X-linked inhibitor of apoptosis protein expression and the regulation of apoptosis during human placental development

In this study, we have examined the expression and potential role of X-linked inhibitor of apoptosis protein (XIAP), Fas, and Fas ligand (FasL) in the regulation of apoptosis throughout placental development. Protein expression was determined by Western blot analysis and immunohistochemistry, whereas apoptotic cell death was assessed by DNA fragmentation analysis and TUNEL. The XIAP was present in trophoblast throughout placental development, but its content significantly decreased during late pregnancy, when apoptosis was maximal. The FasL content was low during early placental development but increased coincidentally to the decrease in XIAP during the third trimester. Our data also suggest that placental apoptosis is the culmination of the relative expression of these cell-death and -survival proteins, a phenomenon that is cell type-specific and dependent on cytodifferentiation and the stage of placental development. Moreover, the induction of syncytiotrophoblast apoptosis may involve the concomitant up-regulation of FasL for Fas activation and the removal of downstream inhibition of the apoptotic cascade by XIAP.     

Regulation of FasL/Fas in human trophoblasts: possible implications for chorioamnionitis

Chorioamnionitis is a common cause of premature birth and is associated with significant morbidity and mortality in the mother and infant. Preterm birth shares similarities with rejection of the fetal allograft, which is characterized by increased apoptosis of placental trophoblasts. We hypothesized that there is increased trophoblast apoptosis in chorioamnionitis and that this increased apoptosis is mediated by the Fas ligand (FasL)/Fas pathway. To test our hypothesis, we examined placental villous tissues from patients with chorioamnionitis and used the TUNEL assay to demonstrate enhanced trophoblast apoptosis in patients with chorioamnionitis. When the same samples were stained for Fas, there was increased trophoblast Fas expression in patients with chorioamnionitis. To define the mechanisms responsible for this increase in trophoblast apoptosis, we cultured villous explants from uncomplicated term placentas with proinflammatory cytokines and demonstrated a marked increase in trophoblast apoptosis. By blocking FasL, we reduced tumor necrosis factor alpha-induced and interferon gamma-induced apoptosis. These data suggest that chorioamnionitis is associated with increased trophoblast apoptosis and enhanced trophoblast Fas expression. As a complement to our in vivo study, we demonstrated that cytokine-induced trophoblast apoptosis is mediated in part by the FasL/Fas pathway, suggesting that cytokines promote sensitivity to Fas-mediated apoptosis. These mechanisms may be important in perpetuating inflammation in the placental microenvironment and may contribute to the pathogenesis of chorioamnionitis.     

The expression of Fas/FasL and apoptosis in yak placentomes

To clarify the status and distribution of Fas and Fas-Ligand (FasL) in yak's placentomes, immunohistochemistry (IHC) was carried out to analyze the expression and location of Fas and FasL in paraffin embedded sections. The area of positive stained sites was selected and measured using image analyses software (Image Pro-Plus 6.0). So the positive index (PI) was calculated to estimate the intensity of protein expression according to the percentage of positive area in corresponding compartment of the placentomes. In cotyledonary villi, Fas mainly presented on the villous trophoblast cells in early pregnancy. The positive index reached a maximum of 20.7±8.8 at the third month of pregnancy. Then Fas was declined rapidly along with the progress of gestation and the value was 2.8±1.3 after the 7th month of pregnancy. However, in caruncular crypts, Fas was mainly localized to isolated cells or clustered cells of the uterine stroma underlying the caruncular epithelium. The intensity was lower and the positive index was changed between 4.7±0.9 and 8.5±1.6 throughout gestation. For FasL, it gave a distinct immunostained distribution. In cotyledonary villi, FasL was localized dominantly and strongly in the cytoplasm of binuclear, mononuclear and trinuclear trophoblast giant cells (TGC). The positive index of FasL maintained a moderate level all through the gestation. In caruncular crypts, the expression of FasL was weak and the positive index was declined. Only in the first two months, maternal uterine epithelial cells intensely expressed FasL and the index reached to the maximum of 19.8±5.2. The result of subcellular localization of Fas ligand using immunoelectron microscopy technology indicated that FasL was subcellular located in some intracellular vesicles of TGC. This means the vesicles of trophoblast giant cells itself can express FasL. By the TUNEL method, apoptosis was detected in yak placentomes. The amount of apoptotic cells was rare. The fetal chorionic trophoblast cells and caruncular crypt epithelium cells demonstrated higher percentage of apoptosis in middle pregnancy, which suggested that apoptosis plays an important role in placental cellular regeneration. In addition, the apoptosis of maternal caruncular stromal cells provides a local mechanism for maternal immunotolerance to the fetus and this mechanism was mediated by Fas-FasL pathway.     

Expression of Fas/Fas ligand by decidual leukocytes in hydatidiform mole

Complete hydatidiform moles are entirely paternally derived and, therefore, represent a complete intrauterine allograft that might be expected to provoke an altered maternal immune response compared with that of normal pregnancy. Uterine decidua contains a large leukocyte population, of which 10%-20% are T lymphocytes. Fas ligand (FasL) expression by placental trophoblast may induce apoptosis of Fas+ lymphocytes, thereby facilitating immune tolerance and survival of the molar trophoblast. Our previous studies have shown an increase in activated CD4+ decidual T cells in molar pregnancy compared with normal pregnancy. This study was designed to characterize and quantitate Fas/FasL expression by decidual leukocytes in complete and partial hydatidiform mole compared with that in normal early pregnancy using single and double immunohistochemical labeling (i.e., avidin-biotin-peroxidase and avidin-biotin-alkaline phosphatase). A significant increase was found in Fas and FasL expression by decidual CD4+ T cells in complete (Fas+, P = 0.0106; FasL+, P = 0.0081) and partial (Fas+, P = 0.0131; FasL+, P = 0.0051) hydatidiform moles, as was a significant decrease in Fas expression by decidual CD8+ T cells in complete (P = 0.0137) and partial (P = 0.0202) hydatidiform mole compared with normal early pregnancy. The implications of altered Fas/FasL status of decidual T-cell subsets in hydatidiform mole are also discussed.     

Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system

The first known hormonal signal of the conceptus during implantation is human chorionic gonadotropin (hCG). Interestingly, increased apoptosis in human endometrium coincides with the implantation window. Factors from the fetal or placental origin as well as maternal hormonal factors are likely to have a potential role in the regulation of apoptotic signaling molecules. We hypothesized that hCG may be a placental link for the development of local maternal immunotolerance. Fas-Fas ligand (FasL) system is one of the apoptotic signaling pathways, shown to be important in the development of local immune tolerance during and after implantation. We report that hCG treatment decreases cell proliferation and increases apoptosis in endometrial cells. Moreover, hCG stimulates FasL mRNA and protein expression without affecting Fas mRNA in these cells. Interestingly, in coculture experiments, hCG-treated endometrial cells induce an increase in T cell apoptosis. Our in vivo results reveal that cells of early pregnancy decidua express strong FasL immunoreactivity, and decidual areas containing interstitial cytotrophoblasts have numerous TUNEL-positive cells. Compared with decidual areas devoid of interstitial cytotrophoblasts, we observed in decidual areas containing interstitial cytotrophoblasts clearly less amount of TUNEL-positive cells. These results suggest that hCG may be a link in the development of peritrophoblastic immune tolerance and may facilitate the trophoblast invasion by regulating proapoptotic molecules such as FasL in endometrial cells.     

Effects of Maternal Smoking on the Placental Expression of Genes Related to Angiogenesis and Apoptosis during the First Trimester

Objective

Maternal cigarette smoking is reportedly associated with miscarriage, fetal growth restriction and placental abruption, and is paradoxically associated with a decreased risk of developing preeclampsia. In the present study, we investigated the gene expression levels of villous tissues in early gestation. We compared the expression levels of the genes related to angiogenesis and apoptosis in the villous tissues obtained from smoking and non-smoking pregnant women.

Materials and Methods

We collected villous tissue samples from 57 women requesting surgical termination due to non-medical reasons at 6–8 weeks of gestation. The maternal cigarette smoking status was evaluated by the level of serum cotinine and patients were divided into active smokers and non-smokers by the serum cotinine level. The placental levels of VEGFA, PGF, FLT1, HIF1A, TP53, BAX and BCL2 mRNA were quantified by real time PCR.

Results

The gene expression level of PGF and HIF1A in the active smoker group was significantly higher than that in the non-smoker group. We did not observe any significant differences in the VEGFA or FLT1 expression between the groups. In active smoker group, the gene expression levels of TP53 and BAX were significantly higher than those in the non-smoker group. The ratio of BAX/BCL2 mRNA in the active smoker group was significantly higher than that in the non-smoker group.

Conclusions

Our findings revealed that smoking might affect the placenta during early pregnancy. Maternal cigarette smoking in early pregnancy may be associated with villus hypoxia, which may influence angiogenesis and apoptosis.     

Fetal growth restriction triggered by polycyclic aromatic hydrocarbons is associated with altered placental vasculature and AhR-dependent changes in cell death

Maternal cigarette smoking is considered an important risk factor associated with fetal intrauterine growth restriction (IUGR). Polycyclic aromatic hydrocarbons (PAHs) are well-known constituents of cigarette smoke, and the effects of acute exposure to these chemicals at different gestational stages have been well established in a variety of laboratory animals. In addition, many PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a cellular xenobiotic sensor responsible for activating the metabolic machinery. In this study, we have applied a chronic, low-dose regimen of PAH exposure to C57Bl/6 female mice before conception. This treatment caused IUGR in day 15.5 post coitum (d15.5) fetuses and yielded abnormalities in the placental vasculature, resulting in significantly reduced arterial surface area and volume of the fetal arterial vasculature of the placenta. However, examination of the small vasculature within the placental labyrinth of PAH-exposed dams revealed extensive branching and enlargement of these vessels, indicating a possible compensatory mechanism. These alterations in vascularization were accompanied by reduced placental cell death rates, increased expression levels of antiapoptotic Xiap, and decreased expression of proapoptotic Bax, cleaved poly(ADP-ribose) polymerase-1, and active caspase-3. AhR-deficient fetuses were rescued from PAH-induced growth restriction and exhibited no changes in the labyrinthine cell death rate. The results of this investigation suggest that chronic exposure to PAHs is a contributing factor to the development of IUGR in human smokers and that the AhR pathway is involved.     

Fas, FasL, Bcl-2, and Bax in the endometrium of rhesus monkey during the menstrual cycle

To study possible role and regulation of apoptosis occurred in primate endometrium, the expression of apoptosis-related molecules, Fas, FasL, B cell lymphoma/leukaemia-2 (Bcl-2), and Bax were analyzed in relation to occurrence of apoptosis and proliferation in the cycling endometrium of the rhesus monkey using immunohistochemistry and Western blot. The cell apoptosis and proliferation were evaluated by means of in situ 3'-end labeling and Ki67 immunostaining, respectively. The results showed that the expressions of Fas, Fas ligand (FasL), Bcl-2, and Bax were co-localized predominantly in the epithelial cells of the endometrium. Modest Fas staining with no obvious change was detected throughout the menstrual cycle, while the levels of FasL and Bax protein in the epithelial cells increased in the secretory phase when apoptosis was most prevalent. In contrast, epithelial immunostaining for Bcl-2 was maximal during the proliferative phase and decreased in the secretory phase. Bcl-2 immunoreactivity was also detected in some immunocytes. The coordinated expression of Fas, FasL, Bcl-2, and Bax in the cycling endometrium of the rhesus monkey suggests that the cyclic changes in endometrial growth and regression may be regulated by the balance of these factors under the action of ovary steroids.     

Effect of maternal chronic hypoxic exposure during gestation on apoptosis in fetal rat heart

Chronic hypoxia during pregnancy is one of the most common insults to fetal development. We tested the hypothesis that maternal hypoxia induced apoptosis in the hearts of near-term fetal rats. Pregnant rats were divided into two groups, normoxic control and continuous hypoxic exposure (10.5% O2) from day 15 to 21 of gestation. Hearts were isolated from fetal rats of 21-day gestational age. Maternal hypoxia increased hypoxia-inducible factor-1alpha protein in fetal hearts. Chronic hypoxia significantly increased the percentage and size of binucleated myocytes and increased apoptotic cells from 1.4 +/- 0.14% to 2.7 +/- 0.3% in the fetal heart. In addition, the active cleaved form of caspase 3 was significantly increased in the hypoxic heart, which was associated with an increase in caspase 3 activity. There was a significant increase in Fas protein levels in the hypoxic heart. Chronic hypoxia did not change Bax protein levels but significantly decreased Bcl-2 proteins. In addition, chronic hypoxia significantly suppressed expression of heat shock protein 70. However, chronic hypoxia significantly increased expression of the anti-apoptotic protein 14-3-3, among other 14-3-3 isoforms. Chronic hypoxia differentially regulated beta-adrenoreceptor (beta-AR) subtypes with an increase in beta1-AR levels but no changes in beta2-AR. The results demonstrate that maternal hypoxia increases apoptosis in fetal rat heart, which may be mediated by an increase in Fas and a decrease in Bcl-2 proteins. Chronic hypoxia-mediated increase in beta1-AR and decrease in heat shock proteins may also play an important role in apoptosis in the fetal heart.     

Osteoprotegerin Induces Apoptosis of Osteoclasts and Osteoclast Precursor Cells via the Fas/Fas Ligand Pathway

Osteoprotegerin (OPG) is known to inhibit differentiation and activation of osteoclasts (OCs) by functioning as a decoy receptor blocking interactions between RANK and RANKL. However, the exact role of OPG in the survival/apoptosis of OCs remains unclear. OPG caused increased rates of apoptosis of both OCs and osteoclast precursor cells (OPCs). The expression of Fas and activated caspase-8 was increased by both 20 ng/mL and 40 ng/mL of OPG, but was markedly decreased at 80 ng/mL. Interestingly, we noted that while levels of Fas ligand (FasL) increased with increasing doses of OPG, the soluble form of FasL in the supernatant decreased. The results of a co-immunoprecipitation assay suggested that the decrease of sFasL might be caused by the binding of OPG. This would block the inhibition of the apoptosis of OCs and OPCs. Furthermore, changes in expression levels of Bax/Bcl-2, cleaved-caspase-9, cleaved-caspased-3 and the translocation of cytochrome c, illustrated that OPG induced apoptosis of OCs and OPCs via the classic Fas/FasL apoptosis pathway, and was mediated by mitochondria. Altogether, our results demonstrate that OPG induces OCs and OPCs apoptosis partly by the Fas/FasL signaling pathway.     

Proliferation of villous trophoblast of the human placenta in normal and abnormal pregnancies.

The proliferation of villous trophoblast in the human placenta was estimated throughout normal gestation and in term placentae from preeclamptic and smoking mothers by two different methods. These were: 1) labeling of DNA producing cells by bromodeoxyuridine (BrdU) followed by immunohistochemistry using a monoclonal anti-BrdU antibody, and 2) immunohistochemical identification of all proliferating cells by the monoclonal antibody Ki67. Both methods revealed comparable results. In uncomplicated pregnancies there was a remarkable decrease in the labeling indices from early gestation to term. This was the result of a diminution of the number of Langhans' cells, although the cell division rate within the Langhans' cell layer remained nearly constant throughout gestation. A prolongation of the cell cycle in the cytotrophoblast cells at term was indicated by an increase in the Ki67/BrdU ratio. Compared with normal term placentae, there was an increase in the trophoblast proliferation rate in preeclampsia, but not in placentae from smoking mothers. Moreover, the number of Langhans' cells was diminished in placentae from smokers. The results indicate that there are different pathogenetic mechanisms of placental impairment in preeclampsia and in maternal smoking. In preeclampsia an injury to the syncytiotrophoblast seems to lead to a repair hyperplasia of the cytotrophoblast, whereas in maternal smoking, there seems to be a direct toxic effect on the cytotrophoblastic cells.     

Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25-31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 +/- 0.34% on gestational day 27 to 3.34 +/- 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.     

Increased immortalization‐upregulated protein 2 (IMUP‐2) by hypoxia induces apoptosis of the trophoblast and pre‐eclampsia

In regulation of the developmental process, the balance between cellular proliferation and cell death is critical. Placental development tightly controls this mechanism, and increased apoptosis of placental trophoblasts can cause a variety of gynecological diseases. Members of the immortalization‐upregulated protein (IMUP) family are nuclear proteins implicated in SV40‐mediated immortalization and cellular proliferation; however, the mechanisms by which their expression is regulated in placental development are still unknown. We compared IMUP‐2 expression in normal and pre‐eclamptic placental tissues and evaluated the function of IMUP‐2 in HTR‐8/SVneo trophoblast cells under hypoxic conditions. IMUP‐2 was expressed in syncytiotrophoblasts and syncytial knots of the placental villi. IMUP‐2 expression was significantly higher in preterm pre‐eclampsia patients than in patients who went to term (P < 0.001); however, we observed no differences in IMUP‐2 expression between normal term patients with and without pre‐eclampsia. Hypoxic conditions increased apoptosis of HTR8/SVneo trophoblast cells and induced IMUP‐2 expression. Also, apoptosis of HTR‐8/SVneo trophoblast cells was increased after IMUP‐2 gene transfection. These results suggest that IMUP‐2 expression is specifically elevated in preterm pre‐eclampsia and under hypoxic conditions, and that IMUP‐2 induces apoptosis of the trophoblast. Therefore, IMUP‐2 might have functional involvement in placental development and gynecological diseases such as pre‐eclampsia. J. Cell. Biochem. 110: 522–530, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptosis and chemoresistance in human ovarian cancer: is Xiap a determinant?

Cisplatin-induced apoptosis in epithelial ovarian cancer cells is in part a consequence of suppressed Xiap expression and upregulation of the Fas/FasL system. Changes in the expression of these 'cell death' and 'cell survival' genes lead to activation of caspase-3, and cleavage of MDM2 and FAK. Failure of cancer cells to maintain a balance in the expression of these genes in favor of apoptotic cell death may be an important factor of chemoresistance. Xiap may be a novel target for gene therapy of human ovarian epithelial cancer and, dependent on P53 status, expression of Xiap antisense alone or in combination with wild-type P53 sense may offer a new approach for the treatment of the chemoresistant cancer.     

Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis.     

Lysophosphatidylcholine-induced apoptosis in H19-7 hippocampal progenitor cells is enhanced by the upregulation of Fas Ligand

Lysophospholipids regulate a wide array of biological processes including apoptosis and neutrophil migration. Fas/Apo-1 and its ligand (FasL) participate in neuronal cell apoptosis causing various neurological diseases. Here, we use hippocampal neuroprogenitor cells to investigate how lysophosphatidylcholine (LPC) induces apoptosis in H19-7 hippocampal progenitor cells via Fas/Fas ligand-mediated apoptotic signaling pathway. Exposed cells with LPC presented on apoptotic morphology, positive TUNEL staining, and DNA fragmentation. We found that the expression of FasL was increased after LPC treatment. Furthermore, LPC-induced H19-7 cell apoptosis was decreased by agonistic anti-FasL antibody. In addition to promotion of caspase cascade activity by LPC, the administration of the caspase inhibitor, DEVD-fmk, prevented H19-7 cell apoptosis. LPC also increased the activation of nuclear factor-κB (NF-κB), which in turn, significantly increased FasL mRNA level. The increase in FasL mRNA level by NF-κB transfection was significantly decreased in the presence of IκB-SR, a super-repressor of IκB. Taken together, these results demonstrate that LPC has the ability to induce apoptosis in H19-7 cells through the upregulation of FasL expression via NF-κB activation.