Apoptosis In Placental Trophoblasts Research Articles

Research on the Improvement of Endothelial Cell Function and Trophoblast Apoptosis in Rats with Preeclampsia by Tanshinone IIA

Background Preeclampsia is a pregnancy complication characterized by high blood pressure and signs of damage to another organ system, often the kidneys. Recent studies suggest that endothelial dysfunction and trophoblast apoptosis are involved in the pathogenesis of preeclampsia. Purpose To investigate the effects of tanshinone IIA (TIIA) on clinical symptoms, vascular endothelial function, and placental trophoblast apoptosis in preeclampsia rats. Materials and Methods Twenty-four pregnant Sprague-Dawley rats, between 8 and 10 weeks old, were randomly divided and allocated into three groups: Control, model, and treatment, with each group consisting of 8 rats. The control group received normal saline injections via the tail vein; the model group received NG-nitro-l-arginine methyl ester (l-NAME) to induce preeclampsia, followed by normal saline injections; and the treatment group received TIIA (10 mg/kg) via tail vein injection after preeclampsia induction with l-NAME. Various parameters were measured, including tail artery systolic pressure, 24-hour proteinuria, offspring and placental weight, levels of endothelial nitric oxide synthase (eNOS), endothelin-1 (ET-1), placental growth factor (PLGF), and soluble FMS-like tyrosine kinase 1 (sFlt-1) in serum, and expression of B-cell lymphoma-2 (Bcl-2) and Bcl-2-related X protein (Bax) in placental trophoblasts. Results TIIA treatment led to decreased tail artery systolic pressure and 24-hour urine protein levels, increased body and placental weights of offspring, and favorable changes in serum levels of ET-1, sFlt-1, eNOS, and PLGF. Moreover, TIIA treatment resulted in decreased Bax levels and apoptosis in placental trophoblasts, along with increased Bcl-2 levels. Conclusion TIIA can improve the clinical symptoms of preeclampsia in rats induced by l-NAME, alleviate the apoptosis of placental trophoblast cells, and improve vascular endothelial function.

Transcutaneous Auricular Vagus Nerve Stimulation Ameliorates Preeclampsia-Induced Apoptosis of Placental Trophoblastic Cells Via Inhibiting the Mitochondrial Unfolded Protein Response.

Preeclampsia is a serious obstetric complication. Currently, there is a lack of effective preventive approaches for this disease. Recent studies have identified transcutaneous auricular vagus nerve stimulation (taVNS) as a potential novel non-pharmaceutical therapeutic modality for preeclampsia. In this study, we investigated whether taVNS inhibits apoptosis of placental trophoblastic cells through ROS-induced UPRmt. Our results showed that taVNS promoted the release of acetylcholine (ACh). ACh decreased the expression of UPRmt by inhibiting the formation of mitochondrial ROS (mtROS), presumably through M3AChR. This reduced the release of pro-apoptotic proteins (cleaved caspase-3, NF-κB-p65, and cytochrome C) and helped preserve the morphological and functional integrity of mitochondria, thus reducing the apoptosis of placental trophoblasts, improving placental function, and relieving preeclampsia. Our study unravels the potential pathophysiological mechanism of preeclampsia. In-depth characterization of the UPRmt is essential for developing more effective therapeutic strategies for preeclampsia targeting mitochondrial function.

MiR 19a promotes proliferation and inhibits apoptosis of placental trophoblasts via PTEN pathway

Purpose: To screen the micro-ribonucleic acids (miRNAs) in the pathological processes of preeclampsia (PE), and to assess their effects on the proliferation and apoptosis of HTR-8/SVneo cells.
 Methods: The miRNAs in PE patients and healthy puerperae were screened out using miRNA microarray assay, and verified through low-throughput quantitative reverse transcription- polymerase chain reaction (qRT-PCR). The effects of expression level of miR-19a on proliferation and cycle of HTR-8/SVneo cells were determined using cell counting kit (CCK)-8 assay and propidium iodide (PI) staining, respectively. The influence of the expression level of miR-19a on apoptosis of HTR-8/SVneo cells was evaluated using Annexin V-fluorescein isothiocyanate (FITC)/PI staining. Subsequently, luciferase reporter gene assay, CCK-8 assay and Annexin V-FITC/PI staining were conducted, respectively.
 Results: Serum miR-19a was significantly decreased in PE patients, and a high level of miR-19a led to increase in the proliferation of HTR-8/SVneo cells. Decreased level of miR-19a arrested HTR-8/SVneo cell cycle at G1 phase, while increase in the miR-19a level repressed the apoptosis of HTR-8/SVneo cells. The PTEN was a direct target of miR-19a, and overexpression of miR-19a promoted the activation of Akt signaling pathway, thereby significantly decreasing the expression levels of downstream phosphorylated p21/p21 (p-p21/p21), p-p27/p27 and p-glycogen synthase kinase-3 beta (GSK3β)/GSK3β. Compared with those in cells transfected with empty vectors, the pro-proliferative and anti-apoptotic effects of miR-19a weakened in HTR-8/SVneo cell lines with overexpressed PTEN.
 Conclusion: MiR-19a targets PTEN in order to activate Akt signaling pathway, thereby promoting proliferation, and inhibiting apoptosis of HTR-8/SVneo cells. This provides new drug targets that can be investigated for future treatment of PE.

RBM10 Regulates Embryonic Trophoblast Injury via Endoplasmic Reticulum Stress

Background: Previous studies shown that RNA binding motif proteins (RBM) participate in regulating various physiological processes such as cell autophagy, proliferation, and apoptosis, and are abnormally highly expressed in placental trophoblast cells intervened by hypoxia in vitro, but their molecular mechanisms regulating placental trophoblast damage remain unclear. This study aims to investigate the role and molecular mechanism of RBM10 in regulating hypoxia-induced placental trophoblast injury through endoplasmic reticulum stress. Methods: CCK-8 cell proliferation assay and Transwell cell invasion assay were applied to detect the proliferation and invasion ability of normal, hypoxic and RBM10 up-regulated plus hypoxic embryonic trophoblast cells, respectively. The expression of endoplasmic reticulum stress-related proteins (ERN-1) and C/EBP homologous protein (CHOP), apoptosis-related proteins B cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Caspase-3, and autophagy-related proteins including microtubule-associated protein 1 light chain 3 (LC-3), Beclin-1 and P62 were also detected by western blot assays. The effects of hypoxia and overexpression of RBM10 on placental trophoblast apoptosis were examined using flow cytometry. Results: We found that the growth and invasion ability of placental trophoblast cells treated with hypoxia were significantly decreased (p < 0.05), and the upregulation of RBM10 further led to the decrease of the growth and invasion ability of hypoxic placental trophoblast cells. In addition, hypoxia promoted the expression of endoplasmic reticulum stress-related proteins (p < 0.05), which triggered apoptosis and autophagy of embryonic trophoblast cells. The data showed that embryonic trophoblast cells regulated cell injury by stimulating endoplasmic reticulum stress after hypoxia. after upregulation of RBM10 expression, the expression levels of endoplasmic reticulum stress-related proteins ERN-1 and CHOP were further increased (p < 0.05), and the apoptosis rate of embryonic trophoblast cells was further increased (p < 0.05). Conclusions: Overall, our findings suggest that post-hypoxia mediates autophagy in embryonic trophoblast cells through stimulation of endoplasmic reticulum stress, thereby promoting apoptosis. Overexpression of RBM10 levels regulates the proliferative, apoptotic capacity of trophoblast cells by affecting cellular endoplasmic reticulum stress. RBM10 plays an important role in regulating hypoxia-induced autophagy and apoptosis in trophoblast cells, and RBM10 upregulation can further stimulate endoplasmic reticulum stress-mediated autophagy and apoptosis in trophoblast cells.

Autophagic flux inhibition, apoptosis, and mitochondrial dysfunction in bile acids‐induced impairment of human placental trophoblast

Intrahepatic cholestasis of pregnancy (ICP) is a common pregnancy-specific disease, characterized by increased bile acid levels and adverse fetal outcomes. We previously reported excessive bile acids led to dysfunction of placental trophoblasts in ICP. However, the detailed mechanism is still unclear. Autophagy is fundamental process for protecting cell survival against adverse conditions. Here, we evaluated the effect of increased concentration of bile acids on autophagy in trophoblasts in vitro and in vivo. First, we demonstrated that the autophagy substrate p62/sequestosome-1 was accumulated in placental tissues from patients with ICP and in human trophoblasts treated with hydrophobic bile acids, including chenodeoxycholic acid and deoxycholic acid. Furthermore, we found that treatment with hydrophobic bile acids impaired autophagic flux in both time- and concentration-dependent manners, by suppressing the AMP-activated protein kinase/unc-51-like kinase 1 autophagic signaling pathway. Notably, trophoblasts were prone to apoptotic cell death upon starvation along with bile-acids treatment in vitro or in an ICP mouse model in vivo. Additionally, we revealed mitochondrial dysfunction was the predominant biological process in excessive bile acids induced trophoblast impairment under starvation by proteomic assay. Collectively, our study proposed a complex interaction of excessive bile acids induced autophagic flux, mitochondrial dysfunction, and cellular apoptosis in placental trophoblasts may play a critical role in the pathogenesis of ICP.

Novel piRNA Regulates PIWIL1 to Modulate the Behavior of Placental Trophoblast Cells and Participates in Preeclampsia.

Objectives This study is aimed at investigating the role of PIWIL1/piRNA in the development of preeclampsia. Methods High-throughput sequencing was performed in 5 preeclampsia and 5 normal placentas to get a piRNA expression profile. WGCNA network was constructed to find hub piRNAs. Through target gene prediction and protein interaction network analysis, we found the potential relationship between the key genes and PIWIL1. Subsequently, we detected the expression of PIWIL1 in 35 preeclampsia and 29 normal placental tissues. Overexpression and inhibition of PIWIL1 in HTR-8/SVneo trophoblast cells were achieved by transfecting an overexpression vector and siRNAs, respectively. Cell proliferation, apoptosis, and invasion were assessed using CCK-8, flow cytometric, and transwell assays, respectively. Results It was found that a total of three piRNAs were upregulated in preeclampsia (pir-hsa-1256314, uniq_271431, and uniq_277797). And two target genes with the highest connectivity (FXR1 and DDX6) both pointed to PIWIL1. PIWIL1 expression was significantly lower in preeclampsia. In vitro studies linked PIWIL1 expression to trophoblast overgrowth. Overexpression of PIWIL1 remarkably promoted cell proliferation and invasion and inhibited apoptosis of HTR-8/SVneo cells and vice versa. Conclusions PIWIL1/piRNA may be involved in the pathogenesis of preeclampsia by inhibiting the proliferation and invasion and promoting the apoptosis of placental trophoblasts. This study was registered with the China Clinical Trials Registry (http://www.clinicaltrials.gov): registration number ChiCTR1900027479.

Circ_0060731 mediated miR-21–5p-PDCD4/ESR1 pathway to induce apoptosis of placental trophoblasts in intrahepatic cholestasis of pregnancy

PurposeIntrahepatic cholestasis of pregnancy (ICP) is a pregnancy complication. However, the pathogenesis of ICP is currently unclear. MethodsWe analyzed the placenta samples of 10 normal and 10 ICP pregnant women. the expressions of circ0060731, miR-21–5p, and their downstream target genes PDCD4, ESR1, and apoptotic protein cleaved-caspase3 were detected in the cell model. ResultsThe expression of Circ_0060731, PDCD4, ESR1, and caspase-3 was higher in the ICP placenta tissue than in the control group, and the expression of miR-21–5p was lower in the ICP group than in the control group. In HTR8/Svneo cells treated with TCA, the expression/levels of Circ_0060731, PDCD4, ESR1, and caspase-3 were significantly higher in the ICP group than in the control group, and miR-21–5p was significantly lower in the ICP group than in the control group. Lentiviral knockdown of miR-21–5p significantly increased the expressions of its downstream genes of PDCD4 and ESR1, and also increased cell apoptosis. Overexpression of miR-21–5p significantly reduced the expression of PDCD4 and ESR1 and reduced cell apoptosis. The dual-luciferase experiment showed that both PDCD4 and ERS1 were the target genes of miR-21–5p. ConclusionCirc_0060731 mediated miR-21–5p-PDCD4/ESR1 pathway could induce apoptosis of placental trophoblasts in intrahepatic cholestasis of pregnancy.

Gestational exposure to environmental cadmium induces placental apoptosis and fetal growth restriction via Parkin-modulated MCL-1 degradation

Heavy metal cadmium (Cd), a classical environmental pollutant, causes placental apoptosis and fetal growth restriction (FGR), whereby the mechanism remains unclear. Here, our human case-control study firstly showed that there was a positive association of Parkin mitochondrial translocation, MCL-1 reduction, placental apoptosis, and all-cause FGR. Subsequently, Cd was administered to establish in vitro and in vivo models of placental apoptosis or FGR. Our models demonstrated that Parkin mitochondrial translocation was observed in Cd-administrated placental trophoblasts. Meaningfully, Parkin siRNA (siR) dramatically mitigated Cd-triggered apoptosis in placental trophoblasts. Mdivi-1 (M-1), an inhibitor for Parkin mitochondrial translocation, mitigated Cd-induced apoptosis in placental trophoblasts, which further ameliorated the effect of attenuated placental sizes in Cd-exposed mice. Furthermore, the interaction of MCL-1 with Parkin or Ub in Cd-stimulated cells was stronger than that in controls. MG132, an inhibitor for proteasome, abolished MCL-1 degradation in Cd-stimulated cells. Importantly, Parkin siR and M-1 memorably abolished the ubiquitin-dependent degradation of MCL-1 in placental trophoblasts. Interestingly, mito-TEMPO and melatonin, two mitochondria-targeted antioxidants, obviously rescued Cd-caused mitochondrial membrane potential (MMP) decrease, Parkin mitochondrial translocation, MCL-1 degradation, and apoptosis in placental trophoblasts. In conclusion, cadmium induces placental apoptosis and FGR via mtROS-mediated Parkin-modulated degradation of MCL-1.

Fas regulates the apoptosis and migration of trophoblast cells by targeting NF-κB.

Placental trophoblast apoptosis is a major pathological feature of preeclampsia. Fas has been reported to be highly expressed in the placentas of patients with preeclampsia. However, the role and underlying mechanisms of Fas in the pathogenesis of preeclampsia have not been elucidated. In the present study, the expression of Fas in JAR human choriocarcinoma cells was overexpressed and knocked down to determine the function and possible mechanism of Fas in trophoblast cells in the progression of preeclampsia. The results of flow cytometry, Cell Counting Kit-8 and Transwell assays indicated that the overexpression of Fas promoted apoptosis, suppressed viability and impaired the migration of the human trophoblast cells. In addition, western blotting revealed that the overexpression of Fas increased the expression of nuclear factor kB (NF-kB), Bax, tumor necrosis factor α (TNF-α) and interleukin-2 (IL-2), and decreased the expression of Bcl-2 at the protein level in trophoblast cells. By contrast, the knockdown of Fas decreased the apoptosis of trophoblast cells and increased their viability and migration. In addition, the knockdown of Fas suppressed the expression of NF-κB, Bax, TNF-α and IL-2, and increased the expression of Bcl-2. Notably, the overexpression of NF-κB p65 attenuated the Fas knockdown-induced inhibition of apoptosis and acceleration of migration of the trophoblast cells. The overexpression of NF-κB in trophoblast cells also reversed the reduction in Bax expression and increase in Bcl-2 expression induced by Fas knockdown in trophoblast cells. These results indicate that Fas regulates the apoptosis and migration of trophoblast cells by targeting NF-κB, which suggests that the silencing of Fas is a promising therapeutic strategy for preeclampsia.

Palmitoleate Protects against Zika Virus-Induced Placental Trophoblast Apoptosis.

Zika virus (ZIKV) infection in pregnancy is associated with the development of microcephaly, intrauterine growth restriction, and ocular damage in the fetus. ZIKV infection of the placenta plays a crucial role in the vertical transmission from the maternal circulation to the fetus. Our previous study suggested that ZIKV induces endoplasmic reticulum (ER) stress and apoptosis of placental trophoblasts. Here, we showed that palmitoleate, an omega-7 monounsaturated fatty acid, prevents ZIKV-induced ER stress and apoptosis in placental trophoblasts. Human trophoblast cell lines (JEG-3 and JAR) and normal immortalized trophoblasts (HTR-8) were used. We observed that ZIKV infection of the trophoblasts resulted in apoptosis and treatment of palmitoleate to ZIKV-infected cells significantly prevented apoptosis. However, palmitate (saturated fatty acid) did not offer protection from ZIKV-induced ER stress and apoptosis. We also observed that the Zika viral RNA copies were decreased, and the cell viability improved in ZIKV-infected cells treated with palmitoleate as compared to the infected cells without palmitoleate treatment. Further, palmitoleate was shown to protect against ZIKV-induced upregulation of ER stress markers, C/EBP homologous protein and X-box binding protein-1 splicing in placental trophoblasts. In conclusion, our studies suggest that palmitoleate protects placental trophoblasts against ZIKV-induced ER stress and apoptosis.

Syncytin-A Knockout Induces Placental Developmental Abnormalities Partially through Calpain1-Apoptosis-Inducing Factor-Mediated Trophoblast Apoptosis

Abstract Objective: Structural abnormalities and dysfunction of the placenta contribute to pregnancy-related complications, such as preeclampsia. Syncytin-A (synA) has been reported to be expressed in the placenta. The contribution of synA to developmental abnormalities and dysfunction of the placenta remains elusive. In this study, we aimed to explore the role of synA in placental development and functions. Methods: SynA-knockout mice were generated using the CRISPR-Cas9 method, and the phenotypes of the placenta and fetus of synA-knockout mice were observed. Real-time quantitative polymerase chain reaction (PCR) and routine PCR were employed to detect the genotypes of the offspring. CD31 immunohistochemistry was used to evaluate the vessel density of the placenta, and the protein levels of key molecules were measured by western blotting. Results: SynA knockout caused fetal death. Furthermore, synA-knockout mice showed placental developmental abnormalities, indicated by a thinner labyrinth layer, thicker spongiotrophoblast layer, lower blood vessel density, and significantly higher numbers of apoptotic trophoblasts, when compared with wild-type littermates. Mechanistically, synA ablation induced apoptosis-inducing factor (AIF) cleavage and nuclear localization and promoted placental trophoblast apoptosis. In addition, synA knockout increased the calpain1 protein levels. The calpain1 inhibitor calpeptin blocked synA knockout-induced AIF cleavage, partially restoring the placental structural abnormalities of synA-knockout mice. Conclusions: SynA knockout leads to placental developmental abnormalities by inducing trophoblastic apoptosis via the calpain1-AIF pathway.

Down\u2010regulation of the Sp1 transcription factor by an increase of microRNA-4497\xa0in human placenta is associated with early recurrent miscarriage

BackgroundThe pathophysiological mechanism of recurrent miscarriage (RM) is unclear. The goals of this study were to determine the role of microRNA-4497 overexpression in placental villus tissues in early RM; To identify the potential target mRNAs of miRNA-4497; And to investigate the microRNA-4497-mediated regulatory mechanisms in placental trophoblasts.MethodsBioinformatics analysis was performed to identify the candidate target genes of miRNA-4497. The protein expression of Sp1 transcription factor (SP1), chemokine (C-X-C motif) receptor 5 (CXCR5) and bone morphogenetic protein 8a (BMP8A) were determined in the villus tissues of the RM and normal groups by Western blotting and immunohistochemistry. Cultured 293T cells were co-transfected with the miRNA-4497 agomir or luciferase reporter vectors containing the wild-type or mutant 3’-UTRs of the target mRNAs to verify the regulatory role of miRNA-4497.ResultsBioinformatics analysis suggested that SP1, CXCR5 and BMP8A mRNAs are potential targets of miRNA-4497. The expression of SP1, CXCR5 and BMP8A proteins in the chorionic villus tissues of RM placentas were significantly decreased compared to those in the normal controls. Moreover, SP1 protein levels were inversely correlated with the levels of miRNA-4497 in the placentas of RM patients and normal controls. The expression of SP1 mRNA and protein were down-regulated in HTR-8/SVneo cells after forced overexpression of the miRNA-4497 agomir. The results of the co-transfection assay showed that mutation of the miRNA-4497-binding sites in the 3’-untranslated region (3’-UTR) of SP1 led to a recovery of luciferase activity upon overexpression of miRNA-4497, suggesting that SP1 could be a direct target of miRNA-4497.ConclusionsAn increased miRNA-4497 level in the placental villus tissues associated with recurrent miscarriage may down-regulate SP1 expression. The negative regulation of SP1 by miRNA-4497 may potentially contribute to the pathogenesis of recurrent miscarriage through promotion of trophoblast apoptosis. These findings provide novel information on the regulation of placental trophoblast apoptosis, and could be useful for the development of new therapeutic strategies for better management of recurrent miscarriage.

Zika virus infection induces endoplasmic reticulum stress and apoptosis in placental trophoblasts

Zika virus (ZIKV) infection to a pregnant woman can be vertically transmitted to the fetus via the placenta leading to Congenital Zika syndrome. This is characterized by microcephaly, retinal defects, and intrauterine growth retardation. ZIKV induces placental trophoblast apoptosis leading to severe abnormalities in the growth and development of the fetus. However, the molecular mechanism behind ZIKV-induced apoptosis in placental trophoblasts remains unclear. We hypothesize that ZIKV infection induces endoplasmic reticulum (ER) stress in the trophoblasts, and sustained ER stress results in apoptosis. HTR-8 (HTR-8/SVneo), a human normal immortalized trophoblast cell and human choriocarcinoma-derived cell lines (JEG-3 and JAR) were infected with ZIKV. Biochemical and structural markers of apoptosis like caspase 3/7 activity and percent apoptotic nuclear morphological changes, respectively were assessed. ZIKV infection in placental trophoblasts showed an increase in the levels of CHOP mRNA and protein expression, which is an inducer of apoptosis. Next, we also observed increased levels of ER stress markers such as phosphorylated forms of inositol-requiring transmembrane kinase/endoribonuclease 1α (P-IRE1α), and its downstream target, the spliced form of XBP1 mRNA, phosphorylated eukaryotic initiation factor 2α (P-eIF2α), and activation of cJun N-terminal Kinase (JNK) and p38 mitogen activated protein kinase (MAPK) after 16–24 h of ZIKV infection in trophoblasts. Inhibition of JNK or pan-caspases using small molecule inhibitors significantly prevented ZIKV-induced apoptosis in trophoblasts. Further, JNK inhibition also reduced XBP1 mRNA splicing and viral E protein staining in ZIKV infected cells. In conclusion, the mechanism of ZIKV-induced placental trophoblast apoptosis involves the activation of ER stress and JNK activation, and the inhibition of JNK dramatically prevents ZIKV-induced trophoblast apoptosis.

Zika virus infection induces endoplasmic reticulum stress and apoptosis in placental trophoblasts

Zika virus (ZIKV) infection in pregnant woman can be vertically transmitted to the fetus via the placenta leading to Congenital Zika syndrome (CZS). CZS is characterized by microcephaly, retinal defects and intrauterine growth retardation. ZIKV also causes placental pathology leading to severe compromise in the growth and development of the fetus. Further, the molecular mechanism behind ZIKV‐induced apoptosis in placental trophoblasts is unknown. We hypothesize that accumulation of viral proteins in the endoplasmic reticulum could lead to sustained endoplasmic reticulum stress (ER stress) and trigger apoptotic events.MethodsHTR‐8, a human normal immortalized trophoblast cell and human choriocarcinoma derived cell lines (JEG‐3 and JAR) were infected with 0.1–1MOI ZIKV. Apoptosis was assessed by characteristic nuclear morphology staining with DAPI and caspase 3/7 activity.ResultsWe observed an increase in the mRNA levels of CHOP and the spliced form of XBP1 gene, 16–24h post infection in trophoblast. We also observed an increase in the levels of ER stress markers such as p‐IRE1α, p‐eif2α, and activation of c‐Jun N‐terminal Kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) after 16–24h of ZIKV infection in trophoblast. As prolonged ER stress can cause apoptosis, we observed a dramatic increase in trophoblast apoptosis 48h post infection. Mechanistically, inhibition of JNK by SP600125 or pan caspase by Z‐VAD‐FMK significantly blocked ZIKV‐induced apoptosis in trophoblast. In conclusion, the mechanism of ZIKV‐induced placental trophoblast apoptosis involves the activation of ER stress and MAPK activation.Support or Funding InformationNIH: P20GM104320, Layman Seed award, UNL, Food for Health Research Initiative, UNL

MiR-200c regulates apoptosis of placental trophoblasts in preeclampsia rats through Wnt/β-catenin signaling pathway.

To investigate the influence of micro ribonucleic acid 200c (miR-200c) on the apoptosis of placental trophoblasts in a rat model of preeclampsia (PE). PE model in rats was established for extracting placental trophoblasts. Overexpression or knockdown of miR-200c was achieved by transfection of miR-200c mimics or inhibitor. Flow cytometry was carried out to detect the apoptotic rate of placental trophoblasts. Dual-luciferase reporter gene assay was performed to detect the interaction of miR-200c with WNT1. Western blotting was applied to determine the changes of protein levels in placental trophoblasts. The expression level of miR-200c in placental trophoblasts of PE group was significantly higher than that in control group. The apoptosis rate was (22.45 ± 2.62)%, (6.58 ± 1.28)%, and (9.57 ± 1.35)% in miR-200c mimic group, miR-200c inhibitor group, and control group, respectively, showing statistically significant differences. MiR-200c overexpression downregulated the expression level of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), but upregulated expression levels of apoptotic proteins Bcl-2-associated X protein (Bax) and active Caspase-3. MiR-200c suppressed WNT1 expression through the interaction with the 3'-untranslated region (3'-UTR) of WNT1. The expressions of WNT1 and β-catenin were up-regulated after miR-200c overexpression, which was reversed by the Wnt/β-catenin pathway activator. MiR-200c is involved in the development and progression of PE through the Wnt/β-catenin signaling pathway.

Saturated Free Fatty Acids Induce Trophoblast Lipoapoptosis

Obesity during pregnancy increases the risk for maternal complications like gestational diabetes, preeclampsia, and maternal inflammation. Maternal obesity also increases the risk of childhood obesity, fetalintrauterine growth restriction (IUGR) and diabetes to the children. Earlier studies demonstrated placental trophoblast apoptosis occurs in patients with preeclampsia and IUGR due to hypoxia. Increased circulating free fatty acids (FFAs) in obesity due to adipose lipolysis induces lipoapoptosis tohepatocytes, cholangiocytes, and pancreatic‐β‐cells. During the third trimester of pregnancy, there is an increase in maternal lipolysis and release of FFAs into the circulation. It is currently unknown if increased FFAs during maternal obesity cause placental cell lipoapoptosis. Palmitate, the predominant circulating FFA, has been shown to induce blastocyst lipoapoptosis and exposure of blastocysts before implantation to palmitate results in a smaller sized fetus. Here, we hypothesis the involvement of trophoblastlipoapoptosis in placental dysfunction during maternal obesity.MethodsApoptosis was assessed by characteristic nuclear morphology staining with DAPI, and caspase 3/7 activity assay. Cleaved PARP and cleaved caspase 3 were examined by Immunoblot.ResultsTreatment of trophoblastcell lines, JEG‐3 and JAR cells with palmitate (PA) or stearate (SA)‐induced trophoblastlipoapoptosis as evidenced by a significant increase in apoptotic nuclearmorphology changes and caspase 3/7 activity. We observed that saturated FFAs caused a concentration‐dependent increase in placental cell lipoapoptosis. We also observed that palmitoleate, a mono‐unsaturated omega‐7 fatty acid mitigated placental cell lipoapoptosis caused due to PA exposure.In conclusion, we show that saturated FFAs induce trophoblast lipoapoptosis. The signaling mechanism of FFA‐induced trophoblast lipoapoptosis are now under investigation.Support or Funding InformationNIH:P20GM104320

Role of the Placental Vitamin D Receptor in Modulating Feto-Placental Growth in Fetal Growth Restriction and Preeclampsia-Affected Pregnancies.

Fetal growth restriction (FGR) is a common pregnancy complication that affects up to 5% of pregnancies worldwide. Recent studies demonstrate that Vitamin D deficiency is implicated in reduced fetal growth, which may be rescued by supplementation of Vitamin D. Despite this, the pathway(s) by which Vitamin D modulate fetal growth remains to be investigated. Our own studies demonstrate that the Vitamin D receptor (VDR) is significantly decreased in placentae from human pregnancies complicated by FGR and contributes to abnormal placental trophoblast apoptosis and differentiation and regulation of cell-cycle genes in vitro. Thus, Vitamin D signaling is important for normal placental function and fetal growth. This review discusses the association of Vitamin D with fetal growth, the function of Vitamin D and its receptor in pregnancy, as well as the functional significance of a placental source of Vitamin D in FGR. Additionally, we propose that for Vitamin D to be clinically effective to prevent and manage FGR, the molecular mechanisms of Vitamin D and its receptor in modulating fetal growth requires further investigation.

Are placental Fas and Fas ligand gene polymorphisms associated with preeclampsia?

ObjectiveIncreased placental trophoblastic apoptosis has been reported in pregnancies complicated by preeclampsia. Fas–Fas ligand is one of the major signal transduction pathways of apoptosis. The objective of this study was to determine if placental Fas and Fas ligand gene polymorphisms differ between patients with and without preeclampsia. Study designForty-five singleton placentas were studied. Twenty-three placentas were from preeclamptic pregnancies and 22 were from normotensive controls. Genotyping was performed for Fas-1377, Fas-691, Fas-670, Fas ligand-844, Fas ligand-1174, Fas ligand-2777. Chi-square and Fisher’s exact tests were used for statistical analysis. ResultsThere were no significant differences in maternal age, parity or race between the two groups. There were no significant differences in genotypes or allele frequencies for the Fas-1377, Fas-691, Fas-670, Fas ligand-844, Fas ligand-1174 and Fas ligand-2777. ConclusionImmune intolerance of maternal and placental interaction plays an important role in the pathogenesis of preeclampsia. Our findings do not support the role of placental Fas and Fas ligand gene polymorphisms in the pathogenesis of preeclampsia.

Hypoxia Induces Autophagy in Primary Human Trophoblasts

Autophagy is a highly regulated and dynamic process that maintains cellular homeostasis and plays a prosurvival role in most cells. Although hypoxia has been shown to induce apoptosis in placental trophoblasts, the hypoxic effect on autophagy has not been studied. We hypothesized that autophagy plays a prosurvival role in the placental trophoblasts by antagonizing hypoxia-induced apoptosis. Our data show that the expression of Light chain 3-II (LC3-II), an autophagic marker and cleaved poly(ADP-ribose) polymerase, an apoptosis marker, are inversely related in cultured trophoblasts. Exposure to rapamycin or hypoxia inactivated mammalian target of rapamycin, as reflected by reduced phosphorylation of ribosomal protein S6, indicating that mammalian target of rapamycin regulates autophagy in cultured cytotrophoblasts. Bafilomycin prevented the degradation of cargo and increased LC3-II and p62 in cytotrophoblasts exposed to hypoxia, revealing enhanced autophagic flux. Importantly, bafilomycin enhanced expression of autophagy-related protein 7 (Atg7), parallel to the increased apoptosis measured by cleaved poly(ADP-ribose) polymerase. LY294002, a phosphatidylinositol 3-kinase inhibitor, increased apoptosis in the trophoblasts under hypoxia or standard conditions. Silencing of Atg7 decreased both apoptosis and LC3-II in the trophoblasts, suggesting a dual role of Atg7 in both autophagy and apoptosis. We conclude that there is a cross talk between autophagy and apoptosis in the placental trophoblasts; autophagy plays a prosurvival role and Atg7 has roles in both autophagy and apoptosis under hypoxia.

PP126. Are placental fas and fas ligand gene polymorphisms associated with preeclampsia?

Increased placental trophoblastic apoptosis has been reported in pregnancies complicated by preeclampsia. Fas-Fas ligand is one of the major signal transduction pathways of apoptosis. To determine if placental Fas and Fas ligand gene polymorphisms differ between patients with and without preeclampsia. Forty-five singleton placentas were studied. Twenty-three placentas were from preeclamptic pregnancies and 22 were from normotensive controls. The study was approved by IRB. Genotyping was performed for Fas-1377, Fas-691, Fas-670, Fas ligand-844, Fas ligand-1174, Fas ligand-2777. Chi-square and Fisher's exact tests were used for statistical analysis. There were no significant differences in maternal age, parity or race between the two groups. There were no significant differences in genotypes or allele frequencies for the placental Fas-1377, Fas-691, Fas-670, Fas ligand-844, Fas ligand-1174 and Fas ligand-2777. Immune intolerance of maternal and placental interaction plays an important role in the pathogenesis of preeclampsia. Our findings do not support the role of placental Fas and Fas ligand gene polymorphisms in the pathogenesis of preeclampsia. The heterogeneous and complex etiology of preeclampsia makes it unrealistic to expect a single nucleotide polymorphism to explain the pathogenesis of this pregnancy complication. This relatively understudied area warrants further investigation.