Portal de Periódicos da CAPES

Glyceryl Trinitrate Inhibits Hypoxia/Reoxygenation-Induced Apoptosis in the Syncytiotrophoblast of the Human Placenta

2007; Elsevier BV; Volume: 170; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2007.060665

1525-2191

Louiza Belkacemi, Shannon Bainbridge, Michelle A. Dickinson, Graeme N. Smith, Charles H. Graham,

Neonatal Respiratory Health Research

Damage of the placenta resulting from ischemia-reperfusion is important to the pathophysiology of preeclampsia. Here we investigated whether low concentrations of glyceryl trinitrate (GTN), a nitric oxide mimetic with anti-apoptotic properties, inhibit hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of chorionic villous explants from human placentas. Compared with villi analyzed immediately after delivery or maintained under normoxic conditions, villi exposed to a 6-hour cycle of hypoxia/reoxygenation exhibited greater numbers of syncytiotrophoblasts with terminal dUTP nick-end labeling (TUNEL)-positive nuclei in the syncytiotrophoblast. This increased number of TUNEL-positive nuclei was paralleled by higher levels of 4-hydroxynonenal (marker of lipid peroxidation), nitrotyrosine residues, and active caspase-3 and polyADP-ribose polymerase expression. Morphological analysis of explants exposed to hypoxia/reoxygenation revealed apoptotic and aponecrotic features similar to those of chorionic villi from preeclamptic pregnancies. Treatment with GTN during the hy-poxia/reoxygenation cycle blocked the increases in the number of TUNEL-positive nuclei and in the levels of 4-hydroxynonenal, nitrotyrosine, and active caspase-3. Incubation with GTN also attenuated the hypoxia/reoxygenation-induced polyADP-ribose polymerase expression and the apoptotic and aponecrotic morphological alterations. These results suggest that small concentrations of nitric oxide protect chorionic villi from hypoxia/reoxygenation-induced damage and provide a rationale for the use of low doses of nitric oxide mimetics in the treatment and/or prevention of preeclampsia. Damage of the placenta resulting from ischemia-reperfusion is important to the pathophysiology of preeclampsia. Here we investigated whether low concentrations of glyceryl trinitrate (GTN), a nitric oxide mimetic with anti-apoptotic properties, inhibit hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of chorionic villous explants from human placentas. Compared with villi analyzed immediately after delivery or maintained under normoxic conditions, villi exposed to a 6-hour cycle of hypoxia/reoxygenation exhibited greater numbers of syncytiotrophoblasts with terminal dUTP nick-end labeling (TUNEL)-positive nuclei in the syncytiotrophoblast. This increased number of TUNEL-positive nuclei was paralleled by higher levels of 4-hydroxynonenal (marker of lipid peroxidation), nitrotyrosine residues, and active caspase-3 and polyADP-ribose polymerase expression. Morphological analysis of explants exposed to hypoxia/reoxygenation revealed apoptotic and aponecrotic features similar to those of chorionic villi from preeclamptic pregnancies. Treatment with GTN during the hy-poxia/reoxygenation cycle blocked the increases in the number of TUNEL-positive nuclei and in the levels of 4-hydroxynonenal, nitrotyrosine, and active caspase-3. Incubation with GTN also attenuated the hypoxia/reoxygenation-induced polyADP-ribose polymerase expression and the apoptotic and aponecrotic morphological alterations. These results suggest that small concentrations of nitric oxide protect chorionic villi from hypoxia/reoxygenation-induced damage and provide a rationale for the use of low doses of nitric oxide mimetics in the treatment and/or prevention of preeclampsia. Preeclampsia is a disease of human pregnancy characterized by a systemic maternal inflammatory response associated with endothelial dysfunction, hypertension, and proteinuria. This condition affects 5 to 7% of all pregnancies and is the main cause of perinatal mortality and morbidity in developed countries. There is also evidence that the risk of subsequent cardiovascular disease is significantly increased in women affected by preeclamptic pregnancies.1Marín R Gorostidi M Portal CG Sanchez M Sanchez E Alvarez J Long-term prognosis of hypertension in pregnancy.Hypertens Pregnancy. 2000; 19: 199-209Crossref PubMed Scopus (95) Google Scholar, 2Nisell H Lintu H Lunell NO Mollerstrom G Pettersson E Blood pressure and renal function seven years after pregnancy complicated by hypertension.Br J Obstet Gynaecol. 1995; 102: 876-881Crossref PubMed Scopus (145) Google Scholar Although the pathophysiology of preeclampsia has not been fully defined, there is evidence that placental oxidative stress attributable to abnormal uteroplacental blood circulation plays a critical role. In preeclampsia, the transformation that normally leads to spiral arterioles with large diameters is defective, and consequently, placental perfusion is compromised.3Brosens IA Robertson WB Dixon HG The role of the spiral arteries in the pathogenesis of preeclampsia.Obstet Gynecol Annu. 1972; 1: 177-191PubMed Google Scholar, 4Roberts JM Lain KY Recent insights into the pathogenesis of pre-eclampsia.Placenta. 2002; 23: 359-372Abstract Full Text PDF PubMed Scopus (505) Google Scholar Furthermore, it has been suggested that uteroplacental blood flow in preeclampsia is intermittent or pulsatile, likely attributable to the persistent sensitivity of the maladapted spiral arterioles to maternal vasopressor molecules.5Hung TH 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 (231) Google Scholar, 6Hung TH 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; 90: 1274-1281Crossref PubMed Scopus (339) Google Scholar It has been postulated that the abnormally decreased and intermittent perfusion of the intervillous space of the placenta results in oxidative damage and the release of apoptotic and aponecrotic placental tissue into the maternal circulation.7Sargent IL Germain SJ Sacks GP Kumar S Redman CW Trophoblast deportation and the maternal inflammatory response in pre-eclampsia.J Reprod Immunol. 2003; 59: 153-160Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar The presence of large amounts of syncytiotrophoblast microfragments in the maternal circulation is thought to promote the maternal systemic inflammatory response and endothelial dysfunction characteristic of preeclampsia.8Redman CW Sargent IL Placental debris, oxidative stress and pre-eclampsia.Placenta. 2000; 21: 597-602Abstract Full Text PDF PubMed Scopus (437) Google Scholar Indeed, an increased prevalence of apoptotic nuclei has been reported in the syncytiotro-phoblast of placentas from pregnancies complicated by preeclampsia.9Allaire AD Ballenger KA Wells SR McMahon MJ Lessey BA Placental apoptosis in preeclampsia.Obstet Gynecol. 2000; 96: 271-276Crossref PubMed Scopus (392) Google Scholar Using an in vitro model of hypoxia/reoxygenation (H/R) that replicates the oxidative stress that placental tissues undergo during preeclampsia, Hung and colleagues6Hung TH 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; 90: 1274-1281Crossref PubMed Scopus (339) Google Scholar demonstrated that the syncytiotrophoblast of normal chorionic villi exposed to H/R undergoes apoptotic and aponecrotic changes similar to those observed in the syncytiotrophoblast of chorionic villi from preeclamptic pregnancies. In a recent study we demonstrated that carbon monoxide (CO) is able to inhibit the H/R-induced apoptosis of the syncytiotrophoblast in chorionic villi from term human placentas.10Bainbridge 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 Nitric oxide (NO), like CO, is a small polyvalent molecule that plays a role in regulating multiple biological functions. It induces vasodilation, regulates platelet adhesion, is involved in various aspects of vascular remodeling, acts as a neurotransmitter, and is a mediator of cell growth and apoptosis. Many cell types, including trophoblast cells, produce NO.11Myatt L Eis AL Brockman DE Kossenjans W Greer I Lyall F Inducible (type II) nitric oxide synthase in human placental villous tissue of normotensive, pre-eclamptic and intrauterine growth-restricted pregnancies.Placenta. 1997; 18: 261-268Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 12Thomsen LL Miles DW Happerfield L Bobrow LG Knowles RG Moncada S Nitric oxide synthase activity in human breast cancer.Br J Cancer. 1995; 72: 41-44Crossref PubMed Scopus (604) Google Scholar, 13Bredt DS Hwang PM Snyder SH Localization of nitric oxide synthase indicating a neural role for nitric oxide.Nature. 1990; 347: 768-770Crossref PubMed Scopus (2699) Google Scholar Recent studies have shown that NO protects cultured extravillous trophoblast cells from apoptosis through a mechanism involving the activation of soluble guanylyl cyclase (sGC).14Dash PR Cartwright JE Baker PN Johnstone AP Whitley GS Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation.Exp Cell Res. 2003; 287: 314-324Crossref PubMed Scopus (67) Google Scholar Thus, in the present study we used a well established explant model6Hung TH 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; 90: 1274-1281Crossref PubMed Scopus (339) Google Scholar to determine whether low concentrations of the NO mimetic glyceryl trinitrate (GTN; nanomolar to micromolar range) are able to attenuate the changes associated with the apoptotic and aponecrotic effects of H/R in the syncytiotrophoblast of term chorionic villi. These changes were assessed by a variety of approaches including the terminal dUTP nick-end labeling (TUNEL) assay and immunodetection of 4-hydroxynonenal (4-HNE, a marker of lipid peroxidation), nitrotyrosine residues, caspase-3, and polyADP-ribose polymerase (PARP). Morphological alterations were assessed by light and electron microscopy. Human term placentas (n = 13) were obtained from nonlaboring normal pregnancies immediately after cesarean deliveries at Kingston General Hospital. Collection of placentas was done with the approval of the Queen's University Research Ethics Board. After the removal of the basal plate from placental lobules, tissue cubes of ∼2 cm3 were dissected from at least seven randomly selected sites free of calcification across the placenta. The tissue was transferred to the laboratory in a sterile sealed container in ice-cold phosphate-buffered saline (PBS). Chorionic villi (5 to 10 mg) from the collected tissue were dissected on ice, rinsed once with ice-cold PBS, and twice with CMRL-1066 culture medium (Invitrogen, Burlington, ON, Canada). Five explants were cultured in individual Costar Netwell supports (15-mm diameter, 74-μm mesh; Cole-Parmer, Anjou, QC, Canada) in 1.2-ml culture medium supplemented with 5% heat-inactivated fetal bovine serum, 2.2 mg/ml NaHCO3, 100 μg/ml streptomycin sulfate, 100 IU/ml penicillin G, 1 μg/ml insulin, and 2 μg/ml l-glutamine (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). For H/R exposures, explants (total of 65) were treated with or without GTN (1 μmol/L or 1 nmol/L; Sabex, Boucherville, QC, Canada) and incubated for 3 hours in an atmosphere of 0.5% O2 (3.8 mm Hg), 5% CO2, and 94.5% N2 maintained by means of a ProOx O2 regulator (Biospherix Inc., Redfield, NY) in a humidified Plexiglas chamber at 37°C. Tissues were then transferred to medium continuously flushed with 21% O2/5% CO2/balance N2 and were incubated in a standard incubator at 5% CO2 and 21% O2 (159 mm Hg) for 3 additional hours. These reoxygenation conditions were in accordance to the method of Hung and colleagues6Hung TH 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; 90: 1274-1281Crossref PubMed Scopus (339) Google Scholar and were chosen to maximize oxidative stress. Additional controls consisted of explants (n = 5 placentas, for a total of 25 explants) kept in normoxic conditions (5% O2, 90% N2, and 5% CO2) for 6 hours. These oxygen concentrations (38 mm Hg) are similar to those measured within the intervillous space of the term placenta.15Fujikura T Yoshida J Blood gas analysis of placental and uterine blood during cesarean delivery.Obstet Gynecol. 1996; 87: 133-136Crossref PubMed Scopus (38) Google Scholar, 16Soothill PW Nicolaides KH Rodeck CH Campbell S Effect of gestational age on fetal and intervillous blood gas and acid-base values in human pregnancy.Fetal Ther. 1986; 1: 168-175Crossref PubMed Scopus (247) Google Scholar At times 0, 3, and 6 hours, explants were collected from all experimental groups and either flash-frozen for molecular analysis or fixed in 4% paraformaldehyde or 2% paraformaldehyde/0.5% glutaraldehyde for histological analysis. Paraformaldehyde-fixed explants were embedded in paraffin and cut into serial sections. To assess apoptosis of the syncytiotrophoblast layer, a fluorescence TUNEL assay was performed according to the manufacturer's instructions (In Situ Cell Death Detection kit; Roche Molecular Biochemicals, Laval, QC, Canada). In brief, deparaffinized, dewaxed, and rehydrated sections were pretreated with 20 μg/ml proteinase K (Sigma-Aldrich) in 10 mmol/L Tris-HCl for 15 minutes, blocked with 10% normal goat serum, and then stained for TUNEL using a reaction mixture containing fluorescein-dUTP. Negative controls consisted of sections incubated without terminal deoxynucleotidyl transferase (TdT). All sections were blinded by a third party and observed with a Leica inverted microscope (Leica Microsystems, Heidelberg, Germany) using a ×20 objective lens. Three fields containing a minimum of 900 syncytiotrophoblast nuclei were randomly selected in each section, and digital images of the TUNEL-stained (green) and 4,6-diaminodino-2-phenylindole (DAPI)-stained (blue) sections were captured and deconvoluted using Slidebook 4.1 software (Intelligent Imaging Innovations Inc., Denver, CO). TUNEL-positive (apoptotic) and DAPI-stained (total) nuclei were counted in the syncytiotrophoblast layer using Image-Pro Plus 5.1 software (Media Cybernetics Inc., Silver Spring, MD). The apoptotic index in each section was calculated as the percentage of TUNEL-positive syncytiotrophoblast nuclei divided by the total number of DAPI-stained syncytiotrophoblast nuclei. Double-fluorescence labeling was used to determine the localization of TUNEL-positive nuclei and 4-HNE within chorionic villous explants. In brief, deparaffinized, de-waxed, and rehydrated sections were pretreated in 20 μg/ml proteinase K (Sigma) in 10 mmol/L Tris-HCl for 15 minutes, blocked with 10% normal goat serum, and stained for TUNEL. Sections were then sequentially incubated with a mouse monoclonal antibody against 4-HNE (1:300; Japan Institute for the Control of Ageing, Shizuka, Japan) for 2 hours and after several washes, the sections were incubated with a fluorescent goat anti-mouse secondary antibody (1:600; 555-nm excitation wavelength, 565-nm emission wavelength; Alexa, Invitrogen). The sections were then subjected to TUNEL staining as per the manufacturer's instructions. All sections were mounted using Vectashield (Vector Laboratories, Burlington, ON, Canada) and observed with a Leica inverted confocal microscope (Leica Microsystems) at a magnification of ×60 with simultaneous excitation and detection of both dyes. Paraffin-embedded placental sections were randomly selected and deparaffinized by heating at 40°C for 20 minutes followed by sequential incubations in Hemo-D and decreasing concentrations of ethanol (each for 2 to 3 minutes). Endogenous peroxidase activity was quenched in 3% hydrogen peroxide in methanol, and nonspecific binding was blocked by incubating the sections in 5% normal goat serum. After extensive washes with PBS, the sections were reacted with a rabbit polyclonal anti-nitrotyrosine antibody (1:500; Upstate Biotechnology Inc., Lake Placid, NY) for 1 hour or with a rabbit polyclonal anti-PARP p85 fragment antibody (1:500; Promega, Nepean, ON, Canada) for 4 hours at room temperature followed by the addition of biotinylated anti-rabbit secondary antibody (1:200; Vector Laboratories). Further processing of the sections for the detection of nitrotyrosine residues or the p85 fragment of PARP was performed according to the instructions provided with the Vectastain Elite ABC kit (Vector Laboratories). Colorimetric detection was achieved using diaminobenzidine as chromogen and hydrogen peroxide as substrate for horseradish peroxidase. Quantification of the p85-fragmented PARP staining was performed using Image-Pro Plus 5.1 software. To further assess oxidative stress and activation of apoptosis, the levels of 4-HNE, nitrotyrosine residues, active caspase-3, and PARP were determined by Western blot analysis of extracts of flash-frozen villous explants (n = 5 to 7 placentas). In brief, explants were cut and lysed using a buffer containing 2% sodium dodecyl sulfate, 10 mmol/L Tris (pH 7.5), and 0.15 mmol/L NaCl. Lysates were homogenized and subjected to DNA shearing (10 times with a 25-gauge needle) and centrifugation. The culture supernatants were collected and stored at −80°C until use. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resolved proteins were transferred onto an Immobilon-P membrane (Millipore Corp., Bedford, MA). After a 1-hour incubation in blocking solution consisting of 5% dry milk and 0.05% Tween 20 in PBS (PBS-T), the membranes were incubated with mouse monoclonal antibody against 4-HNE (1:500; Japan Institute for the Control of Ageing), rabbit polyclonal anti-nitrotyrosine antibody (1:1000; Upstate Biotechnology), goat polyclonal antibody against caspase-3 (1:1000; R&D Research, Minneapolis, MN), or rabbit polyclonal anti-PARP (1:500; Promega) for 1 to 2 hours at room temperature followed by three 5-minute washes in PBS-T. The membranes were then incubated with secondary goat anti-mouse (1:4000; Vector Laboratories Inc.) or rabbit anti-goat IgG (1:2000; Vector Laboratories Inc.) labeled with horseradish peroxidase. After three additional 5-minute washes with PBS-T, the blots were developed using a chemiluminescence Western blotting plus kit (Perkin Elmer, Burlington, ON, Canada), and subsequently exposed onto X-OMAT Kodak film (Eastman-Kodak, Rochester, NY). Uniformity of loading of protein extracts was determined by probing with a monoclonal anti-β-actin antibody (clone AC-15, 1:8000; Bio-Rad, Mississauga, ON, Canada). Relative intensities of bands were determined by densitometry using AlphaErase software (Alpha Innotech Corp., San Leandro, CA). Explants fixed in 2% paraformaldehyde/0.5% glutaraldehyde and embedded in araldite resin were used for light and electron microscopic analysis. Semithin sections (1 μm) were prepared for light microscopy and for the selection of areas to conduct further ultrastructural analysis at the electron microscopic level. Morphological assessments at the light microscopic level were performed using toluidine blue-stained sections. For electron microscopic analysis, ultrathin sections (70 nm) were cut and counterstained with uranyl acetate and lead citrate. An additional control for the ultrastructural analysis consisted of chorionic villi obtained from placentas of preeclamptic women. Preeclampsia was defined by the development of elevated maternal blood pressure (>140/90 mm Hg) and proteinuria (≥300 mg/24 hours). Morphological analysis of tissues was performed using a Hitachi 7000 transmission electron microscope at magnifications of ×3500 to ×15,000. Statistical significance was determined using one-way analysis of variance, when more than two groups were compared, followed by a post hoc Tukey-Kramer test. Nonpaired and paired Student's t-tests were used when only two groups were compared. All statistical tests were two-sided. Data are presented as the mean ± SE, and were considered significant at P < 0.05. Explants fixed immediately after delivery and dissection had very few TUNEL-positive nuclei in the syncytiotrophoblast, with a mean apoptotic index of 1.1 ± 0.4% (Figure 1, A and A′). After 3 hours of incubation in 0.5% O2 without subsequent reoxygenation, the apoptotic index increased to 11.8 ± 1.6% in the untreated control villi (P < 0.001; Figure 1, B and B′) and to 13.0 ± 1.5% and 11.7 ± 2.4% (P < 0.01 for both), respectively, in the explants treated with 1 μmol/L or 1 nmol/L GTN (Figure 1, C, C′, D, and D′). Apoptotic indices were not significantly different in control and GTN-treated explants after the 3-hour incubation in hypoxia alone (Figure 2).Figure 2Percentage of TUNEL-positive nuclei in untreated versus GTN-treated villous explants after a 6-hour H/R insult or after a 6-hour incubation in 5% O2. Treatment of explants with GTN significantly inhibited syncytiotrophoblast apoptosis after a 6-hour exposure to H/R. Relatively low levels of apoptosis were observed in control explants incubated under normoxic conditions (5% O2) with or without GTN. Asterisk indicates a significant difference (P < 0.001) between untreated explants after the 6-hour H/R exposure versus untreated explants after 3 hours of hypoxia alone. The H/R-exposed group consisted of explants isolated from 13 placentas whereas the group exposed to 5% O2 consisted of explants isolated from five placentas. Data are presented as means ± SE.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Compared with the apoptotic index in the syncytiotrophoblast of untreated control explants at the end of the 3-hour incubation in hypoxia, a subsequent 3-hour incubation in 21% oxygen resulted in a further 2.1-fold increase in the apoptotic index (P < 0.001; Figure 1, Figure 2). In contrast, this increase in TUNEL staining after the 3-hour reoxygenation period did not occur in the explants incubated with either 1 μmol/L or 1 nmol/L GTN (Figure 1, Figure 2). Compared with explants processed immediately after delivery, the number of TUNEL-positive nuclei in the syncytiotrophoblast of explants incubated under normoxic conditions (5% O2) for 3 hours did not exhibit a statistically significant increase (Figure 2). However, after 6 hours in normoxia, the apoptotic index in untreated explants increased significantly from 0.3 to 5.4% (P < 0.05). Both untreated control and GTN-treated explants maintained at 5% O2 throughout the incubation period exhibited similar levels of apoptosis at 6 hours (5.4 ± 0.8%, 5.3 ± 1.0%, and 6.7 ± 3.4%, respectively; Figure 2). The localization of 4-HNE- and TUNEL-positive nuclei within chorionic villi was determined by immunofluorescence labeling (Figure 3). In placental explants processed immediately after delivery, the immunofluorescence staining intensity for 4-HNE was very low and present only in the syncytiotrophoblast (Figure 3A). In contrast, staining for 4-HNE was much greater in untreated explants subjected to a cycle of H/R (Figure 3B). This increase in 4-HNE expression in the untreated explants was paralleled by an increase in the number of TUNEL-positive nuclei in these samples. However, regions of the syncytiotrophoblast that showed intense 4-HNE staining did not always contain TUNEL-positive nuclei. This may be attributable to the fact that generation of reactive oxygen species and the subsequent peroxidation of lipids occur early during the H/R stress, whereas DNA fragmentation is a later event in the apoptotic process. Staining for 4-HNE and TUNEL was markedly attenuated in explants exposed to H/R in the presence of 1 μmol/L or 1 nmol/L GTN (Figure 3, C and D). Explants incubated with preimmune IgG exhibited virtually no detectable levels of fluorescence (data not shown). Results of the immunofluorescence staining for 4-HNE were confirmed by Western blot analysis. As shown in Figure 3E, compared with the levels of 4-HNE protein adduct in extracts of explants processed immediately after delivery, 4-HNE levels in chorionic villi increased by almost fourfold after H/R (P < 0.05). However, compared with explants processed immediately after delivery, the levels of 4-HNE in the GTN-treated explants did not increase significantly. Explants processed immediately after delivery exhibited barely detectable levels of nitrotyrosine staining (Figure 4A) or cleaved PARP immunostaining (not shown). However, after H/R explants exhibited intense immunostaining for both nitrotyrosine (Figure 4B) and cleaved PARP (Figure 5A). Most of the staining for nitrotyrosine and PARP was localized to the syncytiotrophoblast, with some PARP staining also localizing to the stroma. However, immunostaining for both nitrotyrosine and cleaved PARP was markedly attenuated in explants exposed to H/R in the presence of GTN (Figure 4, Figure 5). Exposure of explants to 5% O2 for 6 hours with or without GTN resulted in relatively low levels of PARP immunostaining (Figure 5, E–G). Replacement of the primary antibody with nonimmune rabbit IgG resulted in the absence of staining in explants exposed to H/R or maintained in 5% O2 (Figure 5, D and H).Figure 5Immunohistochemical and Western blot analysis of the p85 fragment of PARP. Compared with untreated explants exposed to H/R (A), treatment of H/R-exposed explants with GTN resulted in decreased syncytiotrophoblast immunoreactivity for PARP (B and C). Lower levels of PARP immunostaining were observed in explants exposed to 5% O2 in the absence or presence of GTN throughout the 6-hour incubation period (E–G). Negative controls consisted of sections incubated with nonimmune rabbit IgG (D and H). Combined immunohistochemical staining intensities from five different placentas, as determined by image analysis using ImagePro, revealed significantly lower PARP staining in the syncytiotrophoblast exposed to H/R in the presence of GTN as compared with similar explants exposed to H/R in the absence of GTN (I; single and double asterisks = P < 0.05 and P < 0.01, respectively; one-way analysis of variance followed by Tukey-Kramer test). I: Similar analysis revealed much lower PARP staining intensity in the syncytiotrophoblast of villi maintained under normoxic conditions (5% O2) but not significant differences in the staining intensities in control versus GTN-treated explants. Data are presented as means ± SE. J: PARP immunohistochemistry results from explants exposed to H/R were further confirmed by Western blot analysis. Scale bars = 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Quantitative analysis of PARP staining of the syncytiotrophoblast layer was confirmed by image analysis using ImagePro software. Results revealed that, compared with untreated explants after H/R, treatment with GTN (1 μmol/L and 1 nmol/L) led to decreased levels of syncytiotrophoblast PARP staining after H/R (P < 0.05; Figure 5I). In explants maintained under normoxic conditions (5% O2) for 6 hours, the levels of cleaved PARP immunostaining in the syncytiotrophoblast were low and were not affected by GTN treatment. Levels of nitrotyrosine residues were assessed to determine whether endogenous NO leads to peroxynitrite formation and protein nitration after H/R. Nitrotyrosine immunoblotting revealed the presence of three nitrated fragments of 66, 32, and 16 kd in tissue samples exposed to H/R without GTN (Figure 4E). In contrast, the GTN-treated samples revealed only two bands at 32 and 16 kd. Significantly higher levels of nitrotyrosine residues were present in tissues exposed to H/R without GTN than in tissues exposed to H/R in the presence of 1 nmol/L GTN (P < 0.05) as determined by densitometric analysis (Figure 4E). Cleaved PARP immunostaining results were also confirmed by Western blot analysis (Figure 5J). Densitometric analysis revealed that the expression of active fragment 85 for PARP was significantly higher in tissue samples exposed to H/R alone than in samples exposed to H/R in the presence of 1 nmol/L GTN (P < 0.05). PARP levels in control tissues exposed to 5% O2 for 6 hours were low and unaffected by GTN treatment (not shown). Active caspase-3 levels were assessed because this enzyme is a downstream mediator of apoptosis. Expression of cleaved caspase-3 revealed two polypeptides of 16 and 18 kd (Figure 6). An additional active caspase-3 polypeptide fragment of 20 kd was present only in lysates from untreated explants exposed to H/R. Densitometric analysis revealed that the combined levels of these active caspase-3 polypeptide fragments were significantly increased in explants exposed to H/R without GTN than in explants processed immediately after delivery (P < 0.05; Figure 6). In contrast, this increase in the levels of active caspase-3 fragments did not occur in explants exposed to H/R in the presence of 1 μmol/L or 1 nmol/L GTN (Figure 6). Light microscopic examination of semithin sections of chorionic villi fixed immediately after delivery revealed syncytial nuclei with irregular shapes and normal distribution of chromatin and intact syncytial membranes (Figure 7A). However, after the H/R insult, the untreated explants exhibited several morphological features characteristic