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ORIGINAL RESEARCH |
From the Department of Medicine, Monash University, Box Hill Hospital, Box Hill, Victoria, Australia.
Address reprint requests to: Robert L. Medcalf, MD, Department of Medicine, Monash University, Box Hill Hospital, Arnold Street, Box Hill 3128, Victoria, Australia; E-mail: robert.medcalf{at}med.monash.edu.au.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Placental tissue was obtained from normal pregnancies after cesarean delivery. Placental explants were incubated with increasing concentrations of extracellular magnesium (range 0–2.0 mM). Placental apoptosis was evaluated by tissue morphology, DNA fragmentation, cytokeratin-18 neoepitope formation, and cleavage of plasminogen activator inhibitor type 2.
RESULTS: Physiologic concentrations of extracellular magnesium stimulated placental apoptosis. Magnesium stimulated apoptosis within the physiologic range (0.8–1.2 mM) (n = 6, P < .001) and was associated with cleavage of plasminogen activator inhibitor type 2 and cytokeratin-18 neoepitope formation. These data implicate caspase activation in the transduction of the magnesium-induced apoptotic signal. Therapeutic concentrations of vitamin C, vitamin E, and acetylcysteine (all at 25 µg/mL) inhibited DNA fragmentation and attenuated cleavage of plasminogen activator inhibitor type 2 and cytokeratin-18 neoepitope formation.
CONCLUSION: Magnesium-induced placental apoptosis is a potent mechanism of placental degeneration in vitro and may represent an important regulator of placental tissue dynamics in vivo. The ability of antioxidants to prevent magnesium-induced placental apoptosis implicates oxidation–reduction-dependent signaling events in this process. Furthermore, these findings provide a basis for further studies of antioxidants in mitigating the adverse effects of preeclampsia.
Placentas from pregnancies complicated by fetal growth restriction and preeclampsia have increased apoptosis.1 Perturbation of metal ions has gained attention as a mediator of degenerative diseases2,3 and a regulator of apoptosis.4 Normal human pregnancy is associated with a lowering of maternal serum magnesium concentrations.5 Extracellular magnesium may be an important regulator of placental function, because fetal growth correlates negatively with maternal serum magnesium levels and preeclampsia is associated with increased maternal serum magnesium concentrations.5
Magnesium can also increase nitric oxide activity,6 an important component of oxidative stress,7 and in vitro studies have demonstrated that physiologic levels of magnesium cause trophoblast death.8 Preeclampsia has been hypothesized to be due to increased oxidative stress,9 and the risk of preeclampsia has been reduced by maternal use of antioxidant vitamins.10
The aims of this study were to determine the effect of a physiologic concentration of extracellular magnesium on the induction of apoptosis in cultured term human placental tissue and the role of antioxidants in this process. We tested the hypotheses that increasing the extracellular magnesium concentration within the physiologic range stimulated placental apoptosis and that this process could be prevented by antioxidants.
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MATERIALS AND METHODS |
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The placental tissue was prepared for culture as described by Polliotti et al.11 Tissue viability was assessed by the trypan blue dye exclusion assay, as well as the secretion of plasminogen activator inhibitor type 2, which is an indicator of normal placental function.10 Tissue explants (150 mg) were cultured in quadruplicate from each placenta in 2 mL Hanks balanced salt solution supplemented with 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, with or without magnesium treatment (0–2.0 mM). The effect of magnesium on placental apoptosis was determined by the degree of oligosomal DNA fragmentation, tissue morphologic changes, and cleavage of the intracellular proteins cytokeratin-18 and plasminogen activator inhibitor type 2.
DNA fragmentation, a hallmark of apoptosis, is readily visualized as a series of low molecular weight DNA oligosomal fragments on agarose gels after gel electrophoresis.12 Quantitation of DNA fragmentation in ethidium bromide-stained agarose gels was performed by densitometry.
Tissue morphologic changes were examined with hematoxylin-eosin staining. Cytokeratin-18 neoepitope formation and cleavage of plasminogen activator inhibitor type 2 was detected by immunohistochemistry and/or Western blotting.14 Antibody for cytokeratin-18 neoepitope (M30 CytoDEATH) was obtained from Boehringer Mannheim (Mannheim, Germany); antiplasminogen activator inhibitor type 2 antibody was obtained from American Diagnostica (Greenwich, CT).
To investigate the involvement of oxidation reduction in magnesium-induced placental apoptosis, placental explants were treated with 25 µg/mL of the antioxidants vitamin C (David Bull Laboratories, Melbourne, Australia), vitamin E (
-tocopherol, Sigma, St. Louis, MO), or acetylcysteine (David Bull Laboratories). The effect of these antioxidants on magnesium-induced apoptosis was evaluated by DNA fragmentation and Western blotting, as described above.
All statistical assessments were determined using the Statistical Package for Social Sciences, version 9.0, for Windows (SPSS Inc., Chicago, IL). Data were presented as the mean ± standard deviation. Statistical significance was defined as P < .05.
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RESULTS |
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). Little or no apoptosis was observed in placenta cultures without magnesium treatment.
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, arrow) and formation of cytokeratin-18 neoepitope (Figure 1Aiii, arrow). Cleavage of plasminogen activator inhibitor type 213 and cytokeratin-18 neoepitope formation14 are mediated by activated caspases, suggesting the involvement of caspases in magnesium-induced placental apoptosis. These results were reproduced using tissue obtained from four different placentas.
To obtain additional evidence that placental apoptosis was induced by magnesium, histologic analysis of placental tissue was performed. After 30 hours’ incubation, trophoblast nuclei remained evenly dispersed in explants cultured in the absence of additional MgSO4, which was not structurally different from freshly prepared tissue (Figure 1Bi, ii, arrows). After 30 hours’ treatment with 1 mM MgSO4, the placental tissue developed syncytial knot-like regions (Figure 1Biii, arrow). Syncytial knot formation is a process mediated by an apoptotic-like mechanism that involves clustering of pyknotic trophoblast nuclei.15 Placenta treated with MgSO4 also developed a shrunken morphology consistent with apoptosis in comparison to the slightly edematous appearance of tissue cultured for 30 hours without magnesium treatment (Figure 1Bii,iii ).
Immunohistochemical studies demonstrated extensive plasminogen activator inhibitor type 2 antigen in the trophoblast region (Figure 1Ci, arrows). Magnesium-induced apoptosis did not alter the distribution of this protein, suggesting that cleaved plasminogen activator inhibitor type 2 was confined to trophoblast structures. Cytokeratin-18 neoepitope expression was observed in localized trophoblast regions in placental tissue cultured with 1 mM MgSO4 (Figure 1Cii, arrows) but was absent in placental tissue cultured for 30 hours without magnesium (data not shown). To confirm tissue viability and integrity, the expression pattern of HuR, a ubiquitously expressed protein associated with the regulation of mRNA stability,16 was studied in placental tissue incubated with 1 mM MgSO4. HuR expression was detected in the nuclei and cytoplasm of most villous structures (Figure 1Ciii). Similar results were obtained from four separate experiments using different placental tissue.
We next determined the effect of different concentrations of magnesium on placental apoptosis. Placental tissue was extracted and cultured in the presence or absence of 10% fetal calf serum. Experiments in the absence of fetal calf serum were undertaken because serum is a source of magnesium and influences cell survival. Increasing the concentration of magnesium in culture medium from 0–2 mM MgSO4 increased apoptotic DNA fragmentation during 24 hours of incubation. This was evident in the presence and absence of fetal calf serum (Figure 2a). This experiment was repeated using explants prepared from six different placentas. The degree of apoptosis was quantitated and presented graphically in Figure 2b. The physiologic range of magnesium is between 0.7 and 1.2 mM, and we observed an increase in the degree of placental apoptosis when the magnesium level was increased from 0.8 to 1.2 mM. This particular finding, although presented in Figure 2b, is also shown graphically in Figure 2c to highlight the effect of magnesium at these physiologic concentrations (n = 6, P < .001). Hence, an increase in the magnesium concentration within the physiologic range increased placental apoptosis. These experiments were also repeated using different types of culture media (Dulbecco’s modified Eagles medium and Roswell Park Memorial Institute medium). The pattern of apoptotic DNA fragmentation was similar between the different media at comparable magnesium concentrations (data not shown).
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). This result was reproduced using placental extracts prepared from four different placentas.
Antioxidants have been shown to prevent the onset of preeclampsia10 and can modulate apoptosis in vitro.17,18 We examined the effect of three different antioxidants on magnesium-induced placental apoptosis. As shown in Figure 3, treatment with the antioxidants vitamin C, vitamin E, and acetylcysteine for 30 hours attenuated magnesium-induced placental apoptosis. Indeed, all three antioxidants attenuated magnesium-induced apoptotic DNA fragmentation, cytokeratin 18 neoepitope formation, and cleavage of plasminogen activator inhibitor type 2. Quantitation of the degree of protection conferred by these agents showed that all three antioxidants reduced the degree of magnesium-induced apoptosis by more than 50% (n = 3).
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DISCUSSION |
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The extracellular magnesium ion concentration is tightly regulated and known to be perturbed under pathologic conditions.19 Serum magnesium levels are reduced in normal human pregnancy and elevated in preeclampsia.5 Our results suggest that increased maternal serum magnesium levels may activate caspases and cause placental apoptosis by way of a oxidation–reduction-regulated mechanism. We propose that increased serum magnesium promotes nitric oxide activity,6 an important component of placental oxidative stress,7 that is associated with placental apoptosis20 and known to be elevated in preeclampsia.21
The potential in vivo stimulation of placental apoptosis by extracellular magnesium is an important clinical issue. Magnesium administered therapeutically has recently been associated with fetal cardiac injury and increased neonatal death.22 Although changes in magnesium levels may normally be tolerated, it is possible that, in some individuals, a failure of magnesium homeostasis may occur and perturb placental function, resulting in a pathologic impact on fetal cardiovascular function.
Magnesium-induced placental apoptosis is a potent mechanism of placental degeneration in vitro and may represent an important regulator of placental tissue dynamics in vivo. The ability of antioxidants to prevent magnesium-induced placental apoptosis implicates oxidation–reduction-sensitive signaling events in this process and also provides further support for the use of antioxidants in the treatment of preeclampsia.
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Footnotes |
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We thank Professor Shaun Brennecke for his advice, Professor Gab Kovacs for the clinical specimens, and the Box Hill Hospital Department of Pathology for the histologic evaluations.
Received December 13, 2000. Received in revised form March 12, 2001. Accepted March 23, 2001.
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REFERENCES |
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2. Nelson N. Metal ion transporters and homeostasis. EMBO J 1999;18:4361–71.[Medline]
3. Bush AI. Metals and neuroscience. Curr Opin Chem Biol 2000;4:184–91.[Medline]
4. Tsao N, Lei HY. Activation of the Na(+)/H(+) antiporter, Na+/HCO3(-)/CO3(2-) cotransporter, or Cl(-)/HCO3(-) exchanger in spontaneous thymocyte apoptosis. J Immunol 1996;157:1107–16.[Abstract]
5. Sanders R, Konijnenberg A, Huijgen HJ, Wolf H, Boer K, Sanders GT. Intracellular and extracellular, ionized and total magnesium in pre-eclampsia and uncomplicated pregnancy. Clin Chem Lab Med 1999;37:55–9.[Medline]
6. Yang ZW, Gebrewold A, Nowakowski M, Altura BT, Altura BM. Mg(2+)-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO. Am J Physiol Regul Integr Comp Physiol 2000;278:R628–39.
7. Many A, Hubel CA, Fisher SJ, Roberts JM, Zhou Y. Invasive cytotrophoblasts manifest evidence of oxidative stress in preeclampsia. Am J Pathol 2000;156:321–31.
8. Lueck J, Aladjem S. Effect of therapeutic levels of magnesium sulphate, methyldopa, hydralazine and phenobarbitone on normal term human trophoblast in vitro. Placenta 1982;3:39–44.[Medline]
9. Roberts JM, Hubel CA. Is oxidative stress the link in the two-stage model of pre-eclampsia? Lancet 1999;354: 788–9.[Medline]
10. Chappell LC, Seed PT, Briley AL, Kelly FJ, Lee R, Hunt BJ, et al. Effect of antioxidants on the occurrence of preeclampsia in women at increased risk: A randomised trial. Lancet 1999;354:810–6.[Medline]
11. Polliotti BM, Abramowsky C, Schwartz DA, Keesling SS, Lee GR, Caba J, et al. Culture of first-trimester and full-term human chorionic villus explants: Role of human chorionic gonadotropin and human placental lactogen as a viability index. Early Pregnancy 1995;1:270–80.
12. Gong J, Traganos F, Darzynkiewicz Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 1994;218:314–9.[Medline]
13. Jensen PH, Fladmark KE, Gjertsen BT, Vintermyr OK. Caspase I-related protease inhibition retards the execution of okadaic acid- and camptothecin-induced apoptosis and PAI-2 cleavage, but not commitment to cell death in HL-60 cells. Br J Cancer 1999;79:1685–91.[Medline]
14. Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B, et al. Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 1999;187:567–72.[Medline]
15. Huppertz B, Frank HG, Reister F, Kingdom J, Korr H, Kaufmann P. Apoptosis cascade progresses during turnover of human trophoblast: analysis of villous cytotrophoblast and syncytial fragments in vitro. Lab Invest 1999;79: 1687–702.[Medline]
16. Maurer F, Tierney M, Medcalf RL. An AU-rich sequence in the 3'-UTR of plasminogen activator inhibitor type 2 (PAI-2) mRNA promotes PAI-2 mRNA decay and provides a binding site for nuclear HuR. Nucleic Acids Res 1999;27:1664–73.
17. Mason RP, Leeds PR, Jacob RF, Hough CJ, Zhang KG, Mason PE, et al. Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells. J Neurochem 1999;72:1448–56.[Medline]
18. Patel RP, Moellering D, Murphy-Ullrich J, Jo H, Beckman JS, Darley-Usmar VM. Cell signaling by reactive nitrogen and oxygen species in atherosclerosis. Free Radic Biol Med 2000;28:1780–94.[Medline]
19. Vink R, Cernak I. Regulation of intracellular free magnesium in central nervous system injury. Front Biosci 2000; 5:D656–65.[Medline]
20. Nakatsuka M, Asagiri K, Noguchi S, Habara T, Kudo T. Nafamostat mesylate, a serine protease inhibitor, suppresses lipopolysaccharide-induced nitric oxide synthesis and apoptosis in cultured human trophoblasts. Life Sci 2000;67:1243–50.[Medline]
21. Nasiell J, Nisell H, Blanck A, Lunell NO, Faxen M. Placental expression of endothelial constitutive nitric oxide synthase mRNA in pregnancy complicated by preeclampsia. Acta Obstet Gynecol Scand 1998;77:492–6.[Medline]
22. Scudiero R, Khoshnood B, Pryde PG, Lee K, Wall S, Mittendorf R. Perinatal death and tocolytic magnesium sulfate. Obstet Gynecol 2000;96:178–182.
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