Autophagy induced by HIF1α overexpression supports trophoblast invasion by supplying cellular energy.

Extravillous trophoblasts (EVTs) characterize the invasion of the maternal decidua under low oxygen and poor nutrition at the early feto-maternal interface to establish a successful pregnancy. We previously reported that autophagy in EVTs was activated under 2% O2 in vitro, and autophagy activation was also observed in EVTs at the early feto-maternal interface in vivo. Here, we show that autophagy is an energy source for the invasion of EVTs. Cobalt chloride (CoCl2), which induces hypoxia inducible factor 1α (HIF1α) overexpression, activated autophagy in HTR8/SVneo cells, an EVT cell line. The number of invading HTR8-ATG4B(C74A) cells, an autophagy-deficient EVT cell line, was markedly reduced by 81 percent with the CoCl2 treatment through the suppression of MMP9 level, although CoCl2 did not affect the cellular invasion of HTR8-mStrawberry cells, a control cell line. HTR8-ATG4B(C74A) cells treated with CoCl2 showed a decrease in cellular adenosine triphosphate (ATP) levels and a compensatory increase in the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2RX7), which is stimulated with ATP, whereas HTR8-mStrawberry cells maintained cellular ATP levels and did not affect P2RX7 expression. Furthermore, the decreased invasiveness of HTR8-ATG4B(C74A) cells treated with CoCl2 was neutralized by ATP supplementation to the level of HTR8-ATG4B(C74A) cells treated without CoCl2. These results suggest that autophagy plays a role in maintaining homeostasis by countervailing HIF1α-mediated cellular energy consumption in EVTs.

Impaired autophagy by soluble endoglin, under physiological hypoxia in early pregnant period, is involved in poor placentation in preeclampsia.

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.

Hyperglycemia perturbs biochemical networks in human trophoblast BeWo cells.

Determining the effects of hyperglycemia on gene expression in placental trophoblast is important to gain a better understanding of how diabetes adversely affects pregnancy. In this study, we examined whether exposure to high glucose during forskolin-induced differentiation affects gene expression in differentiated trophoblasts. Human trophoblast BeWo cells were differentiated under low glucose (LG: 11 mM) or high glucose (HG: 25 mM) conditions. Gene expression was analyzed using a GeneChip system and the obtained data were analyzed using Ingenuity Pathways Analysis. In HG conditions, there were marked alterations in gene expression in differentiated BeWo cells compared with LG conditions. In particular, BeWo cells responded to HG with major changes in the expression levels of cell cycle- and metabolism-related genes. We selected the aromatase gene for further investigation of the molecular mechanisms. Mannitol or 3-O-methylglucose did not mimic the expression changes caused by HG, indicating that the effect of glucose was not due to a difference in osmotic pressure, and that glucose metabolism plays an essential role in inducing the HG effects. Cotreatment with N-acetylcysteine reduced the effect of HG on aromatase gene expression, suggesting that hyperglycemia may perturb biochemical networks because of the elevation of oxidative stress. Overall, our results will aid further understanding of the effect of diabetes on the regulation of trophoblast differentiation and function.

Granulysin produced by uterine natural killer cells induces apoptosis of extravillous trophoblasts in spontaneous abortion.

Immune changes are known to occur in recurrent spontaneous abortion, but it is unclear whether either maternal natural killer (NK) cells or T cells attack fetus-derived trophoblasts. To clarify the immunological causes of spontaneous abortion, we examined the relationship between cytotoxic granule proteins in decidual lymphocytes, such as granulysin, granzyme B, and perforin, and the induction of apoptosis in extravillous trophoblasts (EVTs). The number of granulysin-positive CD56(bright) NK cells increased significantly in the decidua basalis during spontaneous abortion compared with normal pregnancy; however, granzyme B- and perforin-positive cells did not change. Interestingly, the expression of granulysin was also detected in the nuclei of EVTs in spontaneous abortion samples. When IL-2-stimulated CD56(bright) NK cells were cocultured with EVT cells (HTR-8/SV40neo), granulysin was found initially in the cytoplasm and then accumulated in the nuclei of the HTR-8/SV40neo cells. Furthermore, transfected cells expressing a GFP-granulysin fusion protein induced apoptosis in HTR-8/SV40neo cells independently of caspases. Our results suggest that granulysin-positive uterine NK cells attack EVTs; subsequently, the uNK-derived granulysin actively accumulates in the nuclei of EVTs, causing the death of EVTs due to apoptosis. These data support a new apoptosis pathway for trophoblasts via uNK-derived granulysin, suggesting that granulysin is involved in spontaneous abortion.

Blocking on the CXCR4/mTOR signalling pathway induces the anti-metastatic properties and autophagic cell death in peritoneal disseminated gastric cancer cells.

Patients with advanced gastric carcinoma, especially peritoneal dissemination, have a poor prognosis even after any treatment. Chemokines are now known to play an important role in cancer growth and metastasis. We recently reported that the chemokine CXCL12 plays an important role in the development of peritoneal carcinomatosis from gastric carcinoma. In this study, we investigated signalling pathway involved in the peritoneal carcinomatosis induced by chemokine CXCL12. Akt was rapidly and strongly phosphorylated by chemokine CXCL12. CXCL12 also induced the activation of p70S6K (S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) included in mammalian target of rapamycin (mTOR) pathways which are located downstream of Akt, resulting in enhancements of metastatic properties such as MMP production, cell migration and cell growth in peritoneal disseminated gastric cancer, NUGC4 cells. Furthermore, mTOR inhibitor rapamycin not only drastically inhibited migration and MMP production, but also induced type II programmed cell death, autophagic cell death. In the present study, we have shown for the first time that the mTOR pathway plays a central role in the development of peritoneal carcinomatosis, and blocking this pathway induces autophagic cell death in disseminated gastric cancer. Therefore, blocking on the CXCR4/mTOR signalling pathway may be useful for the future development of a more effective therapeutic strategy for gastric cancer involved in peritoneal dissemination.

Fetal hydrops associated with congenital pulmonary myofibroblastic tumor.

Abstract We report on a fetus with a congenital pulmonary myofibroblastic tumor, the prenatal detection of which with imaging modalities has not been reported up until now. A 32-year-old woman was referred to our hospital at 29 weeks' gestation because of severe fetal hydrops. Sonograms and magnetic resonance imaging showed a large solid tumor in the left thorax. The fetus died in utero the next day. Autopsy confirmed that the tumor was confined to the lower lobe of the left lung, and circulatory insufficiency from compression by the tumor was considered to be the cause of fetal hydrops and demise. Histologic examination revealed that the tumor was composed of uniform short spindle cells with no atypia and a large number of vessels. In addition, with immunohistochemical studies, the tumor cells were stained for calponin but not for cluster differentiation (CD)-31, CD-34, alpha-smooth muscle actin or S-100.

Massive cystic lymphangiomas of a fetus.

We present a fetus with progressive massive subcutaneous lymphangiomas leading to intrauterine death. A 28-year-old woman was referred to our hospital because of a precordial cystic mass of the fetus. An ultrasound revealed lymphangiomas extending from bilateral axillae to the anterior chest wall. At 18 weeks' gestation, amniocentesis was performed and the karyotype of the fetus was found to be normal 46, XY. Thereafter the lesions increased in size gradually and spread over the body. Amniotic fluid decreased, pericardial, and pleural effusion appeared, and cardiomegaly became evident. The fetus died in utero at 25 weeks' gestation. Postmortem examination revealed a male fetus surrounded with multicystic soft masses spreading over the body, and syndactyly (left third and fourth fingers) was present. Histologically, a number of irregularly dilated lymphatics extended through subcutaneous tissues to the skeletal muscles. No communications between the cysts and the thoracic or abdominal cavity existed, and no lymphatic dilations in the viscera were confirmed. As far as we know, such conditions have rarely been reported. Considering that in previous literature, a favorable prognosis of a fetus with an atypically located (lateral cervical or non-cervical) lymphangioma with a normal karyotype has been reported, our case may be included in a distinct pathological entity. When we find a lymphangioma in a fetus, careful follow-up by ultrasound is mandatory.

Monochorionic triplet pregnancy complicated by severe fetofetal transfusion.

We report two cases of fetofetal transfusion in monochorionic triamniotic triplet pregnancies. Case 1: At 23 weeks' gestation an amnioreduction was carried out. Three days later, the donor triplet died in utero. Immediately after that, a cardiotocogram of the triplet who had been thought to be unaffected by the fetofetal transfusion, showed a non-reassuring fetal status. Although cesarean section was carried out, none of the triplets survived. Case 2: At 24 weeks' gestation a woman was transferred to our center because of fetofetal transfusion in monochorionic triplets. Cesarean section was carried out. However, the recipient died on the 75th day after birth, and the others had neurological problems. Previous reports on fetofetal transfusion in triplets are very limited. The prognosis of this condition has been reported to be severe, irrespective of chorionicity, gestational age at delivery, maternal age, and parity. When managing a monochorionic or dichorionic triplet pregnancy, serial and careful ultrasound examination is mandatory to find early symptoms of this serious condition.

Two cases of a fetus with sirenomelia sequence.

We report two cases of a fetus with sirenomelia sequence which showed oligohydramnios and a single umbilical artery. The first case was of a single fetus with symelia apus and only one leg. Prenatal diagnosis of this case was possible. The second case was of a dichorionic-diamniotic twin pregnancy in which one fetus had symelia dipus with two fused lower extremities. Prenatal diagnosis of the condition was not made. In both cases, the fetuses died shortly after birth from respiratory distress due to severe pulmonary hypoplasia. Absence of urinary tract, imperforate anus, and spine deformity were confirmed in both cases. Although prenatal diagnosis of symelia dipus seems difficult, this condition must be considered in a fetus with severe oligohydramnios.