Failure of Postnatal Ductus Arteriosus Closure in Prostaglandin Transporter–Deficient Mice
Background— Prostaglandin E2 (PGE2) plays a major role both in maintaining patency of the fetal ductus arteriosus and in closure of the ductus arteriosus after birth. The rate-limiting step in PGE2 signal termination is PGE2 uptake by the transporter PGT.
Methods and Results— To determine the role of PGT in ductus arteriosus closure, we used a gene-targeting strategy to produce mice in which PGT exon 1 was flanked by loxP sites. Successful targeting was obtained because neither mice hypomorphic at the PGT allele (PGT Neo/Neo) nor global PGT knockout mice (PGT−/−) exhibited PGT protein expression; moreover, embryonic fibroblasts isolated from targeted mice failed to exhibit carrier-mediated PGE2 uptake. Although born in a normal mendelian ratio, no PGT−/− mice survived past postnatal day 1, and no PGT Neo/Neo mice survived past postnatal day 2. Necropsy revealed patent ductus arteriosus with normal intimal thickening but dilated cardiac chambers. Both PGT Neo/Neo and PGT−/− mice could be rescued through the postnatal period by giving the mother indomethacin before birth. Rescued mice grew normally and had no abnormalities by gross and microscopic postmortem analyses. In accordance with the known role of PGT in metabolizing PGE2, rescued adult PGT−/− mice had lower plasma PGE2 metabolite levels and higher urinary PGE2 excretion rates than wild-type mice.
Conclusion— PGT plays a critical role in closure of the ductus arteriosus after birth by ensuring a reduction in local and/or circulating PGE2 concentrations.
Received March 8, 2009; accepted December 1, 2009.
Prostaglandin E2 (PGE2) modulates many physiological functions.1 In particular, PGE2 maintains patency of the ductus arteriosus (DA) in utero.2,3 Disruption of any of several steps in PGE2 signaling or signal termination results in patent DA (PDA) after birth.2,4–8
Clinical Perspective on p 536
Our laboratory identified the PG transporter PGT,9 which we have proposed to be responsible for the PGE2 uptake step in signal termination.10,11 Its broad tissue expression, high affinity for PGE2, and strong expression in the lung suggest that it mediates the well-described single-pass metabolic pulmonary clearance.12,13 Recently, we coexpressed PGT and 15-hydroxy prostaglandin dehydrogenase (PGDH), showing that the membrane uptake step is rate limiting for overall PGE2 catabolism.11
To test the hypothesis that PGT plays a central role in controlling pericellular PGE2 concentrations10 and thus signaling via PGE2 (EP) receptors, we deleted mouse PGT in vivo using gene targeting methods. Our results indicate that targeted deletion of mouse PGT leads to a persistent DA, which in turn results in neonatal mortality.
Construction of Targeting Vector and Conditional PGT Knockout Mice
A 2.2-kb region containing PGT exon 1 was targeted for deletion (Figure 1). A 13-kb mouse genomic DNA fragment containing PGT exon 1 was subcloned from a mouse 129 Sv/Ev lambda genomic library. The neomycin resistance cassette (Neo), flanked by both FRT and loxP sites, was inserted 490 bp downstream of exon 1. A third loxP site was inserted 1650 bp upstream of exon 1. The targeting vector was linearized with Not1 and transfected by electroporation of iTL1 (129Sv/Ev) ES cells. After selection in G418, surviving colonies were expanded, and polymerase chain reaction (PCR) analysis was performed to identify recombinant clones. The correctly targeted ES cell lines were microinjected into C57Bl/6J blastocysts. Chimeric mice were generated and gave germline transmission of conditional PGT knockout mice, ie, trilox conditional alleles present on a mixed 129Sv and C57Bl/6J genetic background.14
Breeding of the Mice and PCR Genotyping
Potential founder animals were screened by PCR and further confirmed by Southern blotting (Figure 1). Mouse tail DNA was purified (Qiagen, Valencia, Calif) and amplified 35 to 40 cycles. F0 heterozygous trilox conditional alleles (WT/Neo; Figure 1A, line 1 and 2) were detected by PCR with 2 different primer pairs (pairs 1 and 2 in Table I of the online-only Data Supplement). The WT allele was detected either by primer pair 3 (Figure 1A, line 1; AA′; product 2.8 kb) or by primer pair 4 (Figure 1A, line 1; BB′; product 1.0 kb). The product from primer pair 3 in Neo/Neo mice was >5 kb and was not amplified in these conditions.
The Neo gene was then excised by crossing F0 (WT/Neo) with a Rosa 26 FLPe transgenic mouse (129S4/SvJaeSorGt(ROSA) 26Sortm1(FLP1)Dym/J, stock No. 003946; Jackson Laboratories, Bar Harbor, Me),15 leaving 2 loxP sites at the targeted locus (Figure 1A, line 3; loxP). The resulting WT/loxP heterozygous (F1) mice were intercrossed to generate homozygous loxP/loxP mice (F2). The loxP allele was detected by PCR with primer pair 5, which flanks the (5′-most) third loxP site.
Exon 1 was subsequently excised by crossing loxP/loxP mice with an EIIa Cre transgenic mouse16 (B6.FVB-Tg [EIIa-cre] C5379Lmgd/J; stock No. 003724, Jackson Laboratories) to generate the F3 PGT exon 1–null allele mice (Figure 1A, line 4). These mice were intercrossed to generate WT/WT (PGT+/+), WT/null (heterozygotes, ie, PGT+/−), and null/null (PGT−/−). PCR results are shown in Figure 1B. The WT allele was detected as a 2.8-kb fragment, whereas the null allele was detected at 0.6 kb, ie, after excision of 2.2 kb of exon 1. Because the 2.8-kb fragment was barely detectable in heterozygotes (+/−; Figure 1B, middle lane), another primer set flanking exon 1 was designed, which resulted in a 1.0-kb fragment (Figure 1B, bottom).
Southern Blot Analysis
Genomic DNA (10 μg) from the liver of PGT+/+, PGR+/−, and PGT−/− mice was digested with Hpa1 and used for Southern blot analysis for the PGT alleles (Figure 1C) using standard methods. Hybridization was performed with a 5′ external probe (P in Figure 1A, line 1), which had been amplified from C57Bl/6J genomic DNA (forward primer, 5′-GGGGAACTATCTGAAGAGGTAACTGTCAAG-3′; reverse primer, 5′-GGCAAACTCATGGCAAATGCTG-3′). This probe recognized a 9.8-kb fragment in WT mice and a 7.9-kb fragment in null-allele mice.
Generation of PGT−/− Mouse Embryonic Fibroblasts and Determination of 3H-PGE2 Uptake by PGT
We crossed indomethacin-rescued PGT−/− females with PGT+/− males or intercrossed PGT+/− mice and euthanized the pregnant females. Embryos at day 14.5 were dissected away from the uterus and decidua. The head was removed for PCR analysis, and the abdominothoracic contents and blood clots were removed. The remaining tissue was minced, trypsinized at 37°C for 15 minutes, and triturated vigorously. Cell suspensions were washed, plated, and fed with DMEM supplemented with 10% FBS. After overnight incubation, floating cells and debris were removed, and fresh medium was added. The resulting mouse embryonic fibroblast (MEF) cultures were passaged once every 2 to 3 days.
3H-PGE2 uptake was determined in PGT−/− MEFs using previously described methods9 in the presence or absence of an additional 10 μmol/L unlabeled PGE2 for 10 minutes. The PGT-mediated uptake was calculated by subtracting the diffusional uptakes, ie, uptakes from samples containing 10 μmol/L unlabeled PGE2.
Hematoxylin and Eosin Staining of DA and Immunohistochemical Assessment of PGT Expression in Neonatal Mouse Lung and DA
PGT Neo/Neo, PGT+/+, and PGT−/− mice at postnatal days 1 and 2 were examined for morphological abnormalities. After a normal vaginal birth, animals that had died a natural death or animals that were euthanized at 11 hours were placed in 10% neutral buffered formalin overnight and processed for paraffin embedding. Serial transverse sections (5 μm) were cut and mounted on microscope slides. One of every 5 sections was stained with hematoxylin and eosin. Deparaffinized torso sections were also examined for elastin with Verhoeff elastic stain, which was visualized with 2% ferric chloride followed by 5% sodium thiosulfate, and counterstained with van Gieson solution.
Sections of neonatal mouse lung and DA and of adult kidney were subjected to immunohistochemical analysis using standard methods as previously described10,17 with rabbit anti-mouse PGT antibody overnight at 4°C (1:1000 dilution for lung, 1:500 for adult kidney, and 1:400 dilution for ductus). For negative controls, the primary antibody was omitted.
Plasma PGE2 Metabolite and Urinary PGE2 Excretion in WT and Adult Rescued PGT−/− Mice
We collected blood via cardiac puncture from 5- to 7-month old PGT−/− (n=5) and age-matched PGT+/+ (n=6) mice into EDTA and indomethacin (final concentration, 10 μmol/L). Plasma was stored at −80°C until assay. In separate experiments, using metabolic cages, we collected urine from PGT+/+ (n=5) and PGT−/− (n=4) mice at 3 to 5 months of age and determined daily urinary PGE2 excretion. All of the analyses were done with the PGE2 monoclonal enzyme immunoassay kit (catalog No. 514010) and plasma PGE2 metabolite kit (catalog No. 514531) from Cayman Chemical (Ann Arbor, Mich).
Quantitative Real-Time PCR
Kidneys, hearts, and lungs of adult mice (PGT−/−, n=5; PGT+/+, n=6) and fetus bodies at embryonic day 19 (PGT−/−, n=2; PGT+/+, n=4) were frozen and homogenized with a liquid nitrogen–cooled mortar and pestle, after which RNA was isolated with the RNeasy Mini Kit (Qiagen). For real-time PCR, the QuantiTect SYBRGreen reverse-transcription PCR kit (Qiagen) and 120 ng total RNA were used in the PCR reactions as follows: 1 cycle of 50°C for 30 minutes and 1 cycle of 95°C for 16 minutes; 40 cycles of 95°C for 15 seconds, 55°C to 58°C for 30 seconds, and 72°C for 30 seconds; and 1 cycle of 95°C for 15 seconds, 60°C for 15 seconds, and 95°C for 15 seconds. The relative delta delta Ct value was used in the resulting calculation. Some primers were purchased from Qiagen (GeneGlobe products): mGAPDH (QT01658692), mPGT (QT00140567), mCox-1 (QT00155330), mCox-2 (QT00165347), and mEP2 (QT00115276). Primers for mEP4 were custom made by Invitrogen (Carlsbad, Calif; forward primer, ATGGTCATCTTACTCATCGCC; reverse primer, GCAAATCTGGGTTTCTGCTG). The data were normalized to mGAPDH mRNA; the expression of each gene of interest (PGT, cyclooxygenase 1 [COX1], COX2, EP2, and EP4) from each mouse was then normalized to the level of expression in mouse 6 (one of the adult WT mice) or 1Q-3 (one of the WT embryos).
Rescue of PGT Neo/Neo and PGT−/− Mice by Indomethacin
Pregnant female mice bearing PGT Neo/Neo or PGT−/− fetuses were administered indomethacin (Indocin 1 mg, Merck & Co, Inc, Upper Gwynedd, Pa) orally in a single dose at 3 mg/kg body weight 3 to 9 hours before parturition.
Data are expressed as mean±SEM. Comparisons were made using the t test or the Wilcoxon nonparametric test. Differences were considered significant at P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All of the authors have read and agree to the manuscript as written.
PGT-Targeted Mice Lack PGT Protein Expression in the Lung and PGE2 Uptake in Embryonic Fibroblasts
We confirmed the lack of PGT protein expression in PGT Neo/Neo (Figure 2) and PGT−/− mice (data not shown) by immunohistochemistry on neonatal lung, which is the tissue with the highest PGT expression in the normal animal.9,18,19 PGT is expressed in the type II alveolar cells lining the alveolar spaces in the PGT+/+ mice (Figure 2A and Figure I of the online-only Data Supplement20); these cells also express PGDH.21 In contrast, the lungs of PGT Neo/Neo mice had no discernible PGT protein expression.
In separate experiments (Figure 2B), MEFs from PGT+/+ mice exhibited carrier-mediated 3H-PGE2 uptake as evidenced by a 30% augmented uptake when the competitor for PGT (unlabeled PGE2) was absent. In contrast, MEFs from PGT−/− mice failed to demonstrate carrier-mediated 3H-PGE2 uptake. Together, these results demonstrate successful knockout of PGT expression in the targeted animals.
Normal Mendelian Birth Ratios in PGT-Targeted Mice
We examined whether intercrossing WT/Neo heterozygotes resulted in a normal mendelian ratio of F0 mice. Genotypes were determined in newborn pups from 79 pups (8 litters); the genotyping results showed 21 PGT+/+ mice (27%), 39 PGT+/Neo mice (49%), and 19 Neo/Neo mice (24%). In separate experiments, we genotyped 63 newborn pups (6 litters) from PGT+/−×PGT+/− crosses. The genotyping results were 18 PGT+/+ (29%), 33 PGT+/− (52%), and 12 PGT−/− (19%). Thus, both mice in which the Neo cassette is retained in PGT intron 1 and mice completely lacking PGT exon 1 were born in a normal mendelian ratio.
PDA in PGT Neo/Neo and PGT−/− Mice
Despite normal mendelian birth ratios in newborn pups, no PGT−/− mice survived past postnatal day 1, and no PGT Neo/Neo mice survived past postnatal day 2. Cross sections of torsos of PGT+/+ mice (n=3) at postnatal day 1 showed normal closure of the DA (Figure 3a, arrow). On high-power examination, intimal thickening in the form of proliferation of luminal endothelium and migration of medial smooth muscle cells was apparent in the DA of these mice (Figure 3d).22
In contrast, PGT Neo/Neo mice (n=5) at postnatal day 1 or 2 showed PDA (Figure 3b, arrow; the PDA connects the main pulmonary artery to the descending aorta). Similarly, PGT−/− mice (n=5) failed to close the DA at postnatal day 1 (Figure 3c, arrow). On high-power examination, DAs from both PGT Neo/Neo and PGT−/− mice showed a single endothelial layer (Figures 3e and 3f, respectively) with an open lumen that was covered by a layer of normal intimal thickening.
Just before birth (embryonic day 19), the endothelium and underlying intimal layers of the DA of PGT-targeted mice were histologically normal (Figure 4), indicating that targeting PGT did not induce intrinsic structural malformations of the DA vasculature.
Microscopic examination of the hearts of PGT Neo/Neo and PGT−/−mice that had died at postnatal day 1 from PDA revealed dilated cardiac chambers (Figures 5b and 5c, respectively) compared with those of WT mice (Figure 5a), consistent with left-to-right shunt and volume-overload congestive heart failure.
PGT Expression in the DA
We immunolabeled sections of mouse torso using a rabbit polyclonal antiserum directed against mouse PGT. As shown in Figure 6, there was strong labeling in smooth muscle cells of the DA intimal thickening in postnatal day 1 WT mice (Figure 6a; n=3). In contrast, there was no such PGT labeling in the negative control (data not shown) or in the DA of postnatal day 1 PGT Neo/Neo mice (Figure 6b; n=3).
Rescue of Both PGT Neo/Neo and PGT−/− Mice by Indomethacin
To test the hypothesis that high levels of PGE2 in the postpartum period lead to PDA in PGT-targeted mice, we administered the nonselective COX inhibitor indomethacin to pregnant mice several hours before birth (ie, at embryonic day 19) to lower PGE2 concentrations in the newborn pups. All of the pups subjected to maternal indomethacin rescue, including PGT−/−, survived the neonatal period. Histological examination of a rescued 2 week-old PGT Neo/Neo mouse demonstrated a normally closed DA (converted into the ligamentum arteriosum22) (data not shown).
Blood PGE2 Metabolite Concentration and Urinary PGE2 Excretion in Indomethacin-Rescued PGT−/− Mice Compared With PGT+/+ Mice
To test the hypothesis that PGE2 is not metabolized at a normal rate in PGT−/− mice, we measured plasma PGE2 metabolite concentrations in 5- to 7-month-old PGT+/+ mice (n=6) compared with those of age-matched PGT−/− mice that were products of maternal indomethacin rescue (n=5). Plasma PGE2 metabolite concentrations were 2644±751 pg/mL in the PGT+/+-type mice and 856±295 pg/mL in the adult PGT−/− mice (P=0.027 by 1-tailed t test; P=0.022 by Wilcoxon 2-sample test). These results are consistent with a failure to metabolize PGE2 in the PGT−/− mice.
In separate experiments, we determined 24-hour urinary PGE2 excretion in adult mice that were the product of maternal indomethacin rescue. Urinary PGE2 levels were significantly higher in PGT−/− mice compared with PGT+/+ mice (3073±756 pg/d, n=4, versus 1497±187 pg/d, n=5, respectively; P<0.05 by unpaired t test). These results are also consistent with a failure of PGT-null mice to metabolize PGE2.
Modulation of the PGE2 Signaling Pathway in PGT−/− Mice
PGT−/− mice grew normally and were histologically normal at necropsy (n=5 adult mice; data not shown). We measured mRNA levels for PGT, COX-1, COX-2, and the PGE2 receptors EP2 and EP4 in mouse embryos just before birth and in kidney, lung, and heart from adult mice. Table II of the online-only Data Supplement shows that in PGT−/− mice, PGT mRNA levels were significantly decreased to background noise values in whole embryos and in all 3 tissues from rescued adult animals. In the rescued adult PGT−/− mice compared with PGT+/+ mice, lung tissue revealed a statistically significant decrease in COX-1 mRNA, heart revealed a statistically significant increase in EP2 mRNA, and kidney revealed a statistically significant increase in EP4 mRNA.
Separately, we carried out immunocytochemical labeling of COX-1 and COX-2 in the lungs and kidneys of PGT−/− and PGT+/+ mice. These studies revealed no discernible difference in COX-1 or COX-2 expression (Figure II of the online-only Data Supplement).
These studies reveal that mice hypomorphic (Neo/Neo) or null (PGT−/−) at the PGT locus fail to close the DA during postnatal days 1 to 2, resulting in PDA. PDA causes cardiac biventricular chamber dilatation, consistent with the presence of a postnatal left-to-right shunt. Morphologically, the endothelium and internal elastic lamina of the DA of PGT Neo/Neo and PGT−/− embryos just before birth appeared normal. PGT-targeted mice established a normal intimal thickening but failed to constrict the DA after birth. Both PGT Neo/Neo and PGT−/− mice could be rescued through the postnatal period by administration of indomethacin to the mother several hours before birth. Adult rescued PGT-null mice had significantly lower plasma PGE2 metabolite levels and significantly higher urinary PGE2 excretion rates than WT mice, consistent with their failure to metabolize systemic PGE2.
The DA connects the fetal pulmonary artery and descending aorta. Although the DA closes immediately after birth in most cases, it remains open in some infants, a condition known as PDA.23 Nonselective COX inhibitors such as indomethacin have long been used to successfully treat PDA in patients,24 results that are consistent with a large body of literature pointing to a central role of PGE2 in maintaining DA patency.8,23,25–27
A current model postulates 2 separate roles for PGE2 in DA closure. In late fetal life and continuing after birth, PGE2 controls formation of the intimal cushion (or thickening) via EP4 receptors.3,4,7,22,28,29 Postnatally, loss of the placenta as a source of PGE230 and PGE2 metabolism, especially within the pulmonary circulation,12 result in falling circulating PGE2 levels. These falling PGE2 levels induce constriction of the cushion-containing DA.3,7,28,30 The present data are consistent with this model; in other words, targeting PGT does not interfere with intimal thickening, but rather the persistence of PGE2 opposes the constrictor mechanism(s) activated by oxygen.
Although PGT transports PGE2 rapidly and with high affinity9 and although our laboratory had previously built a strong circumstantial case that PGT is the major route for reuptake and metabolism of PGE2,11,31,32 the possibility had remained that either prostanoids might not be the “preferred” PGT substrates or other as-yet undiscovered PG uptake carriers could substitute for PGT. The present results render neither of these 2 possibilities tenable.
At least in the mouse, disrupting PGE2 signaling in any of several ways causes PDA. Although genetic disruptions of Cox-1 or Cox-2 alone or pharmacological disruption of both Cox isoforms has been reported to produce variable effects on postnatal ductus closure,5,8,33–36 genetic disruption of both Cox isoforms uniformly causes postnatal PDA.5,8 Moreover, targeted deletion of the PGE2-specific receptor EP44,7 and targeted deletion of intracellular PGE2 oxidation, which is catalyzed by PGDH,6 also result in PDA. The present results, that targeted deletion of PGT also results in PDA, position PGT within the overall PGE2 signaling pathway (Figure 7).
After birth, systemic PGE2 concentrations fall30,37,38 with loss of the placenta as a source of PGE230 and metabolism of PGE2, especially within the pulmonary circulation.12 Given that the lung is perfused after birth, that both PGT (present results) and PGDH are strongly expressed in the neonatal lung,39 and that PGT is rate limiting for delivering PGE2 to the cytoplasmic PGDH,11 PGT would be well positioned to reduce systemic PGE2 concentrations and to initiate closure of the DA after birth.
Because of technical limitations imposed by the extremely small plasma volume of fetal mice, we could not determine whether PGT−/− mice have elevated plasma PGE2 levels in utero. Although exogenous PGE2 added to the postnatal sheep ductus in vitro has been shown to regulate genes that regulate calcium availability,36 we found that whole-embryo mRNA levels for Cox-1 and Cox-2 and for EP2 and EP4 receptors showed no difference between WT and PGT-null mice (Table II of the online-only Data Supplement); however, indomethacin-rescued adult PGT−/− mice demonstrated downregulation of lung Cox-1 mRNA and upregulation of heart EP2 mRNA and of kidney EP4 mRNA (Table II of the online-only Data Supplement). This difference raises the possibility that the PGT-null fetus is not exposed to abnormally high PGE2 plasma levels, perhaps because the fetal lungs, although rich in PGT expression (Figure 6), are not perfused in utero.
In addition to controlling circulating PGE2 levels, PGT also operates at a local level. PGT is often coexpressed in the same cells as COX,17 and reconstitution experiments have demonstrated PGE2 synthesis and release on the one hand and PGT-mediated reuptake and PGDH-mediated oxidation on the other in the same cell.10,40 These findings have prompted us to advance a “local release-reuptake model,” analogous to that of neurotransmitter signaling at the synaptic cleft, in which PGT constrains prostaglandins to a highly confined environment, thus facilitating precise autocrine/paracrine signaling.10 Although there is some disagreement as to the extent to which the DA of the term or postnatal mouse expresses one or both isoforms of COX,5,8,34,36 the ductus clearly expresses PGE synthase34 and exhibits clear labeling for PGT (present results). The vascular endothelium also strongly expresses PGT.41–43 Taken together, these findings suggest that PGT-mediated PGE2 uptake occurs not only systemically in the pulmonary circulation but also locally in close proximity to the ductus. Further experiments using the isolated mouse ductus obtained from PGT−/− compared with WT mice would likely clarify further the influence of PGT of autocrine PGE2 signaling in this tissue.
Targeted deletion of PGT gene expression in the mouse results in PDA. These results indicate that PGT plays a key role not only in terms of general prostaglandin metabolism but also specifically in terms of modulating PGE2 signaling.
We thank the Einstein Mouse Genetics Course for inspiring the gene targeting and the Einstein Cancer Center for assistance. We also thank Dr Rani Sellers for phenotyping the PGT global knockout mice.
Source of Funding
This work was supported by National Institutes of Health grants RO1-DK49688 and P50-DK064236.
Schneider DJ, Moore JW. Patent ductus arteriosus. Circulation. 2006; 114: 1873–1882.
Segi E, Sugimoto Y, Yamasaki A, Aze Y, Oida H, Nishimura T, Murata T, Matsuoka T, Ushikubi F, Hirose M, Tanaka T, Yoshida N, Narumiya S, Ichikawa A. Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice. Biochem Biophys Res Commun. 1998; 246: 7–12.
Loftin CD, Trivedi DB, Tiano HF, Clark JA, Lee CA, Epstein JA, Morham SG, Breyer MD, Nguyen M, Hawkins BM, Goulet JL, Smithies O, Koller BH, Langenbach R. Failure of ductus arteriosus closure and remodeling in neonatal mice deficient in cyclooxygenase-1 and cyclooxygenase-2. Proc Natl Acad Sci U S A. 2001; 98: 1059–1064.
Reese J, Paria BC, Brown N, Zhao X, Morrow JD, Dey SK. Coordinated regulation of fetal and maternal prostaglandins directs successful birth and postnatal adaptation in the mouse. Proc Natl Acad Sci U S A. 2000; 97: 9759–9764.
Kanai N, Lu R, Satriano JA, Bao Y, Wolkoff AW, Schuster VL. Identification and characterization of a prostaglandin transporter. Science. 1995; 268: 866–869.
Nomura T, Chang HY, Lu R, Hankin J, Murphy RC, Schuster VL. Prostaglandin signaling in the renal collecting duct: release, reuptake, and oxidation in the same cell. J Biol Chem. 2005; 280: 28424–28429.
Nomura T, Lu R, Pucci ML, Schuster VL. The two-step model of prostaglandin signal termination: in vitro reconstitution with the prostaglandin transporter and prostaglandin 15 dehydrogenase. Mol Pharmacol. 2004; 65: 973–978.
Lakso M, Pichel JG, Gorman JR, Sauer B, Okamoto Y, Lee E, Alt FW, Westphal H. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc Natl Acad Sci U S A. 1996; 93: 5860–5865.
Bao Y, Pucci ML, Chan BS, Lu R, Ito S, Schuster VL. Prostaglandin transporter PGT is expressed in cell types that synthesize and release prostanoids. Am J Physiol Renal Physiol. 2002; 282: F1103–F1110.
Kalina M, Socher R. Endocytosis in cultured rat alveolar type II cells: effect of lysosomotropic weak bases on the processes. J Histochem Cytochem. 1991; 39: 1337–1348.
Smith GC. The pharmacology of the ductus arteriosus. Pharmacol Rev. 1998; 50: 35–58.
Yokoyama U, Minamisawa S, Quan H, Ghatak S, Akaike T, Segi-Nishida E, Iwasaki S, Iwamoto M, Misra S, Tamura K, Hori H, Yokota S, Toole BP, Sugimoto Y, Ishikawa Y. Chronic activation of the prostaglandin receptor EP4 promotes hyaluronan-mediated neointimal formation in the ductus arteriosus. J Clin Invest. 2006; 116: 3026–3034.
Yokoyama U, Minamisawa S, Quan H, Akaike T, Suzuki S, Jin M, Jiao Q, Watanabe M, Otsu K, Iwasaki S, Nishimaki S, Sato M, Ishikawa Y. PGE2-activated Epac promotes neointimal cushion formation of the rat ductus arteriosus by a process distinct from that of PKA. J Biol Chem. 2008; 283: 28702–28709.
Schuster VL. Prostaglandin transport. Prostaglandins Other Lipid Mediat. 2002; 68–69: 633–647.
Mitchell MD, Brunt J, Clover L, Walker DW. Prostaglandins in the umbilical and uterine circulations during late pregnancy in the ewe. J Reprod Fertil. 1980; 58: 283–287.
Pucci ML, Endo S, Nomura T, Lu R, Khine C, Chan BS, Bao Y, Schuster VL. Coordinate control of prostaglandin E2 synthesis and uptake by hyperosmolarity in renal medullary interstitial cells. Am J Physiol Renal Physiol. 2006; 290: F641–F649.
Topper JN, Cai J, Stavrakis G, Anderson KR, Woolf EA, Sampson BA, Schoen FJ, Falb D, Gimbrone MA Jr. Human prostaglandin transporter gene (hPGT) is regulated by fluid mechanical stimuli in cultured endothelial cells and expressed in vascular endothelium in vivo. Circulation. 1998; 98: 2396–2403.
Dekker RJ, Boon RA, Rondaij MG, Kragt A, Volger OL, Elderkamp YW, Meijers JC, Voorberg J, Pannekoek H, Horrevoets AJ. KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. Blood. 2006; 107: 4354–4363.
McCormick SM, Eskin SG, McIntire LV, Teng CL, Lu CM, Russell CG, Chittur KK. DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells. Proc Natl Acad Sci U S A. 2001; 98: 8955–8960.
Prostaglandins are small signaling molecules that control multiple bodily functions. Many people manipulate their prostaglandin levels without knowing it because nonsteroidal antiinflammatory drugs such as aspirin act by blocking prostaglandin synthesis. Among their actions, prostaglandins help to keep open a blood vessel in the fetus called the ductus arteriosus and to close the ductus appropriately after birth. The present study focuses on the mechanism by which prostaglandin signaling is shut off. Previous experiments using cells grown in glass dishes have demonstrated that a carrier protein called PGT transports prostaglandins from the blood into the cell interior, where an enzyme inactivates them. The prediction from these studies would be that inactivating or blocking PGT in an experimental animal or human being would cause prostaglandin levels to rise, resulting in abnormal prostaglandin signaling from 1 cell to another. This study used genetic engineering methods to inactivate all PGT carriers in mice. Mice lacking PGT from conception failed to close their ductus arteriosus normally at birth, resulting in their death on or about the first day of life. The results have implications for humans because failure to close the ductus arteriosus after birth is a common congenital disorder.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.862946/DC1.