were treated with atRA at 1 µmol/L in dimethyl sulfoxide (DMSO) for 6, 12, or 24 hours. Control cells were treated with DMSO alone. *, P < .01 compared to DMSO at the same time point. B, Amnion cells were treated with atRA (1 µmol/L) in the presence of cycloheximide (Chx, 1 µg/mL). Control cells were treated with DMSO + cycloheximide. *, P < .001 compared to DMSO + cycloheximide at the same time point. The results were analyzed statistically by 2-factor analysis of variance (ANOVA) for treatment over time.

Intramembranous Absorption and Gene Expression Profiles in Ovine Fetuses

Control AFV was 1110 ± 125 mL while IMA rate was 667 ± 159 mL/d (Table 2). Urine drainage significantly reduced IMA rate and AFV, whereas intra-amniotic infusion increased both IMA rate and AFV. In the urine drainage with replacement group, the increase in AFV and reduction in IMA rate were nonsignificant. Combined data from the 4 experimental groups showed that AFV was positively correlated with IMA rate (r = .69, P < .01).

Table 2.

Amniotic Fluid Dynamics in Ovine Fetuses Under Experimental Conditions.

Volume/Flow Rate	Control, n = 4	Urine Drainage, n = 4	Urine Replacement, n = 4	Intra-Amniotic Fluid Infusion, n = 4	Statistics (ANOVA)
Amniotic fluid volume, mL	1110 ± 125	300 ± 77	1449 ± 220	3351 ± 365	P < 10⁻⁵
Intramembranous absorption rate, mL/d	667 ± 159	101 ± 120	515 ± 29	1366 ± 274	P < .002

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Abbreviation: ANOVA, analysis of variance.

In the amnion, VEGF mRNA levels were significantly elevated during intra-amniotic infusion (Figure 4). The VEGF levels were similarly increased in the urine drainage and urine replacement groups. These changes in VEGF mRNA levels were associated with similar expression profiles for RARβ mRNA (Figure 4), although the changes in RARβ were not statistically significant. In the chorion, there were no significant changes in expression levels of VEGF or RARβ in response to altered IMA rate or AFV (Figure 4). When data from all 4 groups under different experimental conditions of IMA rate were combined, a significant positive correlation occurred between VEGF and RARβ mRNA levels in the amnion (r = .698, P < .01; Figure 5A). This positive relationship was found in the amnion only and not in the chorion (Figure 5B).

Figure 5. — Regression analysis between retinoic acid receptor β (RARβ) and vascular endothelial growth factor (VEGF₁₆₄) messenger RNA (mRNA) levels in the amnion (A) and chorion (B) of ovine fetuses subjected to experimental modifications in intramembranous absorption rate. Bivariate regression analysis was performed on all animals in 4 experimental groups combined (n = 16). Pearson correlation coefficient is represented by the r value. Amnion data were log transformed to normalize variances. The analysis showed a significant positive correlation between VEGF and RARβ mRNA levels in the amnion (P < .01). The relationship between VEGF and RARβ mRNA levels in the chorion was not significant. Filled circle, control; cross, urine drainage; open triangle, urine replacement; filled triangle, intra-amniotic infusion.

The expression of RALDH2 was detected in both amnion and chorion. The mRNA levels decreased in the urine drainage group in the amnion and the urine replacement group in the chorion (Figure 4).

Intramembranous Absorption Relationships With VEGF and RA Receptors

Relationships between IMA rate or AFV and the RARα, RARβ, and RARγ isoforms were examined, and no significant correlations were detected in either the amnion or chorion. Bivariate linear regression analysis showed that VEGF mRNA levels in the amnion similarly were not correlated with IMA rate or AFV (data not shown).

Discussion

The present study demonstrated the existence of an RA pathway in sheep fetal membranes. This finding is comparable to the RA molecular pathway described in the human amnion.²⁶ The presence of all members of the RA receptor family (RAR and RXR) as well as RALDH2 (the RA catalytic enzyme) in both amnion and chorion indicated that the ovine fetal membranes are potential target tissues for retinoid action. Our observations that RARβ was inducible by atRA in ovine amnion cells together with the ineffectiveness of cycloheximide to abolish the RARβ induction, confirmed the ligand-inducible characteristics of RARβ as direct activation and not protein synthesis dependent. The presence of RARE-DR5 sequences in the sheep RARβ promoter further supports the notion that RARβ is a direct retinoid inducible gene in ovine amnion.

In association with the RA-induced upregulation of RARβ, our studies demonstrated that atRA was capable of stimulating VEGF gene expression in amnion cells as has been demonstrated in other cell types.^24,25 Since the effect of atRA was not inhibited by cycloheximide, the stimulation of VEGF is most likely direct and not secondary through protein synthesis. This is further substantiated by identification of the RARE-DR5 sequence in the sheep VEGF promoter. The induction of VEGF paralleled that of RARβ suggesting that atRA was able to simultaneously enhance the transcription of both RARβ and VEGF. These findings suggest the amnion as a target tissue for RA signaling possibly to modulate amnion cellular function.

In this study, we observed that experimentally induced increases in IMA rate led to upregulation of VEGF in the amnion, consistent with our previous findings.^16,17 However, VEGF mRNA levels similarly increased when IMA rate was reduced or unchanged. This indicates that although VEGF may function as a permeability factor to regulate amniotic fluid uptake, additional factors most likely participate in modulating intramembranous transport. Further, VEGF may act indirectly through other mechanisms to modify IMA rate. Conversely, the absence of correlation between VEGF mRNA level in the amnion and IMA rate under experimental conditions of altered intramembranous transport does not corroborate the notion that VEGF plays a critical role in this regulation. During intra-amniotic fluid infusion, IMA rate was elevated and amnion VEGF expression levels increased. Importantly, when IMA and AFV were experimentally modified, a significant positive correlation was found between VEGF and RARβ transcript levels in the amnion. Thus, the changes in VEGF expression could be related to the transcriptional modification of RARβ activity in response to changes in IMA rate. These results are consistent with the assumption that the RA molecular pathway in the amnion may function to induce VEGF during conditions of altered amniotic fluid transport across the fetal placental surface. The identification of vitamin A and retinol-binding proteins in amniotic fluid by other investigators³⁷ supports the potential role of retinoids in amniotic fluid regulation.

In this study, the absence of significant changes in either VEGF or RARβ mRNA levels in the chorion during experimental modifications of IMA rate, as well as the lack of relationship between chorion VEGF and RARβ expressions, suggest that the chorion is most likely not a target tissue for the retinoid pathway.

In the amnion, relationships between RA nuclear receptor expression and IMA rate or AFV were not demonstrated. This finding reaffirms the notion that the RAR nuclear factors are transcriptional regulators of amnion-expressed genes including the VEGF gene rather than as mediators at the translational and functional levels.

In conclusion, the present study demonstrated the presence of an RA molecular pathway in sheep fetal membranes wherein the RAR and RXR nuclear receptors as well as the metabolic enzyme are expressed. In addition, RA is capable of stimulating RARβ and VEGF expression in amnion cells. The observation of a positive relationship between VEGF and RARβ mRNA levels in fetuses during altered IMA rates suggests that RA, presumably acting through VEGF in the amnion, could participate in amniotic fluid regulation during late gestation. Further, the retinoid pathway in fetal membranes could be a useful experimental model for understanding the pathophysiology of amniotic fluid homeostasis.

Acknowledgments

We wish to acknowledge the members of the Sheep Research Facility at OHSU for their technical support in the animal studies. L.B. and V.S. wish to acknowledge the “fondation de l’Université Clermont Auvergne” for its support on this collaborative research project.

Authors’ Note: All authors contributed to the design, experimental data collection, data analysis, and preparation of the manuscript. R.A.B., D.F.A., and C.Y.C. were responsible for the ovine fetal sheep studies, amnion cell experiments, and VEGF analysis. M.R., L.B., and V.S. were responsible for the retinoic acid receptor experiments and analysis. All authors have reviewed and approved the final version of the manuscript. The contents of this study are solely the responsibility of the authors and do not necessarily represent the official view of NIH/NICHD.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by NIH grant R01 HD061541 from the National Institute of Child Health and Human Development. Dr M Rouzaire was supported by an MENSR grant.

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[bibr31-1933719118765979] 31. Cheung CY, Anderson DF, Brace RA. Aquaporins in ovine amnion: responses to altered amniotic fluid volumes and intramembranous absorption rates. Physiol Rep. 2016;4(14):e12868. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Retinoic Acid Pathway Regulation of Vascular Endothelial Growth Factor in Ovine Amnion

Cecilia Y Cheung, PhD

Debra F Anderson, PhD

Marion Rouzaire, PhD

Loïc Blanchon, PhD

Vincent Sapin, PhD

Robert A Brace, PhD

Abstract

Introduction