Carbon Monoxide Inhibits Hypoxia/Reoxygenation-Induced Apoptosis and Secondary Necrosis in Syncytiotrophoblast
2006; Elsevier BV; Volume: 169; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.060184
ISSN1525-2191
AutoresShannon Bainbridge, Louiza Belkacemi, Michelle A. Dickinson, Charles H. Graham, Graeme N. Smith,
Tópico(s)Neonatal Health and Biochemistry
ResumoPre-eclampsia, a hypertensive disorder of pregnancy, affects 5 to 7% of pregnancies. Oxidative stress-induced placental injury and subsequent release of placental debris into the maternal circulation are key pathogenic events in the progression of pre-eclampsia. Women who smoke cigarettes throughout pregnancy are 33% less likely to develop this disorder than nonsmoking women. We postulated that elevated carbon monoxide concentrations in serum of smoking women inhibits apoptosis and debris shedding of trophoblast cells exposed to ischemia-reperfusion injury because carbon monoxide has cytoprotective effects on endothelial and smooth muscle cells in culture. This may be responsible for the reduced risk of pre-eclampsia in smoking women. To assess the cytoprotective properties of carbon monoxide within placental tissue, carbon monoxide treatments were administered to in vitro hypoxia/reoxygenation-insulted villous explants cultured from term human placenta. Induction of apoptosis was assessed using molecular and morphological approaches. Placental villous explants treated with carbon monoxide demonstrated 60% less hypoxia/reoxygenation-induced apoptosis in the differentiated syncytiotrophoblast layer compared with untreated explants undergoing a similar insult. In addition, retention of intact syncytial membranes was observed in carbon monoxide-treated explants. These observations indicate that carbon monoxide has potent antiapoptotic properties within human placenta and may hold therapeutic potential in the treatment of pre-eclampsia. Pre-eclampsia, a hypertensive disorder of pregnancy, affects 5 to 7% of pregnancies. Oxidative stress-induced placental injury and subsequent release of placental debris into the maternal circulation are key pathogenic events in the progression of pre-eclampsia. Women who smoke cigarettes throughout pregnancy are 33% less likely to develop this disorder than nonsmoking women. We postulated that elevated carbon monoxide concentrations in serum of smoking women inhibits apoptosis and debris shedding of trophoblast cells exposed to ischemia-reperfusion injury because carbon monoxide has cytoprotective effects on endothelial and smooth muscle cells in culture. This may be responsible for the reduced risk of pre-eclampsia in smoking women. To assess the cytoprotective properties of carbon monoxide within placental tissue, carbon monoxide treatments were administered to in vitro hypoxia/reoxygenation-insulted villous explants cultured from term human placenta. Induction of apoptosis was assessed using molecular and morphological approaches. Placental villous explants treated with carbon monoxide demonstrated 60% less hypoxia/reoxygenation-induced apoptosis in the differentiated syncytiotrophoblast layer compared with untreated explants undergoing a similar insult. In addition, retention of intact syncytial membranes was observed in carbon monoxide-treated explants. These observations indicate that carbon monoxide has potent antiapoptotic properties within human placenta and may hold therapeutic potential in the treatment of pre-eclampsia. Pre-eclampsia (PE) affects 5 to 7% of all pregnancies and is a leading cause of maternal morbidity and mortality world wide.1Roberts JM Lain KY Recent insights into the pathogenesis of pre-eclampsia.Placenta. 2002; 23: 359-372Abstract Full Text PDF PubMed Scopus (504) Google Scholar, 2Walker JJ Pre-eclampsia.Lancet. 2000; 356: 1260-1265Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar PE is clinically characterized by maternal hypertension and proteinuria, typically presenting in the third trimester. The pathological processes responsible for this disorder are thought to originate early in gestation during placentation.1Roberts JM Lain KY Recent insights into the pathogenesis of pre-eclampsia.Placenta. 2002; 23: 359-372Abstract Full Text PDF PubMed Scopus (504) Google Scholar, 2Walker JJ Pre-eclampsia.Lancet. 2000; 356: 1260-1265Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 3Redman CW Sargent IL Placental debris, oxidative stress and pre-eclampsia.Placenta. 2000; 21: 597-602Abstract Full Text PDF PubMed Scopus (430) Google Scholar Trophoblast cells of the human placenta normally invade and remodel the maternal spiral arterioles at weeks 12 to 16 of gestation to expand vessel capacity and optimize nutrient and oxygen delivery to the developing fetus. In PE, the structural changes to the spiral arterioles are incomplete or absent.1Roberts JM Lain KY Recent insights into the pathogenesis of pre-eclampsia.Placenta. 2002; 23: 359-372Abstract Full Text PDF PubMed Scopus (504) Google Scholar, 4Brosens 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, 5Lim KH Zhou Y Janatpour M McMaster M Bass K Chun SH Fisher SJ Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia.Am J Pathol. 1997; 151: 1809-1818PubMed Google Scholar The result is abnormally high vascular resistance with pulsatile blood flow to the placenta because the unaltered small diameter spiral arterioles remain responsive to vasoactive compounds in the maternal circulation.6Hung 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 (227) Google Scholar, 7Meekins J Pijnenborg R Hanssens M McFadyen I van Asshe A A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies.Br J Obstet Gynaecol. 1994; 101: 669-674Crossref PubMed Scopus (782) Google Scholar This sets up an environment for ischemia-reperfusion injury. Ischemia-reperfusion injury is the result of generated free radicals on reintroduction of molecular oxygen into ischemic tissues. The outcome is widespread lipid and protein oxidative modifications, mitochondrial injury, and tissue apoptosis and necrosis.8Collard CD Gelman S Pathophysiology, clinical manifestations, and prevention of ischemia-reperfusion injury.Anesthesiology. 2001; 94: 1133-1138Crossref PubMed Scopus (505) Google Scholar, 9Vaage J Valen G Pathophysiology and mediators of ischemia-reperfusion injury with special reference to cardiac surgery. 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This syncytium readily undergoes apoptosis and necrosis under conditions of oxidative stress.6Hung 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 (227) Google Scholar, 11Hung 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 (331) Google Scholar As a result, placental debris is shed into the maternal circulation along with the generated free radicals and cytokines, each contributing to a heightened maternal systemic inflammatory response.3Redman CW Sargent IL Placental debris, oxidative stress and pre-eclampsia.Placenta. 2000; 21: 597-602Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 12Sacks GP Seyani L Lavery S Trew G Maternal C-reactive protein levels are raised at 4 weeks gestation.Hum Reprod. 2004; 19: 1025-1030Crossref PubMed Scopus (126) Google Scholar, 13Morris JM Gopaul NK Endresen MJ Knight M Linton EA Dhir S Anggard EE Redman CW Circulating markers of oxidative stress are raised in normal pregnancy and pre-eclampsia.Br J Obstet Gynaecol. 1998; 11: 1195-1199Crossref Scopus (191) Google Scholar, 14Knight M Redman CW Linton EA Sargent IL Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies.Br J Obstet Gynaecol. 1998; 105: 632-640Crossref PubMed Scopus (412) Google Scholar, 15Granger JP Alexander BT Llinas MT Bennett WA Khalil RA Pathophysiology of preeclampsia: linking placental ischemia/hypoxia with microvascular dysfunction.Microcirculation. 2002; 9: 147-160Crossref PubMed Scopus (283) Google Scholar Elevated shedding of debris is postulated to be the pivotal step in the pathophysiology of PE because it transforms a placental disorder into a systemic maternal disease. The release of placental debris into the maternal circulation elicits, along with the heightened maternal inflammatory response, endothelial dysfunction resulting in the clinical disease.3Redman CW Sargent IL Placental debris, oxidative stress and pre-eclampsia.Placenta. 2000; 21: 597-602Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 16Granger JP Alexander BT Llinas MT Bennett WA Khalil RA Pathophysiology of hypertension during preeclampsia linking placental ischemia with endothelial dysfunction.Hypertension. 2001; 38: 718-722Crossref PubMed Google Scholar At present, timely delivery is the only treatment for PE and is usually associated with prematurity of the infant and increased neonatal morbidity/mortality. Several factors predispose women to develop PE including PE in a previous pregnancy, family history of PE, diabetes, and conception with a new sexual partner.17Duckitt K Harrington D Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies.BMJ. 2005; 330: 565Crossref PubMed Scopus (1228) Google Scholar, 18Eskenazi B Fenster L Sidney S A multivariate analysis of risk factors for preeclampsia.JAMA. 1991; 266: 237-241Crossref PubMed Scopus (426) Google Scholar, 19Smith G Walker M Tessier J Millar K Increased incidence of preeclampsia in women conceiving by intrauterine insemination with donor versus partner sperm for treatment of primary infertility.Am J Obstet Gynecol. 1997; 177: 455-458Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar In contrast, a reduced risk for PE is found in women smoking cigarettes throughout pregnancy. The reduction in risk is 33% compared to nonsmoking women.20Bainbridge SA Sidle EH Smith GN Direct placental effects of cigarette smoke protect women from pre-eclampsia: the specific roles of carbon monoxide and antioxidant systems in the placenta.Med Hypotheses. 2005; 64: 17-27Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar The mechanism through which smoking confers protection from PE is unknown. However women who use snuff, a form of smokeless tobacco, do not have a reduced risk for PE but rather an increased incidence compared with controls.21England L Levine R Mills J Klebanoff M Yu K Cnattingius S Adverse pregnancy outcomes in snuff users.Am J Obstet Gynecol. 2003; 189: 939-943Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar It is hypothesized that exposure to elevated concentrations of exogenous carbon monoxide (CO), one of the combustible by-products of cigarette smoke, mediates the observed protective effects of smoking on PE. Environmental pollution and cigarette smoking are major exogenous sources of CO exposure. Carbon monoxide is also endogenously produced throughout the body with important physiological functions including platelet aggregation, anti-inflammatory actions, and smooth muscle relaxation.22Christova T Diankova Z Setchenska M Heme oxygenase-carbon monoxide signalling pathway as a physiological regulator of vascular smooth muscle cells.Acta Physiol Pharmacol Bulg. 2000; 25: 9-17PubMed Google Scholar, 23Vreman HJ Mahoney JJ Stevenson DK Carbon monoxide and carboxyhemoglobin.Adv Pediatr. 1995; 42: 303-325PubMed Google Scholar, 24Ryter SW Otterbein LE Carbon monoxide in biology and medicine.Bioessays. 2004; 26: 270-280Crossref PubMed Scopus (327) Google Scholar Heme oxygenase, the key enzyme involved in CO generation, is widely distributed throughout tissues and organs, including the placenta.25Bainbridge SA Smith GN HO in pregnancy.Free Radic Biol Med. 2005; 38: 979-988Crossref PubMed Scopus (71) Google Scholar, 26Lyall F Barber A Myatt L Bulmer JN Robson SC Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.FASEB J. 2000; 14: 208-219PubMed Google Scholar There is increasing evidence that CO is required for normal placental development and function; endogenous CO production may regulate trophoblast migration and organization1Roberts JM Lain KY Recent insights into the pathogenesis of pre-eclampsia.Placenta. 2002; 23: 359-372Abstract Full Text PDF PubMed Scopus (504) Google Scholar, 25Bainbridge SA Smith GN HO in pregnancy.Free Radic Biol Med. 2005; 38: 979-988Crossref PubMed Scopus (71) Google Scholar, 26Lyall F Barber A Myatt L Bulmer JN Robson SC Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.FASEB J. 2000; 14: 208-219PubMed Google Scholar and play a hemodynamic role.27Bainbridge SA Farley AE McLaughlin BE Graham CH Marks GS Nakatsu K Smith GN Carbon monoxide decreases perfusion pressure in isolated human placenta.Placenta. 2002; 23: 563-569Abstract Full Text PDF PubMed Scopus (60) Google Scholar Further, women who have PE have significantly decreased CO concentrations in their exhaled breath compared with healthy pregnant women,28Baum M Schiff E Kreiser D Dennery PA Stevenson DK Rosenthal T Seidman DS End-tidal carbon monoxide measurements in women with pregnancy-induced hypertension and preeclampsia.Am J Obstet Gynecol. 2000; 183: 900-903Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 29Kreiser D Baum M Seidman DS Fanaroff A Shah D Hendler I Stevenson DK Schiff E Druzin ML End tidal carbon monoxide levels are lower in women with gestational hypertension and pre-eclampsia.J Perinatol. 2004; 24: 213-217Crossref PubMed Scopus (52) Google Scholar suggesting an endogenous regulatory role of this gaseous compound in the maintenance of healthy pregnancies. 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The objective of the current study was to determine whether CO had anti-apoptotic properties in syncytiotrophoblast of term human placenta after a hypoxia/reoxygenation (H/R) insult. This research study and use of human placental tissues was approved by the Queen's University (Kingston, ON, Canada) research ethics board before initiation. Villous explant culture medium contained 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 in CMRL-1066 (Invitrogen, Burlington, ON, Canada) supplemented with 5% heat-inactivated fetal calf serum.40Siman CM Sibley CP Jones CJ Turner MA Greenwood SL The functional regeneration of syncytiotrophoblast in cultured explants of term placenta.Am J Physiol. 2001; 280: R1116-R1122Google Scholar All reagents, unless otherwise indicated, were purchased from Sigma-Aldrich Ltd. (Oakville, ON, Canada). CO-saturated culture medium was prepared on the day of experiment by bubbling CO gas (99.5%; Praxair, Toronto, ON, Canada) through 30 ml of normal culture medium for 30 minutes. Three serial dilutions of the CO-saturated medium in normal medium were prepared and stored in air-tight sealed bottles. CO concentrations in all solutions were measured using gas-solid chromatography as described previously.27Bainbridge SA Farley AE McLaughlin BE Graham CH Marks GS Nakatsu K Smith GN Carbon monoxide decreases perfusion pressure in isolated human placenta.Placenta. 2002; 23: 563-569Abstract Full Text PDF PubMed Scopus (60) Google Scholar Term human placentas (n = 13) were collected immediately after elective cesarean section from nonlaboring, nonsmoking, low-risk women at the Kingston General Hospital (Kingston, ON, Canada). Cubes of 2.5 cm of placenta, devoid of calcifications or tears, were dissected from 7 to 10 random sites across each placenta. The chorionic plate and basal plate were removed from each piece of tissue. The tissue was transferred to the laboratory in a sealed bottle containing ice-cold phosphate-buffered saline (PBS).6Hung 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 (227) Google Scholar Chorionic villous explants (5 to 10 mg each) were dissected on ice from the placental tissue cubes and rinsed 2× with ice-cold PBS and 3× with explant culture medium. Five explants were cultured in individual Costar Netwell supports (15-mm diameter, 74 μm mesh; Cole-Parmer, Anjou, QC, Canada) in 1.2 ml of culture medium11Hung 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 (331) Google Scholar and exposed H/R as previously described6Hung 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 (227) Google Scholar (Figure 1). Initially, explants were cultured under hypoxic conditions (pO2 in medium = 12 to 16 mmHg6Hung 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 (227) Google Scholar) for 3 hours then oxygenation for 3 hours using gas phase 21% O2/5% CO2/balance N2 (pO2 in medium = 133 to 152 mmHg). A subset of villous explants (n = 6 placentas, 30 explants) were subjected to a repeat of this gaseous cycle. The untreated group (n = 13 placentas, 65 explants) was bathed with normal culture medium and the CO-treated group (n = 13 placentas, 65 explants) was bathed in the CO-infused culture medium. Both treated and untreated explant culture controls were kept under normoxic conditions throughout the entire 6-hour experimental period. A normoxic environment was established using a 5% O2/90% N2/5% CO2 gas mixture resulting in pO2 of 45 to 62 mmHg6Hung 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 (227) Google Scholar in the culture medium, a concentration that is similar to the oxygen tension measured within the intervillous space of term placenta.41Fujikura 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, 42Soothill 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 (242) Google Scholar All explants were incubated at 37°C throughout the experiment. At time 0, 3, 6, and 12 hours (in the subset of repeated H/R insult experiments) explants were collected from all experimental groups and either flash-frozen for molecular analysis or fixed in paraformaldehyde for histological analysis. Explants collected at 0, 3, 6, and 12 hours were fixed using 4% paraformaldehyde, embedded in paraffin, and cut into serial sections. To assess apoptosis of the syncytiotrophoblast layer, an immunofluorescence TUNEL assay was performed according to the manufacturer's instructions (In Situ Cell Death Detection kit; Roche Molecular Biochemicals, Laval, QC, Canada). Negative controls were sections incubated without terminal deoxynucleotidyl transferase (TdT). All sections were mounted with an aqueous medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vectashield Mounting Medium; Vector Laboratories, Burlington, ON, Canada). All sections were observed by a blinded third party using a Leica inverted microscope (Leica Microsystems, Richmond Hill, ON, Canada) at a magnification of ×20. Digital images of the TUNEL (green) and DAPI (blue) staining were captured at three randomly selected fields, providing a minimum of 900 syncytiotrophoblast nuclei per slide. Digital images were captured and deconvolved using Slidebook 4.1 software (Intelligent Imaging Innovations, Inc., Denver, CO). TUNEL-positive nuclei (apoptotic nuclei) and DAPI-stained nuclei (total nuclei) were counted in the syncytiotrophoblast cell 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 syncytiotrophoblast nuclei stained TUNEL-positive divided by the total number of DAPI-stained nuclei found within the syncytiotrophoblast. Paraffin-embedded placental sections were randomly selected and deparaffinized by heating them at 40°C for 20 minutes followed by sequential incubations in Hemo-D and decreasing ethanol baths (each for 2 × 3 minutes). Endogenous peroxidase activity was quenched using 3% hydrogen peroxide and nonspecific binding was limited using 5% normal goat serum. The sections were reacted with a rabbit polyclonal anti-PARP p85 fragment antibody (1:500; Promega, Nepean, ON, Canada) for 4 hours at room temperature. A secondary anti-rabbit horseradish peroxidase conjugate (1:200; Vector Laboratories) was then added to the sections. Additional processing of the sections for the detection of the p85 fragment of PARP was performed according to the instructions provided with the Vectashield Elite ABC kit (Vector Laboratories). Colorimetric detection was achieved using diaminobenzidine as the peroxidase substrate. Quantification of the p85-fragmented PARP staining was performed using Image-Pro Plus 5.1 software. Western blot analysis (n = 6) of protein extracts from flash-frozen was used to confirm immunohistochemical staining for fragmented PARP. The membranes were probed with the anti-PARP p85 fragment antibody (1:250), stripped, and sequentially probed with anti-β-actin monoclonal antibody (1:10,000; Bio-Rad, Mississauga, ON, Canada). The p85 PARP fragment band densities were quantified using densitometric analysis (Image-Pro Plus 5.1 software) and normalized to their corresponding β-actin band densities. Initial morphological assessments were completed using toluidine blue-stained semithin sections (1 μm) at a magnification of ×40. Explants collected for electron microscope (EM) analysis were processed as previously described.43Watson AL Skepper JN Jauniaux E Burton GJ Susceptibility of human placental syncytiotrophoblastic mitochondria to oxygen-mediated damage in relation to gestational age.J Clin Endocrinol Metab. 1998; 83: 1697-1705Crossref PubMed Scopus (134) Google Scholar Briefly, these explants were fixed in 2% paraformaldehyde/0.5% glutaraldehyde and embedded in epoxy resin. Ultra-thin sections (70 nm) were cut and counterstained with uranyl acetate and lead citrate. Morphological visualization of the explants was performed using transmission EM (Hitachi 7000; Hitachi, Pleasanton, CA) at magnifications between ×3500 to ×15,000. Morphological analyses were focused on the syncytium as a whole. Specific attention was paid to the integrity of the syncytial membrane, chromatin condensation, and vacuole formation. To assess the resemblance of ultrastructural syncytial damage caused through the in vitro H/R insult used in this study to that observed in vivo within PE placental tissue, villous explants were additionally dissected from term placentas of women diagnosed with PE (maternal hypertension, blood pressure ≥140/90; proteinuria, ≥0.3 g of urine protein/24 hours), as previously described. These explants were immediately fixed for semithin sectioning and EM analysis and subjected to similar morphological analysis detailed above. Villous tissue integrity was analyzed by measuring the release of lactate dehydrogenase (LDH) into the culture medium after 6 hours of explant incubation. LDH concentration was assessed at the Kingston General Hospital core laboratory using a commercially available kit (l-lactate enzymatic colorimetric kit; Roche Diagnostics, Indianapolis, IN) that measures the reduction of NAD to NADH, a reaction that couples the conversion of l-lactate to pyruvate in the presence of LDH. The rate of NADH formation is directly proportional to the catalytic LDH activity and is determined by measuring the increase in absorbance at 340 nm. All data sets were analyzed for Gaussian distribution using the D'Agostinon
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