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Correspondence to: Leslie Myatt, Dept. of Obstetrics and Gynecology, U. of Cincinnati College of Medicine, PO Box 670526, Cincinnati, OH 45267.
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Summary |
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Several isoforms of superoxide dismutase (SOD), including copper/zinc (cytosolic) and manganese (mitochondrial), exist. In the human placenta, SOD may prevent excessive superoxide accumulation and any potential deleterious oxidative effects. In pre-eclampsia, increased levels of lipid peroxide and decreased SOD activity have been described in the placenta. Oxidative stress such as occurs in pre-eclampsia can alter expression of SOD isoforms. The objective of this study was to localize the copper/zinc and manganese SOD isoforms in the placenta using immunohistochemistry and to compare localization and intensity of immunostaining in tissues from normotensive pregnancies with those from pregnancies complicated by pre-eclampsia and/or intrauterine growth restriction (IUGR). Western blotting with specific antibodies recognized a 17-kD copper/zinc and a 23-kD manganese SOD subunit in placental homogenates. Intense immunostaining for the manganese SOD isoform was seen in villous vascular endothelium, but only faint staining was found in the syncytiotrophoblast or villous stroma. In serial sections, intense immunostaining for copper/zinc SOD was seen in certain cells of the villous stroma but only faint immunostaining in syncytiotrophoblast and vascular endothelium. No apparent differences in localization or intensity of immunostaining for either isoform were seen between tissues of normotensive or pre-eclamptic pregnancies, with or without IUGR. The different cellular localizations of the SOD isoforms suggest that they fulfill different functional roles within the placenta.(J Histochem Cytochem 45:1433-1438, 1997)
Key Words: superoxide dismutase, placenta, pre-eclampsia, intrauterine growth restriction, superoxide, pregnancy
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Introduction |
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In the absence of autonomic innervation, blood flow in the human placenta must be determined by humoral mechanisms and by autocrine/paracrine factors produced in the placental vasculature itself (
The action of superoxide is normally limited by its low lipid solubility, its limited membrane transport, and also by its removal by SOD, the rate constant of which is 2 x 109 M-1s-1 (
SOD exists in several isoforms, including the cellular copper/zinc (cytosolic, 32 kD) and the manganese (mitochondrial, 80 kD) isoforms (
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Materials and Methods |
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All procedures were performed under protocols approved by the Institutional Review Board of the University of Cincinnati Medical Center and the Ethical Committee of Glasgow Royal Infirmary. Placental villous tissue (1 x 1 x 1 cm) was collected immediately after delivery from normotensive pregnancies (n = 5) and from pregnancies complicated by either pre-eclampsia (n = 5), intrauterine growth restriction (IUGR; n = 5), or pre-eclampsia plus IUGR (n = 4). Villous tissue was then immediately flash-frozen in liquid nitrogen and stored at -70C until processed. Pre-eclampsia is defined as a blood pressure of 140/90 mmHg on at least two occasions at least 6 hr apart occurring after Week 20 of gestation and accompanied by proteinuria (>300 mg/liter in a 24-hr urine collection) and/or edema. IUGR is defined as a fetal weight less than the fifth percentile using standardized Scottish birthweight tables. The gestational ages of the patients were 30.9 ± 1.7 weeks (normotensive), 35.4 ± 2.6 weeks (pre-eclampsia), 35.5 ± 1.3 weeks (IUGR), and 30.5 ± 1.9 weeks (pre-eclampsia plus IUGR). Because all pathological tissues were pre term, the normotensive group was age-matched to the group with earliest gestation.
Serial sections of villous tissue were cut at 7 µm and immunostained using a monoclonal antibody to Cu/Zn SOD (Sigma; St Louis, MO) or a polyclonal antibody to Mn SOD (Fitzgerald; Concord, MA) with the Vectastain ABC Elite Kit (Vector Laboratories; Burlingame, CA) using DAB reagent. These tissues were incubated in primary antibody for 1.5 hr at 37C, followed by incubation in the secondary antibody. Sections were then blocked for endogenous peroxidase for 3 min using 3% H2O2 in water. Saponin (0.1%) was included in all washes and antibody solutions up to and including the secondary antibody incubation, after which it was excluded from all reagent mixtures. Sections were finally counterstained with hematoxylin and mounted in PBS/glycerol (1:9). The control incubations contained no primary antibody. All sections were then examined by one observer who was blinded to tissue identity, and the localization and intensity of immunostaining for either the Cu/Zn or the Mn SOD were noted.
For Western blot analysis, fresh placental villous tissue was homogenized in 0.32 M sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM DTT, pH 7.8, with a polytron, three times for 10 sec on ice. The homogenates were centrifuged at 1000 x g for 15 min to remove debris, and the protein concentration of the supernatant was determined. Five µg total protein was loaded on 15% polyacrylamide gels (1 mm x 10 cm x 10 cm). Proteins were separated at 20 mAmps/plate at 4C and transferred to nitrocellulose membranes using a BioRad transblot apparatus (BioRad; Melville, NY). Membrane blots were blocked with 5% nonfat dry milk in 0.1% Tween-20 in Tris/saline buffer [50 mM Tris-HCl, pH 7.5, 500 mM NaCl (TTBS)] and incubated in either monoclonal anti-human Cu/Zn SOD antibody 1:250 (IgG1, ascites fluid; Sigma Immunochemicals, St Louis, MO) or polyclonal sheep anti-human Mn SOD, 1:100 (IgG fraction, preabsorbed with human tissue and serum polymers; Calbiochem-Novabiochem International, San Diego, CA) in TTBS for 1 hr at room temperature. Subsequently, membranes were treated with biotin-conjugated goat anti-mouse IgG, 1:1000 (Sigma ImmunoChemicals) or biotin-conjugated rabbit anti-sheep IgG 1:200 (Vector Laboratories) containing 1% goat serum or 1% rabbit serum, respectively. Protein bands were detected with the Vectastain ABC and DAB Peroxidase Substrate kits (Vector Laboratories). Washes in between treatment steps consisted of two quick rinses followed by three washes of 10 min each in TTBS.
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Results |
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Examination of placental villous tissue taken from a normotensive term pregnancy (Figure 1) showed intense immunostaining for the Mn SOD isoform in the endothelium of the villous vasculature (Figure 1A) but only faint staining for Mn SOD in the syncytiotrophoblast and in the stromal cells of the villous core. This was in contrast to immunostaining for Cu/Zn SOD in the same tissue, where on a consecutive section (Figure 1B), intense immunostaining was observed in certain cells of the villous stroma. Faint positive immunostaining for Cu/Zn SOD was also seen in syncytiotrophoblast and vascular endothelium. However in vascular endothelium the intensity of Cu/Zn SOD immunostaining was much less than that of the Mn SOD isoform. In the absence of either primary antibody (Figure 1C), no immunostaining was apparent on the consecutive section of this or any other sample.
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Examination of placental villous tissue taken from pregnancies complicated by either pre-eclampsia, IUGR, or pre-eclampsia plus IUGR showed that there were no apparent differences in localization and intensity of immunostaining for either Mn SOD or Cu/Zn SOD compared to tissues from normotensive pregnancies. Therefore, immunostaining for Mn SOD in the pre-eclamptic (Figure 2A) IUGR (Figure 2C), or pre-eclampsia plus IUGR (Figure 2E) tissue was still seen most intensely in the villous vascular endothelium and faintly in the syncytiotrophoblast and stroma. Moreover, the Cu/Zn isoform of SOD in the pre-eclampsia (Figure 2B) and IUGR (Figure 2D) samples, or in the pre-eclampsia plus IUGR (Figure 2F) samples, was seen again primarily most intensely in certain villous stromal cells, with weaker positive immunostaining in syncytiotrophoblast and vascular endothelium. In these pathological tissues, weak diffuse immunostaining for Cu/Zn SOD was also seen throughout the stroma. Again, in the absence of primary antibody (data not shown) no immunostaining was seen in the villous tissue from any of these pathological pregnancies. On Western blots (Figure 3), single immunostained bands of 17 kD (Cu/Zn SOD) and 23 kD (Mn SOD) were recognized by the SOD antibodies.
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Discussion |
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Using specific antibodies to both the Cu/Zn and Mn SOD isoforms, we have demonstrated the presence of both isoforms in human villous tissue by immunohistochemistry and Western blotting. It is apparent that the two isoforms demonstrate different cellular localizations within the placenta, which suggests that they may fulfill different physiological roles. The Mn SOD isoform appears to be mainly localized to fetal vascular endothelium, suggesting that it may play a role in scavenging superoxide at the level of the endothelium or elsewhere in the vasculature, thus preventing deleterious effects of superoxide on the vasculature. However, it may also play a crucial role in controlling the half-life of endothelium-derived NO (via regulation of superoxide concentrations) and thus in regulating vascular reactivity. Indeed, our previous studies, in which infusion of SOD into the fetal placental vasculature of the perfused placental cotyledon resulted in vasodilatation (
In contrast to the Mn SOD isoform, the Cu/Zn SOD isoform is localized mainly in the villous stromal tissue. At this location, Cu/Zn SOD may protect the villous tissue against the deleterious effects of superoxide generated by fetal macrophages (Hofbauer cells) or from other sources within villous tissue. Interestingly, neither the Mn SOD nor the Cu/Zn SOD isoform appeared to be present in syncytiotrophoblasts at the intensity found in the vascular endothelium and the stroma. Although this may be a relative difference in expression, this finding is surprising, given that
The recognition that the different SOD isoforms are present at different cellular locations within villous tissue cautions against gross measurements of SOD activity in whole placental homogenates in relation to different pathological states of pregnancy. Although placental SOD activity increases throughout gestation and is accompanied by a decrease in placental lipid peroxide concentrations (
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Footnotes |
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1 Presented in part at the 43nd Annual Meeting of the Society of Gynecological Investigation, Philadelphia, PA, March 20-23, 1996.
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Acknowledgments |
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Supported by grants from NIH (HL47860; LM) and Action Research and by the Scottish Hospital Endowments Research Trust (FL, IAG).
Received for publication October 21, 1996; accepted May 23, 1997.
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