Placenta (2004), 25, 518–529 doi:10.1016/j.placenta.2003.10.017
Norepinephrine Transporter (NET), Serotonin Transporter (SERT), Vesicular Monoamine Transporter (VMAT2) and Organic Cation Transporters (OCT1, 2 and EMT) in Human Placenta from Pre-eclamptic and Normotensive Pregnancies B. Bottalico, I. Larsson, J. Brodszki, E. Hernandez-Andrade, B. Cassle´n, K. Marsa´l and S. R. Hansson* Department of Obstetrics and Gynecology, Lund University Hospital, Klinikgatan, 221 85, Lund, Scania, Sweden Paper accepted 14 October 2003
Pre-eclampsia is one of the most common causes of perinatal and maternal morbidity and mortality. High blood pressure and proteinuria are important clinical signs of pre-eclampsia. Sympathetic overactivity and elevated level of circulating vaso active substances, such as monoamines has been shown. Extracellular concentrations of monoamines are normally kept low by specific transporter proteins of which many are expressed in the placenta. In this study we used in situ hybridization and real-time PCR to study the gene expression of monoamine transporters, such as NET, SERT, VMAT2, EMT and OCT1/2, in normal as well as in pre-eclamptic placentae. We demonstrated high expression of NET mRNA in the trophoblast cells of the anchoring villi and a lower expression intensity in the chorionic villi. SERT mRNA was mainly detected in chorionic villi. VMAT2 mRNA was not detected in the central part of the placenta but was present in the spiral arteries of placenta bed biopsies, in cytokeratin positive cells. EMT mRNA was mainly detected in the intra lobular septa and together with OCT1 and OCT2 mRNAs also expressed in scattered cells of placental vessel adventitias. Moreover, quantitative analysis showed a significant lower expression of NET and EMT mRNAs in pre-eclamptic placentae as compared to the control group. A defective gene expression or function of these monoamines transporters might explain the elevated concentrations of monoamines in pre-eclamptic patients. Monoamine transporters may serve as a protective mechanism preventing vasoconstriction in the placental vascular bed and thereby securing a stable blood flow to the fetus. Placenta (2004), 25, 518–529 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Pre-eclampsia (toxemia) is today still one of the most common causes of perinatal and maternal morbidity and mortality. The overall incidence is 3–7 per cent of pregnancies, but mainly primigravidae are affected. The clinical manifestations of pre-eclampsia appear after 20 weeks of gestation and are characterized by low circulating blood volume, haemoconcentration, high blood pressure and proteinuria. Severe cases of pre-eclampsia furthermore involve pathologic activation of the coagulation and fibrinolytic systems. Without intervention the condition may develop into severe epileptic seizures— eclampsia. Only symptomatic treatment of the condition is available and termination of the pregnancy remains the only curative intervention [1]. Removal of the placenta is believed to be the underlying mechanism for resolution of the symptoms [2]. *
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The aetiology is still not known, but defective invasion by placental trophoblast cells into the muscle layers of the spiral arteries seems to be a background key factor. This leads to altered placental blood flow, which in addition may result in intra uterine growth restriction (IUGR). A generalized inflammatory process in vascular endothelial cells is a hallmark of pre-eclampsia [2–5]. Placental derived factors have been suggested, but so far no specific culprit has been found. The hypertension is due to peripheral vasoconstriction and elevated resistance. Increased activity in the sympathetic nervous system or elevated concentrations of circulating vaso-active substances, are both important factors in the pathophysiology of hypertension. In fact, sympathetic overactivity is a significant finding in pre-eclampsia [6]. Norepinephrine (NE) is the principal neurotransmitter in the sympathetic nervous system and also a hormone released by the adrenal medulla during stress. Increased blood levels of monoamines such as norepinephrine (NE) [7,8] and serotonin (5-HT) [9] have been shown in pre-eclampsia as well as in IUGR. 2003 Elsevier Ltd. All rights reserved.
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Table 1. Clinical characteristics of included subjects and offspring
N Maternal age (years) Parity (nulliparity) Gestation length (days/weeks) Systolic pressure (mmHg) Diastolic pressure (mmHg) Proteinuria (g) Gestational weight (g)/sex F : M Apgar scores (1 min) Apgar scores (5 min) Apgar scores (10 min)
Normal pregnant
Pre-eclampsia
7 32.31.4 20.4 (0) 274.73.9/(39.2) 118.73.8 77.13.1 ND 3723160.7/3 : 4 9.10.1 10.00 100
10 31.41.8 1.70.5 (1 of 10) 251.48.6 (35.9)* 145.14.1* 100.32.5* 2.40.8* 2908305.7/1 : 9* 9.00.1 9.70.2 9.90.1
The results are expressed as mean. ND=not detected, F=female, M=male. * Significant differences between the groups, P140/90 mmHg and proteinuria of >0.03 g/l, or a rise of blood pressure >20 mmHg from the first trimester of pregnancy. Patients with essential hypertension and renal or other systemic diseases were excluded.
Tissue sampling and handling Tissue samples were taken immediately after delivery from the placenta and in the case of Ceasarean section from the uterine wall. Two specific areas within the placenta were sampled: (1) a section, 10104 mm, from the maternal surface, at the entrance of spiral arteries and (2) a cube 101010 mm from the central part of the placenta consisting of villi. Placenta bed biopsies were collected from the uterine wall during Caesarean section in order to study gene expression in the modified spiral arteries. Collected samples were snap frozen on dry ice and stored at 80(C. Tissue sections (12 µm), were thaw mounted on to silanized slides and stored at 80(C until hybridization. Fresh frozen tissue rather than fixative-treated
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tissue was used in order to maximize the sensitivity for mRNA detection. Thawing of tissue did not occur prior to sectioning to ensure best possible tissue integrity.
Placenta (2004), Vol. 25
washes to remove excess probe, slides were apposed to Kodak Hyperfilm Biomax MR for 3 days, then coated with nuclear track emulsion (NTB-3, Kodak). After a 4 week exposure at 4(C, slides were developed in Dektol (Kodak), fixed and counterstained with a Giemsa stain.
RNA probes For the human NET mRNA, a probe was used corresponding to 561 NT (316–877), Genbank accession no. NM001043 [28]. For the human 5HTT mRNA, a probe was used corresponding to 475 NT (1845–2320), Genbank accession no. NM00104 [32]. For the vesicular monoamine transporter (VMAT2), corresponding to 486 NT (1300–1786), Genbank accession no. L23205 [12]. For the human OCT1 mRNA, a probe was used corresponding to 460 NT (20–480), Genbank accession no. NM_003057 [21–23]. For the human OCT2 mRNA, a probe was used corresponding to 505 NT (50–555), Genbank accession no. NM_003058 [21,22]. For the human EMT (OCT3) mRNA, a probe was used corresponding to 440 NT (1300–1740), Genbank accession no. NM_021977 [24]. DNA templates were generated by polymerase chain reaction (PCR) from the different cDNAs using bipartite primers consisting of either a T7 RNA promoter and a downstream gene-specific sequence (anti-sense) or a T3 RNA promoter and an upstream gene-specific primer (sense). PCR reactions using 1 ng cDNA, 1 µ primers, 200 µ dNTPs, 3 m MgCl2, 10 m Tris, pH 8.3, 50 m KCl, 2.5 units Taq polymerase (Boerhinger Mannheim) were amplified at 95(C for 1 min, 70(C (NET), 60(C (SERT), 65(C (VMAT2), 62(C (OCT1), 66(C (OCT2), 63(C (EMT) for 1 min and 72(C for 2.5 min for 30 cycles with a final extension at 72(C for 10 min. DNA templates were purified from agarose gels using GeneClean (Bio101) and thereafter sequenced using a cycle sequencing reaction kit (ABI PRISM, Big dye). Complementary RNA (cRNA) probes were transcribed from 25 ng of gel-purified DNA template using 35S-UTP (Dupont NEN, 1300 Ci/mmol) and either T3 or T7 RNA polymerase according to manufacturer’s instructions (Ambion MAXIscript) to generate sense and antisense probes, respectively.
RNA Hybridization Tissue sections were fixed, dehydrated and delipidated as previously described [33]. Sections were hybridized (20–24 h, 55(C) with 2106 cpm of denatured 35S-cRNA probe per 80 µl hybridization buffer consisting of 20 m Tris–HCl (pH 7.4), 1 m EDTA (pH 8.0), 300 m NaCl, 50 per cent formamide, 10 per cent dextran sulphate, 1Denhardt’s, 25 mg/ml yeast tRNA, 100 µg/ml salmon sperm DNA, 250 µg/ml total yeast RNA (fraction XI, Sigma), 150 m dithiothreitol (DTT), 0.15 per cent sodium thiosulfate (NTS), and 0.15 per cent sodium dodecyl sulphate (SDS). Following
RNA extractions Total RNA was extracted from frozen tissue using Trizol (Gibco BRL) according to manufacturer’s instructions. Proteoglycan and polysaccharide contaminations were removed by adding isopropanol followed by salt precipitation with 0.8 sodium citrate and 1.2 sodium chloride. The quality of RNA samples were determined by electrophoresis through a 1.5 per cent agaros/2 per cent formalin denaturing gel with a 1 MOPS buffer (Intergen company). RNA loading mix (GenHunter) was used to verify the 18S and 28S RNA bands under UV light. Only samples with visible 18S/28S bands were included for further analysis.
cDNA synthesis RNA was reverse transcribed according to protocols from applied biosystems. In a 50 µl reaction containing: 0.5 µg total RNA, and a final concentrations of 1 TaqMan RT buffer, 5.5 m MgCl2, 500 µ dNTPs, 2,5 µ random hexamers, 0.4 U/µl RNase inhibitor, and 1.25 U/µl MultiScribe Reverse Transcriptase. The reactions were incubated at 25(C for 10 min, at 48(C for 30 min and then 5 min of inactivation at 95(C. The samples were stored at 20(C until further use.
Real-time PCR amplification Gene transcripts were quantified using real-time PCR on ABI PRISM 7000 sequence detection system (Applied Biosystems). Primers and probes were designed using the Primer Express software program or alternatively ordered from Assays on-Design/Demande (Applied Biosystems). Each primer pair was located on different exons of the investigated gene in order to avoid genomic DNA contamination (Table 2). Oligonucleotide probes labelled with fluorogenic dye, 6 carboxyfluorescein (Fam) and quenched with 6 carboxytetramethylrhodamine (Tamra) (Table 2). PCR reactions were carried out in a 25 µl final volume containing final concentrations: 1 Universal PCR Master Mix (Applied Biosystems), 0.5 µ TaqMan probe, 0.9 µ of forward and reverse primers respectively, and 1 µl of 10 ng/µl of a DNA aliquot. For transcripts analysed with pre-manufactured probes the reactions were carried out in a 25 µl final volume containing final concentrations: 1 Universal PCR Master Mix (Applied Biosystems), 1 Assaymix (Applied Biosystems), 0.25 µ probe, 0.9 µ of forward and reverse primers respectively, and
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Table 2. Data on primers and probes used for real-time PCR amplification mRNA
Accession number
Size (NT)
Primers, forward (F) and reverse (R)/ assay on demand number
h-NET
NM_001043
75
h-SERT hVMAT-2 h-EMT h-OCT1
NM_001045 L23205 NM_021977 NM_003057
