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Troglitazone attenuates hypoxia-induced injury in cultured term human trophoblasts
American Journal of Obstetrics and Gynecology (2004) 191, 2154e9
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Troglitazone attenuates hypoxia-induced injury in cultured term human trophoblasts Uriel Elchalal, MD, Rachel G. Humphrey, MD, Steven D. Smith, BS, Chaobin Hu, PhD, Yoel Sadovsky, MD, D. Michael Nelson, MD, PhD* Departments of Obstetrics and Gynecology and Cell Biology and Physiology, Washington University School of Medicine, St Louis, Mo Received for publication December 12, 2003; revised May 1, 2004; accepted May 5, 2004
KEY WORDS Placenta Hypoxia Troglitazone Peroxisome proliferator activated receptoreg Apoptosis
Objective: The purpose of this study was to test the hypothesis that the thiazolidinedione troglitazone, a peroxisome proliferator activated receptoreg ligand, attenuates hypoxia-induced trophoblast injury. Study design: Cytotrophoblasts from 4 term human placentas were cultured in the presence or absence of 10 mmol/L troglitazone in either 20% oxygen (standard conditions) or 1% oxygen (hypoxic conditions) for variable periods before cell harvest. Medium behuman chorionic gonadotropin and human placental lactogen were analyzed by enzyme-linked immunosorbent assay. Apoptosis was quantified by cytokeratin-18 cleavage products staining; p53 expression was examined by Western blot analysis. Results: behuman chorionic gonadotropin and human placental lactogen levels were R2-fold higher in troglitazone-exposed cells at 16 hours of hypoxia, compared with vehicle control cells (P !.05). The apoptotic index was reduced by R30% (P !.001), and the expression of p53 was 2-fold lower (P !.02) in troglitazone-exposed cells under hypoxia for %16 hours but not different after O24 hours of low oxygen. Conclusion: Troglitazone attenuates the influence of acute hypoxia on cultured term human trophoblasts. Ó 2004 Elsevier Inc. All rights reserved.
Optimal fetal growth depends on placental function. Positioned to regulate maternal fetal exchange, the trophoblast bilayer is subject to transient hypoxia Supported by National Institutes of Health grant HD 29190 (D.M.N.), National Institutes of Health ES 11597 (Y.S.), and Society for Maternal-Fetal Medicine Scholarship Award (R.G.H.). Presented, in part, at the 50th Annual Meeting of the Society for Gynecologic Investigation, Washington, DC, March 27-30, 2003. * Reprint requests: D. Michael Nelson, MD, PhD, Department of Obstetrics/Gynecology, Washington University School of Medicine, 4566 Scott Ave, St. Louis, MO, 63110-1094. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.05.009
in normal pregnancies. Approximately 25% of the 4 million pregnancies per year in the United States are at risk for chronic placental hypoxia, and virtually every pregnancy is at risk for acutely lower oxygen tension in the intervillous space. The mechanisms by which hypoxia alters trophoblast function are poorly understood but involve apoptosis and necrosis.1 Understanding the effect of hypoxia on the placenta is of utmost importance because fetal size approximately doubles in the last 2 months of pregnancy, which is a process that is dependent largely on placental function.
Elchalal et al The nuclear receptor peroxisome proliferator activated receptor gamma (PPARg), a member of the nuclear receptor super-family of transcription factors, regulates gene expression through the binding of target gene promoters as homodimers or heterodimers with retinoid X-receptor alpha.2 Thiazolidinediones (such as troglitazone, rosiglitazone, and pioglitazone) are activators of PPARg and were developed as a class of antidiabetic drugs for the treatment of type 2 diabetes mellitus.3 In addition, PPARg activation by these drugs modulates cellular differentiation. We previously showed that troglitazone induces differentiation in cultured human trophoblasts.4 We also showed that hypoxia hinders differentiation5 and enhances apoptosis6 in cultured trophoblasts from term human placentas. Importantly, we6 and others7 found that cytotrophoblasts were more susceptible than syncytiotrophoblasts to the apoptotic effects of hypoxia. Because the PPARg ligand troglitazone enhances trophoblast differentiation and because enhancement of trophoblast differentiation diminishes hypoxic damage and apoptosis in trophoblasts, we tested the hypothesis that exposure of primary term human trophoblasts to troglitazone would attenuate the impact of hypoxia on these cells.
Material and methods Trophoblast isolation and culture Informed consent for the use of human placentas was obtained by a protocol that was approved by the Institutional Review Board of Washington University School of Medicine. Placentas were obtained immediately after term singleton deliveries after uncomplicated pregnancies. Cytotrophoblasts were isolated by the trypsin-DNase, percoll (Sigma Chemical Company, St. Louis, Mo) gradient centrifugation method described by Kliman et al8 with modifications5,6,9 and were frozen for later use. Cytotrophoblasts were cultured in Dulbecco’s modified Eagle medium that was supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah). Nonadherent cells and syncytial fragments were removed after 4 hours by being washed 3 times with phosphatebuffered saline solution (PBS).4 The cultures received fresh media with or without 10 mmol/L troglitazone or DMSO vehicle control before incubation in an aerobic or anaerobic chamber (Forma Scientific, Marietta, Ohio). They were then incubated for 0,16, 24, 48, or 72 hours before cell harvest at 72 hours under 5% carbon dioxide and either 20% oxygen (standard conditions) or %1% oxygen (hypoxic conditions). Culture conditions are given in Figure 1.
Hormone assays Medium betaehuman chorionic gonadotropin (b-hCG) and human placental lactogen (hPL) levels (n = 4
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Figure 1 A schematic presentation of culture paradigms used in our study.
specimens) were determined in duplicates by enzymelinked immunosorbent assay (DRG International, Mountainside, NJ), according to the manufacturer’s instructions. Colorimetric assay was performed with a microplate reader (Anthos htIII; Anthos Labtec Instruments, Salzburg, Austria) at 450 nm. Hormone levels were expressed in milli-international units of bhCG per milliliter of media or micrograms of hPL of milliliter of media, which were then normalized to the protein content. Protein concentration was determined by Biorad DC protein assay (Biorad, Hercules, Calif).
Immunocytochemistry Apoptosis was assessed by immunostaining for the cleavage products of cytokeratin 18.10 To create a positive control for apoptotic trophoblasts, we exposed cultures to 1 mmol/L staurosporine, 10 ng/mL tumor necrosis factorea and 25 mg/mL etoposide, which was designated to be a ‘‘death cocktail.’’ Duplicate cultures (n = 4) for each paradigm were fixed in ice cold methanol for 20 minutes, blocked with 1% bovine serum albumin, and 0.1% Tween in PBS for 30 minutes, and incubated in the same buffer for 2 hours at room temperature with mouse anti-cleaved cytokeratin 18 (M30; dilution of 1:50; Roche Diagnostics, Mannheim, Germany). After 3 washes in PBS, the cells were incubated for 2 hours with anti-mouse fluorescein isothiocyanate (Sigma Chemical Company) in room temperature. Controls included the omission of the primary antibody. After 3 PBS washes, cell nuclei were counterstained with propidium iodide (Sigma Chemical Company); sections were rinsed in PBS, mounted with GelMount (Biomeda, Foster City, Calif), and viewed under a microscope (TE2000U; Nikon, Japan) that was equipped with epifluorescence optics and a digital image capture system (Micropublisher camera and QCapture
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Elchalal et al software; QImaging, Burnaby, British Columbia, Canada). Images of 5 random 0.25-mm2 microscopic fields were captured; the cells were stained for cytokeratin 18; the cleavage products were quantified, and total nuclei for each field were recorded. The apoptotic index was defined as the percentage of cytokeratin 18 neoepitope positive cells that were stained in green, divided by the total number of nuclei stained in red.
Western immunoblot Western analysis (n = 4 analyses) was performed as described,6 which were modified in the following manner: Cells were lysed in a buffer that contained 10 mmol/L HEPES, 7.5 mmol/L MgCl2, 1% Triton X-100, 2 mmol/L EGTA, 1 mmol/L DTT, and protease inhibitor cocktail (Sigma Chemical Company). Protein aliquots of 10 to 20 mg/lane were electrophoresed at 100 V for 2 hours on sodium dodecylsulfate-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, Mass) overnight at 4(C, blocked with 5% skim milk for 1 hour, and incubated with antibodies to p53 (1:400 dilution; monoclonal mouse anti-human p53; RDI, Flanders, NJ) or b-actin (1:1000 dilution; polyclonal goat anti-actin immunoglobulin G; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif), washed twice for 10 minutes with tris-buffered saline solution ([TBST] 0.75% Tween 20), and incubated for 1 hour with the corresponding horseradish peroxidase-linked secondary antibodies (1:2000; Santa Cruz Biotechnology, Inc), then washed twice for 10 minutes with TBST. Membranes were processed for chemiluminescence with the Amersham ECL kit (Amersham Pharmacia Biotech, Inc, Piscataway, NJ), and chemiluminescence was analyzed with Epichemi 3 darkroom (UVP BioImaging System, Upland, CA). Protein density was normalized to b-actin expression.
Statistical analysis Data are expressed as mean G SD, and comparisons were made by paired t-test; a probability value of !.05 considered significant.
Results Figure 2 The effect of troglitazone on trophoblast secretion of b-hCG (A) and hPL (B). Media were collected from the final 48 to 72 hours in culture and were assayed for each biochemical marker of trophoblast differentiation. Cells were harvested at 72 hours and normalize to protein levels. This is a representative experiment from 1 of 4 placentas that were studied with identical qualitative results that were obtained in each experiment. The asterisk denotes P !.05; the open bars represent controls; the closed bars represent troglitazone.
We initially determined the effect of increasing the duration of hypoxia on the release of the biochemical markers of trophoblast differentiation,b-hCG and hPL (Figure 2). As expected, exposure to hypoxia for up to 48 hours diminished the release of b-hCG and hPL. Both hormones were undetectable in cultures that were exposed to hypoxia for O48 hours. On exposure to troglitazone in standard conditions, b-hCG increased from 200,277 mIU/100 mg to 498,480 mIU/100 mg protein (P !.05), and hPL release increased from 1963
Elchalal et al to 2868 mg/L (P !.01) between 48 and 72 hours in cultures, compared with vehicle control. Importantly, after 16 hours of hypoxia, b-hCG and hPL levels were R2-fold higher in troglitazone-exposed cells compared with vehicle control (P !.05), which suggests that PPARg activation diminished the influence of hypoxia on trophoblast differentiation. This effect of troglitazone disappeared after 24 hours, when levels of both b-hCG and hPL were not significantly different between troglitazone and DMSO-exposed cells that were incubated in hypoxic conditions. These data indicate that troglitazone can attenuate the adverse effects of short-term hypoxia on biochemical differentiation of cultured trophoblasts. We next determined whether troglitazone influenced the process of hypoxia-induced apoptosis in cultured trophoblasts. During the apoptotic cascade, effector caspases degrade cytokeratin-18 in trophoblasts to expose neoepitopes as cytokeratin-18 cleavage products. We used M30, a specific antibody that is raised against these neoepitopes in the cytokeratin-18 cleavage products, because it is specific for caspase-mediated apoptosis.10 We found that, in cultures without troglitazone, increasing the duration of hypoxia progressively increased the apoptotic indices, as determined by the apoptotic index (Figure 3, A). Exposure to troglitazone significantly reduced the apoptotic index for cultures in standard conditions without hypoxia (from 36% to 22%; P !.0001). Importantly, troglitazone also reduced the level of apoptosis compared with vehicle control (from 54% to 39%; P !.001) in cultures grown in hypoxia for 16 hours (Figure 3, B). This effect of troglitazone was again observed at 24 hours (P !.02). There was no difference in the apoptotic index of cultures that were exposed to hypoxia for R48 hours in the presence or absence of troglitazone. These data provide further support of the positive influence of troglitazone in the protection of trophoblast from acute hypoxia-induced apoptosis. We and others have shown that hypoxia-induced apoptosis in trophoblast is characterized by an increase in p53 expression.11 The p53 protein is a transcription factor that, when activated, regulates a cassette of transcripts that regulate cell differentiation and apoptosis. This protein also plays a pivotal role in the cellular response to exogenous stimuli (such as hypoxia), which halts the cell cycle and allows repair of DNA or promotes apoptosis, if excess cellular injury occurs.12 We therefore assessed the influence of troglitazone on expression of p53 in hypoxic trophoblasts. When compared with cultures that were exposed to vehicle control, the addition of troglitazone reduced the expression of p53 expression by 2-fold in cells that were exposed to 16 hours of hypoxia (P !.02), but troglitazone did not alter p53 expression in cultures that were exposed to 72 hours of hypoxia (Figure 4).
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Figure 3 A, Photomicrographs of primary trophoblast cultures that were exposed to 16 hours or 72 hours of hypoxia in the presence of control or troglitazone and stained by for the cleavage products of cytokeratin 18 (green) indicative of apoptosis and for nuclei with propidium iodide exposure (red). B, Quantified apoptotic index from 4 primary cultures of all paradigms that were studied. The cross bar denotes 50 mm; the asterisk denotes P ! .05, control (open bars) versus troglitazone (closed bars).
Comment Hypoxia hinders differentiation and enhances apoptosis in cultured term human trophoblast, as identified by lower b-hCG and hPL levels and by increased cytokeratin 18 cleavage and p53 expression in cells that were
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Figure 4 Representative immunoblot of p53 and b-actin expression (A) and densitometric analysis of p53 normalized to b-actin (B) of protein extracts from 4 primary human trophoblast cultures that were exposed to 16 hours or 72 hours of hypoxia in control (open bars) or troglitazone (closed bars). COS7 cells provided a positive control for p53 and bactin.
Elchalal et al exposed to hypoxia for up to 72 hours. Our data show that the PPARg ligand troglitazone attenuates the adverse effects of short-term hypoxia (up to 16 hours) on cultured term human trophoblasts. This protective effect of troglitazone disappears when the duration of hypoxia is O24 hours. Taken together, the results demonstrate that exposure of human trophoblasts to troglitazone can modulate trophoblast response beneficially to short-term hypoxia in vitro. PPARg plays a key role in differentiation of trophoblast in both the mouse13 and human.4,14,15 Mice that express mutant PPARg (exhibit defective vascularization of trophoblast in the labyrinthine placenta, which results in embryonic lethality at day 10.5.13 Activation of PPARg by troglitazone in primary cultured human trophoblasts enhances cellular biochemical and morphologic differentiation.4 The effect of PPARg activation is both ligand and tissue dependent.4,13,15-19 For example, PPARg activity is enhanced in primary human trophoblasts by oxidized lipids14 and inhibited by 15-deoxy-D12,14prostaglandinJ2.4 Activation of PPARg in other tissues (such as adipose) enhances differentiation,13,14 yet activation of the PPARg pathway inhibits osteoclast differentiation.15 The enhancement of trophoblast differentiation by troglitazone and the knowledge that hypoxia limits differentiation of term trophoblast laid the groundwork for the hypothesis that was tested in the present study. Although we demonstrated that troglitazone attenuates the influence of hypoxia on trophoblasts, this effect was observed only during relatively short (!16 hours) exposure to severe hypoxia. When trophoblasts were exposed to hypoxia for R24 hours, the effect of troglitazone was no longer observed. These findings may not be applicable to first-trimester trophoblasts, which exhibit a different response to hypoxia and are naturally exposed to a low oxygen tension.20 Two putative mechanisms likely are involved in the attenuation of apoptosis by troglitazone when term trophoblasts are exposed to hypoxia. The first mechanism is by the effect of troglitazone on differentiation of cytotrophoblasts into syncytiotrophoblasts, because the latter phenotype is less susceptible to hypoxic-induced apoptosis.6,7 The second mechanism may be through a direct effect of troglitazone on apoptosis by the inhibition of apoptosis activators or the activation of mechanisms that involve the inhibitors of apoptosis. In support of this mechanism, we found that troglitazone administration is associated with a reduced level of the pro-apoptotic protein p53. Additional mechanisms may also play a role. The short-lived protection that is offered by troglitazone for trophoblast that has been exposed to a severe hypoxic insult suggests that the beneficial effect of this ligand is overridden by proapoptotic mechanisms after a critical time period. Our low oxygen paradigm represents extreme hypoxia (FiO2, %1%) of cultured primary term trophoblasts. We
Elchalal et al and others have shown that this FiO2 reproducibly enhances apoptosis in cultured trophoblasts from term placentas.21 We speculate that the protection that is offered by troglitazone might persist longer if a lesser degree of hypoxia was used. We chose term trophoblasts because we wanted to understand how oxygen modulates placental function in the last trimester of pregnancy when fetal demands for gas exchange and nutrition are high and fetal size doubles. The pO2 in the intervillous blood in uncomplicated pregnancies is 40 to 80 mm Hg, yet a wide range of oxygen tensions may be present in placentas from pregnancies that are complicated by hypoperfusion and growth restriction. For example, umbilical blood samples in intrauterine growth restriction show that pO2 levels may be !15 mm Hg in severely affected pregnancies.22-24 Clearly, an in vitro system of cultured trophoblast may not represent physiologic conditions of trophoblast accurately in vivo. Nevertheless, our study implies that pharmacologic activation of PPARg can protect trophoblast partially from adverse effects of short-term exposure to extreme hypoxia. Knowing that villous trophoblasts in the placentas of women with intrauterine growth restriction show higher than normal levels of apoptosis,11,25 we speculate that PPARg activation may prove useful in the attenuation of placental injury in selected pregnancies. We thank Tim Schaiff, PhD, Ms Lori Rideout, and Ms Linda Dioneda for their help with the figures and the manuscript preparation.
References 1. Huppertz B, Kingdom J, Caniggia I, Desoye G, Black S, Korr H, et al. Hypoxia favors necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation. Placenta 2003;24:181-90. 2. Rocchi S, Auwerx J. Peroxisome proliferator-activated receptorgamma: a versatile metabolic regulator. Ann Med 1999;31:342-51. 3. Lehman JM, Moore LB, Smith-Oliver TA, Wilkinson WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferators-activated receptor PPAR. J Biol Chem 1995;270:12953-6. 4. Schaiff WT, Carlson MG, Smith SD, Levy R, Nelson DM, Sadovsky Y. Peroxisome proliferator-activated receptor-gamma modulates differentiation of human trophoblast in a ligand-specific manner. J Clin Endocrinol Metab 2000;85:3874-81. 5. Nelson DM, Johnson RD, Smith SD, Anteby EY, Sadovsky Y. Hypoxia limits differentiation and up-regulates expression and activity of prostaglandin H synthase 2 in cultured trophoblast from term human placenta. Am J Obstet Gynecol 1999;180:896-902. 6. Levy R, Smith SD, Chandler K, Sadovsky Y, Nelson DM. Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor. Am J Physiol Cell Physiol 2000;278:982-8. 7. Crocker IP, Cooper S, Ong SC, Baker PN. Differences in apoptotic susceptibility of cytotrophoblasts and syncytiotrophoblasts in normal pregnancy to those complicated with preeclampsia and intrauterine growth restriction. Am J Pathol 2003;162:637-43.
2159 8. Kliman HJ, Nestler JE, Semasi E, Sango JM, Strauss JF III. Purification, characterization and in-vitro differentiation of cytotrophoblasts from human term placenta. Endocrinology 1986;118:1567-82. 9. Guilbert LJ, Winkler-Lowen B, Sherburne R, Rote NS, Li H, Morrish DW. Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments. Placenta 2002;23:175-83. 10. Kadyrov M, Kaufman P, Huppertz B. Expression of a cytokeratin 18 neo-epitope is a specific marker for trophoblast apoptosis in human placenta. Placenta 2001;22:44-8. 11. Levy R, Smith SD, Yussuf K, Huntter PC, Kraus FT, Sadovsky Y, et al. Trophoblast apoptosis from pregnancies complicated by fetal growth restriction is associated with enhanced p53 expression. Am J Obstet Gynecol 2002;186:1056-61. 12. Stempien-Oteero A, Karsan A, Cornejo CJ, Xiang H, Eunson T, Morrison RS, et al. Mechanism of hypoxia-induced endothelial cell death. J Cell Biol 1999;274:8039-45. 13. Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR, et al. PPARg is required for placental, cardiac, and adipose tissue development. Mol Cell 1999;4:585-95. 14. Schild RL, Schaiff WT, Carlson MG, Cronbach EJ, Nelson DM, Sadovsky Y. The activity of PPAR gamma in primary human trophoblasts is enhanced by oxidized lipids. J Clin Endocrinol Metab 2002;87:1105-10. 15. Mbalaviele G, Abu-Amer Y, Meng A, Jaiswal R, Beck S, Pittenger MF, et al. Activation of peroxisome proliferator-activated receptor-gamma pathway inhibits osteoclast differentiation. J Biol Chem 2000;275:14388-93. 16. Capparuccia L, Marzioni D, Giordano A, Fazioli F, De Nictolis M, Busso N, et al. PPARgamma expression in normal human placenta, hydatidiform mole and choriocarcinoma. Mol Hum Reprod 2002;8:574-9. 17. Keelan JA, Sato TA, Marvin KW, Lander J, Gilmour RS, Mitchell MD. 15-Deoxy-Delta(12, 14)-prostaglandin J(2), a ligand for peroxisome proliferator-activated receptor-gamma, induces apoptosis in JEG3 choriocarcinoma cells. Biochem Biophys Res Commun 1999;262:579-85. 18. Waite LL, Person EC, Zhou Y, Lim KH, Scanlan TS, Taylor RN. Placental peroxisome proliferator-activated receptor-gamma is upregulated by pregnancy serum. J Clin Endocrinol Metab 2000;85:3808-14. 19. Tarrade A, Schoonjans K, Guibourdenche J, Bidart JM, Vidaud M, Auwerx J, et al. PPAR gamma/RXR alpha heterodimers are involved in human CG beta synthesis and human trophoblast differentiation. Endocrinology 2001;142:4504-14. 20. Jauniaux E, Hempstock J, Greenwold N, Burton GJ. Trophoblastic oxidative stress in relation to temporal and regional differences in maternal placental blood flow in normal and abnormal early pregnancies. Am J Pathol 2003;162:115-25. 21. Kilani RT, Mackova M, Davidge ST, Guilbert LJ. Effect of oxygen levels in villous trophoblast apoptosis. Placenta 2003;24:826-34. 22. Soothill PW, Nicolaides KH, Rodeck CH, Campbell S. The effect of gestational age on blood gas and acid-base values in human pregnancies. Fetal Ther 1986;1:166-73. 23. Kingdom JCP, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta 1997;18: 613-21. 24. Sibley CP, Pardi G, Cetin I, Todros T, Piccoli E, Kaufmann P, et al. Pathogenesis of intrauterine growth restriction (IUGR): conclusions derived from a European union Biomed 2 concerted action project ‘‘importance of oxygen supply in intrauterine growth restricted pregnancies’’: a workshop report. Placenta 2002;16:75S-9S. 25. Leung DN, Smith SC, To KF, Sahota DS, Baker PN. Increased placental apoptosis in pregnancies complicated by preeclampsia. Am J Obstet Gynecol 2001;184:1249-50.
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Troglitazone attenuates hypoxia-induced injury in cultured term human trophoblasts Uriel Elchalal, MD, Rachel G. Humphrey, MD, Steven D. Smith, BS, Chaobin Hu, PhD, Yoel Sadovsky, MD, D. Michael Nelson, MD, PhD* Departments of Obstetrics and Gynecology and Cell Biology and Physiology, Washington University School of Medicine, St Louis, Mo Received for publication December 12, 2003; revised May 1, 2004; accepted May 5, 2004
KEY WORDS Placenta Hypoxia Troglitazone Peroxisome proliferator activated receptoreg Apoptosis
Objective: The purpose of this study was to test the hypothesis that the thiazolidinedione troglitazone, a peroxisome proliferator activated receptoreg ligand, attenuates hypoxia-induced trophoblast injury. Study design: Cytotrophoblasts from 4 term human placentas were cultured in the presence or absence of 10 mmol/L troglitazone in either 20% oxygen (standard conditions) or 1% oxygen (hypoxic conditions) for variable periods before cell harvest. Medium behuman chorionic gonadotropin and human placental lactogen were analyzed by enzyme-linked immunosorbent assay. Apoptosis was quantified by cytokeratin-18 cleavage products staining; p53 expression was examined by Western blot analysis. Results: behuman chorionic gonadotropin and human placental lactogen levels were R2-fold higher in troglitazone-exposed cells at 16 hours of hypoxia, compared with vehicle control cells (P !.05). The apoptotic index was reduced by R30% (P !.001), and the expression of p53 was 2-fold lower (P !.02) in troglitazone-exposed cells under hypoxia for %16 hours but not different after O24 hours of low oxygen. Conclusion: Troglitazone attenuates the influence of acute hypoxia on cultured term human trophoblasts. Ó 2004 Elsevier Inc. All rights reserved.
Optimal fetal growth depends on placental function. Positioned to regulate maternal fetal exchange, the trophoblast bilayer is subject to transient hypoxia Supported by National Institutes of Health grant HD 29190 (D.M.N.), National Institutes of Health ES 11597 (Y.S.), and Society for Maternal-Fetal Medicine Scholarship Award (R.G.H.). Presented, in part, at the 50th Annual Meeting of the Society for Gynecologic Investigation, Washington, DC, March 27-30, 2003. * Reprint requests: D. Michael Nelson, MD, PhD, Department of Obstetrics/Gynecology, Washington University School of Medicine, 4566 Scott Ave, St. Louis, MO, 63110-1094. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.05.009
in normal pregnancies. Approximately 25% of the 4 million pregnancies per year in the United States are at risk for chronic placental hypoxia, and virtually every pregnancy is at risk for acutely lower oxygen tension in the intervillous space. The mechanisms by which hypoxia alters trophoblast function are poorly understood but involve apoptosis and necrosis.1 Understanding the effect of hypoxia on the placenta is of utmost importance because fetal size approximately doubles in the last 2 months of pregnancy, which is a process that is dependent largely on placental function.
Elchalal et al The nuclear receptor peroxisome proliferator activated receptor gamma (PPARg), a member of the nuclear receptor super-family of transcription factors, regulates gene expression through the binding of target gene promoters as homodimers or heterodimers with retinoid X-receptor alpha.2 Thiazolidinediones (such as troglitazone, rosiglitazone, and pioglitazone) are activators of PPARg and were developed as a class of antidiabetic drugs for the treatment of type 2 diabetes mellitus.3 In addition, PPARg activation by these drugs modulates cellular differentiation. We previously showed that troglitazone induces differentiation in cultured human trophoblasts.4 We also showed that hypoxia hinders differentiation5 and enhances apoptosis6 in cultured trophoblasts from term human placentas. Importantly, we6 and others7 found that cytotrophoblasts were more susceptible than syncytiotrophoblasts to the apoptotic effects of hypoxia. Because the PPARg ligand troglitazone enhances trophoblast differentiation and because enhancement of trophoblast differentiation diminishes hypoxic damage and apoptosis in trophoblasts, we tested the hypothesis that exposure of primary term human trophoblasts to troglitazone would attenuate the impact of hypoxia on these cells.
Material and methods Trophoblast isolation and culture Informed consent for the use of human placentas was obtained by a protocol that was approved by the Institutional Review Board of Washington University School of Medicine. Placentas were obtained immediately after term singleton deliveries after uncomplicated pregnancies. Cytotrophoblasts were isolated by the trypsin-DNase, percoll (Sigma Chemical Company, St. Louis, Mo) gradient centrifugation method described by Kliman et al8 with modifications5,6,9 and were frozen for later use. Cytotrophoblasts were cultured in Dulbecco’s modified Eagle medium that was supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah). Nonadherent cells and syncytial fragments were removed after 4 hours by being washed 3 times with phosphatebuffered saline solution (PBS).4 The cultures received fresh media with or without 10 mmol/L troglitazone or DMSO vehicle control before incubation in an aerobic or anaerobic chamber (Forma Scientific, Marietta, Ohio). They were then incubated for 0,16, 24, 48, or 72 hours before cell harvest at 72 hours under 5% carbon dioxide and either 20% oxygen (standard conditions) or %1% oxygen (hypoxic conditions). Culture conditions are given in Figure 1.
Hormone assays Medium betaehuman chorionic gonadotropin (b-hCG) and human placental lactogen (hPL) levels (n = 4
2155
Figure 1 A schematic presentation of culture paradigms used in our study.
specimens) were determined in duplicates by enzymelinked immunosorbent assay (DRG International, Mountainside, NJ), according to the manufacturer’s instructions. Colorimetric assay was performed with a microplate reader (Anthos htIII; Anthos Labtec Instruments, Salzburg, Austria) at 450 nm. Hormone levels were expressed in milli-international units of bhCG per milliliter of media or micrograms of hPL of milliliter of media, which were then normalized to the protein content. Protein concentration was determined by Biorad DC protein assay (Biorad, Hercules, Calif).
Immunocytochemistry Apoptosis was assessed by immunostaining for the cleavage products of cytokeratin 18.10 To create a positive control for apoptotic trophoblasts, we exposed cultures to 1 mmol/L staurosporine, 10 ng/mL tumor necrosis factorea and 25 mg/mL etoposide, which was designated to be a ‘‘death cocktail.’’ Duplicate cultures (n = 4) for each paradigm were fixed in ice cold methanol for 20 minutes, blocked with 1% bovine serum albumin, and 0.1% Tween in PBS for 30 minutes, and incubated in the same buffer for 2 hours at room temperature with mouse anti-cleaved cytokeratin 18 (M30; dilution of 1:50; Roche Diagnostics, Mannheim, Germany). After 3 washes in PBS, the cells were incubated for 2 hours with anti-mouse fluorescein isothiocyanate (Sigma Chemical Company) in room temperature. Controls included the omission of the primary antibody. After 3 PBS washes, cell nuclei were counterstained with propidium iodide (Sigma Chemical Company); sections were rinsed in PBS, mounted with GelMount (Biomeda, Foster City, Calif), and viewed under a microscope (TE2000U; Nikon, Japan) that was equipped with epifluorescence optics and a digital image capture system (Micropublisher camera and QCapture
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Elchalal et al software; QImaging, Burnaby, British Columbia, Canada). Images of 5 random 0.25-mm2 microscopic fields were captured; the cells were stained for cytokeratin 18; the cleavage products were quantified, and total nuclei for each field were recorded. The apoptotic index was defined as the percentage of cytokeratin 18 neoepitope positive cells that were stained in green, divided by the total number of nuclei stained in red.
Western immunoblot Western analysis (n = 4 analyses) was performed as described,6 which were modified in the following manner: Cells were lysed in a buffer that contained 10 mmol/L HEPES, 7.5 mmol/L MgCl2, 1% Triton X-100, 2 mmol/L EGTA, 1 mmol/L DTT, and protease inhibitor cocktail (Sigma Chemical Company). Protein aliquots of 10 to 20 mg/lane were electrophoresed at 100 V for 2 hours on sodium dodecylsulfate-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, Mass) overnight at 4(C, blocked with 5% skim milk for 1 hour, and incubated with antibodies to p53 (1:400 dilution; monoclonal mouse anti-human p53; RDI, Flanders, NJ) or b-actin (1:1000 dilution; polyclonal goat anti-actin immunoglobulin G; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif), washed twice for 10 minutes with tris-buffered saline solution ([TBST] 0.75% Tween 20), and incubated for 1 hour with the corresponding horseradish peroxidase-linked secondary antibodies (1:2000; Santa Cruz Biotechnology, Inc), then washed twice for 10 minutes with TBST. Membranes were processed for chemiluminescence with the Amersham ECL kit (Amersham Pharmacia Biotech, Inc, Piscataway, NJ), and chemiluminescence was analyzed with Epichemi 3 darkroom (UVP BioImaging System, Upland, CA). Protein density was normalized to b-actin expression.
Statistical analysis Data are expressed as mean G SD, and comparisons were made by paired t-test; a probability value of !.05 considered significant.
Results Figure 2 The effect of troglitazone on trophoblast secretion of b-hCG (A) and hPL (B). Media were collected from the final 48 to 72 hours in culture and were assayed for each biochemical marker of trophoblast differentiation. Cells were harvested at 72 hours and normalize to protein levels. This is a representative experiment from 1 of 4 placentas that were studied with identical qualitative results that were obtained in each experiment. The asterisk denotes P !.05; the open bars represent controls; the closed bars represent troglitazone.
We initially determined the effect of increasing the duration of hypoxia on the release of the biochemical markers of trophoblast differentiation,b-hCG and hPL (Figure 2). As expected, exposure to hypoxia for up to 48 hours diminished the release of b-hCG and hPL. Both hormones were undetectable in cultures that were exposed to hypoxia for O48 hours. On exposure to troglitazone in standard conditions, b-hCG increased from 200,277 mIU/100 mg to 498,480 mIU/100 mg protein (P !.05), and hPL release increased from 1963
Elchalal et al to 2868 mg/L (P !.01) between 48 and 72 hours in cultures, compared with vehicle control. Importantly, after 16 hours of hypoxia, b-hCG and hPL levels were R2-fold higher in troglitazone-exposed cells compared with vehicle control (P !.05), which suggests that PPARg activation diminished the influence of hypoxia on trophoblast differentiation. This effect of troglitazone disappeared after 24 hours, when levels of both b-hCG and hPL were not significantly different between troglitazone and DMSO-exposed cells that were incubated in hypoxic conditions. These data indicate that troglitazone can attenuate the adverse effects of short-term hypoxia on biochemical differentiation of cultured trophoblasts. We next determined whether troglitazone influenced the process of hypoxia-induced apoptosis in cultured trophoblasts. During the apoptotic cascade, effector caspases degrade cytokeratin-18 in trophoblasts to expose neoepitopes as cytokeratin-18 cleavage products. We used M30, a specific antibody that is raised against these neoepitopes in the cytokeratin-18 cleavage products, because it is specific for caspase-mediated apoptosis.10 We found that, in cultures without troglitazone, increasing the duration of hypoxia progressively increased the apoptotic indices, as determined by the apoptotic index (Figure 3, A). Exposure to troglitazone significantly reduced the apoptotic index for cultures in standard conditions without hypoxia (from 36% to 22%; P !.0001). Importantly, troglitazone also reduced the level of apoptosis compared with vehicle control (from 54% to 39%; P !.001) in cultures grown in hypoxia for 16 hours (Figure 3, B). This effect of troglitazone was again observed at 24 hours (P !.02). There was no difference in the apoptotic index of cultures that were exposed to hypoxia for R48 hours in the presence or absence of troglitazone. These data provide further support of the positive influence of troglitazone in the protection of trophoblast from acute hypoxia-induced apoptosis. We and others have shown that hypoxia-induced apoptosis in trophoblast is characterized by an increase in p53 expression.11 The p53 protein is a transcription factor that, when activated, regulates a cassette of transcripts that regulate cell differentiation and apoptosis. This protein also plays a pivotal role in the cellular response to exogenous stimuli (such as hypoxia), which halts the cell cycle and allows repair of DNA or promotes apoptosis, if excess cellular injury occurs.12 We therefore assessed the influence of troglitazone on expression of p53 in hypoxic trophoblasts. When compared with cultures that were exposed to vehicle control, the addition of troglitazone reduced the expression of p53 expression by 2-fold in cells that were exposed to 16 hours of hypoxia (P !.02), but troglitazone did not alter p53 expression in cultures that were exposed to 72 hours of hypoxia (Figure 4).
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Figure 3 A, Photomicrographs of primary trophoblast cultures that were exposed to 16 hours or 72 hours of hypoxia in the presence of control or troglitazone and stained by for the cleavage products of cytokeratin 18 (green) indicative of apoptosis and for nuclei with propidium iodide exposure (red). B, Quantified apoptotic index from 4 primary cultures of all paradigms that were studied. The cross bar denotes 50 mm; the asterisk denotes P ! .05, control (open bars) versus troglitazone (closed bars).
Comment Hypoxia hinders differentiation and enhances apoptosis in cultured term human trophoblast, as identified by lower b-hCG and hPL levels and by increased cytokeratin 18 cleavage and p53 expression in cells that were
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Figure 4 Representative immunoblot of p53 and b-actin expression (A) and densitometric analysis of p53 normalized to b-actin (B) of protein extracts from 4 primary human trophoblast cultures that were exposed to 16 hours or 72 hours of hypoxia in control (open bars) or troglitazone (closed bars). COS7 cells provided a positive control for p53 and bactin.
Elchalal et al exposed to hypoxia for up to 72 hours. Our data show that the PPARg ligand troglitazone attenuates the adverse effects of short-term hypoxia (up to 16 hours) on cultured term human trophoblasts. This protective effect of troglitazone disappears when the duration of hypoxia is O24 hours. Taken together, the results demonstrate that exposure of human trophoblasts to troglitazone can modulate trophoblast response beneficially to short-term hypoxia in vitro. PPARg plays a key role in differentiation of trophoblast in both the mouse13 and human.4,14,15 Mice that express mutant PPARg (exhibit defective vascularization of trophoblast in the labyrinthine placenta, which results in embryonic lethality at day 10.5.13 Activation of PPARg by troglitazone in primary cultured human trophoblasts enhances cellular biochemical and morphologic differentiation.4 The effect of PPARg activation is both ligand and tissue dependent.4,13,15-19 For example, PPARg activity is enhanced in primary human trophoblasts by oxidized lipids14 and inhibited by 15-deoxy-D12,14prostaglandinJ2.4 Activation of PPARg in other tissues (such as adipose) enhances differentiation,13,14 yet activation of the PPARg pathway inhibits osteoclast differentiation.15 The enhancement of trophoblast differentiation by troglitazone and the knowledge that hypoxia limits differentiation of term trophoblast laid the groundwork for the hypothesis that was tested in the present study. Although we demonstrated that troglitazone attenuates the influence of hypoxia on trophoblasts, this effect was observed only during relatively short (!16 hours) exposure to severe hypoxia. When trophoblasts were exposed to hypoxia for R24 hours, the effect of troglitazone was no longer observed. These findings may not be applicable to first-trimester trophoblasts, which exhibit a different response to hypoxia and are naturally exposed to a low oxygen tension.20 Two putative mechanisms likely are involved in the attenuation of apoptosis by troglitazone when term trophoblasts are exposed to hypoxia. The first mechanism is by the effect of troglitazone on differentiation of cytotrophoblasts into syncytiotrophoblasts, because the latter phenotype is less susceptible to hypoxic-induced apoptosis.6,7 The second mechanism may be through a direct effect of troglitazone on apoptosis by the inhibition of apoptosis activators or the activation of mechanisms that involve the inhibitors of apoptosis. In support of this mechanism, we found that troglitazone administration is associated with a reduced level of the pro-apoptotic protein p53. Additional mechanisms may also play a role. The short-lived protection that is offered by troglitazone for trophoblast that has been exposed to a severe hypoxic insult suggests that the beneficial effect of this ligand is overridden by proapoptotic mechanisms after a critical time period. Our low oxygen paradigm represents extreme hypoxia (FiO2, %1%) of cultured primary term trophoblasts. We
Elchalal et al and others have shown that this FiO2 reproducibly enhances apoptosis in cultured trophoblasts from term placentas.21 We speculate that the protection that is offered by troglitazone might persist longer if a lesser degree of hypoxia was used. We chose term trophoblasts because we wanted to understand how oxygen modulates placental function in the last trimester of pregnancy when fetal demands for gas exchange and nutrition are high and fetal size doubles. The pO2 in the intervillous blood in uncomplicated pregnancies is 40 to 80 mm Hg, yet a wide range of oxygen tensions may be present in placentas from pregnancies that are complicated by hypoperfusion and growth restriction. For example, umbilical blood samples in intrauterine growth restriction show that pO2 levels may be !15 mm Hg in severely affected pregnancies.22-24 Clearly, an in vitro system of cultured trophoblast may not represent physiologic conditions of trophoblast accurately in vivo. Nevertheless, our study implies that pharmacologic activation of PPARg can protect trophoblast partially from adverse effects of short-term exposure to extreme hypoxia. Knowing that villous trophoblasts in the placentas of women with intrauterine growth restriction show higher than normal levels of apoptosis,11,25 we speculate that PPARg activation may prove useful in the attenuation of placental injury in selected pregnancies. We thank Tim Schaiff, PhD, Ms Lori Rideout, and Ms Linda Dioneda for their help with the figures and the manuscript preparation.
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