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Exposure to cigarette smoke is known to induce disease during pregnancy. Recent evidence showed that exposure to secondhand smoke (SHS) negatively impacts fetal and placental weights, leading to the development of intrauterine growth restriction (IUGR). Electronic cigarettes (eCigs) represent a phenomenon that has recently emerged, and their use is also steadily rising. Even so, the effects of SHS or eCigs during gestation remain limited. In the present study, we wanted to characterize the effects of SHS or eCig exposure at two different important gestational points during mouse pregnancy. C57/Bl6 mice were exposed to SHS or eCigs via a nose-only delivery system for 4 days (from 14.5 to 17.5 gestational days (dGA) or for 6 days (from 12.5 dGA to 17.5 dGA)). At the time of necropsy (18.5 dGA), placental and fetal weights were recorded, maternal blood pressure was determined, and a dipstick test to measure proteinuria was performed. Placental tissues were collected, and inflammatory molecules in the placenta were identified. Treatment with SHS showed the following: (1) a significant decrease in placental and fetal weights following four days of exposure, (2) higher systolic and diastolic blood pressure following six days of exposure, and (3) increased proteinuria after six days of exposure. Treatment with eCigs showed the following: (1) a significant decrease in placental weight and fetal weight following four or six days of exposure, (2) higher systolic and diastolic blood pressure following six days of exposure, and (3) increased proteinuria after six days of exposure. We also observed different inflammatory markers associated with the development of IUGR or PE. We conclude that the detrimental effects of SHS or eCig treatment coincide with the length of maternal exposure. These results could be beneficial in understanding the long-term effects of SHS or eCig exposure in the development of placental diseases.

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are placental pathologies known to complicate pregnancy and cause neonatal disorders. To date, there is a limited number of studies on the genetic similarity of these conditions. DNA methylation is a heritable epigenetic process that can regulate placental development. Our objective was to identify methylation patterns in placental DNA from normal, PE and IUGR-affected pregnancies. DNA was extracted, and bisulfite was converted, prior to being hybridized for the methylation array. Methylation data were SWAN normalized and differently methylated regions were identified using applications within the USEQ program. UCSC's Genome browser and Stanford's GREAT analysis were used to identify gene promoters. The commonality among affected genes was confirmed by Western blot. We observed nine significantly hypomethylated regions, two being significantly hypomethylated for both PE and IGUR. Western blot confirmed differential protein expression of commonly regulated genes. We conclude that despite the uniqueness of methylation profiles for PE and IUGR, the similarity of some methylation alterations in pathologies could explain the clinical similarities observed with these obstetric complications. These results also provide insight into the genetic similarity between PE and IUGR and suggest possible gene candidates plausibly involved in the onset of both conditions.

Background: Gestational diabetes mellitus (GDM) is associated with important factors that influence fetal development. Sphingolipids are known to be associated with the development of diabetes. Our objective was to examine ceramide, a key sphingolipid, hyperosmolarity, and apoptosis in placentas from GDM patients treated with insulin or diet., Methods: Ceramide levels were assessed in placental tissues using immunohistochemistry. Immunoblot was performed to quantify serine palmitoyltransferase (SPT), the rate-limiting enzyme in ceramide biosynthesis, NFAT5, SMIT, AR, caspase 3 and the X-linked inhibitor of apoptosis. Trophoblast cells were treated with insulin or ceramide and assessments for mitochondrial respiration, caspase 3 and XIAP were also performed., Results: Immunohistochemistry showed increased ceramides in the placental villous trophoblasts of the insulin-treated GDM patients. Nuclear SPT was upregulated only in the insulin-treated GDM placenta when compared to controls. Nuclear NFAT5 was also increased in the GDM placenta. Active caspase 3 was elevated in placentas from both insulin- and diet-treated GDM patients. Mitochondrial respiration was decreased in trophoblasts treated with ceramide. Active caspase was not changed while XIAP protein was increased in trophoblasts treated with ceramide., Conclusions: Our findings confirm the presence of ceramide in the human placenta of control and GDM patients. Furthermore, we conclude that ceramide is increased in the placental trophoblast during insulin treatment and that its upregulation correlates with elevated NFAT5, SMIT, increased apoptosis and decreased trophoblast mitochondrial respiration.

Cigarette smoke is known to be a risk for the development of intrauterine growth restriction (IUGR). Our objective was to assess the effects of secondhand smoke (SHS) during pregnancy and to what extent it regulates the activation of mTOR family members and murine trophoblast invasion. Mice were treated to SHS for 4 days. Placental and fetal weights were recorded at the time of necropsy. Immunohistochemistry was used to determine the level of placental trophoblast invasion. Western blots were utilized to assess the activation of caspase 3, XIAP, mTOR, p70 and 4EBP1 in treated and control placental lysates. As compared to controls, treated animals showed: (1) decreased placental (1.4-fold) and fetal (2.3-fold) weights (p < 0.05); (2) decreased trophoblast invasion; (3) significantly decreased active caspase 3 (1.3-fold; p < 0.02) and increased active XIAP (3.6-fold; p < 0.05) in the placenta; and (4) a significant decrease in the activation of placental mTOR (2.1-fold; p < 0.05), p70 (1.9-fold; p < 0.05) and 4EBP1 (1.3-fold; p < 0.05). Confirmatory in vitro experiments revealed decreased trophoblast invasion when SW71 cells were treated with 0.5 or 1.0 % cigarette smoke extract (CSE). Similar to primary smoking, SHS may induce IUGR via decreased activation of the mTOR family of proteins in the placenta. Increased activation of the placental XIAP protein could be a survival mechanism for abnormal trophoblast cells during SHS exposure. Further, CSE reduced trophoblast invasion, suggesting a direct causative effect of smoke on susceptible trophoblast cells involved in IUGR progression. These results provide important insight into the physiological consequences of SHS exposure and smoke-mediated placental disease.

The Pyruvate kinase isozymes M2 (PKM2) protein is a metabolic enzyme that regulates the final step of glycolysis. This enzyme is present in highly proliferating cells and is expressed in the placenta. We recently demonstrated upregulated placental PKM2 during human intrauterine growth restriction (IUGR). Our current objective was to determine PKM2 regulation of trophoblast invasion, trophoblast PKM2 localization as well as mTOR protein expression, and to determine effects of activation of PKM2 during IUGR. Human placental tissues were obtained and analyzed by immunohistochemistry and western blot. Trophoblast cells were cultured in normoxic and hypoxic conditions and real time cell invasion and PKM2 protein were determined during activation (Fructose-6-bisphosphate; FBP6) or inhibition (Shikonin) of PKM2. In vivo studies determined the effects of PKM2 activation on placental and fetal weights. IUGR samples had elevated levels of p-PKM2. Different trophoblast PKM2 localization and expression was observed during normoxia and hypoxia. Decreased trophoblast invasion and PKM2 expression was observed during mTOR inhibition. Protection from decreased placental and fetal weights was observed by PKM2 activation. We conclude that PKM2 regulates trophoblast cell invasion depending on its subcellular location. Our results suggest that PKM2 regulation in trophoblast cells is more directly affected during hypoxia and its expression is regulated by mTOR activity. Additionally, we conclude that activation of PKM2 could reverse and/or rescue the deceased placental and fetal weights observed during IUGR. These results suggest that PKM2 could be a mediator of trophoblast cell invasion and its abundance influences the development of complicated pregnancies like IUGR. [ABSTRACT FROM AUTHOR]

Problem Receptor for advanced glycation end products (RAGE) is a receptor implicated in the modulation of inflammation. Inflammation has been associated with pregnancy pathologies including preeclampsia (PE), intrauterine growth restriction (IUGR), and gestational diabetes mellitus (GDM). Our objective was to examine placental RAGE expression in PE, IUGR, and GDM complications. Method of study Human placental tissues were obtained for RAGE determination using Q-PCR, immunohistochemistry, and Western blot. Invasive trophoblast cells were cultured and treated with AGES for RAGE activation studies. Results Compared to control placenta, we observed: (i) decreased RAGE gene expression during GDM, (ii) increased RAGE protein in the PE placenta, and (iii) decreased RAGE protein in the IUGR placenta. In trophoblast cells exposed AGEs, we observed: (i) decreased trophoblast invasion, (ii) increased c-Jun N-terminal kinases (JNK) and Extracellular signal-regulated kinases (ERK), and (iii) increased TNF-α and IL-1β secretion. Conclusion We conclude that placental RAGE is activated during PE and that RAGE-mediated inflammation in the trophoblast involves increased pro-inflammatory cytokine secretion. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin ( mTOR) is a protein that regulates cell growth in response to altered nutrient and growth factor availability. Our objective was to assess activated mTOR and its intracellular intermediates p70, and 4 EBP1 in placental and invasive trophoblast cells in a hypoxia-induced model of intrauterine growth restriction ( IUGR) in rats. Rats were treated with hypoxia (9%) for 4 days. Placental and fetal weights, as well as conceptus numbers were recorded at the time of necropsy. Immunohistochemistry was used to determine the level of trophoblast invasion and apoptosis. Western blots were used to determine the activation of mTOR, p70, and 4 EBP1 in the placenta and the uterine mesometrial compartment. We observed (1) decreased placental (21%) and fetal (24%) weights ( P < 0.05); (2) decreased trophoblast invasion; (3) significantly increased active 4 EBP1 (28%; P < 0.05) in invasive trophoblast cells yet no changes in the activation of mTOR and p70 proteins; and (4) a significant decrease in the activation of mTOR (48%; P < 0.05) with no differences in p70 or 4 EBP1 activation in the placenta. We conclude that the development of IUGR is correlated with decreased activation of the mTOR protein in the placenta and increased 4 EBP1 activity in the invading trophoblast. These results provide important insight into the physiological relevance of these pathways. Furthermore, modification of these and other related targets during gestation may alleviate IUGR severity. [ABSTRACT FROM AUTHOR]

Impaired DNA damage responses are associated with several diseases, including pregnancy complications. Recent research identified an ATM-kinase dependent function for the nuclear isoform of the receptor for advanced glycation end-products (RAGE) during double strand break (DSB)-repair. RAGE contributes to end-resectioning of broken DNA sites by binding with the MRE11-Rad50-Nbs1 (MRN) complex. Placental research is limited regarding the impact of genomic instability and the mechanism for potential repair. We tested the hypothesis regarding the involvement of RAGE during the repair of placental DNA-DSBs. We first identified that the pregnancy complications of PE and preterm labor (PTL) experience loss of genomic integrity and an in vitro trophoblast cell model was used to characterize trophoblast DSBs. Colocalized immunofluorescence of γ-H2AX and RAGE support the potential involvement of RAGE in cellular responses to DNA-DSBs. Immunoblotting for both molecules in PE and PTL placenta samples and in trophoblast cells validated a connection. Co-immunoprecipitation studies revealed interactions between RAGE and pATM and MRE11 during DNA-DSBs. Reduced cellular invasion confirmed the role of genomic instability in trophoblastic function. Collectively, these experiments identified genomic instability in pregnancy complications, the impact of defective DNA on trophoblast function, and a possible RAGE-mediated mechanism during DNA-DSB repair. [ABSTRACT FROM AUTHOR]