Nitrotyrosine Immunostaining Correlates with Increased Extracellular Matrix: Evidence of Postplacental Hypoxia

doi:10.1053/plac.2001.0634

Placenta

Volume 22, Supplement A, April 2001, Pages S56-S62

https://doi.org/10.1053/plac.2001.0634 Get rights and content

Abstract

Nitrotyrosine residues (NT), an index of oxidative stress arising from peroxynitrite formation and action, are found in placental vasculature of pregnancies complicated by pre-eclampsia (PE) or pregestational insulin-dependent diabetes mellitus (IDDM). This study correlates conventional placental pathology with NT immunostaining in 20 cases of perinatal mortality (13 stillbirths and seven cases of neonatal mortality) associated with PE, IDDM, amniotic fluid infection syndrome (AFIS), or from fetal/neonatal demise not related to these conditions (congenital anomalies) (n=five/group). Patients with PE have more decidual arteriolopathy and Tenney–Parker change, while patients with IDDM and ascending infection have more villous cytotrophoblastic hyperplasia. Archival paraffin-embedded placental sections were immunostained for NT for correlation with clinical features and H&E histological findings. The intensity of immunostaining for NT varied from absent (n=7) to 1+ (n=5) or 2+ (n=8). All eight placentae with 2+ staining showed increased villous extracellular matrix (ECM), compared to none of five with 1+ staining and two of seven with no staining (χ²=14.3, P=0.001). There was no statistically significant difference in the percentage of stem villi with luminal vascular abnormalities (5.7 vs 10 vs 35.7 per cent, F=2.3,P =0.1). Our data show that increased production of reactive oxygen species by placental tissue may be associated with increased extracellular matrix, itself produced by fibroblasts under the influence of oxygen. NT immunostaining may therefore help differentiate those cases of perinatal morbidity/mortality associated with post-placental hypoxia provided that the secondary impact of intrauterine fetal death can be excluded by future studies.

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Histological evidence of oxidative stress and premature senescence in preterm premature rupture of the human fetal membranes recapitulated in vitro
2014, American Journal of Pathology
Citation Excerpt :
Detected bands were analyzed densitometrically using the ImageJ software version 1.46r (NIH, Bethesda, MD), and results were normalized to β-actin (A5441; Sigma, St. Louis, MO). To document oxidative stress in clinical specimens and in tissues treated with CSE in vitro, we used IHC to localize 3-nitrotyrosine (3-NT) based on the principle that ROS produce highly reactive peroxynitrite radicals that react with exposed tyrosine residues on proteins, resulting in a stable polypeptide-bound 3-NT.22–27 IHC was performed using the anti–3-NT antibody (39B6; catalog no. ab61392; Abcam) at a dilution of 1:200.
Preterm prelabor rupture of the membranes (pPROM) may lead to preterm births (PTBs). We investigated premature senescence of fetal membranes in women with pPROM and spontaneous PTB with intact membranes (<34 weeks) and the inducibility fetal membrane senescence phenotype by oxidative stress in vitro. IHC was performed for p53, p21, and phospho (p)-p38 mitogen-activated protein kinase (MAPK) as markers of senescence phenotype in pPROM, PTBs, and term births. Term fetal membranes were exposed to cigarette smoke extract to induce oxidative stress. Western blots documented p-p53 and p-p38 MAPK. Transmission electron microscopy assessed cellular morphologic features in clinical and cigarette smoke extract–treated membranes. A total of 80% of pPROM cells and >60% of term cells were positive for all three senescence phenotype markers, and concentrations were higher than in PTBs (P < 0.05). p53 staining was comparable in membranes from PTB and term birth pregnancies, whereas only <30% and <45% of cells were positive for p21 and p38 MAPK, respectively. In vitro cigarette smoke extract exposure increased p-p38 MAPK without any detectable change in p-p53 MAPK. Enlargement of organelles consistent with senescence phenotype was evident in pPROM and term membranes in vivo and after cigarette smoke extract treatment in vitro but was less apparent in PTBs. Histologic and biochemical resemblance of pPROM and term membranes suggests premature senescence of the membranes is a mechanistic feature in pPROM, and this can be phenocopied in an in vitro model.
Mammalian Ste20-like protein kinase 3 plays a role in hypoxia-induced apoptosis of trophoblast cell line 3A-sub-E
2011, International Journal of Biochemistry and Cell Biology
Citation Excerpt :
Peroxynitrite (ONOO−), a highly reactive pro-oxidant, can be formed from the interaction of NO and O2− (Wang et al., 2000; Xia et al., 1996) and quickly modify proteins at tyrosine residues to form nitrotyrosine (NTyr) (Noris et al., 2004; Webster et al., 2008). Therefore, the formation of NTyr may indicate the elevation of NO and oxidative stress in response to hypoxia (Fujita et al., 2008; Stanek et al., 2001) and can be readily determined by the appearance of a major nitrated protein at around 55 kDa by immunoblotting (Satoh et al., 2008). The formation of NTyr in 3A cells increased in response to hypoxia in a time-dependent manner (Fig. 2A, hypoxia panel).
Mammalian Ste20-like protein kinase 3 (Mst3) is a key player in inducing apoptosis in a variety of cell types and has recently been shown to participate in the signaling pathway of hypoxia-induced apoptosis of human trophoblast cell line 3A-sub-E (3A). It is believed that oxidative stress may occur during hypoxia and induce the expression of Mst3 in 3A cells via the activation of c-Jun N-terminal protein kinase 1 (JNK1). This hypothesis was demonstrated by the suppressive effect of dl-α-lipoic acid, a reactive oxygen species scavenger, in hypoxia-induced responses of 3A cells such as Mst3 expression, nitrotyrosine formation, JNK1 activation and apoptosis. Similar results were also observed in trophoblasts of human placental explants in both immunohistochemical studies and immunoblot analyses. These suggested that the activation of Mst3 might trigger the apoptotic process in trophoblasts by activating caspase 3 and possibly other apoptotic pathways. The role of nitric oxide synthase (NOS) and NADPH oxidase (NOX) in hypoxia-induced Mst3 up-regulation was also demonstrated by the inhibitory effect of N^G-nitro-l-arginine and apocynin, which inhibits NOS and NOX, respectively. Oxidative stress was postulated to be induced by NOS and NOX in 3A cells during hypoxia. In conclusion, hypoxia induces oxidative stress in human trophoblasts by activating NOS and NOX. Subsequently, Mst3 is up-regulated and plays an important role in hypoxia-induced apoptosis of human trophoblasts.
Membrane microscopic chorionic pseudocysts are associated with increased amount of placental extravillous trophoblasts
2010, Pathology
To further study the pathogenesis of membrane microscopic chorionic pseudocysts (MCP), a presumptive placental chronic hypoxic lesion, this analysis retrospectively assessed the amount, maturity, proliferation, and apoptosis of membrane and chorionic disc extravillous trophoblasts in placentas with MCP.
Sections of 50 consecutive placentas with MCP (study group) and 50 placentas without MCP from pregnancies matched for gestational age (control group) were immunostained for human placental lactogen (hPL, a marker of trophoblastic maturity), M30 (a marker of irreversible apoptosis in epithelial cells), and E-cadherin and Ki-67 (double immunostaining, epithelial and proliferation markers, respectively). Positivity was evaluated quantitatively and analysed statistically.
A normal placenta features up to a seven cell thick membrane extravillous trophoblast layer and up to five cell islands or placental septa per chorionic disc section. Study group placentas featured 2.2 times more cell islands or placental septa (p < 0.01), and had membrane extravillous trophoblast layers 1.6 times thicker than control group placentas (p < 0.001). The degree of apoptosis, proliferation, and maturation in membrane and placental septa or cell island extravillous trophoblasts did not show statistically significant differences between the study group and the control group (p > 0.05).
The increased extravillous trophoblast accumulation and secretory activity is associated with MCP formation. The findings place MCP among other placental lesions associated with extravillous trophoblast accumulation and possibly dysfunction.
Determination of non-bilayer phospholipid arrangements and their antibodies in placentae and sera of patients with hypertensive disorders of pregnancy
2006, Placenta
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Further analysis of a larger number of IUGR samples without PE will be necessary to fully evaluate this possibility. There are several studies that suggest that apoptotic cell antigens are recognized as the target of autoantibodies [22,32] and that these antigens could be related to an increase in oxidative stress in the placenta of preeclamptic patients [33–36]. Placentas from patients with PE exhibited strong immunostaining for the NPA in the apical and basal syncytiotrophoblast membranes when compared to those from control subjects suggesting that there are some differences in the metabolic stages or in the cell death stages of these syncytiotrophoblasts.
Studies suggest that preeclampsia (PE) originates in the placenta and is associated with deficient trophoblast invasion of spiral arteries. The direct cause remains unknown, but preeclampsia is often associated with circulating factors that can induce generalized endothelial dysfunction. Antiphospholipid antibodies (APA) in circulation are also associated with vascular diseases. Although the quantification of APA is not currently used as a prognostic of the risk of PE, studies suggest that thrombophilias play a role in PE pathogenesis. In fact, the pathology of placentae from PE and Antiphospholipid syndrome patients is similar; atherosis, thrombosis and infarction, and endothelium activation represent the pathological mechanisms.
We identified a new antibody which recognizes non-bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, and which triggered an autoimmune-like disease in mice. We evaluated the presence of NPA in the placentae and in sera, and whether NPA induced NPA antibodies in patients with hypertensive disorders of pregnancy (HDP). Results showed increased levels of NPA in the syncytiotrophoblast, extravillous cytotrophoblast, syncytial knots and the amnion epithelial cell membranes of the placenta, as well as increases in NPA and NPA antibodies in sera from HDP patients, when compared with controls. This suggests that NPA derived from placenta could be one of multiple factors associated with pregnancy pathologies.
Accumulation of advanced glycation end products in women with preeclampsia: Possible involvement of placental oxidative and nitrative stress
2006, Placenta
Citation Excerpt :
We observed that the intensity of immunostaining of nitrotyrosine, a footprint of peroxynitrite, in the placentas from preeclamptic women was greater than that in the placentas from healthy women. This observation supports previous reports [46–48]. Formation and action of peroxynitrite, particularly in the endothelium, may result in vascular damage that contributes to the increased placental vascular resistance [46,49,50].
Advanced glycation end products (AGEs) are known to cause oxidative damage in various cells by binding with its receptor, RAGE. We measured the serum level of AGEs and examined the AGEs, RAGE, and the other biomarkers of oxidative stress in the placentas from preeclamptic women.
Competitive ELISA was carried out to measure the AGEs in serum. Western blotting was performed to analyze AGEs and RAGE in the placenta. Immunohistochemical analyses were performed to examine the localization of AGEs, RAGE, and other biomarkers of oxidative stress in the placenta.
The mean level of serum AGEs in preeclamptic women was significantly higher than that in healthy non-pregnant women or healthy pregnant women. Western blotting revealed that the level of AGEs or RAGE in preeclamptic placenta was significantly higher than that in normal placenta. Immunohistochemical analyses showed that levels of nitrotyrosine and nitroguanosine, which are formed by reactive nitrogen species, in preeclamptic placenta were higher than those in normal placenta. Accumulation of 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine indicated enhanced oxidative modifications of lipids and DNA in preeclamptic placenta.
The AGE–RAGE system, which is upregulated in preeclampsia, is likely to be involved in the oxidative stress of preeclampsia.
Increased matrix metalloproteinases 2 and 9 in placenta of diabetic rats at midgestation
2005, Placenta
Citation Excerpt :
The increased MMPs found in the placental tissue from diabetic rats are likely to lead to an increased proliferative and remodelling activity, leading to an increased number of immature villi and increased vascularization, structural alterations that are observed in the studied diabetic model. Besides, the diabetic pathology leads to nitrosative and oxidative stress in different tissues, and hence to the production of NO and reactive oxygen species (ROS) [42,43,19]. Both NO and ROS are involved in the formation of peroxynitrite, a potent oxidant that induces damage through the inhibition of cellular respiration, protein nitrosylation, lipid peroxidation, DNA breakdown and disruption of signalling pathways [44].
Matrix metalloproteinases (MMPs) are involved in placental remodelling throughout pregnancy. Diabetes mellitus induces alterations in tissue production of NO, a regulator of MMPs activity. The present work evaluates placental and fetal MMPs and NO levels during midpregnancy in neonatal streptozotocin-induced diabetic rats. MMP-2 and MMP-9 immunolabelling was increased both in the labyrinth zone (p < 0.001) and in the giant trophoblast cells of the junctional zone (p < 0.001) from diabetic placenta, when compared with controls. Also MMP-2 (p < 0.01) and MMP-9 (p < 0.005) activities were increased in both maternal and fetal sides of diabetic placenta when related to controls. In both sides of the diabetic placenta, nitrate/nitrite concentrations (which indicate NO production) were higher than in controls (p < 0.05). An intense immunostaining for nitrotyrosine, indicating peroxynitrite-induced damage, was found in both labyrinth (p < 0.001) and junctional zones (p < 0.001) of diabetic placenta. Enhanced MMP-2 activity (p < 0.05) and NO production were also higher in the fetuses from diabetic rats when compared to controls (p < 0.005). These findings demonstrate alterations in MMPs and NO in the feto-placental unit of diabetic rats, anomalies that are likely to be involved in the developmental alterations induced by maternal diabetes.

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^f1: To whom correspondence should be addressed at: Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0529, USA.

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Pathology of the human placenta

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The villous cytotrophoblast as an index of placental ischaemia

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The incidence and significance of Hofbauer cells in the mature human placenta

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Nitrotyrosine Immunostaining Correlates with Increased Extracellular Matrix: Evidence of Postplacental Hypoxia