Nuclear Factor-κB, p38, and Stress-Activated Protein Kinase Mitogen-Activated Protein Kinase Signaling Pathways Regulate Proinflammatory Cytokines and Apoptosis in Human Placental Explants in Response to Oxidative Stress
2007; Elsevier BV; Volume: 170; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.061035
ISSN1525-2191
AutoresTereza Cindrová‐Davies, Olivera Spasić-Bošković, Eric Jauniaux, D. Stephen Charnock‐Jones, Graham J. Burton,
Tópico(s)Biomarkers in Disease Mechanisms
ResumoPreeclampsia is a potentially fatal complication of human pregnancy characterized by hypertension, proteinuria, and edema. Placental oxidative stress is a key element in the pathogenesis of the syndrome and results in the release of a cocktail of factors, including proinflammatory cytokines and apoptotic debris, that in turn cause activation of the maternal endothelium. The intermediary molecular mechanisms underlying this release are unknown, but they represent a potential target for therapeutic interventions. We examined activation of signaling pathways during hypoxia-reoxygenation of villous explants in vitro. Hypoxia-reoxygenation activated the p38 and stress-activated protein kinase mitogen-activated protein kinase (MAPK) and the nuclear factor-κB pathways. Downstream consequences included increased tissue concentrations and secretion of tumor necrosis factor-α and interleukin-1β, increased expression of cyclooxygenase-2, and increased apoptosis. Administration of vitamins C and E to explants blocked activation of the p38 and stress-activated protein kinase MAPK and nuclear factor-κB pathways. Vitamin administration or p38 pathway inhibition also reduced cyclooxygenase-2 expression, tumor necrosis factor-α and interleukin-1β secretion, and the levels of apoptosis. We conclude that oxidative stress is a potent inducer of placental synthesis and release of proinflammatory factors. Most of these effects are mediated through the p38 MAPK and nuclear factor-κB pathways and can be effectively blocked by vitamins C and E in vitro. Preeclampsia is a potentially fatal complication of human pregnancy characterized by hypertension, proteinuria, and edema. Placental oxidative stress is a key element in the pathogenesis of the syndrome and results in the release of a cocktail of factors, including proinflammatory cytokines and apoptotic debris, that in turn cause activation of the maternal endothelium. The intermediary molecular mechanisms underlying this release are unknown, but they represent a potential target for therapeutic interventions. We examined activation of signaling pathways during hypoxia-reoxygenation of villous explants in vitro. Hypoxia-reoxygenation activated the p38 and stress-activated protein kinase mitogen-activated protein kinase (MAPK) and the nuclear factor-κB pathways. Downstream consequences included increased tissue concentrations and secretion of tumor necrosis factor-α and interleukin-1β, increased expression of cyclooxygenase-2, and increased apoptosis. Administration of vitamins C and E to explants blocked activation of the p38 and stress-activated protein kinase MAPK and nuclear factor-κB pathways. Vitamin administration or p38 pathway inhibition also reduced cyclooxygenase-2 expression, tumor necrosis factor-α and interleukin-1β secretion, and the levels of apoptosis. We conclude that oxidative stress is a potent inducer of placental synthesis and release of proinflammatory factors. Most of these effects are mediated through the p38 MAPK and nuclear factor-κB pathways and can be effectively blocked by vitamins C and E in vitro. Preeclampsia is the most important complication of human pregnancy worldwide and a major contributor to maternal and fetal morbidity and mortality. Despite much research, the pathophysiology remains elusive, although there is strong evidence that generation of placental oxidative stress is a key intermediary event.1Roberts JM Hubel CA Is oxidative stress the link in the two-stage model of pre-eclampsia?.Lancet. 1999; 354: 788-789Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 2Redman CW Sargent IL Latest advances in understanding preeclampsia.Science. 2005; 308: 1592-1594Crossref PubMed Scopus (2098) Google Scholar The stress is thought to induce the placenta to release a cocktail of factors, including proinflammatory cytokines, antiangiogenic factors, and apoptotic debris, which culminates in an enhanced maternal inflammatory response.3Roberts JM Endothelial dysfunction in preeclampsia.Semin Reprod Endocrinol. 1998; 16: 5-15Crossref PubMed Scopus (517) Google Scholar However, the molecular mechanisms linking oxidative stress to release of these factors are not known. In part, this is due to the constraint that placental tissues are only available for study at the end of pregnancy, when the pathology is well established or secondary pathology may have been superimposed. Early-onset preeclampsia, the most severe form of the syndrome, has its origins in the first trimester of pregnancy.4Burton GJ Hung T-H Hypoxia-reoxygenation: a potential source of placental oxidative stress in normal pregnancy and preeclampsia.Fetal Maternal Med Rev. 2003; 14: 97-117Crossref Scopus (45) Google Scholar During this period, fetal extravillous trophoblast cells migrate into the endometrium, and their presence is associated with the physiological conversion of the maternal spiral arteries into dilated, flaccid conduits that ensure an inviolable blood supply to the conceptus. Trophoblast invasion is impaired in preeclampsia for a number of possible reasons, but the net result is absent, or severely reduced, conversion of the arteries.5Brosens JJ Pijnenborg R Brosens IA The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature.Am J Obstet Gynecol. 2002; 187: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar We recently proposed that the retention of smooth muscle within the vessel walls leads to persistence of vasoreactivity and to intermittent perfusion of the intervillous space.4Burton GJ Hung T-H Hypoxia-reoxygenation: a potential source of placental oxidative stress in normal pregnancy and preeclampsia.Fetal Maternal Med Rev. 2003; 14: 97-117Crossref Scopus (45) Google Scholar, 6Burton GJ Jauniaux E Placental oxidative stress: from miscarriage to preeclampsia.J Soc Gynecol Investig. 2004; 11: 342-352Crossref PubMed Scopus (552) Google Scholar Consequently, the placental tissues are exposed to fluctuating oxygen concentrations and hence suffer repeated ischemia-reperfusion type injuries. In support of this theory, we and others have demonstrated that villi sampled from normal placentas delivered by caesarean section show increased oxidative stress when subjected to hypoxia-reoxygenation (H/R) compared with controls maintained under hypoxia alone.7Hung T-H Skepper JN Burton GJ In vitro ischemia-reperfusion injury in term human placenta as a model for oxidative stress in pathological pregnancies.Am J Pathol. 2001; 159: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar These changes closely mimic those reported in preeclampsia and could be attenuated with desferrioxamine, the electron spin trap α-phenyl-N-tert-butylnitrone,4Burton GJ Hung T-H Hypoxia-reoxygenation: a potential source of placental oxidative stress in normal pregnancy and preeclampsia.Fetal Maternal Med Rev. 2003; 14: 97-117Crossref Scopus (45) Google Scholar, 7Hung T-H Skepper JN Burton GJ In vitro ischemia-reperfusion injury in term human placenta as a model for oxidative stress in pathological pregnancies.Am J Pathol. 2001; 159: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar or carbon monoxide.8Bainbridge SA Belkacemi L Dickinson M Graham CH Smith GN Carbon monoxide inhibits hypoxia/reoxygenation-induced apoptosis and secondary necrosis in syncytiotrophoblast.Am J Pathol. 2006; 169: 774-783Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Hypoxia-reoxygenation also proved a potent stimulus for apoptotic changes within the syncytiotrophoblast8Bainbridge SA Belkacemi L Dickinson M Graham CH Smith GN Carbon monoxide inhibits hypoxia/reoxygenation-induced apoptosis and secondary necrosis in syncytiotrophoblast.Am J Pathol. 2006; 169: 774-783Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 9Hung T-H Skepper JN Charnock-Jones DS Burton GJ Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in preeclampsia.Circ Res. 2002; 28: 1274-1281Crossref Scopus (341) Google Scholar and induced secretion of tumor necrosis factor (TNF)-α.10Hung T-H Charnock-Jones DS Skepper JN Burton GJ Secretion of tumor necrosis factor-alpha from human placental tissues induced by hypoxia-reoxygenation causes endothelial cell activation in vitro: a potent mediator of the inflammatory response in preeclampsia.Am J Pathol. 2004; 164: 1049-1061Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar Although these studies established that hypoxia-reoxygenation is a more physiological stimulus than hypoxia alone for generating the placental changes associated with preeclampsia, they did not explore the signaling pathways involved. Evidence of the involvement of oxidative stress in the pathogenesis of preeclampsia stimulated several clinical trials to test the prophylactic benefits of antioxidant vitamins C and E in women at risk of preeclampsia. Although a beneficial effect of antioxidants was suggested in a small randomized trial of women at high risk of preeclampsia,11Chappell LC Seed PT Briley AL Kelly FJ Lee R Hunt BJ Parmar K Bewley SJ Shennan AH Steer PJ Poston L Effect of antioxidants on the occurrence of pre-eclampsia in women at increased risk: a randomised trial.Lancet. 1999; 354: 810-816Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar a recent multicenter randomized clinical trial (VIP trial) showed no benefit in prevention of the disease.12Poston L Briley AL Seed PT Kelly FJ Shennan AH Vitamin C and vitamin E in pregnant women at risk for pre-eclampsia (VIP trial): randomised placebo-controlled trial.Lancet. 2006; 367: 1145-1154Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar By contrast, we have previously demonstrated that vitamins can prevent hypoxia-reoxygenation-induced secretion of free fetal DNA from villous explants, suggesting that they can limit trophoblast damage in vitro.13Tjoa M-L Cindrova-Davies T Spasic-Boskovic O Bianchi DW Burton GJ Trophoblastic oxidative stress and the release of cell-free feto-placental DNA.Am J Pathol. 2006; 169: 400-404Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar In view of these conflicting in vivo and in vitro findings, we have identified the signaling pathways activated by acute hypoxia-reoxygenation in vitro and tested the effects of antioxidant vitamins C and E on these pathways and the downstream consequences induced. Antibodies to the phosphorylated and total forms of p38, stress-activated protein kinase (SAPK), IκB, nuclear factor-κB (NF-κB), Hsp27, TNF-α, cleaved caspase-3, and cleaved caspase-9 were from Cell Signaling Technology (Beverly, MA). Anti-cyclooxygenase (COX)-2 was from Cayman Chemical (Ann Arbor, MI). Anti-4-hydroxy-2-nonenal (HNE) antibody was from Axxora (Nottingham, UK) and anti-Hsp90 from Stressgen Bioreagents Corp. (York, UK). The horseradish peroxidase-conjugated secondary antibodies were from Amersham Biosciences (Buckinghamshire, UK). Alexa 488 and Alexa 568 fluorescently labeled antibodies were from Molecular Probes Invitrogen Detection Technologies (Leiden, The Netherlands), and biotinylated secondary antibodies were from Vector Laboratories (Peterborough, UK). The PD169316 inhibitor was from Calbiochem (San Diego, CA); ascorbic acid, SB202190, and Trolox (water-soluble vitamin E) were from Sigma (Poole, UK). Secreted TNF-α was detected by colorimetric enzyme-linked immunosorbent assay using Quantikine kits from R&D Systems (Oxon, UK). Placentas (n = 12) were collected from normal-term singleton pregnancies delivered by elective caesarean section with informed written consent of the patients and permission of the Local Research Ethics Committee. Villous samples were taken midway between the chorionic and basal plates from the periphery of lobules free of visible infarction, calcification, hematoma, or tears. After a brief rinse in ice-cold phosphate-buffered saline, samples were placed into ice-cold transport medium (TCS large vessel endothelial cell basal medium; TCS CellWorks, Milton Keynes, UK) containing 2% fetal bovine serum, heparin, hydrocortisone, human epidermal growth factor, human basic fibroblast growth factor, 25 μg/ml gentamicin, and 50 ng/ml amphotericin B, 1 mmol/L vitamin C, and 1 mmol/L Trolox) that had been equilibrated with 5% O2/90% N2/5% CO2. Following transport to the laboratory on ice, placental samples were further dissected into small pieces (about 5 mm in diameter) in ice-cold culture medium in a glove box under 10% O2/85% N2/5% CO2. Samples were cultured on individual Costar Netwell (24-mm diameter, 500-μm mesh; Corning Life Sciences, Acton, MA) supports in 4 ml of culture medium per well in six-well plates. Approximately 6 to 10 pieces were added to each well, depending on the experimental requirements. Placental explants were incubated in pregassed medium under: 1) normoxic conditions (controls) (10% O2/85% N2/5% CO2) for 7 hours (n = 6) or 16 hours (n = 6), or 2) subjected to hypoxia (0.5% O2/94.5% N2/5% CO2) for 1 hour and subsequent reoxygenation at normoxia (10% O2/85% N2/5% CO2) for the following 6 hours (n = 6) or 15 hours (n = 6) (H/R treatment). All inserts were transferred into previously pregassed medium after 1 hour of incubation. The p38 inhibitor PD169316 was used at a concentration of 10 μmol/L and SB202190 at 30 μmol/L. Vitamin treatment included the addition of 2 mmol/L ascorbic acid and 1 mmol/L Trolox. In all experimental setups, medium was changed in a glove box under a low O2 environment after the first hour of incubation. The p38 inhibitors and vitamins were added at the beginning of each experiment and also when medium was changed. Tissue homogenization to obtain protein lysate and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed as previously described.13Tjoa M-L Cindrova-Davies T Spasic-Boskovic O Bianchi DW Burton GJ Trophoblastic oxidative stress and the release of cell-free feto-placental DNA.Am J Pathol. 2006; 169: 400-404Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar Proteins were revealed and quantified using Image J software (National Institutes of Health, version 1.36b, http://rsb.info.nih.gov/ij/, accessed Sept 2006). Membranes were reprobed with antibody recognizing β-actin to control for protein loading. The values are expressed as a percentage of the control lysate (100%) for each experiment. All data are presented as means ± SEM. Statistical analysis was performed using StatView (SAS Institute Inc., Cary, NC). Normalized Western blot measurements were analyzed using repeated measures analysis of variance. Differences between two groups were evaluated using a paired Student's t-test. In all cases, results were considered significant at P < 0.05. Immunohistochemistry with diaminobenzidine detection was performed according to a protocol described recently.13Tjoa M-L Cindrova-Davies T Spasic-Boskovic O Bianchi DW Burton GJ Trophoblastic oxidative stress and the release of cell-free feto-placental DNA.Am J Pathol. 2006; 169: 400-404Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar For immunofluorescent staining, sections were incubated with primary antibodies overnight as in the colorimetric protocol, washed, and incubated for 1 hour at room temperature with species-specific Alexa 488 or Alexa 568 secondary fluorescent antibodies. Sections were washed in Tris-buffered saline and subsequently mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories). Images were captured using a Leica confocal microscope (LeicaTCS-NT; Leica Instruments GmbH, Wetzlar, Germany). Expression of the heat shock proteins Hsp27 and Hsp90, lipid peroxidation, and the formation of peroxynitrite were used as markers of oxidative stress. H/R strongly increased Hsp27, Hsp90, HNE, and nitrotyrosine concentrations in the explants after 7 and 16 hours of incubation (Figure 1, Figure 5), reflecting an increase of oxidative stress that was localized principally to the syncytiotrophoblast (Figure 5, a–c). The addition of the antioxidant vitamins C and E significantly reduced markers of oxidative stress in H/R-treated samples (Figure 1, Figure 5). All experiments were repeated at least six times.Figure 5Immunostaining against nitrotyrosine (a), HNE (b), Hsp27 (c), P-NF-κB (d), COX-2 (e), and cleaved caspase-9 and M30 (f) localized H/R-induced increase in these markers principally to trophoblast. Scale bar = 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) H/R stimulated phosphorylation of the p38 and SAPK MAPK pathways after 7 and 16 hours of incubation, whereas total p38 and SAPK levels remained unchanged (Figure 2, a and b). The addition of vitamins suppressed phosphorylation of p38 at 7 hours and that of SAPK stress kinase at both time points (Figure 2, a and b). The p38 pathway has been implicated in the production of proinflammatory cytokines, suggesting a possible role in the regulation of NF-κB activity. Phosphorylation and subsequent degradation of the IκB protein allows NF-κB translocation to the nucleus, where it regulates gene expression. Hence, the phosphorylation status of IκB serves to determine activation of this pathway. H/R greatly elevated phosphorylation of IκB at 7 and 16 hours of incubation (Figure 2c). Total IκB expression was increased in H/R-treated samples after 7 hours of incubation but returned to basal level at 16 hours, reflecting IκB degradation. The addition of vitamins significantly suppressed phosphorylation of IκB by H/R (Figure 2c). Immunohistochemistry demonstrated nuclear translocation of phospho-NF-κB to syncytial nuclei, indicative of activation and involvement in the regulation of gene expression (Figure 5d). NF-κB exerts its effects by regulating genes encoding cytokines, chemokines, adhesion molecules, growth factors, and inducible proinflammatory enzymes such as COX-2. Therefore, the tissue expression of COX-2 and the levels of TNF-α and interleukin (IL)-1β were examined. Vitamins blocked the NF-κB pathway, and their effect on these downstream pathways was thus of interest. The p38 MAPK pathway has also been shown to regulate inflammatory cytokines and COX-2.14Kumar S Boehm J Lee JC p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases.Nat Rev Drug Discov. 2003; 2: 717-726Crossref PubMed Scopus (1099) Google Scholar, 15Lee JC Laydon JT McDonnell PC Gallagher TF Kumar S Green D McNulty D Blumenthal MJ Heys JR Landvatter SW Strickler JE McLaughlin MM Siemens IR Fisher SM Livi GP White JR Adams JL Young PR A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.Nature. 1994; 372: 739-746Crossref PubMed Scopus (3169) Google Scholar To test involvement of this pathway, we used the p38 inhibitors PD169316 and SB202190 in conjunction with H/R. Application of both inhibitors yielded very similar results (Figure 3d), and for simplicity, only data from experiments using PD169316 are shown here. Secretion of TNF-α from the explants into the supernatant was measured at 7 and 16 hours by enzyme-linked immunosorbent assay. H/R caused a significant increase in TNF-α secretion, which increased with time (Figure 3a). The addition of vitamins powerfully suppressed this effect at both time points. The p38 inhibitor suppressed TNF-α secretion at 7 hours but had no effect at 16 hours (Figure 3a). Tissue production of IL-1β (Figure 3b) and TNF-α (data not shown) was also stimulated by H/R. The addition of vitamins prevented the H/R stimulation of IL-1β after 7- and 16-hour incubation. PD169316 only reduced tissue concentrations of IL-1β at 7 hours and had no effect at 16 hours (Figure 3b). Secretion of IL-1β was not measured. H/R-induced tissue production of TNF-α increased in a similar manner as that of IL-1β (not shown). H/R also stimulated a significant increase in COX-2 protein concentrations at both time points compared with normoxic controls, and this was localized mainly to the trophoblast (Figure 3, Figure 5). Again, this increase was suppressed by vitamins and p38 kinase inhibitors at 7 and 16 hours (Figure 3, Figure 5). H/R stimulated the activation of molecules required for apoptosis in the placental explants as seen by an increase in the concentrations of cleaved caspase-3 (Figure 4a) and cleaved caspase-9 at 16 hours (cleavage of caspase-3 was undetectable at 7 hours) (Figure 4b). During apoptosis cytokeratins within trophoblast cells are cleaved by active caspase-3 to yield a specific product detected by the M30 antibody.16Kadyrov M Kaufmann P Huppertz B Expression of a cytokeratin 18 neo-epitope is a specific marker for trophoblast apoptosis in human placenta.Placenta. 2001; 22: 44-48Abstract Full Text PDF PubMed Scopus (82) Google Scholar M30 staining confirmed an increase in trophoblast apoptosis following H/R (Figure 5f). Crucially, both PD169316 and vitamins C and E significantly reduced cleavage of both caspases to control levels in placental explants subjected to H/R and reduced the incidence of M30 staining (Figure 4, Figure 5). The aim was to investigate the acute effects of oxidative stress on the human placenta to understand the pathophysiology of the placental changes that underlie preeclampsia. We have demonstrated that placental explants challenged with hypoxia-reoxygenation in vitro show a marked increase in the levels of oxidative stress and activation of the p38 and SAPK MAPK and the NF-κB pathways. The addition of the antioxidant vitamins C and E effectively suppressed concentrations of markers of oxidative stress, levels of apoptosis, and secretion of TNF-α and IL-1β induced by H/R at 7 and 16 hours. They also suppressed the levels of COX-2 and inhibited the phosphorylation of p38, SAPK, and IκB. A p38 inhibitor was used to evaluate the effect of the p38 pathway on the downstream effects of H/R. Inhibition of p38 effectively suppressed H/R-induced COX-2 expression at 7 and 16 hours and the level of apoptosis-associated markers at 16 hours. However, it only suppressed TNF-α and IL-1β expression and secretion at 7 hours and had no effect after 16 hours of culture. We used the pharmacological inhibitor of p38, PD169316, to address these questions. Similar results were obtained using a second pharmacological inhibitor of p38 signaling, SB202190. Both pharmacological agents are frequently used to block the p38 pathway. However, it should be noted that the specificity of the two p38 kinase inhibitors, PD169316 and SB202190, is questionable as they can also inhibit JNK/SAPK and may also inhibit other upstream kinases.17Schlezinger JJ Emberley JK Sherr DH Activation of multiple mitogen-activated protein kinases in pro/pre-B cells by GW7845, a peroxisome proliferator-activated receptor gamma agonist, and their contribution to GW7845-induced apoptosis.Toxicol Sci. 2006; 92: 433-444Crossref PubMed Scopus (20) Google Scholar We are aware of these difficulties, and we used both inhibitors at the lowest possible doses. However, we cannot exclude nonspecific effects of these inhibitors. We performed parallel experiments, in which explants were challenged with 1 mmol/L hydrogen peroxide (H2O2) under normoxic conditions for 7 or 16 hours. Remarkably, H/R and H2O2 treatments resulted in the same changes listed above, and the addition of vitamins was just as effective in suppressing H2O2-induced effects. For simplicity, only H/R effects are presented in this article. The addition of vitamins to H/R-treated samples suppressed most effects of H/R in vitro. Vitamins C and E act in concert to scavenge reactive oxygen species, with vitamin C being required to recycle vitamin E. In agreement with our results, vitamin C has been reported to suppress TNF-α-induced nuclear translocation of NF-κB, NF-κB-dependent reporter transcription, and IκBα phosphorylation in human cell lines and primary endothelial cells.18Cárcamo J Pedraza A Borquez-Ojeda O Golde D Vitamin C suppresses TNF-α-induced NF-κB activation by inhibiting IκBα phosphorylation.Biochemistry. 2002; 41: 12995-13002Crossref PubMed Scopus (227) Google Scholar In another study, the addition of vitamins C and E inhibited intracellular reactive oxygen species production and activation of the NF-κB, PKR, eIF-2α, protein kinase C, and p38 MAPK pathways.19Tan PH Sagoo P Chan C Yates JB Campbell J Beutelspacher SC Foxwell BMJ Lombardi G George AJT Inhibition of NF-κB and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells.J Immunol. 2005; 174: 7633-7644PubMed Google Scholar Intracellular vitamin C has been shown to protect human umbilical vein endothelial cells from H/R-induced apoptosis, preventing loss of mitochondrial membrane potential, the release of cytochrome c, and activation of caspase-9 and caspase-3 during H/R.20Dhar-Mascareño M Carcamo JM Golde DW Hypoxia-reoxygenation-induced mitochondrial damage and apoptosis in human endothelial cells are inhibited by vitamin C.Free Radic Biol Med. 2005; 38: 1311-1322Crossref PubMed Scopus (125) Google Scholar It would thus seem that the beneficial effects of vitamins in our system are mediated through inhibition of NF-κB by vitamin C. Vitamin E is known to inhibit the p38 MAPK pathway in smooth muscle cells and to prevent activation of NADPH oxidase in monocyte mitochondria, resulting in lower levels of intracellular reactive oxygen species.21Cachia O Benna JE Pedruzzi E Descomps E Gougerot-Pocidalo MA Leger CL α-Tocopherol inhibits the respiratory burst in human monocytes: attenuation of p47phox membrane translocation and phosphorylation.J Biol Chem. 1998; 273: 32801-32805Crossref PubMed Scopus (220) Google Scholar α-Tocopherol also inhibits protein kinase C activation necessary for smooth muscle cell activation22Boscoboinik D Szewczyk A Azzi A Alpha-tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity.Arch Biochem Biophys. 1991; 286: 264-269Crossref PubMed Scopus (184) Google Scholar, 23Boscoboinik D Szewczyk A Hensey C Azzi A Inhibition of cell proliferation by alpha-tocopherol: role of protein kinase C.J Biol Chem. 1991; 266: 6188-6194Abstract Full Text PDF PubMed Google Scholar and prevents human platelet aggregation via a protein kinase C-dependent mechanism.24Freedman JE Farhat JH Loscalzo J Keaney Jr, JF Alpha-tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism.Circulation. 1996; 94: 2434-2440Crossref PubMed Scopus (281) Google Scholar In addition, vitamin E down-regulates the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 in endothelial cells,25Cominacini L Garbin U Pasini AF Davoli A Campagnola M Contessi GB Pastorino AM Lo Cascio V Antioxidants inhibit the expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 induced by oxidized LDL on human umbilical vein endothelial cells.Free Radic Biol Med. 1997; 22: 117-127Crossref PubMed Scopus (222) Google Scholar and inhibits cyclooxygenase activity in macrophages from aged mice by reducing peroxynitrite production.26Beharka AA Wu D Serafini M Meydani SN Mechanism of vitamin E inhibition of cyclooxygenase activity in macrophages from old mice: role of peroxynitrite.Free Radic Biol Med. 2002; 32: 503-511Crossref PubMed Scopus (97) Google Scholar, 27Wu D Hayek MG Meydani S Vitamin E and macrophage cyclooxygenase regulation in the aged.J Nutr. 2001; 131: 382S-388SPubMed Google Scholar These effects of vitamin E, namely p38 and COX-2 inhibition and reduced peroxynitrite formation, are in agreement with the findings of our study. Inhibitors of the p38 pathway (PD169316 and SB202190) were equally effective in suppressing apoptosis and COX-2 expression and to some degree that of the proinflammatory cytokines. The p38 pathway has been implicated in the posttranscriptional regulation of TNF-α, IL-1, and COX-2 mRNAs.14Kumar S Boehm J Lee JC p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases.Nat Rev Drug Discov. 2003; 2: 717-726Crossref PubMed Scopus (1099) Google Scholar, 15Lee JC Laydon JT McDonnell PC Gallagher TF Kumar S Green D McNulty D Blumenthal MJ Heys JR Landvatter SW Strickler JE McLaughlin MM Siemens IR Fisher SM Livi GP White JR Adams JL Young PR A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.Nature. 1994; 372: 739-746Crossref PubMed Scopus (3169) Google Scholar In our study, placental explants subjected to oxidative stress showed increased secretion or tissue levels of TNF-α and IL-1β. The function of these cytokines in normal pregnancy and delivery is not fully established. TNF-α, IL-1, and IL-6 have been detected in human amniotic fluid during pregnancy and labor28Santhanam U Avila C Romero R Viguet H Ida N Sakurai S Sehgal PB Cytokines in normal and abnormal parturition: elevated amniotic fluid interleukin-6 levels in women with premature rupture of membranes associated with intrauterine infection.Cytokine. 1991; 3: 155-163Crossref PubMed Scopus (141) Google Scholar, 29Romero R Mazor M Sepulveda W Avila C Copeland D Williams J Tumor necrosis factor in preterm and term labor.Am J Obstet Gynecol. 1992; 166: 1576-1587Abstract Full Text PDF PubMed Scopus (352) Google Scholar and in preterm labor in the presence of chorioamnionitis.30Romero R Brody DT Oyarzun E Mazor M Wu YK Hobbins JC Durum SK Infection and labor. III. Interleukin-1: a signal for the onset of parturition.Am J Obstet Gynecol. 1989; 160: 1117-1123Abstract Full Text PDF PubMed Scopus (585) Google Scholar These
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