Comparison of Bcl-xL protein expression in placental trophoblast cells between pregnancy complicated by severe preeclampsia and normotensive pregnancy

Preeclampsia is one of the main causes of maternal and perinatal mortality and morbidity.The pathogenesis of preeclampsia remains unclear until now. It is believed thatregulation of apoptosis in trophoblast cells plays an important role in the pathophysiologyof preeclampsia. Failure of spiral arteries remodeling will eventually lead to placentalhypoxia lead to excessive trophoblast apoptosis. The molecular mechanism of apoptosisis very complicated involving many signaling molecules included Bcl-2 proteins. The Bcl-2 protein group consists of proapoptosis proteins (Bax) and apoptosis inhibitor proteins(Bcl-2 and Bcl-xL). The aimed of this stuty was to compare the expression of Bcl-xLprotein in placental trophoblast cells of pregnancy complicated by severe preeclampsiawith that normotensive pregnancy. This study was an observational study with crosssectional design involving 43 pregnancy patients with severe preeclampsia and 38normotensive pregnancy who treated in Dr. Sardjito General Hospital, Yogyakarta fromOctober 2011 until March 2012. Placenta samples were obtained from all subjects forBcl-xL protein expression analysis using immunohistochemistry technique. Data wereanalyzed using independent t-test, chi-square test, and logistic regression. A p value<0.05 was considered significant. Significant difference in Bcl-xL protein expressionin trophoblast cells of pregnancy complicated by severe preeclampsia (1.29 ± 0.12)compared to that normotensive pregnancy (1.71 ± 0.14) was reported (p = 0.00). Inaddition, logistic regression test showed that diagnosis of severe preeclampsia had astatistically significant role in Bcl-xL protein expression (p= 0.000). In conclusion, theexpression of Bcl-xL protein is lower in pregnancy complicated by severe preeclampsiacompared to normotensive pregnancy

Trophoblast lineage-specific differentiation and associated alterations in preeclampsia and fetal growth restriction.

The human placenta is a poorly-understood organ, but one that is critical for proper development and growth of the fetus in-utero. The epithelial cell type that contributes to primary placental functions is called "trophoblast," including two main subtypes, villous and extravillous trophoblast. Cytotrophoblast and syncytiotrophoblast comprise the villous compartment and contribute to gas and nutrient exchange, while extravillous trophoblast invade and remodel the uterine wall and vessels, in order to supply maternal blood to the growing fetus. Abnormal differentiation of trophoblast contributes to placental dysfunction and is associated with complications of pregnancy, including preeclampsia (PE) and fetal growth restriction (FGR). This review describes what is known about the cellular organization of the placenta during both normal development and in the setting of PE/FGR. It also explains known trophoblast lineage-specific markers and pathways regulating their differentiation, and how these are altered in the setting of PE/FGR, focusing on studies which have used human placental tissues. Finally, it also highlights remaining questions and needed resources to advance this field

Ekspresja kaspazy-3, Bax i Bcl-2 w łożyskach ciąż leczonych i nieleczonych z powodu wewnątrzmacicznego zahamowania wzrastania płodu

Abstract Background: Fetal growth restriction (FGR) is the reason of high prematurity rate and its later complications. Restriction of utero-placental circulation, which could be changed by IURG treatment, plays the main role in FGR. The results of changes in apoptosis-related genes expression due to FGR treatment may help further in the prevention and treatment of FGR. Material and methods: Caspase-3, Bax and Bcl-2 expressions in normal pregnancies and those complicated by treated and untreated FGR have been compared. The study was conducted in 2005-2006 at the High-Risk Pregnancy Unit of Medical University in Łódź and Kopernik Hospital in Łódź. Caspase-3, Bax and Bcl-2 expressions were assessed by immunohistochemical method. Bcl-2 was assessed in the trophoblast, Bax and caspase-3 in the decidua and the trophoblast. Results: The mean value of Bcl-2 in the trophoblast was 58.8±12.7 in the FGR-untreated group, 37.0±0.5 in the FGR-treated group and 65.7±6.9 in the control group. In the FGR-untreated group the mean value of Bax expression was 60.6±10.7 in the trophoblast and 32.0±7.3 in the decidua. In the FGR-treated group the mean value of Bax expression was 42.2±12.2 in the trophoblast and 20.9±6.4 in the decidua. In the control group the mean value of Bax expression was 13.6±2.2 in the trophoblast and 6.6±6.8 in the decidua. In the FGR-untreated group the mean value of Cpp-32 expression was 40.1±9.1 in the trophoblast and 42.6±.12.5 in the decidua. In the FGR-treated group the mean value of Cpp-32 expression was 21.3±6.8 in the trophoblast and 23.7±5.1 in the decidua. In the control group the mean value of Cpp-32 expression was 13,6±6,3 in trophoblast and 11.6±5.3 in the decidua. Conclusions: Increased expression of pro-apoptotic proteins in the placenta might be one of the reasons for FGR development. The treatment used in the FGR group decreased the process of apoptosis.Streszczenie Hipotrofia wewnątrzmaciczna (FGR) wiąże się z wysokim odsetkiem wcześniactwa i jego późniejszych powikłań. W patogenezie hipotrofii główną rolę odgrywa ograniczenie przepływu maciczno-łożyskowego, co może być modyfikowane przez stosowanie terapii. Uzyskanie zmian zachodzących podczas leczenia w zakresie procesu apoptozy może wpłynąć w przyszłości na sposób zapobiegania i leczenia hipotrofii wewnątrzmacicznej. Materiał i metody: Porównano ekspresję kaspazy-3, Bax i Bcl-2 w ciąży niepowikłanej i powikłanej hipotrofią wewnątrzmaciczną z podziałem na kobiety nieleczone i leczone. Badania były prowadzone w Klinice Patologii Ciąży Uniwersytetu Medycznego w Łodzi w latach 2005-2006. Ekspresję kaspazy-3, Bax i Bcl-2 oceniano metodami immunohistochemicznymi. Białko Bcl-2 było oceniane w trofoblaście, Bax i kaspaza-3 w doczesnej i trofoblaście. Wyniki: W grupie nieleczonych średnia wartość ekspresji Bcl-2 w trofoblaście wynosiła, 58,8±12,7, w grupie leczonych 37,0±10,5, w grupie kontrolnej 65,7±6,9. W grupie nieleczonych średnia wartość Bax w trofoblaście wynosiła 60,6±10,7, w doczesnej natomiast 32,0±7,3. W grupie kobiet leczonych średnia wartość ekspresji w trofoblaście wynosiła 42,2±12,2, w doczesnej natomiast 20,9±6,4. W grupie kontrolnej w trofoblaście wynosiła 13,6±2,2, a w doczesnej 6,6±6,8. W grupie nieleczonych średnia wartość kaspazy-3 mierzonej za pomocą aktywności Cpp-32 wynosiła w trofoblaście 40,1±9,1, a w doczesnej 42,6±12,5. W grupie kobiet leczonych średnia ekspresja Cpp-32 w trofoblaście wynosiła 21,3±6,8, w doczesnej 23,7±5,1. W grupie kontrolnej w trofoblaście wartość ekspresji Cpp-32 oceniano na 13,6±6,3, w doczesnej uzyskano wartości 11,6±5,3. Wnioski: Podwyższona ekspresja białek proapoptotycznych w łożysku może być jednym z powodów rozwoju hipotrofii wewnątrzmacicznej. Leczenie zastosowane w hipotrofii wewnątrzmacicznej obniżyło oceniane parametry apoptozy

A lesson for cancer research : placental microarray gene analysis in preeclampsia

Tumor progression and pregnancy share many common features, such as immune tolerance and invasion. The invasion of trophoblasts in the placenta into the uterine wall is essential for fetal development, and is thus precisely regulated. Its deregulation has been implicated in preeclampsia, a leading cause for maternal and perinatal mortality and morbidity. Pathogenesis of preeclampsia remains to be defined. Microarray-based gene profiling has been widely used for identifying genes responsible for preeclampsia. In this review, we have summarized the recent data from the microarray studies with preeclamptic placentas. Despite the complex of gene signatures, suggestive of the heterogeneity of preeclampsia, these studies identified a number of differentially expressed genes associated with preeclampsia. Interestingly, most of them have been reported to be tightly involved in tumor progression. We have discussed these interesting genes and analyzed their potential molecular functions in preeclampsia, compared with their roles in malignancy development. Further investigations are warranted to explore the involvement in molecular network of each identified gene, which may provide not only novel strategies for prevention and therapy for preeclampsia but also a better understanding of cancer cells. The trophoblastic cells, with their capacity for proliferation and differentiation, apoptosis and survival, migration, angiogenesis and immune modulation by exploiting similar molecular pathways, make them a compelling model for cancer research

Molecular and cellular responses of normal human trophoblast to vascular endothelial growth factor and placenta growth factor

Implantation and growth of the placenta requires extensive angiogenesis in fetal villi and maternal decidua to form vascular structures involved in placental exchange. Vascular development, cell proliferation, differentiation and invasiveness are required for proper development of the placenta. Many growth factors and receptors are involved in these processes. The human placenta is a rich source of angiogenic growth factors and their receptors, which are known to control vascular changes and control trophoblast function [1, 2]. Within the placenta, trophoblast are a source of angiogenic growth factors like transforming growth factor -α and β (TGF-α and β), epidermal growth factor (EGF), fibroblast growth factor (FGF-1 and 2), vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) [3]

Quantification of Placental Dysfunction in Pregnancy Complications

Background The pathogenetic mechanisms behind placental dysfunction-related complications like preeclampsia and intrauterine growth restriction have remained perplexing till now, in part because of lack of well-defined structural and functional molecular characterisation. There is growing evidence that links trophoblast debris and the existence of syncytial nuclear aggregates (SNA) to the pathogenesis of gestational diseases. Characterisation and quantification of structural and functional parameters of placental dysfunction may give researchers a clearer picture of the mechanisms underlying the development of high risk pregnancy. Methods Placental samples were obtained from normal term pregnancies, preterm controls, as well as from pregnancies complicated by preeclampsia (PET), intrauterine growth restriction (IUGR) and PET-IUGR. Formalin-fixed, paraffin-embedded sections were visualised with H&E, stained using immunohistochemistry (IHC) and digitally scanned. Using stereological methodology, volumes of placental SNAs, trophoblasts, villi and capillaries were measured. Three dimensional (3D) volume reconstructions of terminal placental villi with SNAs and fibrinoid degenerations were created. IHC-labelled slides were analysed by image analysis algorithms. Differential expression of placental genes and miRNAs, hypothesised to regulate cell death in placental dysfunction, were quantified using RT-qPCR. BeWo cell lines were carried out for in vitro validation of the effects miRNAs regulating programmed cell death (PCD) using flow cytometry and western blotting. Results Specific morphometric patterns of villous, trophoblasts, SNA and capillary volumes were demonstrated with characteristic higher SNAs and lower capillary volumes in PET placentae with reciprocal patterns in IUGR placentae showing a negative correlation pattern between nuclear aggregates and capillary volumes. Image analysis of immune-labelled slides showed a higher autophagy marker expression in PET and a positive correlation to SNAs as well as a balanced reciprocal expression patterns with apoptosis. Moreover, miR-204 transfected BeWo cells showed a similar balanced reciprocal regulation of autophagy and apoptosis expressions. Conclusion We have demonstrated that applying stereology-based and image analysis on digitised placental sections can be useful in quantifying and dissecting structural and functional patterns in normal and abnormal placental function. 3D reconstruction model are a novel approach towards placental characterisation in normal and complicated pregnancies. The study also showed that miR-204 plays a vital role in the regulation of placental autophagy and apoptosis, critical in the pathophysiology of placental dysfunction

Quantification of Placental Dysfunction in Pregnancy Complications

Background The pathogenetic mechanisms behind placental dysfunction-related complications like preeclampsia and intrauterine growth restriction have remained perplexing till now, in part because of lack of well-defined structural and functional molecular characterisation. There is growing evidence that links trophoblast debris and the existence of syncytial nuclear aggregates (SNA) to the pathogenesis of gestational diseases. Characterisation and quantification of structural and functional parameters of placental dysfunction may give researchers a clearer picture of the mechanisms underlying the development of high risk pregnancy. Methods Placental samples were obtained from normal term pregnancies, preterm controls, as well as from pregnancies complicated by preeclampsia (PET), intrauterine growth restriction (IUGR) and PET-IUGR. Formalin-fixed, paraffin-embedded sections were visualised with H&E, stained using immunohistochemistry (IHC) and digitally scanned. Using stereological methodology, volumes of placental SNAs, trophoblasts, villi and capillaries were measured. Three dimensional (3D) volume reconstructions of terminal placental villi with SNAs and fibrinoid degenerations were created. IHC-labelled slides were analysed by image analysis algorithms. Differential expression of placental genes and miRNAs, hypothesised to regulate cell death in placental dysfunction, were quantified using RT-qPCR. BeWo cell lines were carried out for in vitro validation of the effects miRNAs regulating programmed cell death (PCD) using flow cytometry and western blotting. Results Specific morphometric patterns of villous, trophoblasts, SNA and capillary volumes were demonstrated with characteristic higher SNAs and lower capillary volumes in PET placentae with reciprocal patterns in IUGR placentae showing a negative correlation pattern between nuclear aggregates and capillary volumes. Image analysis of immune-labelled slides showed a higher autophagy marker expression in PET and a positive correlation to SNAs as well as a balanced reciprocal expression patterns with apoptosis. Moreover, miR-204 transfected BeWo cells showed a similar balanced reciprocal regulation of autophagy and apoptosis expressions. Conclusion We have demonstrated that applying stereology-based and image analysis on digitised placental sections can be useful in quantifying and dissecting structural and functional patterns in normal and abnormal placental function. 3D reconstruction model are a novel approach towards placental characterisation in normal and complicated pregnancies. The study also showed that miR-204 plays a vital role in the regulation of placental autophagy and apoptosis, critical in the pathophysiology of placental dysfunction

The role of c-jun n-terminal kinase (JNK) and tumour protein (p53) in HIV associated pre-eclampsia.

Masters Degree. University of KwaZulu-Natal, Durban.Background: In pre-eclampsia, immune maladaptation to the foetal allograft results in impaired trophoblast invasion and defective spiral arterial remodelling with consequential placental oxidative stress. Placental apoptosis can be initiated by various stimuli including hypoxia and oxidative stress and is notably exaggerated in pre-eclampsia. This elevated apoptosis prevents normal replenishment of the syncytiotrophoblast, promotes syncytial degeneration and releases vasoactive or inflammatory factors into the maternal circulation thereby provoking the endothelial dysfunction seen in pre-eclampsia. Since the p53 antigen and JNK are important mediators of apoptosis we determined their expression in HIV associated pre-eclampsia. Method: Blood samples were collected from normotensive pregnant and pre-eclamptic HIV infected and negative women. Buffy coat was extracted and a Bio-plex multiplex assay to quantify expression of phosphorylated-p53 and JNK. Results: Based on pregnancy type, a significant difference in the expression of p53 was noted between the pre-eclamptic vs normotensive pregnant group regardless of HIV status (p=0.0162). Irrespective of pregnancy type, there was also a significant difference found between the HIV positive and HIV negative groups (p=0.0469). Furthermore, there was a significant difference in the expression of p53 between the HIV negative pre-eclamptic vs the HIV negative normotensive group and HIV infected normotensive vs the HIV negative pre-eclamptic group. Despite having no statistical significance (p=0.8701), the expression of JNK was found to be increased in the pre-eclamptic compared to the normotensive pregnant group. Similarly, based on HIV status, regardless of pregnancy type no statistical significance was found (p=0.2227), however, there was a decrease in the expression of JNK in the HIV positive compared to the HIV negative group. xiii Conclusion: These experiments demonstrate a significant increase in the expression of p53 with an upwards trend in the expression of JNK in pre-eclampsia, confirming their influence on trophoblast cell invasion in pre-eclampsia development. This increase in expression of p53 and JNK in pre-eclampsia, holds potential value as a risk indicator of pre-eclamptic development. In contrast, a significant down regulation of p53 with a downwards trend of JNK expression was noted in the HIV positive group, possibly due to immune reconstitution following HAART

Profile of Apoptotic Marker Genes and Histopathology of the Placenta in Pregnancies with Pre-Eclampsia

Background: Pre-eclampsia (PE) is a hypertensive disorder in pregnancy and a significant cause of maternal and perinatal mortality and morbidity. Failure of spiral artery remodeling due to abnormal apoptosis, triggers disturbances in the mother and the baby’s growth. This study aimed to identify the profile of apoptotic marker genes and histopathological features of the placenta in pregnancies with pre-eclampsia.Methods: This study had used case-control method. Samples were taken from normal pregnancies (n=25) and pregnant women with pre-eclampsia (n=25) using a purposive sampling method from several hospitals in Jambi. qRT-PCR was used to examine apoptotic gene expression from placental tissue and hematoxyline eosin staining to view the placenta’s microscopic appearance. The targeted genes were BCL2-associated X (BAX) and B-cell lymphoma 2 (BCL-2). Histopathological changes of the placenta observed were syncytial node, cytotrophoblast, villous edema, hypervascularization, fibrosis stroma, atherosis, infarction, and thrombosis.Results: Relative BAX genes expression were increased once in placenta pre-eclampsia compared to controls, but not statistically significant (p-value>0.05). There was no difference between the decline of BCL-2 gene expression in pre-eclampsia placenta compared to the control (p-value >0.05). Histopathological changes in the placenta were syncytial node and cytotrophoblast (25 of 25), villous edema (19 of 25), hypervascularization (24 of 25), fibrosis stroma (22 of 25), atherosis (12 of 25), infarction (17 of 25), and thrombosis (24 of 25).Conclusion: The expression of BAX genes in pre-eclampsia tends to increase compared to normal pregnancy, and the expression of BCL-2 decreases.  The histopathological features of pre-eclampsia pregnancy placenta are mostly syncytial nodes, cytotrophoblasts, stromal fibrosis, and thrombosis

Beta-2 GPI induced tissue factor and placental apoptosis for the pathophysiology of pregnancy loss in antiphospholipid syndrome

Based on large concurrent studies on human and in vivo results from experimental animals, it is evident that antiphospholipid syndrome (APS) plays a vital role in pregnancy failure in human being. Many underlying pathophysiology including venous thrombosis, thrombocytopenia and placental apoptosis have been demonstrated for the APS-mediated pregnancy loss. On the other hand, Tissue factor (TF) remains considered as a crucial factor for pregnancy morbidity in women with APS globally. Hence, we hypothesize that TF and/or beta-2 glycoprotein – I (β2GPI)-induced TF might play an important role for the increased index of apoptosis in placenta, especially during early stages of fetal development. Further, this could represent as potentially preventable etiology of APS-mediated pregnancy loss in women